BACKGROUND OF THE INVENTION <EOH>1.Field of Invention The present invention relates generally to the field of network communications.","More specifically, the present invention is related to a system and method for traversing firewalls and network address translators (NATs).","2.Discussion of Prior Art NATs and firewalls present a challenge to a network software programming, while their functions and operations are different: firewalls filter information into and out of the private network, while NATs hide or encapsulate a private network behind a single (or few) “real” Internet Protocol addresses.","Their effect on many network applications is the same: The inability to send and receive information when receiving information using UDP (e.g., UDP data-grams coming into the private network).","The inability to send and receive information when opening TCP communications into the private network.","Each of the below described references teach the method of firewalls in general.","However, none of the references provide or suggest the present invention method of ATM over IP traversing firewalls and network address translators (NATs).","U.S. Pat.","No.","5,898,830, assigned to Network Engineering Software describes a system, which allows connectionless traffic across a firewall.","Rule checking is performed on the first packet entering, and if it is determined that the packet needs to be sent, a virtual host sends it to the destination computer.","A time limit is set and so long as the set time limit does not run out, the communication is allowed.","Addressing is accomplished utilizing name based addressing for end-to-end communication, with virtual hosts/DNS servers providing the intermediate address routing information.","A connection type session does not appear to be initiated for the UDP transport.","U.S. Pat.","No.","5,915,087 discloses a firewall system, which allows communication, using a connectionless protocol.","The firewall holds a list of servers located on the private side, and intercepts any communications addressed to the servers.","The firewall then binds a port and notes it in a link table.","The packet is passed to a stack, on the private side, which forwards the packet to the server.","Any communications from the server to the originating client is sent to the firewall, where the originating clients address is determined using the link table.","U.S. Pat.","No.","5,778,174 describes a system, which utilizes an external machine, located on a public network to bypass a router firewall.","A client on the public network connects to the external machine.","A private channel is opened between the external machine and a machine internal to the private network.","The internal machine connects to the destination server, and communication between the client and server is conducted through the external and internal machines.","U.S. Pat.","No.","5,941,988 provides for a proxy system that “glues” together two separate TCP connections terminating at a common host (proxy).","When communications from one connection are received at the proxy, the headers are altered to address the socket at the end of the second connection, and the sequence numbers of the first connection are mapped to the sequence space of the second connection.","The non-patent literature entitled, “A Weakness in the 4.2 BSD Unix TCP/IP Software” describes the spoofing of a trusted host to communicate with a system, having a list of the trusted hosts, from a host that is not on the trusted list.","It should however be noted that the prior art described above fails to provide many features, for example an explicit recitation of opening a connection-oriented session in order to allow connectionless data-grams to pass through a NAT/firewall is not provided.","Additionally, none of the prior art described above uses a proxy server to exchange respective address information between two hosts and the hosts communicating directly via the address information and spoofing the proxy, in order to traverse at least one firewall.","Whatever the precise merits, features and advantages of the above cited references, none of them achieve or fulfills the purposes of the present invention."],["<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides for a method and a system for allowing an incoming UDP packet to traverse a NAT/firewall comprising, opening a TCP connection and utilizing a Raw-IP interface which builds the UDP packet utilizing the parameters of the TCP connection (e.g., session number, port, etc.).","Furthermore, the present system provides for a method and system for allowing communication between two machines, at least one of which is behind a firewall.","Connections are established between each machine and a proxy server sitting on a public network.","The proxy then communicates the port and address information of each machine to the other machine, after which, each machine sends directly to each other using the supplied port and address information, while using the proxy servers port and address information as the source port and address."],["RELATED APPLICATIONS The present application claims the benefit of provisional patent application “Traversing Firewalls and NATs”, Ser.","No.","60/255,422, filed Dec. 14, 2000.In addition, this application incorporates by reference, co-pending U.S. patent application Ser.","No.","09/867,371, filed May 29, 2001.BACKGROUND OF THE INVENTION 1.Field of Invention The present invention relates generally to the field of network communications.","More specifically, the present invention is related to a system and method for traversing firewalls and network address translators (NATs).","2.Discussion of Prior Art NATs and firewalls present a challenge to a network software programming, while their functions and operations are different: firewalls filter information into and out of the private network, while NATs hide or encapsulate a private network behind a single (or few) “real” Internet Protocol addresses.","Their effect on many network applications is the same: The inability to send and receive information when receiving information using UDP (e.g., UDP data-grams coming into the private network).","The inability to send and receive information when opening TCP communications into the private network.","Each of the below described references teach the method of firewalls in general.","However, none of the references provide or suggest the present invention method of ATM over IP traversing firewalls and network address translators (NATs).","U.S. Pat.","No.","5,898,830, assigned to Network Engineering Software describes a system, which allows connectionless traffic across a firewall.","Rule checking is performed on the first packet entering, and if it is determined that the packet needs to be sent, a virtual host sends it to the destination computer.","A time limit is set and so long as the set time limit does not run out, the communication is allowed.","Addressing is accomplished utilizing name based addressing for end-to-end communication, with virtual hosts/DNS servers providing the intermediate address routing information.","A connection type session does not appear to be initiated for the UDP transport.","U.S. Pat.","No.","5,915,087 discloses a firewall system, which allows communication, using a connectionless protocol.","The firewall holds a list of servers located on the private side, and intercepts any communications addressed to the servers.","The firewall then binds a port and notes it in a link table.","The packet is passed to a stack, on the private side, which forwards the packet to the server.","Any communications from the server to the originating client is sent to the firewall, where the originating clients address is determined using the link table.","U.S. Pat.","No.","5,778,174 describes a system, which utilizes an external machine, located on a public network to bypass a router firewall.","A client on the public network connects to the external machine.","A private channel is opened between the external machine and a machine internal to the private network.","The internal machine connects to the destination server, and communication between the client and server is conducted through the external and internal machines.","U.S. Pat.","No.","5,941,988 provides for a proxy system that “glues” together two separate TCP connections terminating at a common host (proxy).","When communications from one connection are received at the proxy, the headers are altered to address the socket at the end of the second connection, and the sequence numbers of the first connection are mapped to the sequence space of the second connection.","The non-patent literature entitled, “A Weakness in the 4.2 BSD Unix TCP/IP Software” describes the spoofing of a trusted host to communicate with a system, having a list of the trusted hosts, from a host that is not on the trusted list.","It should however be noted that the prior art described above fails to provide many features, for example an explicit recitation of opening a connection-oriented session in order to allow connectionless data-grams to pass through a NAT/firewall is not provided.","Additionally, none of the prior art described above uses a proxy server to exchange respective address information between two hosts and the hosts communicating directly via the address information and spoofing the proxy, in order to traverse at least one firewall.","Whatever the precise merits, features and advantages of the above cited references, none of them achieve or fulfills the purposes of the present invention.","SUMMARY OF THE INVENTION The present invention provides for a method and a system for allowing an incoming UDP packet to traverse a NAT/firewall comprising, opening a TCP connection and utilizing a Raw-IP interface which builds the UDP packet utilizing the parameters of the TCP connection (e.g., session number, port, etc.).","Furthermore, the present system provides for a method and system for allowing communication between two machines, at least one of which is behind a firewall.","Connections are established between each machine and a proxy server sitting on a public network.","The proxy then communicates the port and address information of each machine to the other machine, after which, each machine sends directly to each other using the supplied port and address information, while using the proxy servers port and address information as the source port and address.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 illustrates the Intranet to Internet data transfer scenario.","FIG.","2 illustrates the Internet to Intranet data transfer scenario.","FIG.","3 illustrates the Intranet to Intranet data transfer scenario.","FIG.","4 illustrates a bi-directional connection, using TCP and HTTP, communicating indirectly with the proxy.","FIG.","5 illustrates TCP spoofing of the present invention.","FIG.","6 illustrates TCP spoofing of the present invention in the presence of a packet forwarder.","FIG.","7 illustrates the methodology associated with the present invention.","DESCRIPTION OF THE PREFERRED EMBODIMENTS While this invention is illustrated and described in a preferred embodiment, the invention may be produced in many different configurations, forms and materials.","There is depicted in the drawings, and will herein be described in detail, a preferred embodiment of the invention, with the understanding that the present disclosure is to be considered as an exemplification of the principles of the invention and the associated functional specifications for its construction and is not intended to limit the invention to the embodiment illustrated.","Those skilled in the art will envision many other possible variations within the scope of the present invention.","When a communicating device, such as the Internet phone or a Voice-over-IP Gateway or an IETF MGCP Gateway or an ITU-T H.248 Gateway or a PacketCable Residential Gateway or a CPE Gateway (Customer premises equipment Gateway), opens a signaling connection from a private network to a public network, the TCP channel is bi-directional, and therefore the signaling protocol can execute in both directions.","This also allows HTTP to work behind network address translators (NATs) and Firewalls.","Connections are always opened from the private network to the public network; taking advantage of the fact that TCP data communications are bi-directional.","NATs present an additional translation step when communicating.","NATs map the source addresses (in the private network) of the originating computer into a public address and a port number on the public interface of the NAT.","As long as TCP is used, the translation can be done in reverse, as the TCP channel is bi-directional.","Multimedia signaling and media streaming is usually UDP-based for better efficiency, which introduces the problem—the ingress system sends UDP packets to the public interface on the NAT, and the NAT has no automatic method to map this UDP data-gram to the actual computer that is supposed to receive that data-gram.","The solution provided by the present invention is to stream audio and video (and other time-sensitive data) over TCP, but TCP streaming and windowing mechanizing hurts the real-time performance.","The present invention opens a TCP connection as usual (using TCP), and then switches to a Raw-IP interface that sends Raw-IP data-grams that are legal TCP messages using just opened TCP channel parameters (e.g., session number, port, etc.)","To an intermediate system, these messages will look like standard TCP messages, but as they are sent using Raw-IP, the usual timing issues that TCP introduces to real-time media streaming are not in place.","Thus, the present invention uses the protocol software to “spoof” the TCP channel to enable real-time TCP communications.","It is impossible with NATs and Firewalls to open ingress connections (e.g., it is impossible to open a TCP connection to a computer behind a Firewall or the NAT.","Thus: 1.It is impossible to originate communications from the public network into the private network.","2.It is impossible to originate communications from a private network (to a public network) to another private network.","Thus, the present invention uses a server proxy that both communication parties open their TCP channels to (using the previous procedure).","Then, the proxy communicates to each party the other party's source address/port (of the TCP channel).","Finally, each communication element sends information to the other party using the server proxy source address/port.","It should be noted that packets are sent directly between the communicating entities, as the proxy is only used to hold the TCP state to “spoof” the NATs and Firewalls.","In the preferred embodiment of the present invention, full communication is possible between all types of private and public networks, as long as outgoing TCP channel establishment is allowed.","In another embodiment, the server proxy and originating clients use TCP/HTTP, which is universally supported, and in this instance, all information is tunneled over the simulated TCP/HTTP channel.","In the Internet as it exists today, using the small address space provided by IPv4, many networks deploy NAT (network address translation) devices to enlarge the internal address space.","In addition, many networks deploy firewall devices to block intrusions and hacking.","Many firewalls also support integrated NAT capabilities.","The end-result of both types of devices is that ingress traffic (one originating outside the network and destined into the network) is usually blocked, as incoming connections are usually blocked for firewalls and are impossible to complete on NAT devices, and as the originating (outside the NAT) IP host is unaware of the destination internal IP address.","Thus, users cannot place audio/video calls from NAT protected networks (as the audio and video will not penetrate back into the network from the remote called host), and in many cases users behind corporate firewalls are blocked from using such services.","A communications protocol such as the TrulyGlobal™ Protocol (TGP), (as described in the related application, “Communication Protocol”) can be used in conjunction with the present invention to operate over standard HTTP and remote TGP servers to use the HTTP back-channel to send information to the client; and ensuring that all actions carried by TGP traverse both NATs and firewalls.","Intranet, as defined in this application, is a network that is protected by a NAT or a firewall device, and blocks all incoming traffic into the protected network (e.g., TCP connections cannot be initiated into the network, and UDP traffic will be blocked at the entry-point into the network).","Similarly, Internet is defined as a public addressed, unprotected network, where fill IP communication is possible.","In the Intranet to Internet scenario, as illustrated in FIG.","1, a user inside an Intranet is attempting to call a user that is outside the Intranet, and the remote user is in the public Intranet.","The problem encountered is that while the call will be successfully set-up (as the originating host is allowed to open connection to the outside network), audio and/or video data will not be able to get back into the Intranet, hence the caller will not be able to hear and/or see the called device.","Similarly, in an Internet to Intranet scenario, as illustrated in FIG.","2, the caller cannot open a signaling channel at all to the called device, as ingress connections into the Intranet are not allowed.","Lastly, in the Intranet to Intranet scenario, as illustrated in FIG.","3, the same end-effect as the previous scenario happens, e.g., the caller cannot open a signaling channel at all to the called device, as ingress connections into the Intranet from the Internet are not allowed.","The solution provided for by the present invention uses TCP and potentially HTTP, and a service is provided outside the Intranet (in the public Internet) to help both end-points to complete calls.","The first assumption made is that the clients inside the Intranet can initiate TCP or at least TCP/HTTP specifically to the public Internet, so some form of communications is possible.","HTTP can be used to insure safe traversal via HTTP proxies.","Once a TCP/HTTP connection is available, bi-directional communications are possible.","Outwards messages use standard HTTP commands to request resources (using URLs), and incoming information flow returns using the HTTP reply channel (as TCP/HTTP is full-duplex).","Furthermore, at any time, as illustrated by FIG.","4, the caller can initiate a TCP/HTTP connection (or a plain TCP connection) to a service that resides in the public Internet, and that service is responsible to “proxy” the request (using the reply leg of the remote HTTP session) to the called-device.","When both devices have bi-directional connections to the proxy, they can communicate indirectly via the proxy.","While the present invention shows how proxy-based communication can work, the problem when the communication between the two hosts has to flow via the proxy, which adds delay and has limited scalability.","The solution as per the present invention is to spoof the TCP session to allow direct TCP communications between the two machines.","This scenario is illustrated in FIG.","5.When a machine behind a proxy, NAT or firewall establishes a session with the outside world, the session is mapped on the outside of Intranet 1 and 2 on the public interface address(s) to an internal connection between Host 1 and 2 and their gateways to the Internet.","Sending correctly formed TCP packets to that interface will result in the gateway forwarding these packets to the correct host inside the private network.","The sequence is as follows: 1.A session is established from Host 1 in Intranet 1 to the Proxy (AB session).","2.A session is established from Host 2 in Intranet 2 to the Proxy (DC session).","3.The Address BP (public side of session A) is found by inspecting the source address/port of B.","4.The Address of CP (public side of session D) is found by inspecting the source address/port of C. 5.The external mapped addresses are provided to the other hosts, i.e., Host 1 is provided with address/pair CP and Host 2 is provided with address/port pair BP.","TCP session B parameters are provided to host 2.TCP session C parameters are provided to host 1.6.Hosts 1 and 2 will spoof TCP packets for sessions B and C, sent to target address/port pairs BP and CP.","This traffic will go directly between the two networks and not via the Proxy.","In effect, a virtual TCP session C1/B1 is created by combining the two existing TCP sessions C and B.","Once the spoofed TCP session is handed over to Hosts 1 and 2, the Proxy should not send any information on that session, as session parameters may be out-of-date.","The session is kept open for the duration Hosts 1 and 2 requires it, and will be closed by either Host when required.","The proxy is only used for establishing session, and does not use the session for anything else once it is “handed over”.","In some cases the internal network will filter spoofed packets (for security, e.g., hack prevention) and therefore will not let the packets with the spoofed source address leave the internal network.","In such cases, as illustrated by FIG.","6, the TCP connections will be handed over to a packet forwarder (that resides in the same server or a separate server) that handles the packet interchange.","One of the side effects of spoofing the TCP session between Hosts 1 and 2 is that the TCP session parameters can be changed or completely ignored, as long as packets are synthetically correct (as per TCP), they can be sent without consideration to window-sizes, exponential back-off algorithms or slow-start mechanisms.","When such a spoofed TCP session is in place, it can be used to transmit both audio and video with the same time of performance that is expected from UDP.","The session-establishment procedures described above allow any session to be established between any two computers.","This is done as a result of Host 1 calling Host 2 (or the reverse).","The calling host will send a Call-Establishment message to the Proxy, which will (pending, any policy decision) forward the request to the called Host.","The called host will receive the Call Answer transaction over the back-channel of the session it already has with the Proxy, requesting it to answer the call.","If the called host responses positively, one or more media channel(s) will be established between Host 1 and 2, with the help of the proxy as required by the session's parameters (audio only, audio and video, etc).","It should be noted that both IETF SIP and ITU-T H.323 signaling can be used, but are not required.","In one embodiment, the Proxy contains all the required functionality (e.g., signaling a RTP:Address:Port destination instead of a H323:Address:Port destination).","Furthermore, one skilled in the art can recognize that IETF SIP (by manipulating IETF Session Description Protocol (SDP) parameters) and ITU H.323 (by Manipulating ITU-T H.245 OpenLogicalChannel or FastStart parameters) can be used, with minor changes, to accomplish the required signaling.","The present invention is implemented using a raw-IP interfaces that spoofs the TCP sessions.","A limited TCP stack is implemented that creates synthetically correct TCP packets, to insure the packets are interpreted and forwarded correctly by the NATs, proxies and firewalls in the way.","Such a spoofed-TCP stack does not need to support any reliable transmission, as it is only used for real-time sensitive transmission purposes.","FIG.","7 summarizes the methodology 700 associated with the present invention.","In step 702, both hosts establish a connection (e.g., TCP connection or TCP/HTTP connection) with a TCP proxy server.","Next, in step 704, external mapped addresses BP and CP associated with the firewalls of both hosts are identified.","Subsequently, in step 706, the identified external mapped addresses are exchanged between the two hosts.","Lastly, the TCP packets are spoofed to transmit the data (e.g., streaming multimedia data) between the hosts.","Furthermore, the present invention includes a computer program code based product, which is a storage medium having program code stored therein, which can be used to instruct a computer to perform any of the methods associated with the present invention.","The computer storage medium includes any of, but not limited to, the following: CD-ROM, DVD, magnetic tape, optical disc, hard drive, floppy disk, ferroelectric memory, flash memory, ferromagnetic memory, optical storage, charge coupled devices, magnetic or optical cards, smart cards, EEPROM, EPROM, RAM, ROM, DRAM, SRAM, SDRAM, or any other appropriate static or dynamic memory, or data storage devices.","Implemented in computer program code based products are software modules for: aiding in establishing a communication link with a proxy server over a network, wherein a first and second device can access the network over a firewall; inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; notifying said first device regarding said identified external mapped address CP and notifying said second device regarding said identified external mapped address BP; and aiding said first or second device in spoofing TCP packets via transmitting data with said notified external mapped address as the destination address.","Also implemented in computer program based products are software modules for: aiding in establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; notifying said packet forwarder regarding said identified external mapped addresses CP and BP, and forwarding TCP packets via transmitting data with said packet forwarder as said destination address and computer readable program code aiding said packet forwarder in forwarding said data with CP as the destination address, or forwarding TCP packets via transmitting data with said packet forwarder as said destination address and computer readable program code aiding said packet forwarder in forwarding said data with BP as the destination address.","CONCLUSION A system and method has been shown in the above embodiments for the effective implementation of a method and a system for traversing firewalls and network address translations (NATs).","While various preferred embodiments have been shown and described, it will be understood that there is no intent to limit the invention by such disclosure, but rather, it is intended to cover all modifications and alternate constructions falling within the spirit and scope of the invention, as defined in the appended claims.","For example, the present invention should not be limited by type of firewall, type of network address translation device, location of packet forwarder, software/program, computing environment, or specific computing hardware.","The above enhancements are implemented in various computing environments.","For example, the present invention may be implemented on a multi-nodal system (e.g., LAN) or networking system (e.g., Internet, WWW, wireless web).","All programming, and data related thereto are stored in computer memory, static or dynamic, and may be retrieved by the user in any of: conventional computer storage, display (i.e., CRT) and/or hardcopy (i.e., printed) formats.","The programming of the present invention may be implemented by one of skill in the art of network communications."]],"string":"[\n [\n \"Traversing firewalls and nats\",\n \"An incoming UDP packet is allowed to traverse a network address translation (NAT) device or a firewall, wherein first, a TCP connection is opened and a Raw-IP interface is utilized to build the UDP-like packet using the parameters of the TCP connection (e.g., session number, port, etc.)\",\n \"Furthermore, when one of two communicating machines is behind a firewall, a connection is established between each of the machines and a proxy server located in a public network.\",\n \"The proxy then communicates the port and address information while using the proxy server's port and address information as the source port and address, or provides both with an address of an appropriate (potentially based on network proximity) packet forwarder.\"\n ],\n [\n \"1.A method for transmitting data between a first and second device by traversing firewalls, said method comprising the steps of: a. said first and second device establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; b. said proxy server inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; c. said proxy server notifying said first device regarding said identified external mapped address CP and said proxy server notifying said second device regarding said identified external mapped address BP, and d. said first or second device spoofing TCP packets via transmitting data with said notified external mapped address as the destination address.\",\n \"2.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said step of spoofing TCP packets is done via Raw-IP datagrams.\",\n \"3.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said communication link is established via TCP.\",\n \"4.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said communication link is established via TCP/HTTP.\",\n \"5.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said firewall is equipped with a network address translation device (NAT).\",\n \"6.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said network is any of: local area network (LAN), wide area network (WAN), wireless network, or the Internet.\",\n \"7.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said data is streaming multimedia data.\",\n \"8.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, said method comprising the steps of: a. said first and second device establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; b. said proxy server inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; c. said proxy server notifying said packet forwarder regarding said identified external mapped addresses CP and BP, and d. said first device forwarding TCP packets via transmitting data with said packet forwarder as said destination address and said packet forwarder forwarding said data with CP as the destination address, or said second device forwarding TCP packets via transmitting data with said packet forwarder as said destination address and said packet forwarder forwarding said data with BP as the destination address.\",\n \"9.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per claim 8, wherein said step of forwarding TCP packets is done via Raw-IP datagrams.\",\n \"10.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per.\",\n \"Claim 8, wherein said communication link is established via TCP.\",\n \"11.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per claim 8, wherein said communication link is established via TCP/HTTP.\",\n \"12.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per claim 8, wherein said firewall is equipped with a network address translation device (NAT).\",\n \"13.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per claim 8, wherein said network is any of: local area network (LAN), wide area network (WAN), wireless network, or the Internet.\",\n \"14.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per claim 8, wherein said data is streaming multimedia data.\",\n \"15.A system for transmitting data between a first and second device by traversing firewalls, said system comprising: a. a first host and an associated first firewall; b. a second host and an associated second firewall; c. a proxy server that establishes a communication link with said first and second host, identifies from said first and second firewalls external mapped addresses BP and CP respectively, and forwards said CP to said first device and forwards said BP to said second device, whereupon said first and second host utilize said forwarded external mapped addresses to spoof TCP packets and forward data directly between each other.\",\n \"16.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said proxy server forwards TCP packets via Raw-IP datagrams.\",\n \"17.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said communication link is established via TCP.\",\n \"18.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said communication link is established via TCP/HTTP.\",\n \"19.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said firewall is equipped with a network address translation device (NAT).\",\n \"20.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said network is any of: local area network (LAN), wide area network (WAN), wireless network, or the Internet.\",\n \"21.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said data is streaming multimedia data.\",\n \"22.A system for transmitting data between a first and second device by traversing firewalls, said system comprising: a. a first host and an associated first firewall; b. a second host and an associated second firewall; c. a proxy server that establishes a communication link with said first and second host, identifies from said first and second firewalls external mapped addresses BP and CP respectively; d. a packet forwarder receiving BP and CP from said proxy server and using BP and CP to forward incoming communications from said first device to second device with CP as destination address, or forwarding incoming communications from said second device to first device with BP as the destination address.\",\n \"23.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said packet forwarder forwards TCP packets via Raw-IP datagrams.\",\n \"24.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said communication link is established via TCP.\",\n \"25.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said communication link is established via TCP/HTTP.\",\n \"26.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said firewall is equipped with a network address translation device (NAT).\",\n \"27.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said network is any of: local area network (LAN), wide area network (WAN), wireless network, or the Internet.\",\n \"28.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said data is streaming multimedia data.\",\n \"29.An article of manufacture comprising a computer usable medium having computer readable program code embodied therein for assisting in the transmission of data between a first and second device by traversing firewalls, said article further comprising: a. computer readable program code aiding in establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; b. computer readable program code inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; c. computer readable program code notifying said first device regarding said identified external mapped address CP and computer readable program code notifying said second device regarding said identified external mapped address BP, and d. computer readable program code aiding said first or second device in spoofing TCP packets via transmitting data with said notified external mapped address as the destination address.\",\n \"30.An article of manufacture comprising a computer usable medium having computer readable program code embodied therein for aiding in forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, said medium further comprising: a. computer readable program code aiding in establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; b. computer readable program code inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; c. computer readable program code notifying said packet forwarder regarding said identified external mapped addresses CP and BP, and d. computer readable program code forwarding TCP packets via transmitting data with said packet forwarder as said destination address and computer readable program code aiding said packet forwarder in forwarding said data with CP as the destination address, or computer readable program code forwarding TCP packets via transmitting data with said packet forwarder as said destination address and computer readable program code aiding said packet forwarder in forwarding said data with BP as the destination address.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of Invention The present invention relates generally to the field of network communications.\",\n \"More specifically, the present invention is related to a system and method for traversing firewalls and network address translators (NATs).\",\n \"2.Discussion of Prior Art NATs and firewalls present a challenge to a network software programming, while their functions and operations are different: firewalls filter information into and out of the private network, while NATs hide or encapsulate a private network behind a single (or few) “real” Internet Protocol addresses.\",\n \"Their effect on many network applications is the same: The inability to send and receive information when receiving information using UDP (e.g., UDP data-grams coming into the private network).\",\n \"The inability to send and receive information when opening TCP communications into the private network.\",\n \"Each of the below described references teach the method of firewalls in general.\",\n \"However, none of the references provide or suggest the present invention method of ATM over IP traversing firewalls and network address translators (NATs).\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,898,830, assigned to Network Engineering Software describes a system, which allows connectionless traffic across a firewall.\",\n \"Rule checking is performed on the first packet entering, and if it is determined that the packet needs to be sent, a virtual host sends it to the destination computer.\",\n \"A time limit is set and so long as the set time limit does not run out, the communication is allowed.\",\n \"Addressing is accomplished utilizing name based addressing for end-to-end communication, with virtual hosts/DNS servers providing the intermediate address routing information.\",\n \"A connection type session does not appear to be initiated for the UDP transport.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,915,087 discloses a firewall system, which allows communication, using a connectionless protocol.\",\n \"The firewall holds a list of servers located on the private side, and intercepts any communications addressed to the servers.\",\n \"The firewall then binds a port and notes it in a link table.\",\n \"The packet is passed to a stack, on the private side, which forwards the packet to the server.\",\n \"Any communications from the server to the originating client is sent to the firewall, where the originating clients address is determined using the link table.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,778,174 describes a system, which utilizes an external machine, located on a public network to bypass a router firewall.\",\n \"A client on the public network connects to the external machine.\",\n \"A private channel is opened between the external machine and a machine internal to the private network.\",\n \"The internal machine connects to the destination server, and communication between the client and server is conducted through the external and internal machines.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,941,988 provides for a proxy system that “glues” together two separate TCP connections terminating at a common host (proxy).\",\n \"When communications from one connection are received at the proxy, the headers are altered to address the socket at the end of the second connection, and the sequence numbers of the first connection are mapped to the sequence space of the second connection.\",\n \"The non-patent literature entitled, “A Weakness in the 4.2 BSD Unix TCP/IP Software” describes the spoofing of a trusted host to communicate with a system, having a list of the trusted hosts, from a host that is not on the trusted list.\",\n \"It should however be noted that the prior art described above fails to provide many features, for example an explicit recitation of opening a connection-oriented session in order to allow connectionless data-grams to pass through a NAT/firewall is not provided.\",\n \"Additionally, none of the prior art described above uses a proxy server to exchange respective address information between two hosts and the hosts communicating directly via the address information and spoofing the proxy, in order to traverse at least one firewall.\",\n \"Whatever the precise merits, features and advantages of the above cited references, none of them achieve or fulfills the purposes of the present invention.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides for a method and a system for allowing an incoming UDP packet to traverse a NAT/firewall comprising, opening a TCP connection and utilizing a Raw-IP interface which builds the UDP packet utilizing the parameters of the TCP connection (e.g., session number, port, etc.).\",\n \"Furthermore, the present system provides for a method and system for allowing communication between two machines, at least one of which is behind a firewall.\",\n \"Connections are established between each machine and a proxy server sitting on a public network.\",\n \"The proxy then communicates the port and address information of each machine to the other machine, after which, each machine sends directly to each other using the supplied port and address information, while using the proxy servers port and address information as the source port and address.\"\n ],\n [\n \"RELATED APPLICATIONS The present application claims the benefit of provisional patent application “Traversing Firewalls and NATs”, Ser.\",\n \"No.\",\n \"60/255,422, filed Dec. 14, 2000.In addition, this application incorporates by reference, co-pending U.S. patent application Ser.\",\n \"No.\",\n \"09/867,371, filed May 29, 2001.BACKGROUND OF THE INVENTION 1.Field of Invention The present invention relates generally to the field of network communications.\",\n \"More specifically, the present invention is related to a system and method for traversing firewalls and network address translators (NATs).\",\n \"2.Discussion of Prior Art NATs and firewalls present a challenge to a network software programming, while their functions and operations are different: firewalls filter information into and out of the private network, while NATs hide or encapsulate a private network behind a single (or few) “real” Internet Protocol addresses.\",\n \"Their effect on many network applications is the same: The inability to send and receive information when receiving information using UDP (e.g., UDP data-grams coming into the private network).\",\n \"The inability to send and receive information when opening TCP communications into the private network.\",\n \"Each of the below described references teach the method of firewalls in general.\",\n \"However, none of the references provide or suggest the present invention method of ATM over IP traversing firewalls and network address translators (NATs).\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,898,830, assigned to Network Engineering Software describes a system, which allows connectionless traffic across a firewall.\",\n \"Rule checking is performed on the first packet entering, and if it is determined that the packet needs to be sent, a virtual host sends it to the destination computer.\",\n \"A time limit is set and so long as the set time limit does not run out, the communication is allowed.\",\n \"Addressing is accomplished utilizing name based addressing for end-to-end communication, with virtual hosts/DNS servers providing the intermediate address routing information.\",\n \"A connection type session does not appear to be initiated for the UDP transport.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,915,087 discloses a firewall system, which allows communication, using a connectionless protocol.\",\n \"The firewall holds a list of servers located on the private side, and intercepts any communications addressed to the servers.\",\n \"The firewall then binds a port and notes it in a link table.\",\n \"The packet is passed to a stack, on the private side, which forwards the packet to the server.\",\n \"Any communications from the server to the originating client is sent to the firewall, where the originating clients address is determined using the link table.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,778,174 describes a system, which utilizes an external machine, located on a public network to bypass a router firewall.\",\n \"A client on the public network connects to the external machine.\",\n \"A private channel is opened between the external machine and a machine internal to the private network.\",\n \"The internal machine connects to the destination server, and communication between the client and server is conducted through the external and internal machines.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,941,988 provides for a proxy system that “glues” together two separate TCP connections terminating at a common host (proxy).\",\n \"When communications from one connection are received at the proxy, the headers are altered to address the socket at the end of the second connection, and the sequence numbers of the first connection are mapped to the sequence space of the second connection.\",\n \"The non-patent literature entitled, “A Weakness in the 4.2 BSD Unix TCP/IP Software” describes the spoofing of a trusted host to communicate with a system, having a list of the trusted hosts, from a host that is not on the trusted list.\",\n \"It should however be noted that the prior art described above fails to provide many features, for example an explicit recitation of opening a connection-oriented session in order to allow connectionless data-grams to pass through a NAT/firewall is not provided.\",\n \"Additionally, none of the prior art described above uses a proxy server to exchange respective address information between two hosts and the hosts communicating directly via the address information and spoofing the proxy, in order to traverse at least one firewall.\",\n \"Whatever the precise merits, features and advantages of the above cited references, none of them achieve or fulfills the purposes of the present invention.\",\n \"SUMMARY OF THE INVENTION The present invention provides for a method and a system for allowing an incoming UDP packet to traverse a NAT/firewall comprising, opening a TCP connection and utilizing a Raw-IP interface which builds the UDP packet utilizing the parameters of the TCP connection (e.g., session number, port, etc.).\",\n \"Furthermore, the present system provides for a method and system for allowing communication between two machines, at least one of which is behind a firewall.\",\n \"Connections are established between each machine and a proxy server sitting on a public network.\",\n \"The proxy then communicates the port and address information of each machine to the other machine, after which, each machine sends directly to each other using the supplied port and address information, while using the proxy servers port and address information as the source port and address.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 illustrates the Intranet to Internet data transfer scenario.\",\n \"FIG.\",\n \"2 illustrates the Internet to Intranet data transfer scenario.\",\n \"FIG.\",\n \"3 illustrates the Intranet to Intranet data transfer scenario.\",\n \"FIG.\",\n \"4 illustrates a bi-directional connection, using TCP and HTTP, communicating indirectly with the proxy.\",\n \"FIG.\",\n \"5 illustrates TCP spoofing of the present invention.\",\n \"FIG.\",\n \"6 illustrates TCP spoofing of the present invention in the presence of a packet forwarder.\",\n \"FIG.\",\n \"7 illustrates the methodology associated with the present invention.\",\n \"DESCRIPTION OF THE PREFERRED EMBODIMENTS While this invention is illustrated and described in a preferred embodiment, the invention may be produced in many different configurations, forms and materials.\",\n \"There is depicted in the drawings, and will herein be described in detail, a preferred embodiment of the invention, with the understanding that the present disclosure is to be considered as an exemplification of the principles of the invention and the associated functional specifications for its construction and is not intended to limit the invention to the embodiment illustrated.\",\n \"Those skilled in the art will envision many other possible variations within the scope of the present invention.\",\n \"When a communicating device, such as the Internet phone or a Voice-over-IP Gateway or an IETF MGCP Gateway or an ITU-T H.248 Gateway or a PacketCable Residential Gateway or a CPE Gateway (Customer premises equipment Gateway), opens a signaling connection from a private network to a public network, the TCP channel is bi-directional, and therefore the signaling protocol can execute in both directions.\",\n \"This also allows HTTP to work behind network address translators (NATs) and Firewalls.\",\n \"Connections are always opened from the private network to the public network; taking advantage of the fact that TCP data communications are bi-directional.\",\n \"NATs present an additional translation step when communicating.\",\n \"NATs map the source addresses (in the private network) of the originating computer into a public address and a port number on the public interface of the NAT.\",\n \"As long as TCP is used, the translation can be done in reverse, as the TCP channel is bi-directional.\",\n \"Multimedia signaling and media streaming is usually UDP-based for better efficiency, which introduces the problem—the ingress system sends UDP packets to the public interface on the NAT, and the NAT has no automatic method to map this UDP data-gram to the actual computer that is supposed to receive that data-gram.\",\n \"The solution provided by the present invention is to stream audio and video (and other time-sensitive data) over TCP, but TCP streaming and windowing mechanizing hurts the real-time performance.\",\n \"The present invention opens a TCP connection as usual (using TCP), and then switches to a Raw-IP interface that sends Raw-IP data-grams that are legal TCP messages using just opened TCP channel parameters (e.g., session number, port, etc.)\",\n \"To an intermediate system, these messages will look like standard TCP messages, but as they are sent using Raw-IP, the usual timing issues that TCP introduces to real-time media streaming are not in place.\",\n \"Thus, the present invention uses the protocol software to “spoof” the TCP channel to enable real-time TCP communications.\",\n \"It is impossible with NATs and Firewalls to open ingress connections (e.g., it is impossible to open a TCP connection to a computer behind a Firewall or the NAT.\",\n \"Thus: 1.It is impossible to originate communications from the public network into the private network.\",\n \"2.It is impossible to originate communications from a private network (to a public network) to another private network.\",\n \"Thus, the present invention uses a server proxy that both communication parties open their TCP channels to (using the previous procedure).\",\n \"Then, the proxy communicates to each party the other party's source address/port (of the TCP channel).\",\n \"Finally, each communication element sends information to the other party using the server proxy source address/port.\",\n \"It should be noted that packets are sent directly between the communicating entities, as the proxy is only used to hold the TCP state to “spoof” the NATs and Firewalls.\",\n \"In the preferred embodiment of the present invention, full communication is possible between all types of private and public networks, as long as outgoing TCP channel establishment is allowed.\",\n \"In another embodiment, the server proxy and originating clients use TCP/HTTP, which is universally supported, and in this instance, all information is tunneled over the simulated TCP/HTTP channel.\",\n \"In the Internet as it exists today, using the small address space provided by IPv4, many networks deploy NAT (network address translation) devices to enlarge the internal address space.\",\n \"In addition, many networks deploy firewall devices to block intrusions and hacking.\",\n \"Many firewalls also support integrated NAT capabilities.\",\n \"The end-result of both types of devices is that ingress traffic (one originating outside the network and destined into the network) is usually blocked, as incoming connections are usually blocked for firewalls and are impossible to complete on NAT devices, and as the originating (outside the NAT) IP host is unaware of the destination internal IP address.\",\n \"Thus, users cannot place audio/video calls from NAT protected networks (as the audio and video will not penetrate back into the network from the remote called host), and in many cases users behind corporate firewalls are blocked from using such services.\",\n \"A communications protocol such as the TrulyGlobal™ Protocol (TGP), (as described in the related application, “Communication Protocol”) can be used in conjunction with the present invention to operate over standard HTTP and remote TGP servers to use the HTTP back-channel to send information to the client; and ensuring that all actions carried by TGP traverse both NATs and firewalls.\",\n \"Intranet, as defined in this application, is a network that is protected by a NAT or a firewall device, and blocks all incoming traffic into the protected network (e.g., TCP connections cannot be initiated into the network, and UDP traffic will be blocked at the entry-point into the network).\",\n \"Similarly, Internet is defined as a public addressed, unprotected network, where fill IP communication is possible.\",\n \"In the Intranet to Internet scenario, as illustrated in FIG.\",\n \"1, a user inside an Intranet is attempting to call a user that is outside the Intranet, and the remote user is in the public Intranet.\",\n \"The problem encountered is that while the call will be successfully set-up (as the originating host is allowed to open connection to the outside network), audio and/or video data will not be able to get back into the Intranet, hence the caller will not be able to hear and/or see the called device.\",\n \"Similarly, in an Internet to Intranet scenario, as illustrated in FIG.\",\n \"2, the caller cannot open a signaling channel at all to the called device, as ingress connections into the Intranet are not allowed.\",\n \"Lastly, in the Intranet to Intranet scenario, as illustrated in FIG.\",\n \"3, the same end-effect as the previous scenario happens, e.g., the caller cannot open a signaling channel at all to the called device, as ingress connections into the Intranet from the Internet are not allowed.\",\n \"The solution provided for by the present invention uses TCP and potentially HTTP, and a service is provided outside the Intranet (in the public Internet) to help both end-points to complete calls.\",\n \"The first assumption made is that the clients inside the Intranet can initiate TCP or at least TCP/HTTP specifically to the public Internet, so some form of communications is possible.\",\n \"HTTP can be used to insure safe traversal via HTTP proxies.\",\n \"Once a TCP/HTTP connection is available, bi-directional communications are possible.\",\n \"Outwards messages use standard HTTP commands to request resources (using URLs), and incoming information flow returns using the HTTP reply channel (as TCP/HTTP is full-duplex).\",\n \"Furthermore, at any time, as illustrated by FIG.\",\n \"4, the caller can initiate a TCP/HTTP connection (or a plain TCP connection) to a service that resides in the public Internet, and that service is responsible to “proxy” the request (using the reply leg of the remote HTTP session) to the called-device.\",\n \"When both devices have bi-directional connections to the proxy, they can communicate indirectly via the proxy.\",\n \"While the present invention shows how proxy-based communication can work, the problem when the communication between the two hosts has to flow via the proxy, which adds delay and has limited scalability.\",\n \"The solution as per the present invention is to spoof the TCP session to allow direct TCP communications between the two machines.\",\n \"This scenario is illustrated in FIG.\",\n \"5.When a machine behind a proxy, NAT or firewall establishes a session with the outside world, the session is mapped on the outside of Intranet 1 and 2 on the public interface address(s) to an internal connection between Host 1 and 2 and their gateways to the Internet.\",\n \"Sending correctly formed TCP packets to that interface will result in the gateway forwarding these packets to the correct host inside the private network.\",\n \"The sequence is as follows: 1.A session is established from Host 1 in Intranet 1 to the Proxy (AB session).\",\n \"2.A session is established from Host 2 in Intranet 2 to the Proxy (DC session).\",\n \"3.The Address BP (public side of session A) is found by inspecting the source address/port of B.\",\n \"4.The Address of CP (public side of session D) is found by inspecting the source address/port of C. 5.The external mapped addresses are provided to the other hosts, i.e., Host 1 is provided with address/pair CP and Host 2 is provided with address/port pair BP.\",\n \"TCP session B parameters are provided to host 2.TCP session C parameters are provided to host 1.6.Hosts 1 and 2 will spoof TCP packets for sessions B and C, sent to target address/port pairs BP and CP.\",\n \"This traffic will go directly between the two networks and not via the Proxy.\",\n \"In effect, a virtual TCP session C1/B1 is created by combining the two existing TCP sessions C and B.\",\n \"Once the spoofed TCP session is handed over to Hosts 1 and 2, the Proxy should not send any information on that session, as session parameters may be out-of-date.\",\n \"The session is kept open for the duration Hosts 1 and 2 requires it, and will be closed by either Host when required.\",\n \"The proxy is only used for establishing session, and does not use the session for anything else once it is “handed over”.\",\n \"In some cases the internal network will filter spoofed packets (for security, e.g., hack prevention) and therefore will not let the packets with the spoofed source address leave the internal network.\",\n \"In such cases, as illustrated by FIG.\",\n \"6, the TCP connections will be handed over to a packet forwarder (that resides in the same server or a separate server) that handles the packet interchange.\",\n \"One of the side effects of spoofing the TCP session between Hosts 1 and 2 is that the TCP session parameters can be changed or completely ignored, as long as packets are synthetically correct (as per TCP), they can be sent without consideration to window-sizes, exponential back-off algorithms or slow-start mechanisms.\",\n \"When such a spoofed TCP session is in place, it can be used to transmit both audio and video with the same time of performance that is expected from UDP.\",\n \"The session-establishment procedures described above allow any session to be established between any two computers.\",\n \"This is done as a result of Host 1 calling Host 2 (or the reverse).\",\n \"The calling host will send a Call-Establishment message to the Proxy, which will (pending, any policy decision) forward the request to the called Host.\",\n \"The called host will receive the Call Answer transaction over the back-channel of the session it already has with the Proxy, requesting it to answer the call.\",\n \"If the called host responses positively, one or more media channel(s) will be established between Host 1 and 2, with the help of the proxy as required by the session's parameters (audio only, audio and video, etc).\",\n \"It should be noted that both IETF SIP and ITU-T H.323 signaling can be used, but are not required.\",\n \"In one embodiment, the Proxy contains all the required functionality (e.g., signaling a RTP:Address:Port destination instead of a H323:Address:Port destination).\",\n \"Furthermore, one skilled in the art can recognize that IETF SIP (by manipulating IETF Session Description Protocol (SDP) parameters) and ITU H.323 (by Manipulating ITU-T H.245 OpenLogicalChannel or FastStart parameters) can be used, with minor changes, to accomplish the required signaling.\",\n \"The present invention is implemented using a raw-IP interfaces that spoofs the TCP sessions.\",\n \"A limited TCP stack is implemented that creates synthetically correct TCP packets, to insure the packets are interpreted and forwarded correctly by the NATs, proxies and firewalls in the way.\",\n \"Such a spoofed-TCP stack does not need to support any reliable transmission, as it is only used for real-time sensitive transmission purposes.\",\n \"FIG.\",\n \"7 summarizes the methodology 700 associated with the present invention.\",\n \"In step 702, both hosts establish a connection (e.g., TCP connection or TCP/HTTP connection) with a TCP proxy server.\",\n \"Next, in step 704, external mapped addresses BP and CP associated with the firewalls of both hosts are identified.\",\n \"Subsequently, in step 706, the identified external mapped addresses are exchanged between the two hosts.\",\n \"Lastly, the TCP packets are spoofed to transmit the data (e.g., streaming multimedia data) between the hosts.\",\n \"Furthermore, the present invention includes a computer program code based product, which is a storage medium having program code stored therein, which can be used to instruct a computer to perform any of the methods associated with the present invention.\",\n \"The computer storage medium includes any of, but not limited to, the following: CD-ROM, DVD, magnetic tape, optical disc, hard drive, floppy disk, ferroelectric memory, flash memory, ferromagnetic memory, optical storage, charge coupled devices, magnetic or optical cards, smart cards, EEPROM, EPROM, RAM, ROM, DRAM, SRAM, SDRAM, or any other appropriate static or dynamic memory, or data storage devices.\",\n \"Implemented in computer program code based products are software modules for: aiding in establishing a communication link with a proxy server over a network, wherein a first and second device can access the network over a firewall; inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; notifying said first device regarding said identified external mapped address CP and notifying said second device regarding said identified external mapped address BP; and aiding said first or second device in spoofing TCP packets via transmitting data with said notified external mapped address as the destination address.\",\n \"Also implemented in computer program based products are software modules for: aiding in establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; notifying said packet forwarder regarding said identified external mapped addresses CP and BP, and forwarding TCP packets via transmitting data with said packet forwarder as said destination address and computer readable program code aiding said packet forwarder in forwarding said data with CP as the destination address, or forwarding TCP packets via transmitting data with said packet forwarder as said destination address and computer readable program code aiding said packet forwarder in forwarding said data with BP as the destination address.\",\n \"CONCLUSION A system and method has been shown in the above embodiments for the effective implementation of a method and a system for traversing firewalls and network address translations (NATs).\",\n \"While various preferred embodiments have been shown and described, it will be understood that there is no intent to limit the invention by such disclosure, but rather, it is intended to cover all modifications and alternate constructions falling within the spirit and scope of the invention, as defined in the appended claims.\",\n \"For example, the present invention should not be limited by type of firewall, type of network address translation device, location of packet forwarder, software/program, computing environment, or specific computing hardware.\",\n \"The above enhancements are implemented in various computing environments.\",\n \"For example, the present invention may be implemented on a multi-nodal system (e.g., LAN) or networking system (e.g., Internet, WWW, wireless web).\",\n \"All programming, and data related thereto are stored in computer memory, static or dynamic, and may be retrieved by the user in any of: conventional computer storage, display (i.e., CRT) and/or hardcopy (i.e., printed) formats.\",\n \"The programming of the present invention may be implemented by one of skill in the art of network communications.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10450751"}}},{"rowIdx":282,"cells":{"text":{"kind":"list like","value":[["Novel nucleic acids and polypeptides","The present invention provides novel nucleic acids, novel polypeptide sequences encoded by these nucleic acids and uses thereof."],["1.An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-30368, a mature protein coding portion of SEQ ID NO: 1-30368, an active domain of SEQ ID NO: 1-30368, and complementary sequences thereof.","2.An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide hybridizes to the polynucleotide of claim 1 under stringent hybridization conditions.","3.An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide has greater than about 90% sequence identity with the polynucleotide of claim 1.4.The polynucleotide of claim 1 wherein said polynucleotide is DNA.","5.An isolated polynucleotide of claim 1 wherein said polynucleotide comprises the complementary sequences.","6.A vector comprising the polynucleotide of claim 1.7.An expression vector comprising the polynucleotide of claim 1.8.A host cell genetically engineered to comprise the polynucleotide of claim 1.9.A host cell genetically engineered to comprise the polynucleotide of claim 1 operatively associated with a regulatory sequence that modulates expression of the polynucleotide in the host cell.","10.An isolated polypeptide, wherein the polypeptide is selected from the group consisting of: (a) a polypeptide encoded by any one of the polynucleotides of claim 1; and (b) a polypeptide encoded by a polynucleotide hybridizing under stringent conditions with any one of SEQ ID NO: 1-30368.11.A composition comprising the polypeptide of claim 10 and a carrier.","12.An antibody directed against the polypeptide of claim 10.13.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide of claim 1 for a period sufficient to form the complex; and b) detecting the complex, so that if a complex is detected, the polynucleotide of claim 1 is detected.","14.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to the polynucleotide of claim 1 under such conditions; b) amplifying a product comprising at least a portion of the polynucleotide of claim 1; and c) detecting said product and thereby the polynucleotide of claim 1 in the sample.","15.The method of claim 14, wherein the polynucleotide is an RNA molecule and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA polynucleotide.","16.A method for detecting the polypeptide of claim 10 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex; and b) detecting formation of the complex, so that if a complex formation is detected, the polypeptide of claim 10 is detected.","17.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10 under conditions sufficient to form a polypeptide/compound complex; and b) detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.","18.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10, in a cell, under conditions sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and b) detecting the complex by detecting reporter gene sequence expression, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.","19.A method of producing the polypeptide of claim 10, comprising, a) culturing a host cell comprising a polynucleotide sequence selected from the group consisting of a polynucleotide sequence of SEQ ID NO: 1-30368, a mature protein coding portion of SEQ ID NO: 1-30368, an active domain of SEQ ID NO: 1-30368, complementary sequences thereof and a polynucleotide sequence hybridizing under stringent conditions to SEQ ID NO: 1-30368, under conditions sufficient to express the polypeptide in said cell; and b) isolating the polypeptide from the cell culture or cells of step (a).","20.An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 30369-60736, the mature protein portion thereof, or the active domain thereof.","21.The polypeptide of claim 20 wherein the polypeptide is provided on a polypeptide array.","22.A collection of polynucleotides, wherein the collection comprises the sequence information of at least one of SEQ ID NO: 1-30368.23.The collection of claim 22, wherein the collection is provided on a nucleic acid array.","24.The collection of claim 23, wherein the array detects full-matches to any one of the polynucleotides in the collection.","25.The collection of claim 23, wherein the array detects mismatches to any one of the polynucleotides in the collection.","26.The collection of claim 22, wherein the collection is provided in a computer-readable format.","27.A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier.","28.A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising an antibody that specifically binds to a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier."],["<SOH> 2.BACKGROUND <EOH>Technology aimed at the discovery of protein factors (including e.g.","cytokines, such as lymphokines, interferons, CSFs, chemokines, and interleukins) has matured rapidly over the past decade.","The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning).","More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.","Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences."],["<SOH> 3.SUMMARY OF THE INVENTION <EOH>The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.","The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.","The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases.","The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.","These nucleic acid sequences are designated as SEQ ID NO: 1-30368.The polypeptides sequences are designated SEQ ID NO: 30369-60736.The nucleic acids and polypeptides are provided in the Sequence Listing.","In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases.","In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.","The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-30368 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-30368.A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-30368 or a degenerate variant or fragment thereof.","The identifying sequence can be 100 base pairs in length.","The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-30368.The sequence information can be a segment of any one of SEQ ID NO: 1-30368 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-30368.A collection as used in this application can be a collection of only one polynucleotide.","The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array.","In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment.","The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.","The collection can also be provided in a computer-readable format.","This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.","Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.","In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the arL In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.","The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-30368; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-30368.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing (e.g., SEQ ID NO: 30369-60736); (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.","orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.","The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein.","Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-30368; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.","Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.","The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.","host cells) of the invention.","The invention also provides compositions comprising a polypeptide of the invention.","Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.","The invention also provides host cells transformed or transfected with a polynucleotide of the invention.","The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells.","Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.","Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.","These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like.","For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.","In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.","The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.","For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.","Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.","The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.","Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.","In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.","The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample.","Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.","The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected.","The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.","The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.","Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.","The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.","Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.","Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.","The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the invention is identified.","The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies.","In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products.","Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.","The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in the sequence listing).","If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["1.TECHNICAL FIELD The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these-polynucleotides and proteins, for example in therapeutic, diagnostic and research methods.","2.BACKGROUND Technology aimed at the discovery of protein factors (including e.g.","cytokines, such as lymphokines, interferons, CSFs, chemokines, and interleukins) has matured rapidly over the past decade.","The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning).","More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.","Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.","3.SUMMARY OF THE INVENTION The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.","The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.","The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases.","The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.","These nucleic acid sequences are designated as SEQ ID NO: 1-30368.The polypeptides sequences are designated SEQ ID NO: 30369-60736.The nucleic acids and polypeptides are provided in the Sequence Listing.","In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases.","In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.","The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-30368 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-30368.A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-30368 or a degenerate variant or fragment thereof.","The identifying sequence can be 100 base pairs in length.","The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-30368.The sequence information can be a segment of any one of SEQ ID NO: 1-30368 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-30368.A collection as used in this application can be a collection of only one polynucleotide.","The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array.","In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment.","The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.","The collection can also be provided in a computer-readable format.","This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.","Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.","In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the arL In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.","The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-30368; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-30368.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing (e.g., SEQ ID NO: 30369-60736); (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.","orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.","The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein.","Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-30368; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.","Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.","The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.","host cells) of the invention.","The invention also provides compositions comprising a polypeptide of the invention.","Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.","The invention also provides host cells transformed or transfected with a polynucleotide of the invention.","The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells.","Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.","Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.","These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like.","For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.","In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.","The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.","For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.","Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.","The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.","Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.","In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.","The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample.","Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.","The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected.","The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.","The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.","Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.","The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.","Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.","Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.","The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the invention is identified.","The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies.","In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products.","Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.","The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in the sequence listing).","If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.","4.DETAILED DESCRIPTION OF THE INVENTION 4.1 Definitions It must be noted that as used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.","The term “active” refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide.","According to the invention, the terms “biologically active” or “biological activity” refer to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule.","Likewise “immunologically active” or “immunological activity” refers to the capability of the natural, recombinant or synthetic polypeptide to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.","The term “activated cells” as used in this application are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.","The terms “complementary” or “complementarity” refer to the natural binding of polynucleotides by base pairing.","For example, the sequence 5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′.","Complementarity between two single-stranded molecules may be “partial” such that only some of the nucleic acids bind or it may be “complete” such that total complementarity exists between the single stranded molecules.","The degree of complementarity between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.","The term “embryonic stem cells (ES)” refers to a cell that can give rise to many differentiated cell types in an embryo or an adult, including the germ cells.","The term “germ line stem cells (GSCs)” refers to stem cells derived from primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes.","The term “primordial germ cells (PGCs)” refers to a small population of cells set aside from other cell lineages particularly from the yolk sac, mesenteries, or gonadal ridges during embryogenesis that have the potential to differentiate into germ cells and other cells.","PGCs are the source from which GSCs and ES cells are derived The PGCs, the GSCs and the ES cells are capable of self-renewal.","Thus these cells not only populate the germ line and give rise to a plurality of terminally differentiated cells that comprise the adult specialized organs, but are able to regenerate themselves.","The term “expression modulating fragment,” EMF, means a series of nucleotides which modulates the expression of an operably linked ORF or another EMF.","As used herein, a sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF.","EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements).","One class of EMEs are nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.","The terms “nucleotide sequence” or “nucleic acid” or “polynucleotide” or “oligonucleotide” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides.","These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material.","In the sequences herein A is adenine, C is cytosine, T is thymine, G is guanine and N is A, C, G or T (U).","It is contemplated that where the polynucleotide is RNA, the T (thymine) in the sequences provided herein is substituted with U (uracil).","Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.","The terms “oligonucleotide fragment” or a “polynucleotide fragment”, “portion,” or “segment” or “probe” or “primer” are used interchangeably and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides.","The fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides.","Preferably the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from about 17 to 30 nucleotides and most preferably from about 20 to 25 nucleotides.","Preferably the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules.","A fragment or segment may uniquely identify each polynucleotide sequence of the present invention.","Preferably the fragment comprises a sequence substantially similar to any one of SEQ ID NO: 1-30368.Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al.","(Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250).","They may be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art.","Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., both of which are incorporated herein by reference in their entirety.","The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-30368.The sequence information can be a segment of any one of SEQ ID NO: 1-30368 that uniquely identifies or represents the sequence information of that sequence of SEQ ID NO: 1-30368.One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300.In the human genome, there are three billion base pairs in one set of chromosomes.","Because 420 possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosomes.","Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5.When these segments are used in arrays for expression studies, fifteen-mer segments can be used.","The probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences comprise less than approximately 5% of the entire genome sequence.","Similarly, when using sequence information for detecting a single mismatch, a segment can be a twenty-five mer.","The probability that the twenty-five mer would appear in a human genome with a single mismatch is calculated by multiplying the probability for a full match (1÷425) times the increased probability for mismatch at each nucleotide position (3×25).","The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five.","The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.","The term “open reading frame,” ORF, means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein.","The terms “operably linked” or “operably associated” refer to functionally related nucleic acid sequences.","For example, a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence.","While operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g.","repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.","The term “pluripotent” refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism.","A pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.","The terms “polypeptide” or “peptide” or “amino acid sequence” refer to an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and to naturally occurring or synthetic molecules.","A polypeptide “fragment,” “portion,” or “segment” is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids.","The peptide preferably is not greater than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids.","Preferably the peptide is from about 5 to about 200 amino acids.","To be active, any polypeptide must have sufficient length to display biological and/or immunological activity.","The term “naturally occurring polypeptide” refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.","The term “translated protein coding portion” means a sequence which encodes for the full length protein which may include any leader sequence or any processing sequence.","The term “mature protein coding sequence” means a sequence which encodes a peptide or protein without a signal or leader sequence.","The “mature protein portion” means that portion of the protein which does not include a signal or leader sequence.","The peptide may have been produced by processing in the cell which removes any leader/signal sequence.","The mature protein portion may or may not include an initial methionine residue.","The methionine residue may be removed from the protein during processing in the cell.","The peptide may be produced synthetically or the protein may have been produced using a polynucleotide only encoding for the mature protein coding sequence.","The term “derivative” refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.","The term “variant”(or “analog”) refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e.g., recombinant DNA techniques.","Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.","Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the “redundancy” in the genetic code.","Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system.","Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.","Preferably, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements.","“Conservative” amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.","For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.","“Insertions” or “deletions” are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids.","The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.","Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides.","Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention.","For example, such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.","Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression.","For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.","The terms “purified” or “substantially purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like.","In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).","The term “isolated” as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source.","In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same.","The terms “isolated” and “purified” do not encompass nucleic acids or polypeptides present in their natural source.","The term “recombinant,” when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems.","“Microbial” refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems.","As a product, “recombinant microbial” defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation.","Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.","The term “recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence.","An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.","Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.","Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue.","This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.","The term “recombinant expression system” means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally.","Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed.","This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers.","Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed.","The cells can be prokaryotic or eukaryotic.","The term “secreted” includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell.","“Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed.","“Secreted” proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reticulum.","“Secreted” proteins are also intended to include proteins containing non-typical signal sequences (e.g.","Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2):134-143) and factors released from damaged cells (e.g.","Interleukin-1 Receptor Antagonist, see Arend, W. P. et. al.","(1998) Annu.","Rev.","Immunol.","16:27-55).","Where desired, an expression vector may be designed to contain a “signal or leader sequence” which will direct the polypeptide through the membrane of a cell.","Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.","The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent.","Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringent conditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.).","Other exemplary hybridization conditions are described herein in the examples.","In instances of hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligonucleotides), 48° C. (for 17-base oligos), 55° C. (for 20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).","As used herein, “substantially equivalent” can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences.","Typically, such a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less).","Such a sequence is said to have 65% sequence identity to the listed sequence.","In one embodiment, a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 25% (75% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity).","Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% identity, more preferably at least 98% identity, and most preferably at least 99% identity.","Substantially equivalent nucleotide sequences of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code.","Preferably, nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, more preferably at least about 80% sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% identity, more preferably at least about 98% sequence identity, and most preferably at least about 99% sequence identity.","For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent.","For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a spurious stop codon) should be disregarded.","Sequence identity may be determined, e.g., using the Jotun Hein method (Hein, J.","(1990) Methods Enzymol.","183:626-645).","Identity between sequences can also be determined by other methods known in the art, e.g.","by varying hybridization conditions.","The term “totipotent” refers to the capability of a cell to differentiate into all of the cell types of an adult organism.","The term “transformation” means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration.","The term “transfection” refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed.","The term “infection” refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.","As used herein, an “uptake modulating fragment,” UMF, means a series of nucleotides which mediate the uptake of a linked DNA fragment into a cell.","UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below.","The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence.","The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined.","As described above, a UMF will increase the frequency of uptake of a linked marker sequence.","Each of the above terms is meant to encompass all that is described for each, unless the context dictates otherwise.","4.2 Nucleic Acids of the Invention Nucleotide sequences of the invention are set forth in the Sequence Listing.","The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 1-30368; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 30369-60736; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polypeptides of any one of SEQ ID NO: 30369-60736.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a) the complement of any of the nucleotides sequences of SEQ ID NO: 1-30368; (b) nucleotide sequences encoding any one of the amino acid sequences set forth in the Sequence Listing; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptides of SEQ ID NO: 30369-60736.Domains of interest may depend on the nature of the encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic domains, or combinations thereof; domains in immunoglobulin-like proteins include the variable immunoglobulin-like domains; domains in enzyme-like polypeptides include catalytic and substrate binding domains; and domains in ligand polypeptides include receptor-binding domains.","The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA.","The polynucleotides may include all of the coding region of the cDNA or may represent a portion of the coding region of the cDNA.","The present invention also provides genes corresponding to the cDNA sequences disclosed herein.","The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein.","Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.","Further 5′ and 3′ sequence can be obtained using methods known in the art.","For example, full length cDNA or genomic DNA that corresponds to any of the polynucleotides of SEQ ID NO: 1-30368 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any of the polynucleotides of SEQ ID NO: 1-30368 or a portion there of as a probe.","Alternatively, the polynucleotides of SEQ ID NO: 1-30368 may be used as the basis for suitable primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.","The nucleic acid sequences of the invention can be assembled from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases, such as dbEST, gbpri, and UniGene.","The EST sequences can provide identifying sequence information, representative fragment or segment information, or novel segment information for the full-length gene.","The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above.","Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more typically at least about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%, 94%, and even more typically at least about 95%, 96%, 97%, 98%, 99%, sequence identity to a polynucleotide recited above.","Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to any of the nucleotide sequences of SEQ ID NO: 1-30368, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides.","Fragments of, e.g.","15, 17, or 20 nucleotides or more that are selective for (i.e.","specifically hybridize to any one of the polynucleotides of the invention) are contemplated.","Probes capable of specifically hybridizing to a polynucleotide can differentiate polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate human genes from genes of other species, and are preferably based on unique nucleotide sequences.","The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof.","Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NO: 1-30368, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NO: 1-30368 with a sequence from another isolate of the same species.","Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein.","In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.","The nearest neighbor or homology result for the nucleic acids of the present invention, including SEQ ID NO: 1-30368 can be obtained by searching a database using an algorithm or a program.","Preferably, a BLAST which stands for Basic Local Alignment Search Tool is used to search for local sequence alignments (Altshul, S. F. J. Mol.","Evol.","36 290-300 (1993) and Altschul S. F. et al.","J. Mol.","Biol.","21:403-410(1990)).","Alternatively a FASTA version 3 search against Genpept, using Fastxy algorithm.","Species homologs (or orthologs) of the disclosed polynucleotides and proteins are also provided by the present invention.","Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.","The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.","The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids.","These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide.","There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation.","Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature.","These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions).","Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site.","Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous.","Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.","Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues.","Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein.","In a preferred method, polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis.","This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed.","In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183 (1983).","A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res.","10:6487-6500 (1982).","PCR may also be used to create amino acid sequence variants of the novel nucleic acids.","When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant.","PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer.","The product DNA fragments replace the corresponding region in the plasmid and this gives a polynucleotide encoding the desired amino acid variant.","A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al.","Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids.","Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.","Polynucleotides encoding preferred polypeptide truncations of the invention can be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.","The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.","The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA.","Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides of the desired sequence identities.","In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 1-30368, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells.","Also included are the cDNA inserts of any of the clones identified herein.","A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al.","(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).","Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art.","Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.","In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.","Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.","A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.","The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-30368 or a fragment thereof or any other polynucleotides of the invention.","In one embodiment, the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-30368 or a fragment thereof is inserted, in a forward or reverse orientation.","In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF.","Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention.","The following vectors are provided by way of example.","Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).","Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).","The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res.","19, 44854490 (1991), in order to produce the protein recombinantly.","Many suitable expression control sequences are known in the art.","General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990).","As defined herein “operably linked” means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.","Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.","Two appropriate vectors are pKK232-8 and pCM7.Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc.","Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.","Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.","Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TP1gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.","Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others.","The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.","Optionally, the heterologous sequence can encode a fusion protein including an amino terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.","Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.","The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.","Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.","As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).","Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis., USA).","These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.","Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.","Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.","Polynucleotides of the invention can also be used to induce immune responses.","For example, as described in Fan et al, Nat.","Biotech.","17:870-872 (1999), incorporated herein by reference, nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical administration of naked plasmid DNA or following injection, and preferably intramuscular injection of the DNA.","The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.","4.3 Antisense Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1-30368, or fragments, analogs or derivatives thereof.","An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.","In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only a portion thereof.","Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a protein of any of SEQ ID NO: 30369-60736 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-30368 are additionally provided.","In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence of the invention.","The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.","In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence of the invention.","The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).","Given the coding strand sequences encoding a nucleic acid disclosed herein (e.g., SEQ ID NO: 1-30368), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.","The antisense nucleic acid molecule can be complementary to the entire coding region of a mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of a mRNA.","For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a mRNA.","An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.","An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.","For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.","Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.","Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).","The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein according to the invention to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.","The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.","An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.","Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.","For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.","The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.","To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.","In yet another embodiment, the antisense nucleic acid molecule of the invention is an-anomeric nucleic acid molecule.","An-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual-units, the strands run parallel to each other (Gaultier et al.","(1987) Nucleic Acids Res 15: 6625-6641).","The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al.","(1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al.","(1987) FEBS Lett 215: 327-330).","4.4 Ribozymes and PNA Moieties In still another embodiment, an antisense nucleic acid of the invention is a ribozyme.","Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region.","Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave a mRNA transcripts to thereby inhibit translation of a mRNA.","A ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1-30368).","For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an mRNA of SEQ ID NO: 1-30368 (see, e.g., Cech et al.","U.S. Pat.","No.","4,987,071; and Cech et al.","U.S. Pat.","No.","5,116,742).","Alternatively, polynucleotides of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules.","See, e.g., Bartel et al., (1993) Science 261:1411-1418.Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells.","See generally, Helene.","(1991) Anticancer Drug Des.","6: 569-84; Helene.","et al.","(1992) Ann.","N.Y. Acad.","Sci.","660:27-36; and Maher (1992) Bioassays 14: 807-15.In various embodiments, the nucleic acids of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.","For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al.","(1996) Bioorg Med Chem 4: 5-23).","As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.","The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.","The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al.","(1996) above; Perry-O'Keefe et al.","(1996) PNAS 93: 14670-675.PNAs of the invention can be used in therapeutic and diagnostic applications.","For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.","PNAs of the invention can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymnes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B.","(1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al.","(1996), above; Perry-OKeefe (1996), above).","In another embodiment, PNAs of the invention can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.","For example, PNA-DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA.","Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.","PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above).","The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al.","(1996) Nucl Acids Res 24: 3357-63.For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag et al.","(1989) Nucl Acid Res 17: 5973-88).","PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al.","(1996) above).","Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment.","See, Petersen et al.","(1975) Bioorg Med Chem Lett 5: 1119-11124.In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc.","Natl.","Acad.","Sci.","U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc.","Natl.","Acad.","Sci.","84:648-652; PCT Publication No.","WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No.","WO89/10134).","In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm.","Res.","5:539-549).","To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.","4.5 Hosts The present invention further provides host cells genetically engineered to contain the polynucleotides of the invention.","For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods.","The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.","Knowledge of nucleic acid sequences allows for modification of cells to permit, or increase, expression of endogenous polypeptide.","Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the polypeptide at higher levels.","The heterologous promoter is inserted in such a manner that it is operatively linked to the encoding sequences.","See, for example, PCT International Publication No.","WO94/12650, PCT International Publication No.","WO92/20808, and PCT International Publication No.","WO91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.","If linked to the coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.","The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.","Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)).","The host cells containing one of the polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF.","Any host/vector system can be used to express one or more of the ORFs of the present invention.","These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, 293 cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B. subtilis.","The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level.","Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters.","Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.","Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of which is hereby incorporated by reference.","Various mammalian cell culture systems can also be employed to express recombinant protein.","Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981).","Other cell lines capable of expressing a compatible vector are, for example, the C127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.","Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences.","DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.","Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps.","Protein refolding steps can be used, as necessary, in completing configuration of the mature protein.","Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.","Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.","Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or insects or in prokaryotes such as bacteria.","Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.","Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins.","If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein.","Such covalent attachments may be accomplished using known chemical or enzymatic methods.","In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.","As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.","Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.","Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.","These sequence include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.","The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.","Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.","Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.","Here, the naturally occurring sequences are deleted and new sequences are added.","In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome.","The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative, selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.","Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.","The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.","No.","5,272,071 to Chappel; U.S. Pat.","No.","5,578,461 to Sherwin et al.","; International Application No.","PCT/US92/09627 (WO93/09222) by Selden et al.","; and International Application No.","PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.","4.6 Polypeptides of the Invention The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences set forth as any one of SEQ ID NO: 30369-60736 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ID NO: 1-30368 or the corresponding full length or mature protein.","Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ ID NO: 30369-60736 or (c) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions.","The invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ ID NO: 30369-60736 or the corresponding full length or mature protein; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological activity.","Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ ID NO: 30369-60736.Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention.","Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer.","Chem.","Soc.","114, 9245-9253 (1992), both of which are incorporated herein by reference.","Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.","The present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins.","The protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences.","The mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell.","The sequence of the mature form of the protein is also determinable from the amino acid sequence of the full-length form.","Where proteins of the present invention are membrane bound, soluble forms of the proteins are also provided.","In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which they are expressed.","Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.","The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention.","By “degenerate variant” is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence.","Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.","A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention.","At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers.","The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity.","This technique is particularly useful in producing small peptides and fragments of larger polypeptides.","Fragments are useful, for example, in generating antibodies against the native polypeptide.","Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.","The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein.","As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level.","One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.","The invention also relates to methods for producing a polypeptide comprising growing a culture of host cells of the invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown.","For example, the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide.","The polypeptide can be recovered from the culture, conveniently from the culture medium, or from a lysate prepared from the host cells and further purified.","Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein.","In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein.","One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention.","These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography.","See, e.g., Scopes, Protein Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in Molecular Biology.","Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.","The purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides.","These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins.","The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.","In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.","In addition, the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells.","The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for SEQ ID NO: 30369-60736.The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.","The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered.","For example, modifications in the peptide or DNA sequence can be made by those skilled in the art using known techniques.","Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.","For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.","Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat.","No.","4,518,584).","Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.","Regions of the protein that are important for the protein function can be determined by various methods known in the art including the alanine-scanning method which involved systematic substitution of single or strings of amino acids with alanine, followed by testing the resulting alanine-containing variant for biological activity.","This type of analysis determines the importance of the substituted amino acid(s) in biological activity.","Regions of the protein that are important for protein function may be determined by the eMATRIX program.","Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and are useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein.","Such modifications are encompassed by the present invention.","The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.","Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBat™ kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No.","1555 (1987), incorporated herein by reference.","As used herein, an insect cell capable of expressing a polynucleotide of the present invention is “transformed.” The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.","The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.","The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl™ or Cibacrom blue 3GA Sepharose™; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.","Alternatively, the protein of the invention may also be expressed in a form that will facilitate purification.","For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-5-transferase (GST) or thioredoxin (TRX), or as a His-tag.","Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.","), Pharmacia (Piscataway, N.J.) and Invitrogen, respectively.","The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.","One such epitope (“FLAG®”) is commercially available from Kodak (New Haven, Conn.).","Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein.","Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.","The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an “isolated protein.” The polypeptides of the invention include analogs (variants).","This embraces fragments, as well as peptides in which one or more amino acids has been deleted, inserted, or substituted.","Also, analogs of the polypeptides of the invention embrace fusions of the polypeptides or modifications of the polypeptides of the invention, wherein the polypeptide or analog is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent.","Such analogs may exhibit improved properties such as activity and/or stability.","Examples of moieties which may be fused to the polypeptide or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to pancreatic cells, e.g., antibodies to pancreatic cells, antibodies to immune cells such as T-cells, monocytes, dendritic cells, granulocytes, etc., as well as receptor and ligands expressed on pancreatic or immune cells.","Other moieties which may be fused to the polypeptide include therapeutic agents which are used for treatment, for example, immunosuppressive drugs such as cyclosporin, SK506, azathioprine, CD3 antibodies and steroids.","Also, polypeptides may be fused to immune modulators, and other cytokines such as alpha or beta interferon.","4.6.1 Determining Polypeptide and Polynucleotide Identity and Similarity Preferred identity and/or similarity are designed to give the largest match between the sequences tested.","Methods to determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et al., J. Molec.","Biol.","215:403410 (1990), PSI-BLAST (Altschul S. F. et al., Nucleic Acids Res.","vol.","25, pp.","3389-3402, herein incorporated by reference), eMatrix software (Wu et al., J. Comp.","Biol., Vol.","6, pp.","219-235 (1999), herein incorporated by reference), eMotif software (Nevill-Manning et al, ISMB-97, Vol.","4, pp.","202-209, herein incorporated by reference), pFam software (Sonnhammer et al., Nucleic Acids Res., Vol.","26(1), pp.","320-322 (1998), herein incorporated by reference) and the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol.","Biol, 157, pp.","105-31 (1982), incorporated herein by reference).","The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al.","NCB NLM NIH Bethesda, Md.","20894; Altschul, S., et al., J. Mol.","Biol.","215:403-410 (1990).","4.7 Chimeric and Fusion Proteins The invention also provides chimeric or fusion proteins.","As used herein, a “chimeric protein” or “fusion protein” comprises a polypeptide of the invention operatively linked to another polypeptide.","Within a fusion protein the polypeptide according to the invention can correspond to all or a portion of a protein according to the invention.","In one embodiment, a fusion protein comprises at least one biologically active portion of a protein according to the invention.","In another embodiment, a fusion protein comprises at least two biologically active portions of a protein according to the invention.","Within the fusion protein, the term “operatively linked” is intended to indicate that the polypeptide according to the invention and the other polypeptide are fused in-frame to each other.","The polypeptide can be fused to the N-terminus or C-terminus.","For example, in one embodiment a fusion protein comprises a polypeptide according to the invention operably linked to the extracellular domain of a second protein.","In another embodiment, the fusion protein is a GST-fusion protein in which the polypeptide sequences of the invention are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences.","In another embodiment, the fusion protein is an immunoglobulin fusion protein in which the polypeptide sequences according to the invention comprises one or more domains are fused to sequences derived from a member of the immunoglobulin protein family.","The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand and a protein of the invention on the surface of a cell, to thereby suppress signal transduction in vivo.","The immunoglobulin fusion proteins can be used to affect the bioavailability of a cognate ligand.","Inhibition of the ligand/protein interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e,g., cancer as well as modulating (e.g.","promoting or inhibiting) cell survival.","Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction of a polypeptide of the invention with a ligand.","A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques.","For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.","In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.","Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al.","(eds.)","CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).","Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).","A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein of the invention.","4.8 Gene Therapy Mutations in the polynucleotides of the invention gene may result in loss of normal function of the encoded protein.","The invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention.","Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments).","See, for example, Anderson, Nature, supplement to vol.","392, no.","6679, pp.","25-20 (1998).","For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).","Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression).","Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.","Treated cells can then be introduced in vivo for therapeutic purposes.","Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states.","It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.","Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art.","Further, the polypeptides of the present invention can be inhibited by using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific.","The present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.","These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.","Knowledge of DNA sequences provided by the invention allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide.","Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels.","The heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences.","See, for example, PCT International Publication No.","WO 94/12650, PCT International Publication No.","WO 92/20808, and PCT International Publication No.","WO 91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.","If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co-amplificationof the desired protein coding sequences in the cells.","In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.","As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.","Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.","Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.","These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.","The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.","Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.","Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.","Here, the naturally occurring sequences are deleted and new sequences are added.","In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome.","The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.","Markers useful for this purpose include the Herpes Sinplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphonbosyl-transferase (gpt) gene.","The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.","No.","5,272,071 to Chappel; U.S. Pat.","No.","5,578,461 to Sherwin et al.","; International Application No.","PCT/US92/09627 (WO93/09222) by Selden et al.","; and International Application No.","PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.","4.9 Transgenic Animals In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)].","Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.","Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.","Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.","No.","5,557,032, incorporated herein by reference.","Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.","Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.","Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.","No.","5,489,743 and PCT Publication No.","WO94/28122, incorporated herein by reference.","Transgenic animals can be prepared wherein all or part of a promoter of the polynucleotides of the invention is either activated or inactivated to alter the level of expression of the polypeptides of the invention.","Inactivation can be carried out using homologous recombination methods described above.","Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.","The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.","The polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express polypeptides of the invention or that express a variant polypeptide.","Such animals are useful as models for studying the in vivo activities of polypeptide as well as for studying modulators of the polypeptides of the invention.","In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)).","Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.","Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.","Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.","No.","5,557,032, incorporated herein by reference.","Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.","Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.","Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.","No.","5,489,743 and PCT Publication No.","WO94/28122, incorporated herein by reference.","Transgenic animals can be prepared wherein all or part of the polynucleotides of the invention promoter is either activated or inactivated to alter the level of expression of the polypeptides of the invention.","Inactivation can be carried out using homologous recombination methods described above.","Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.","The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.","4.10 Uses and Biological Activity The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified herein.","Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).","The mechanism underlying the particular condition or pathology will dictate whether the polypeptides of the invention, the polynucleotides of the invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment.","Thus, “therapeutic compositions of the invention” include compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or polypeptides of the invention (including full length protein, mature protein and truncations or domains thereof), or compounds and other substances that modulate the overall activity of the target gene products, either at the level of target gene/protein expression or target protein activity.","Such modulators include polypeptides, analogs, (variants), including fragments and fusion proteins, antibodies and other binding proteins; chemical compounds that directly or indirectly activate or inhibit the polypeptides of the invention (identified, e.g., via drug screening assays as described herein); antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies or other binding partners that specifically recognize one or more epitopes of the polypeptides of the invention.","The polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.","4.10.1 Research uses and Utilities The polynucleotides provided by the present invention can be used by the research community for various purposes.","The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a “gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response.","Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.","The polypeptides provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.","Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.","Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.","Methods for performing the uses listed above are well known to those skilled in the art.","References disclosing such methods include without limitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.4.10.2 Nutritional Uses Polynucleotides and polypeptides of the present invention can also be used as nutritional sources or supplements.","Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.","In such cases the polypeptide or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.","In the case of microorganisms, the polypeptide or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.","4.10.3 Cytokine and Cell Proliferation/Differentiation Activity A polypeptide of the present invention may exhibit activity relating to cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.","A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.","Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.","The activity of therapeutic compositions of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco.","Therapeutic compositions of the invention can be used in the following: Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.","137:3494-3500, 1986; Bertagnolli et al., J. Immunol.","145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., 1.Immunol.","149:3778-3783, 1992; Bowman et al., I. Immunol.","152:1756-1761, 1994.Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology.","J. E. e.a.","Coligan eds.","Vol 1 pp.","3.12.1-3.12.14, John Wiley and Sons, Toronto.","1994; and Measurement of mouse and human interleukin-γ, Schreiber, R. D. In Current Protocols in Immunology.","J. E. e.a Coligan eds.","Vol 1 pp.","6.8.1-6.8.8, John Wiley and Sons, Toronto.","1994.Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology.","J. E. e.a Coligan eds.","Vol 1 pp.","6.3.1-6.3.12, John Wiley and Sons, Toronto.","1991; deVries et al., J. Exp.","Med.","173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.","Natl.","Acad.","Sci.","U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6-Nordan, R. In Current Protocols in Immunology.","J. E. Coligan eds.","Vol 1 pp.","6.6.1-6.6.5, John Wiley and Sons, Toronto.","1991; Smith et al., Proc.","Natl.","Aced.","Sci.","U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J.","In Current Protocols in Immunology.","J. E. Coligan eds.","Vol 1 pp.","6.15.1 John Wiley and Sons, Toronto.","1991; Measurement of mouse and human Interleukin 9—Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J.","In Current Protocols in Immunology.","J. E. Coligan eds.","Vol 1 pp.","6.13.1, John Wiley and Sons, Toronto.","1991.Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc.","Natl.","Acad.","Sci.","USA 77:6091-6095, 1980; Weinberger et al., Eur.","J. Immun.","11:405-411, 1981; Takai et al., J. Immunol.","137:3494-3500, 1986; Takai et al., J. Immunol.","140:508-512, 1988.4.10.4 Stem Cell Growth Factor Activity A polypeptide of the present invention may exhibit stem cell growth factor activity and be involved in the proliferation, differentiation and survival of pluripotent and totipotent stem cells including primordial germ cells, embryonic stem cells, hematopoietic stem cells and/or germ line stem cells.","Administration of the polypeptide of the invention to stem cells in vivo or ex vivo is expected to maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering damaged or diseased tissues, transplantation, manufacture of bio-pharmaceuticals and the development of bio-sensors.","The ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.","It is contemplated that multiple different exogenous growth factors and/or cytokines may be administered in combination with the polypeptide of the invention to achieve the desired effect, including any of the growth factors listed herein, other stem cell maintenance factors, and specifically including stem cell factor (SCF), leukemia inhibitory factor (LIF), Flt-3 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatory protein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).","Since totipotent stem cells can give rise to virtually any mature cell type, expansion of these cells in culture will facilitate the production of large quantities of mature cells.","Techniques for culturing stem cells are known in the art and administration of polypeptides of the invention, optionally with other growth factors and/or cytokines, is expected to enhance the survival and proliferation of the stem cell populations.","This can be accomplished by direct administration of the polypeptide of the invention to the culture medium.","Alternatively, stroma cells transfected with a polynucleotide that encodes for the polypeptide of the invention can be used as a feeder layer for the stem cell populations in culture or in vivo.","Stromal support cells for feeder layers may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see U.S. Pat.","No.","5,690,926).","Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention.","This will allow for generation of undifferentiated totipotential/pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types.","These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential mRNA to create cDNA libraries and templates for polymerase chain reaction experiments.","These studies would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.","Expansion and maintenance of totipotent stem cell populations will be useful in the treatment of many pathological conditions.","For example, polypeptides of the present invention may be used to manipulate stem cells in culture to give rise to neuroepithelial cells that can be used to augment or replace cells damaged by illness, autoimmune disease, accidental damage or genetic disorders.","The polypeptide of the invention may be useful for inducing the proliferation of neural cells and for the regeneration of nerve and brain tissue, i.e.","for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders which involve degeneration, death or trauma to neural cells or nerve tissue.","In addition, the expanded stem cell populations can also be genetically altered for gene therapy purposes and to decrease host rejection of replacement tissues after grafting or implantation.","Expression of the polypeptide of the invention and its effect on stem cells can also be manipulated to achieve controlled differentiation of the stem cells into more differentiated cell types.","A broadly applicable method of obtaining pure populations of a specific differentiated cell type from undifferentiated stem cell populations involves the use of a cell-type specific promoter driving a selectable marker.","The selectable marker allows only cells of the desired type to survive.","For example, stem cells can be induced to differentiate into cardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin.","Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In: Principles of Tissue Engineering eds.","Lanza et al., Academic Press (1997)).","Alternatively, directed differentiation of stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.","In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity.","Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson et al.","Proc.","Natl.","Acad.","Sci, U.S.A., 92: 7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines.","The ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi-solid support e.g.","as described by Bernstein et al., Blood, 77: 2316-2321 (1991).","4.10.5 Hematopoiesis Regulating Activity A polypeptide of the present invention may be involved in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell disorders.","Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.","in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (ie., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.","Therapeutic compositions of the invention can be used in the following: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.","Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al.","Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells.","R. I. Freshney, et al.","eds.","Vol pp.","265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al., Proc.","Natl.","Acad.","Sci.","USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A.","In Culture of Hematopoietic Cells.","R. I. Freshney, et al.","eds.","Vol pp.","23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of Hematopoietic Cells.","R. I. Freshney, et al.","eds.","Vol pp.","1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells.","R. I. Freshney, et al.","eds.","Vol pp.","163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J.","In Culture of Hematopoietic Cells.","R. I. Freshney, et al.","eds.","Vol pp.","139-162, Wiley-Liss, Inc., New York, N.Y. 1994.4.10.6 Tissue Growth Activity A polypeptide of the present invention also may be involved in bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of burns, incisions and ulcers.","A polypeptide of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.","Compositions of a polypeptide, antibody, binding partner, or other modulator of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints.","De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.","A polypeptide of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of bone-forming cells.","Treatment of osteoporosis, osteoarthritis, bone degenerative disorders, or periodontal disease, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.)","mediated by inflammatory processes may also be possible using the composition of the invention.","Another category of tissue regeneration activity that may involve the polypeptide of the present invention is tendon/ligament formation.","Induction of tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.","Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.","De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.","The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.","The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.","The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.","The compositions of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e.","for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue.","More specifically, a composition may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome.","Further conditions that may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke.","Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a composition of the invention.","Compositions of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.","Compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues.","Part of the desired effects may be by inhibition or modulation of fibrotic scarring may allow normal tissue to regenerate.","A polypeptide of the present invention may also exhibit angiogenic activity.","A composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.","A composition of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.","Therapeutic compositions of the invention can be used in the following: Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No.","WO95/16035 (bone, cartilage, tendon); International Patent Publication No.","WO95/05846 (nerve, neuronal); International Patent Publication No.","WO91/07491 (skin, endothelium).","Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps.","71-112 (Maibach, H. I. and Rovee, D. T., eds.","), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.","Invest.","Dermatol 71:382-84 (1978).","4.10.7 Immune Stimulating or Suppressing Activity A polypeptide of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.","A polynucleotide of the invention can encode a polypeptide exhibiting such activities.","A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.","These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.","More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp.","and various fungal infections such as candidiasis.","Of course, in this regard, proteins of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.","Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.","Such a protein (or antagonists thereof, including antibodies) of the present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems.","Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein (or antagonists thereof) of the present invention.","The therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig slin sensitization test (Vohr et al., Arch.","Toxocol.","73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol.","Environ.","Health 53: 563-79).","Using the proteins of the invention it may also be possible to modulate immune responses, in a number of ways.","Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.","The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.","Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.","Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased.","Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.","Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).","For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation.","Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.","The administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.","Moreover, a lack of costinulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.","Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.","To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.","The efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.","Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4I g fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc.","Natl.","Acad.","Sci USA, 89:11102-11105 (1992).","In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.","846-847) can be used to determine the effect of therapeutic compositions of the invention on the development of that disease.","Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.","Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self-tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.","Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.","Administration of reagents which block stimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process.","Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.","The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases.","Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.","840-856).","Upregulation of an antigen function (e.g., a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy.","Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response.","For example, enhancing an immune response may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis.","Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.","Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.","The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.","A polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells.","In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class 1 alpha chain protein and P2 microglobulin protein or an MHC class II alpha chain protein and an MHC class II beta chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.","Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell.","Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.","Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.","The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc.","Natl.","Acad.","Sci.","USA 78:2488-2492, 1981; Herrmnann et al., J. Immunol.","128:1968-1974, 1982; Handa et al., J. Immunol.","135:1564-1572, 1985; Takai et al., I. Immunol.","137:3494-3500, 1986; Takai et al., J. Immunol.","140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol.","153:3079-3092, 1994.Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol.","144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology.","J. E. e.a.","Coligan eds.","Vol 1 pp.","3.8.1-3.8.16, John Wiley and Sons, Toronto.","1994.Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.","137:3494-3500, 1986; Takai et al., J. Immunol.","140:508-512, 1988; Bertagnolli et al., J. Immunol.","149:3778-3783, 1992.Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol.","134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260,1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:40374045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc.","Nat.","Acad.","Sci.","USA 88:7548-7551, 1991.4.10.8 Activin/Inhibin Activity A polypeptide of the present invention may also exhibit activin- or inhibin-related activities.","A polynucleotide of the invention may encode a polypeptide exhibiting such characteristics.","Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH).","Thus, a polypeptide of the present invention, alone or in heterodirners with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals.","Administration of sufficient amounts of other inhibins can induce infertility in these mammals.","Alternatively, the polypeptide of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary.","See, for example, U.S. Pat.","No.","4,798,885.A polypeptide of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as, but not limited to, cows, sheep and pigs.","The activity of a polypeptide of the invention may, among other means, be measured by the following methods.","Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc.","Natl.","Acad.","Sci.","USA 83:3091-3095, 1986.4.10.9 Chemotactic/Chemokinetic Activity A polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.","A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.","Chemotactic and chemolinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action.","Chemotactic or chemokinetic compositions (e.g.","proteins, antibodies, binding partners, or modulators of the invention) provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections.","For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.","A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.","Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells.","Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.","Therapeutic compositions of the invention can be used in the following: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.","Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al.","J. Clin.","Invest.","95:1370-1376, 1995; Lind et al.","APMIS 103:140-146, 1995; Muller et al Eur.","J. Immunol.","25:1744-1748; Gruber et al.","J. of Immunol.","152:5860-5867, 1994; Johnston et al.","J. of Immunol.","153:1762-1768, 1994.4.10.10 Hemostatic and Thrombolytic Activity A polypeptide of the invention may also be involved in hemostatis or thrombolysis or thrombosis.","A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.","Compositions may be usefull in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.","A composition of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).","Therapeutic compositions of the invention can be used in the following: Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin.","Pharmacol.","26:131-140, 1986; Burdick et al., Thrombosis Res.","45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79(1991); Schaub, Prostaglandins 35:467-474,1988.4.10.11 Cancer Diagnosis and Therapy Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis.","Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer.","For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy.","Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition.","Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.","Cancer treatments promote tumor regression by inhibiting tumor cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumor growth) and/or prohibiting metastasis by reducing tumor cell motility or invasiveness.","Therapeutic compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Karposi's sarcoma.","Polypeptides, polynucleotides, or modulators of polypeptides of the invention (including inhibitors and stimulators of the biological activity of the polypeptide of the invention) may be administered to treat cancer.","Therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g.","reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.","The composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail.","An anti-cancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery.","The use of anti-cancer cocktails as a cancer treatment is routine.","Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.","In addition, therapeutic compositions of the invention may be used for prophylactic treatment of cancer.","There are hereditary conditions and/or environmental situations (e.g.","exposure to carcinogens) known in the art that predispose an individual to developing cancers.","Under these circumstances, it may be beneficial to treat these individuals with therapeutically effective doses of the polypeptide of the invention to reduce the risk of developing cancers.","In vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment.","These in vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, N.Y. Ch 18 and Ch 21), tumor systems in nude mice as described in Giovanella et al., J. Natl.","Can.","Inst., 52: 921-30 (1974), mobility and invasive potential of tumor cells in Boyden Chamber assays as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays such as induction of vascularization of the chick chorioallantoic membrane or induction of vascular endothelial cell migration as described in Ribatta et al., Intl.","J. Dev.","Biol., 40: 1189-97 (1999) and Li et al., Clin.","Exp.","Metastasis, 17:423-9 (1999), respectively.","Suitable tumor cells lines are available, e.g.","from American Type Tissue Culture Collection catalogs.","4.10.12 RECEPTOR/LIGAND ACTIVITY A polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions.","A polynucleotide of the invention can encode a polypeptide exhibiting such characteristics.","Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses.","Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.","A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.","The activity of a polypeptide of the invention may, among other means, be measured by the following methods: Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc.","Natl.","Acad.","Sci.","USA 84:6864-6868, 1987; Bierer et al., J. Exp.","Med.","168:1145-1156, 1988; Rosenstein et al., J. Exp.","Med.","169:149-160 1989; Stoltenborg et al., J. Immunol.","Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.By way of example, the polypeptides of the invention may be used as a receptor for a ligand(s) thereby transmitting the biological activity of that ligand(s).","Ligands may be identified through binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays, or other methods known in the art.","Studies characterizing drugs or proteins as agonist or antagonist or partial agonists or a partial antagonist require the use of other proteins as competing ligands.","The polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, colorimetric molecules or toxin molecules by conventional methods.","(“Guide to Protein Purification” Murray P. Deutscher (ed) Methods in Enzymology Vol.","182 (1990) Academic Press, Inc. San Diego).","Examples of radioisotopes include, but are not limited to, tritium and carbon-14.Examples of colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules.","Examples of toxins include, but are not limited, to ricin.","4.10.13 DRUG SCREENING This invention is particularly useful for screening chemical compounds by using the novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques.","The polypeptides or fragments employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.","One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof.","Drugs are screened against such transformed cells in competitive binding assays.","Such cells, either in viable or fixed form, can be used for standard binding assays.","One may measure, for example, the formation of complexes between polypeptides of the invention or fragments and the agent being tested or examine the diminution in complex formation between the novel polypeptides and an appropriate cell line, which are well known in the art.","Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include (1) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.","Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.","The sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves.","Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof.","For a review, see Science 282:63-68 (1998).","Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods.","Of particular interest are peptide and oligonucleotide combinatorial libraries.","Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.","For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr.","Opin.","Biotechnol.","8:701-707 (1997).","For reviews and examples of peptidomimetic libraries, see Al-Obeidi et al., Mol.","Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol, 1(1): 114-19 (1997); Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996) (alkylated dipeptides).","Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to bind a polypeptide of the invention.","The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.","In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.","The binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes.","The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention.","Alternatively, the binding molecules may be complexed with imaging agents for targeting and imaging purposes.","4.10.14 ASSAY FOR RECEPTOR ACTIVITY The invention also provides methods to detect specific binding of a polypeptide e.g.","a ligand or a receptor.","The art provides numerous assays particularly useful for identifying previously unknown binding partners for receptor polypeptides of the invention.","For example, expression cloning using mammalian or bacterial cells, or dihybrid screening assays can be used to identify polynucleotides encoding binding partners.","As another example, affinity chromatography with the appropriate immobilized polypeptide of the invention can be used to isolate polypeptides that recognize and bind polypeptides of the invention.","There are a number of different libraries used for the identification of compounds, and in particular small molecules, that modulate (i.e., increase or decrease) biological activity of a polypeptide of the invention.","Ligands for receptor polypeptides of the invention can also be identified by adding exogenous ligands, or cocktails of ligands to two cells populations that are genetically identical except for the expression of the receptor of the invention: one cell population expresses the receptor of the invention whereas the other does not.","The responses of the two cell populations to the addition of ligands(s) are then compared.","Alternatively, an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s).","As still another example, BIAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.","The role of downstream intracellular signaling molecules in the signaling cascade of the polypeptide of the invention can be determined.","For example, a chimeric protein in which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of a protein, whose ligand has been identified, is produced in a host cell.","The cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor.","Known downstream proteins involved in intracellular signaling can then be assayed for expected modifications i.e.","phosphorylation.","Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.","4.10.15 Anti-Inflammtory Activity Compositions of the present invention may also exhibit anti-inflammatory activity.","The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.","Compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1.Compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.","Compositions of this invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.","4.10.16 LEUKEMIAS Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention.","Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.","B. Lippincott Co., Philadelphia).","4.10.17 NERVOUS SYSTEM DISORDERS Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.","Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (iii) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis; (iv) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis; (v) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (vi) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (vii) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.","Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons.","For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful according to the invention: (i) increased survival time of neurons in culture; (ii) increased sprouting of neurons in culture or in vivo; (iii) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (iv) decreased symptoms of neuron dysfunction in vivo.","Such effects may be measured by any method known in the art.","In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al.","(1990, J. Neurosci.","10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al.","(1980, Exp.","Neurol.","70:65-82) or Brown et al.","(1981, Ann.","Rev.","Neurosci.","4:17-42); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.","In specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).","4.10.18 Other Activities A polypeptide of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fingi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.","4.10.19 Identification of Polymorphisms The demonstration of polymorphisms makes possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment.","Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately.","For example, the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.","Polymorphisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymorphism in the DNA.","For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced.","Alternatively, the DNA may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymorphism is extended with one or more labeled nucleotides).","In addition, traditional restriction fragment length polymorphism analysis (using restriction enzymes that provide differential digestion of the genomic DNA depending on the presence or absence of the polymorphism) may be performed.","Arrays with nucleotide sequences of the present invention can be used to detect polymorphisms.","The array can comprise modified nucleotide sequences of the present invention in order to detect the nucleotide sequences of the present invention.","In the alternative, any one of the nucleotide sequences of the present invention can be placed on the array to detect changes from those sequences.","Alternatively a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.","4.10.20 Arthritis and Inflammation The immunosuppressive effects of the compositions of the invention against rheumatoid arthritis are determined in an experimental animal model system.","The experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et al., 1963, Int.","Arch.","Allergy Appl.","Immunol., 23:129.Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA).","The route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture.","The polypeptide is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg.","The control consists of administering PBS only.","The procedure for testing the effects of the test compound would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24.At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall arthritis score may be obtained as described by J. Holoskitz above.","An analysis of the data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.","4.11 Therapeutic Methods The compositions (including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides) of the invention have numerous applications in a variety of therapeutic methods.","Examples of therapeutic applications include, but are not limited to, those exemplified herein.","4.11.1 Example One embodiment of the invention is the administration of an effective amount of the polypeptides or other composition of the invention to individuals affected by a disease or disorder that can be modulated by regulating the peptides of the invention.","While the mode of administration is not particularly important, parenteral administration is preferred.","An exemplary mode of administration is to deliver an intravenous bolus.","The dosage of the polypeptides or other composition of the invention will normally be determined by the prescribing physician.","It is to be expected that the dosage will vary according to the age, weight, condition and response of the individual patient.","Typically, the amount of polypeptide administered per dose will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight, with the preferred dose being about 0.1 μg/kg to 10 mg/kg of patient body weight.","For parenteral administration, polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle.","Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin.","The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient.","The preparation of such solutions is within the skill of the art.","4.12 Pharmaceutical Formulations and Routes of Administration A protein or other composition of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources and including antibodies and other binding partners of the polypeptides of the invention) may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders.","Such a composition may optionally contain (in addition to protein or other active ingredient and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.","The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).","The characteristics of the carrier will depend on the route of administration.","The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, ThF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.","In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question.","These agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), as well as cytokines described herein.","The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or other active ingredient or complement its activity or use in treatment.","Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein or other active ingredient of the invention, or to minimize side effects.","Conversely, protein or other active ingredient of the present invention may be included in formulations of the particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent (such as IL-1Ra, IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents).","A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.","As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.","As an alternative to being included in a pharmaceutical composition of the invention including a first protein, a second protein or a therapeutic agent may be concurrently administered with the first protein (e.g., at the same time, or at differing times provided that therapeutic concentrations of the combination of agents is achieved at the treatment site).","Techniques for formulation and administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.","A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.","When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone.","When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.","In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated.","Protein or other active ingredient of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.","When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein or other active ingredient of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially.","If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.","4.12.1 Routes of Administration Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.","Administration of protein or other active ingredient of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.","Intravenous administration to the patient is preferred.","Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthritic joints or in fibrotic tissue, often in a depot or sustained release formulation.","In order to prevent the scarring process frequently occurring as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops.","Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue.","The liposomes will be targeted to and taken up selectively by the afflicted tissue.","The polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action.","The determination of a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art.","Preferably for wound treatment, one administers the therapeutic compound directly to the site.","Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models.","Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.","4.12.2 Compositions/Formulations Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.","These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.","Proper formulation is dependent upon the route of administration chosen.","When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.","When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.","The tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient of the present invention.","When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.","The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.","When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and preferably from about 1 to 50% protein or other active ingredient of the present invention.","When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.","The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.","A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.","The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.","For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.","Such penetrants are generally known in the art.","For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.","Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.","Pharmaceutical preparations for oral use can be obtained from a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.","Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).","If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.","Dragee cores are provided with suitable coatings.","For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.","Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.","Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.","The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.","In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.","In addition, stabilizers may be added.","All formulations for oral administration should be in dosages suitable for such administration.","For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.","For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.","In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.","Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.","The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.","Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.","The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.","Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.","Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.","Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.","Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.","Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.","Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.","The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.","In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.","Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.","Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.","A pharmaceutical carrier for the hydrophobic compounds of the invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.","The co-solvent system may be the VPD co-solvent system.","VPD is a solution of 3%/o w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.","The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution.","This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.","Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.","Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g.","polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.","Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed.","Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.","Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.","Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.","Various types of sustained-release materials have been established and are well known by those skilled in the art.","Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.","Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed.","The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients.","Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.","Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions.","Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.","The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) or other active ingredient(s) of present invention along with protein or peptide antigens.","The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.","B-lymphocytes will respond to antigen through their surface immunoglobulin receptor.","T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.","MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.","The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.","Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.","The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.","Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like.","Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat.","Nos.","4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.","The amount of protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone.","Ultimately, the attending physician will decide the amount of protein or other active ingredient of the present invention with which to treat each individual patient.","Initially, the attending physician will administer low doses of protein or other active ingredient of the present invention and observe the patient's response.","Larger doses of protein or other active ingredient of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.","It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein or other active ingredient of the present invention per kg body weight.","For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.","When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.","Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.","Topical administration may be suitable for wound healing and tissue repair.","Therapeutically useful agents other than a protein or other active ingredient of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.","Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing or other active ingredient-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.","Such matrices may be formed of materials presently in use for other implanted medical applications.","The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties.","The particular application of the compositions will define the appropriate formulation.","Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.","Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen.","Further matrices are comprised of pure proteins or extracellular matrix components.","Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.","Matrices may be comprised of combinations of any of the above-mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate.","The biocermics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.","Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns.","In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.","A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).","Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).","The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.","In further compositions, proteins or other active ingredients of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.","These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF).","The therapeutic compositions are also presently valuable for veterinary applications.","Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins or other active ingredients of the present invention.","The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.","The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.","For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage.","Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.","Polynucleotides of the present invention can also be used for gene therapy.","Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject.","Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).","Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.","Treated cells can then be introduced in vivo for therapeutic purposes.","4.12.3 Effective Dosage Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose.","More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated.","Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.","For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays.","For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans.","For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the protein's biological activity).","Such information can be used to more accurately determine useful doses in humans.","A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient.","Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).","The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50.Compounds which exhibit high therapeutic indices are preferred.","The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.","The dosage of such compounds lies preferably within a range of circulating concentrations that include the EDso with little or no toxicity.","The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.","The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.","See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.","1 p.1.Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC).","The MEC will vary for each compound but can be estimated from in vitro data.","Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration.","However, HPLC assays or bioassays can be used to determine plasma concentrations.","Dosage intervals can also be determined using MEC value.","Compounds should be administered using a regimen that maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.","In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.","An exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 μg/kg to 25 mg/kg of patient body weight daily, varying in adults and children.","Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.","The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.","4.12.4 Packaging The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.","The pack may, for example, comprise metal or plastic foil, such as a blister pack.","The pack or dispenser device may be accompanied by instructions for administration.","Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.","4.13 Antibodies Also included in the invention are antibodies to proteins, or fragments of proteins of the invention.","The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen.","Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab, and F(ab′)2 fragments, and an Fab expression library.","In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.","Certain classes have subclasses as well, such as IgG1, IgG2, and others.","Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.","Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.","An isolated related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.","The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens.","An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, (for example the amino acid sequence shown in SEQ ID NO: 30369), and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.","Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.","Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.","In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region on the surface of the protein of the invention that is located on the surface of the protein, e.g., a hydrophilic region.","A hydrophobicity analysis of the human related protein sequence will indicate which regions of a related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.","As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation.","See, e.g., Hopp and Woods, 1981, Proc.","Nat.","Acad.","Sci.","USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol.","Biol.","157: 105-142, each of which is incorporated herein by reference in its entirety.","Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.","A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.","Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference).","Some of these antibodies are discussed below.","5.13.1 Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing.","An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.","Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.","Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.","The preparation can further include an adjuvant.","Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.","), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.","Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).","The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum.","Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography.","Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol.","14, No.","8 (Apr.","17, 2000), pp.","25-28).","5.13.2 Monoclonal Antibodies The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product.","In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.","MAbs thus contain an antigen-binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.","Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).","In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.","Alternatively, the lymphocytes can be immunized in vitro.","The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.","Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.","The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.","59-103).","Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.","Usually, rat or mouse myeloma cell lines are employed.","The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.","For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.","Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.","More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.","Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.","51-63).","The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.","Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).","Such techniques and assays are known in the art.","The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal.","Biochem.","107:220 (1980).","Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.","After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods.","Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.","Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.","The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.","The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat.","No.","4,816,567.DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).","The hybridoma cells of the invention serve as a preferred source of such DNA.","Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.","The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat.","No.","4,816,567; Morrison, Nature 368 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.","Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.","5.13.2 Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies.","These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.","Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.","Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.","(See also U.S. Pat.","No.","5,225,539.)","In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.","Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.","In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.","The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr.","Op.","Struct.","Biol., 2:593-596 (1992)).","5.13.3 Human Antibodies Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes.","Such antibodies are termed “human antibodies”, or “fully human antibodies” herein.","Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.","77-96).","Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983.Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.","77-96).","In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol.","Biol., 227:381 (1991); Marks et al., J. Mol.","Biol., 222:581 (1991)).","Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.","Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.","This approach is described, for example, in U.S. Pat.","Nos.","5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.","(Bio/Technology 10, 779-783 (1992)); Lonberg et al.","(Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern.","Rev.","Immunol.","13 65-93 (1995)).","Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.","(See PCT publication WO94/02602).","The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.","The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments.","An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.","The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096.This animal produces B cells which secrete fully human immunoglobulins.","The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.","Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.","An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Pat.","No.","5,939,598.It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.","A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat.","No.","5,916,771.It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.","The hybrid cell expresses an antibody containing the heavy chain and the light chain.","In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.5.13.4 Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Pat.","No.","4,946,778).","In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.","Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab′)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab′)2fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) FV fragments.","5.13.5 Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.","In the present case, one of the binding specificities is for an antigenic protein of the invention.","The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.","Methods for making bispecific antibodies are known in the art.","Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)).","Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure.","The purification of the correct molecule is usually accomplished by affinity chromatography steps.","Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences.","The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.","It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions.","DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism.","For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).","According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.","The preferred interface comprises at least a part of the CH3 region of an antibody constant domain.","In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g.","tyrosine or tryptophan).","Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.","alanine or threonine).","This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.","Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g.","F(ab′)2 bispecific antibodies).","Techniques for generating bispecific antibodies from antibody fragments have been described in the literature.","For example, bispecific antibodies can be prepared using chemical linkage.","Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments.","These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.","The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.","One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody.","The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.","Additionally, Fab′ fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies.","Shalaby et al., J. Exp.","Med.","175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule.","Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.","The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.","Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described.","For example, bispecific antibodies have been produced using leucine zippers.","Kostelny et al., J. Immunol.","148(5):1547-1553 (1992).","The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion.","The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.","This method can also be utilized for the production of antibody homodimers.","The “diabody” technology described by Hollinger et al., Proc.","Natl.","Acad.","Sci.","USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.","The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain.","Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.","Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) diners has also been reported.","See, Gruber et al., J. Immunol.","152:5368 (1994).","Antibodies with more than two valencies are contemplated.","For example, trispecific antibodies can be prepared.","Tutt et al., J. Immunol.","147:60 (1991).","Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.","Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.","CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.","Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.","These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.","Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (° F.).","5.13.6 Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.","Heteroconjugate antibodies are composed of two covalently joined antibodies.","Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat.","No.","4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089).","It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.","For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond.","Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat.","No.","4,676,980.5.13.7 Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer.","For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.","The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).","See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.","Immunol., 148: 2918-2922 (1992).","Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al.","Cancer Research, 53: 2560-2565 (1993).","Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities.","See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).","5.13.8 Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).","Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above.","Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.","A variety of radionuclides are available for the production of radioconjugated antibodies.","Examples include 212Bi, 131I, 90Y, 90Y, and 186Re.","Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).","For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).","Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.","See WO94/11026.In another embodiment, the antibody can be conjugated to a “receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.","4.14 Computer Readable Sequences In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media.","As used herein, “computer readable media” refers to any medium which can be read and accessed directly by a computer.","Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention.","As used herein, “recorded” refers to a process for storing information on computer readable medium.","A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.","A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention.","The choice of the data storage structure will generally be based on the means chosen to access the stored information.","In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium.","The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.","A skilled artisan can readily adapt any number of data processor structuring formats (e.g.","text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.","By providing any of the nucleotide sequences SEQ ID NO: 1-30368 or a representative fragment thereof; or a nucleotide sequence at least 95% identical to any of the nucleotide sequences of SEQ ID NO: 1-30368 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes.","Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium.","The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol.","Biol.","215:403-410 (1990)) and BLAZE (Brutlag et al., Comp.","Chem.","17:203-207 (1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) within a nucleic acid sequence.","Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.","As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention.","The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.","A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention.","As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means.","As used herein, “data storage means” refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.","As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means.","Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif.","A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention.","Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASIN and BLASTA (NPOLYPEPTIDEIA).","A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems.","As used herein, a “target sequence” can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids.","A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database.","The most preferred sequence length of a target sequence is from about 10 to 300 amino acids, more preferably from about 30 to 100 nucleotide residues.","However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.","As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.","There are a variety of target motifs known in the art.","Protein target motifs include, but are not limited to, enzyme active sites and signal sequences.","Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).","4.15 Triple Helix Formation In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA.","Polynucleotides suitable for use in these methods are preferably 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.","Acids Res.","6:3073 (1979); Cooney et al., Science 15241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Olnno, J. Neurochem.","56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).","Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.","Both techniques have been demonstrated to be effective in model systems.","Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.","4.16 Diagnostic Assays and Kits The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label.","In general, methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample.","Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample.","In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample.","In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of the nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample.","Conditions for incubating a nucleic acid probe or antibody with a test sample vary.","Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay.","One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention.","Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol.","1 (1982), Vol.","2 (1983), Vol.","3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).","The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine.","The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed.","Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.","In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.","Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.","In detail, a compartment kit includes any kit in which reagents are contained in separate containers.","Such containers include small glass containers, plastic containers or strips of plastic or paper.","Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.","Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.","), and containers which contain the reagents used to detect the bound antibody or probe.","Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.","One skilled in the art will readily recognize that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.","4.17 Medical Imaging The novel polypeptides and binding partners of the invention are useful in medical imaging of sites expressing the molecules of the invention (e.g., where the polypeptide of the invention is involved in the immune response, for imaging sites of inflammation or infection).","See, e.g., Kunkel et al., U.S. Pat.","No.","5,413,778.Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.","4.18 Screening Assays Using the isolated proteins and polynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any of the nucleotide sequences set forth in SEQ ID NO: 1-30368, or bind to a specific domain of the polypeptide encoded by the nucleic acid.","In detail, said method comprises the steps of: (a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and (b) determining whether the agent binds to said protein or said nucleic acid.","In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.","Likewise, in general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.","Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified.","Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound).","Alternatively, compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound).","Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.","The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents.","The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.","For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention.","Alternatively, agents may be rationally selected or designed.","As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the particular protein.","For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like, capable of binding to a specific peptide sequence, in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide, W.H.","Freeman, N.Y. (1992), pp.","289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.","In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention.","As described above, such agents can be randomly screened or rationally designed/selected.","Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control.","One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA.","Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.","Agents suitable for use in these methods preferably contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.","Acids Res.","6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem.","56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).","Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.","Both techniques have been demonstrated to be effective in model systems.","Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.","Agents that bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent.","Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.","4.19 Use of Nucleic Acids as Probes Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences.","The hybridization probes of the subject invention may be derived from any of the nucleotide sequences SEQ ID NO: 1-30368.Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from of any of the nucleotide sequences SEQ ID NO: 1-30368 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.","Any suitable hybridization technique can be employed, such as, for example, in situ hybridization.","PCR as described in U.S. Pat.","Nos.","4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences.","Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both.","The probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.","Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA probes.","Such vectors are known in the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides.","The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences.","The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques.","These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like.","The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y. Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data.","Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981f).","Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease.","The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.","4.20 Preparation of Support Bound Oligonucleotides Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.","Support bound oligonucleotides may be prepared by any of the methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon.","One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers.","Immobilization can be achieved using passive adsorption (Inouye & Hondo, (1990) J. Clin.","Microbiol.","28(6) 1469-72); using UV light (Nagata et al., 1985; Dahlen et al., 1987; Morrissey& Collins, (1989) Mol.","Cell Probes 3(2) 189-207) or by covalent binding of base modified DNA (Keller et al., 1988; 1989); all references being specifically incorporated herein.","Another strategy that may be employed is the use of the strong biotin-streptavidin interaction as a linker.","For example, Broude et al.","(1994) Proc.","Natl.","Acad.","Sci.","USA 91(8) 3072-6, describe the use of biotinylated probes, although these are duplex probes, that are immobilized on streptavidin-coated magnetic beads.","Streptavidin-coated beads may be purchased from Dynal, Oslo.","Of course, this same linking chemistry is applicableto coating any surface with streptavidin Biotinylated probes may be purchased from various sources, such as, e.g., Operon Technologies (Alameda, Calif.).","Nunc Laboratories (Naperville, Ill.) is also selling suitable material that could be used.","Nunc Laboratories have developed a method by which DNA can be covalently bound to the microwell surface termed Covalink NH.","CovaLink NH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridge-heads for further covalent coupling.","CovaLink Modules may be purchased from Nunc Laboratories.","DNA molecules may be bound to CovaLink exclusively at the 5′-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA (Rasmussen et al., (1991) Anal.","Biochem.","198(1) 138-42).","The use of CovaLink NH strips for covalent binding of DNA molecules at the 5′-end has been described (Rasmussen et al., (1991).","In this technology, a phosphoramidate bond is employed (Chu et al., (1983) Nucleic Acids Res.","1 (8) 6513-29).","This is beneficial as immobilization using only a single covalent bond is preferred.","The phosphoramidate bond joins the DNA to the CovaLink NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm.","To link an oligonucleotide to CovaLink NH via an phosphoramidate bond, the oligonucleotide terminus must have a 5′-end phosphate group.","It is, perhaps, even possible for biotin to be covalently bound to CovaLink and then streptavidin used to bind the probes.","More specifically, the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min.","at 95° C. and cooling on ice for 10 min.","Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm7), is then added to a final concentration of 10 mM 1-MeIm7.A ss DNA solution is then dispensed into CovaLink NH strips (75 ul/well) standing on ice.","Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIm7, is made fresh and 25 ul added per well.","The strips are incubated for 5 hours at 50° C. After incubation the strips are washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3 times, then they are soaked with washing solution for 5 min., and finally they are washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50° C.).","It is contemplated that a further suitable method for use with the present invention is that described in PCT Patent Application WO 90/03382 (Southern & Maskos), incorporated herein by reference.","This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3′-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups carried by the support.","The oligonucleotide is then synthesized on the supported nucleoside and protecting groups removed from the synthetic oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support.","Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphorate.","An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed.","For example, addressable laser-activated photodeprotection may be employed in the chemical synthesis of oligonucleotides directly on a glass surface, as described by Fodor et al.","(1991) Science 251(4995) 767-73, incorporated herein by reference.","Probes may also be immobilized on nylon supports as described by Van Ness et al.","(1991) Nucleic Acids Res.","19(12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier (1988) Anal.","Biochem.","169(1) 104-8; all references being specifically incorporated herein.","To link an oligonucleotide to a nylon support, as described by Van Ness et at (1991), requires activation of the nylon surface via alkylation and selective activation of the 5′-amine of oligonucleotides with cyanuric chloride.","One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described by Pease et al., (1994) PNAS USA 91(11) 5022-6, incorporated herein by reference).","These authors used current photolithographic techniques to generate arrays of immobilized oligonucleotide probes (DNA chips).","These methods, in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5′-protected N-acyl-deoxynucleoside phosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies.","A matrix of 256 spatially defined oligonucleotide probes may be generated in this manner.","4.21 Preparation of Nucleic Acid Fragments The nucleic acids may be obtained from any appropriate source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC inserts, and RNA, including mRNA without any amplification steps.","For example, Sambrook et al.","(1989) describes three protocols for the isolation ofhigh molecular weight DNA from mammalian cells (p. 9.14-9.23).","DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and/or prepared directly from genomic DNA or cDNA by PCR or other amplification methods.","Samples may be prepared or dispensed in multiwell plates.","About 100-1000 ng of DNA samples may be prepared in 2-500 ml of final volume.","The nucleic acids would then be fragmented by any of the methods known to those of skill in the art including, for example, using restriction enzymes as described at 9.24-9.28 of Sambrook et al.","(1989), shearing by ultrasound and NaOH treatment.","Low pressure shearing is also appropriate, as described by Schriefer et at (1.990) Nucleic Acids Res.","18(24) 7455-6, incorporated herein by reference).","In this method, DNA samples are passed through a small French pressure cell at a variety of low to intermediate pressures.","A lever device allows controlled application of low to intermediate pressures to the cell.","The results of these studies indicate that low-pressure shearing is a useful alternative to sonic and enzymatic DNA fragmentation methods.","One particularly suitable way for fragmenting DNA is contemplated to be that using the two base recognition endonuclease, CviJI, described by Fitzgerald et al.","(1992) Nucleic Acids Res.","20(14) 3753-62.These authors described an approach for the rapid fragmentation and fractionation of DNA into particular sizes that they contemplated to be suitable for shotgun cloning and sequencing.","The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends.","A typical reaction conditions, which alter the specificity of this enzyme (CviJI**), yield a quasi-random distribution of DNA fragments form the small molecule pUC19 (2688 base pairs).","Fitzgerald et al.","(1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI** digest ofpUCl9 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z minus M13 cloning vector.","Sequence analysis of 76 clones showed that CviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation.","As reported in the literature, advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed.","Irrespective of the manner in which the nucleic acid fragments are obtained or prepared, it is important to denature the DNA to give single stranded pieces available for hybridization.","This is achieved by incubating the DNA solution for 2-5 minutes at 80-90° C. The solution is then cooled quickly to 2° C. to prevent renaturation of the DNA fragments before they are contacted with the chip.","Phosphate groups must also be removed from genomic DNA by methods known in the art.","4.22 Preparation of DNA Arrays Arrays may be prepared by spotting DNA samples on a support such as a nylon membrane.","Spotting may be performed by using arrays of metal pins (the positions of which correspond to an array of wells in a microtiter plate) to repeated by transfer of about 20 nl of a DNA solution to a nylon membrane.","By offset printing, a density of dots higher than the density of the wells is achieved.","One to 25 dots may be accommodated in 1 mm2, depending on the type of label used.","By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed.","Samples in one subarray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones.","Each of the subarrays may represent replica spotting of the same samples.","In one example, a selected gene segment may be amplified from 64 patients.","For each patient, the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample).","A plate for each of the 64 patients is prepared.","By using a 96-pin device, all samples may be spotted on one 8×12 cm membrane.","Subarrays may contain 64 samples, one from each patient.","Where the 96 subarrays are identical, the dot span may be 1 mm2 and there may be a 1 mm space between subarrays.","Another approach is to use membranes or plates (available from NUNC, Naperville, Ill.) which may be partitioned by physical spacers e.g.","a plastic grid molded over the membrane, the grid being similar to the sort of membrane applied to the bottom of multiwell plates, or hydrophobic strips.","A fixed physical spacer is not preferred for imaging by exposure to flat phosphor-storage screens or x-ray films.","The present invention is illustrated in the following examples.","Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention.","Accordingly, it is intended that the broader aspects of the present invention not be limited to the disclosure of the following examples.","The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention.","Indeed, numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the present preferred embodiments.","Consequently, the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims.","All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.","5.0 EXAMPLES 5.1 Example 1 Novel Nucleic Acid Sequences Obtained From Various Libraries A plurality of novel nucleic acids were obtained from cDNA libraries prepared from various human tissues and in some cases isolated from a genomic library derived from human chromosome using standard PCR, SBH sequence signature analysis and Sanger sequencing techniques.","The inserts of the library were amplified with PCR using primers specific for the vector sequences which flank the inserts.","Clones from cDNA libraries were spotted on nylon membrane filters and screened with oligonucleotide probes (e.g., 7-mers) to obtain signature sequences.","The clones were clustered into groups of similar or identical sequences.","Representative clones were selected for sequencing.","In some cases, the 5′ sequence of the amplified inserts was then deduced using a typical Sanger sequencing protocol.","PCR products were purified and subjected to fluorescent dye terminator cycle sequencing.","Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer to obtain the novel nucleic acid sequences.","In some cases RACE (Rapid Amplification of cDNA Ends) was performed to further extend the sequence in the 5′ direction.","5.2 Example 2 Novel Contigs The novel contigs of the invention were assembled from sequences that were obtained from a cDNA library by methods described in Example 1 above, and in some cases sequences obtained from one or more public databases.","The sequences for the resulting nucleic acid contigs are designated as SEQ ID NO: 1-30368 and are provided in the attached Sequence Listing.","The contigs were assembled using an EST sequence as a seed.","Then a recursive algorithm was used to extend the seed EST into an extended assemblage, by pulling additional sequences from different databases (i.e., Hyseq's database containing EST sequences, dbEST version 115, gb pri 115, and UniGene version 103, and exons from public domain genomic sequences predicted by GenScan) that belong to this assemblage.","The algorithm terminated when there was no additional sequences from the above databases that would extend the assemblage.","Further, the inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST score greater than 300 and percent identity greater than 95%.","The novel predicted polypeptides (including proteins) encoded by the novel polynucleotides (SEQ ID NO: 1-30368) of the present invention are incorporated in the attached Sequence Listing.","A subset the predicted polypeptide sequences contain an unknown amino acid, a stop codon, a possible nucleotide deletion or a possible nucleotide insertion.","These sequences have been shown in their entirety with the special characters in Table 2.Table 2 also shows the corresponding start and stop nucleotide locations to each of SEQ ID NO: 1-30368.Table 2 also indicates the method by which the polypeptide was predicted.","Method A refers to a polypeptide obtained by using a software program called FASTY (available from http://fastabioch.virginia.edu) which selects a polypeptide based on a comparison of the translated novel polynucleotide to known polynucleotides (W. R Pearson, Methods in Enzymology, 183:63-98 (1990), herein incorporated by reference).","Method B refers to a polypeptide obtained by using a software program called GenScan for human/vertebrate sequences (available from Stanford University, Office of Technology Licensing) that predicts the polypeptide based on a probabilistic model of gene structure/compositional properties (C. Burge and S. Karlin, J. Mol.","Biol., 268:78-94(1997), incorporated herein by reference).","Method C refers to a polypeptide obtained by using a Hyseq proprietary software program that translates the novel polynucleotide and its complementary strand into six possible amino acid sequences (forward and reverse frames) and chooses the polypeptide with the longest open reading frame.","The nearest neighbor results for SEQ ID NO: 1-30368 were obtained by a BLASTP version 2.0al 19 MP-WashU search against Genpept release 121 and Geneseq release 200103 (Derwent), using BLAST algorithm.","The nearest neighbor result showed the closest homologue for SEQ ID NO: 1-30368.The nearest neighbor results for SEQ ID NO: 1-30368 are incorporated in the attached Sequence Listing.","Using eMatrix software package (Stanford University, Stanford, Calif.) (Wu et al., J. Comp.","Biol., Vol.","6 pp.","219-235 (1999) herein incorporated by reference), all the sequences were examined to determine whether they had identifiable signature regions.","The attached Sequence Listing provodes the results obtained by eMatrix analysis for each polypeptide as follows: the signature region found in the indicated polypeptide sequences, the description of the signature, the eMatrix p-value(s) and the position(s) of the signature within the polypeptide sequence.","Using the pFam software program (Sonnhammer et al., Nucleic Acids Res., Vol.","26(1) pp.","320-322 (1998) herein incorporated by reference) all the polypeptide sequences were examined for domains with homology to certain peptide domains.","The attached Sequence Listing provides the results obtained by PFAM analysis for each peptide, namely: the name of the domain found, the description, the p-value and the pFam score for the identified domain within the sequence.","Tables 1 and 2 follow.","Table I shows the various tissue sources of SEQ ID NO: 1-30368.Table 2 shows the start and stop nucleotides for the translated amino acid sequence for which each assemblage encodes.","Table 2 also provides a correlation between the amino acid sequences set forth in the Sequence Listing, the nucleotide sequences set forth in the Sequence Listing and the SEQ ID NO: in U.S. Ser.","No.","09/540,217"]],"string":"[\n [\n \"Novel nucleic acids and polypeptides\",\n \"The present invention provides novel nucleic acids, novel polypeptide sequences encoded by these nucleic acids and uses thereof.\"\n ],\n [\n \"1.An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-30368, a mature protein coding portion of SEQ ID NO: 1-30368, an active domain of SEQ ID NO: 1-30368, and complementary sequences thereof.\",\n \"2.An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide hybridizes to the polynucleotide of claim 1 under stringent hybridization conditions.\",\n \"3.An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide has greater than about 90% sequence identity with the polynucleotide of claim 1.4.The polynucleotide of claim 1 wherein said polynucleotide is DNA.\",\n \"5.An isolated polynucleotide of claim 1 wherein said polynucleotide comprises the complementary sequences.\",\n \"6.A vector comprising the polynucleotide of claim 1.7.An expression vector comprising the polynucleotide of claim 1.8.A host cell genetically engineered to comprise the polynucleotide of claim 1.9.A host cell genetically engineered to comprise the polynucleotide of claim 1 operatively associated with a regulatory sequence that modulates expression of the polynucleotide in the host cell.\",\n \"10.An isolated polypeptide, wherein the polypeptide is selected from the group consisting of: (a) a polypeptide encoded by any one of the polynucleotides of claim 1; and (b) a polypeptide encoded by a polynucleotide hybridizing under stringent conditions with any one of SEQ ID NO: 1-30368.11.A composition comprising the polypeptide of claim 10 and a carrier.\",\n \"12.An antibody directed against the polypeptide of claim 10.13.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide of claim 1 for a period sufficient to form the complex; and b) detecting the complex, so that if a complex is detected, the polynucleotide of claim 1 is detected.\",\n \"14.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to the polynucleotide of claim 1 under such conditions; b) amplifying a product comprising at least a portion of the polynucleotide of claim 1; and c) detecting said product and thereby the polynucleotide of claim 1 in the sample.\",\n \"15.The method of claim 14, wherein the polynucleotide is an RNA molecule and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA polynucleotide.\",\n \"16.A method for detecting the polypeptide of claim 10 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex; and b) detecting formation of the complex, so that if a complex formation is detected, the polypeptide of claim 10 is detected.\",\n \"17.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10 under conditions sufficient to form a polypeptide/compound complex; and b) detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.\",\n \"18.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10, in a cell, under conditions sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and b) detecting the complex by detecting reporter gene sequence expression, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.\",\n \"19.A method of producing the polypeptide of claim 10, comprising, a) culturing a host cell comprising a polynucleotide sequence selected from the group consisting of a polynucleotide sequence of SEQ ID NO: 1-30368, a mature protein coding portion of SEQ ID NO: 1-30368, an active domain of SEQ ID NO: 1-30368, complementary sequences thereof and a polynucleotide sequence hybridizing under stringent conditions to SEQ ID NO: 1-30368, under conditions sufficient to express the polypeptide in said cell; and b) isolating the polypeptide from the cell culture or cells of step (a).\",\n \"20.An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 30369-60736, the mature protein portion thereof, or the active domain thereof.\",\n \"21.The polypeptide of claim 20 wherein the polypeptide is provided on a polypeptide array.\",\n \"22.A collection of polynucleotides, wherein the collection comprises the sequence information of at least one of SEQ ID NO: 1-30368.23.The collection of claim 22, wherein the collection is provided on a nucleic acid array.\",\n \"24.The collection of claim 23, wherein the array detects full-matches to any one of the polynucleotides in the collection.\",\n \"25.The collection of claim 23, wherein the array detects mismatches to any one of the polynucleotides in the collection.\",\n \"26.The collection of claim 22, wherein the collection is provided in a computer-readable format.\",\n \"27.A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier.\",\n \"28.A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising an antibody that specifically binds to a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier.\"\n ],\n [\n \"<SOH> 2.BACKGROUND <EOH>Technology aimed at the discovery of protein factors (including e.g.\",\n \"cytokines, such as lymphokines, interferons, CSFs, chemokines, and interleukins) has matured rapidly over the past decade.\",\n \"The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning).\",\n \"More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.\",\n \"Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.\"\n ],\n [\n \"<SOH> 3.SUMMARY OF THE INVENTION <EOH>The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.\",\n \"The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.\",\n \"The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases.\",\n \"The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.\",\n \"These nucleic acid sequences are designated as SEQ ID NO: 1-30368.The polypeptides sequences are designated SEQ ID NO: 30369-60736.The nucleic acids and polypeptides are provided in the Sequence Listing.\",\n \"In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases.\",\n \"In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.\",\n \"The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-30368 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-30368.A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-30368 or a degenerate variant or fragment thereof.\",\n \"The identifying sequence can be 100 base pairs in length.\",\n \"The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-30368.The sequence information can be a segment of any one of SEQ ID NO: 1-30368 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-30368.A collection as used in this application can be a collection of only one polynucleotide.\",\n \"The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array.\",\n \"In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment.\",\n \"The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.\",\n \"The collection can also be provided in a computer-readable format.\",\n \"This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.\",\n \"Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.\",\n \"In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the arL In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.\",\n \"The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-30368; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-30368.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing (e.g., SEQ ID NO: 30369-60736); (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.\",\n \"orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.\",\n \"The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein.\",\n \"Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-30368; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.\",\n \"Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.\",\n \"The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.\",\n \"host cells) of the invention.\",\n \"The invention also provides compositions comprising a polypeptide of the invention.\",\n \"Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.\",\n \"The invention also provides host cells transformed or transfected with a polynucleotide of the invention.\",\n \"The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells.\",\n \"Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.\",\n \"Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.\",\n \"These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like.\",\n \"For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.\",\n \"In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.\",\n \"The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.\",\n \"For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.\",\n \"Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.\",\n \"The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.\",\n \"Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.\",\n \"In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.\",\n \"The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample.\",\n \"Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.\",\n \"The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected.\",\n \"The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.\",\n \"The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.\",\n \"Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.\",\n \"The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.\",\n \"Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.\",\n \"Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.\",\n \"The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the invention is identified.\",\n \"The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies.\",\n \"In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products.\",\n \"Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.\",\n \"The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in the sequence listing).\",\n \"If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"1.TECHNICAL FIELD The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these-polynucleotides and proteins, for example in therapeutic, diagnostic and research methods.\",\n \"2.BACKGROUND Technology aimed at the discovery of protein factors (including e.g.\",\n \"cytokines, such as lymphokines, interferons, CSFs, chemokines, and interleukins) has matured rapidly over the past decade.\",\n \"The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning).\",\n \"More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.\",\n \"Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.\",\n \"3.SUMMARY OF THE INVENTION The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.\",\n \"The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.\",\n \"The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases.\",\n \"The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.\",\n \"These nucleic acid sequences are designated as SEQ ID NO: 1-30368.The polypeptides sequences are designated SEQ ID NO: 30369-60736.The nucleic acids and polypeptides are provided in the Sequence Listing.\",\n \"In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases.\",\n \"In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.\",\n \"The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-30368 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-30368.A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-30368 or a degenerate variant or fragment thereof.\",\n \"The identifying sequence can be 100 base pairs in length.\",\n \"The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-30368.The sequence information can be a segment of any one of SEQ ID NO: 1-30368 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-30368.A collection as used in this application can be a collection of only one polynucleotide.\",\n \"The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array.\",\n \"In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment.\",\n \"The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.\",\n \"The collection can also be provided in a computer-readable format.\",\n \"This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.\",\n \"Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.\",\n \"In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the arL In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.\",\n \"The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-30368; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-30368.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing (e.g., SEQ ID NO: 30369-60736); (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.\",\n \"orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.\",\n \"The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein.\",\n \"Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-30368; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.\",\n \"Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.\",\n \"The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.\",\n \"host cells) of the invention.\",\n \"The invention also provides compositions comprising a polypeptide of the invention.\",\n \"Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.\",\n \"The invention also provides host cells transformed or transfected with a polynucleotide of the invention.\",\n \"The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells.\",\n \"Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.\",\n \"Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.\",\n \"These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like.\",\n \"For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.\",\n \"In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.\",\n \"The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.\",\n \"For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.\",\n \"Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.\",\n \"The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.\",\n \"Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.\",\n \"In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.\",\n \"The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample.\",\n \"Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.\",\n \"The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected.\",\n \"The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.\",\n \"The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.\",\n \"Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.\",\n \"The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.\",\n \"Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.\",\n \"Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.\",\n \"The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the invention is identified.\",\n \"The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies.\",\n \"In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products.\",\n \"Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.\",\n \"The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in the sequence listing).\",\n \"If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.\",\n \"4.DETAILED DESCRIPTION OF THE INVENTION 4.1 Definitions It must be noted that as used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.\",\n \"The term “active” refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide.\",\n \"According to the invention, the terms “biologically active” or “biological activity” refer to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule.\",\n \"Likewise “immunologically active” or “immunological activity” refers to the capability of the natural, recombinant or synthetic polypeptide to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.\",\n \"The term “activated cells” as used in this application are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.\",\n \"The terms “complementary” or “complementarity” refer to the natural binding of polynucleotides by base pairing.\",\n \"For example, the sequence 5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′.\",\n \"Complementarity between two single-stranded molecules may be “partial” such that only some of the nucleic acids bind or it may be “complete” such that total complementarity exists between the single stranded molecules.\",\n \"The degree of complementarity between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.\",\n \"The term “embryonic stem cells (ES)” refers to a cell that can give rise to many differentiated cell types in an embryo or an adult, including the germ cells.\",\n \"The term “germ line stem cells (GSCs)” refers to stem cells derived from primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes.\",\n \"The term “primordial germ cells (PGCs)” refers to a small population of cells set aside from other cell lineages particularly from the yolk sac, mesenteries, or gonadal ridges during embryogenesis that have the potential to differentiate into germ cells and other cells.\",\n \"PGCs are the source from which GSCs and ES cells are derived The PGCs, the GSCs and the ES cells are capable of self-renewal.\",\n \"Thus these cells not only populate the germ line and give rise to a plurality of terminally differentiated cells that comprise the adult specialized organs, but are able to regenerate themselves.\",\n \"The term “expression modulating fragment,” EMF, means a series of nucleotides which modulates the expression of an operably linked ORF or another EMF.\",\n \"As used herein, a sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF.\",\n \"EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements).\",\n \"One class of EMEs are nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.\",\n \"The terms “nucleotide sequence” or “nucleic acid” or “polynucleotide” or “oligonucleotide” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides.\",\n \"These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material.\",\n \"In the sequences herein A is adenine, C is cytosine, T is thymine, G is guanine and N is A, C, G or T (U).\",\n \"It is contemplated that where the polynucleotide is RNA, the T (thymine) in the sequences provided herein is substituted with U (uracil).\",\n \"Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.\",\n \"The terms “oligonucleotide fragment” or a “polynucleotide fragment”, “portion,” or “segment” or “probe” or “primer” are used interchangeably and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides.\",\n \"The fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides.\",\n \"Preferably the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from about 17 to 30 nucleotides and most preferably from about 20 to 25 nucleotides.\",\n \"Preferably the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules.\",\n \"A fragment or segment may uniquely identify each polynucleotide sequence of the present invention.\",\n \"Preferably the fragment comprises a sequence substantially similar to any one of SEQ ID NO: 1-30368.Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al.\",\n \"(Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250).\",\n \"They may be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art.\",\n \"Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., both of which are incorporated herein by reference in their entirety.\",\n \"The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-30368.The sequence information can be a segment of any one of SEQ ID NO: 1-30368 that uniquely identifies or represents the sequence information of that sequence of SEQ ID NO: 1-30368.One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300.In the human genome, there are three billion base pairs in one set of chromosomes.\",\n \"Because 420 possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosomes.\",\n \"Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5.When these segments are used in arrays for expression studies, fifteen-mer segments can be used.\",\n \"The probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences comprise less than approximately 5% of the entire genome sequence.\",\n \"Similarly, when using sequence information for detecting a single mismatch, a segment can be a twenty-five mer.\",\n \"The probability that the twenty-five mer would appear in a human genome with a single mismatch is calculated by multiplying the probability for a full match (1÷425) times the increased probability for mismatch at each nucleotide position (3×25).\",\n \"The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five.\",\n \"The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.\",\n \"The term “open reading frame,” ORF, means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein.\",\n \"The terms “operably linked” or “operably associated” refer to functionally related nucleic acid sequences.\",\n \"For example, a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence.\",\n \"While operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g.\",\n \"repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.\",\n \"The term “pluripotent” refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism.\",\n \"A pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.\",\n \"The terms “polypeptide” or “peptide” or “amino acid sequence” refer to an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and to naturally occurring or synthetic molecules.\",\n \"A polypeptide “fragment,” “portion,” or “segment” is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids.\",\n \"The peptide preferably is not greater than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids.\",\n \"Preferably the peptide is from about 5 to about 200 amino acids.\",\n \"To be active, any polypeptide must have sufficient length to display biological and/or immunological activity.\",\n \"The term “naturally occurring polypeptide” refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.\",\n \"The term “translated protein coding portion” means a sequence which encodes for the full length protein which may include any leader sequence or any processing sequence.\",\n \"The term “mature protein coding sequence” means a sequence which encodes a peptide or protein without a signal or leader sequence.\",\n \"The “mature protein portion” means that portion of the protein which does not include a signal or leader sequence.\",\n \"The peptide may have been produced by processing in the cell which removes any leader/signal sequence.\",\n \"The mature protein portion may or may not include an initial methionine residue.\",\n \"The methionine residue may be removed from the protein during processing in the cell.\",\n \"The peptide may be produced synthetically or the protein may have been produced using a polynucleotide only encoding for the mature protein coding sequence.\",\n \"The term “derivative” refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.\",\n \"The term “variant”(or “analog”) refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e.g., recombinant DNA techniques.\",\n \"Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.\",\n \"Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the “redundancy” in the genetic code.\",\n \"Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system.\",\n \"Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.\",\n \"Preferably, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements.\",\n \"“Conservative” amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.\",\n \"For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.\",\n \"“Insertions” or “deletions” are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids.\",\n \"The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.\",\n \"Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides.\",\n \"Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention.\",\n \"For example, such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.\",\n \"Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression.\",\n \"For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.\",\n \"The terms “purified” or “substantially purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like.\",\n \"In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).\",\n \"The term “isolated” as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source.\",\n \"In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same.\",\n \"The terms “isolated” and “purified” do not encompass nucleic acids or polypeptides present in their natural source.\",\n \"The term “recombinant,” when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems.\",\n \"“Microbial” refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems.\",\n \"As a product, “recombinant microbial” defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation.\",\n \"Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.\",\n \"The term “recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence.\",\n \"An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.\",\n \"Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.\",\n \"Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue.\",\n \"This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.\",\n \"The term “recombinant expression system” means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally.\",\n \"Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed.\",\n \"This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers.\",\n \"Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed.\",\n \"The cells can be prokaryotic or eukaryotic.\",\n \"The term “secreted” includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell.\",\n \"“Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed.\",\n \"“Secreted” proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reticulum.\",\n \"“Secreted” proteins are also intended to include proteins containing non-typical signal sequences (e.g.\",\n \"Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2):134-143) and factors released from damaged cells (e.g.\",\n \"Interleukin-1 Receptor Antagonist, see Arend, W. P. et. al.\",\n \"(1998) Annu.\",\n \"Rev.\",\n \"Immunol.\",\n \"16:27-55).\",\n \"Where desired, an expression vector may be designed to contain a “signal or leader sequence” which will direct the polypeptide through the membrane of a cell.\",\n \"Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.\",\n \"The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent.\",\n \"Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringent conditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.).\",\n \"Other exemplary hybridization conditions are described herein in the examples.\",\n \"In instances of hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligonucleotides), 48° C. (for 17-base oligos), 55° C. (for 20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).\",\n \"As used herein, “substantially equivalent” can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences.\",\n \"Typically, such a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less).\",\n \"Such a sequence is said to have 65% sequence identity to the listed sequence.\",\n \"In one embodiment, a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 25% (75% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity).\",\n \"Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% identity, more preferably at least 98% identity, and most preferably at least 99% identity.\",\n \"Substantially equivalent nucleotide sequences of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code.\",\n \"Preferably, nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, more preferably at least about 80% sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% identity, more preferably at least about 98% sequence identity, and most preferably at least about 99% sequence identity.\",\n \"For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent.\",\n \"For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a spurious stop codon) should be disregarded.\",\n \"Sequence identity may be determined, e.g., using the Jotun Hein method (Hein, J.\",\n \"(1990) Methods Enzymol.\",\n \"183:626-645).\",\n \"Identity between sequences can also be determined by other methods known in the art, e.g.\",\n \"by varying hybridization conditions.\",\n \"The term “totipotent” refers to the capability of a cell to differentiate into all of the cell types of an adult organism.\",\n \"The term “transformation” means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration.\",\n \"The term “transfection” refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed.\",\n \"The term “infection” refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.\",\n \"As used herein, an “uptake modulating fragment,” UMF, means a series of nucleotides which mediate the uptake of a linked DNA fragment into a cell.\",\n \"UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below.\",\n \"The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence.\",\n \"The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined.\",\n \"As described above, a UMF will increase the frequency of uptake of a linked marker sequence.\",\n \"Each of the above terms is meant to encompass all that is described for each, unless the context dictates otherwise.\",\n \"4.2 Nucleic Acids of the Invention Nucleotide sequences of the invention are set forth in the Sequence Listing.\",\n \"The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 1-30368; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 30369-60736; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polypeptides of any one of SEQ ID NO: 30369-60736.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a) the complement of any of the nucleotides sequences of SEQ ID NO: 1-30368; (b) nucleotide sequences encoding any one of the amino acid sequences set forth in the Sequence Listing; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptides of SEQ ID NO: 30369-60736.Domains of interest may depend on the nature of the encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic domains, or combinations thereof; domains in immunoglobulin-like proteins include the variable immunoglobulin-like domains; domains in enzyme-like polypeptides include catalytic and substrate binding domains; and domains in ligand polypeptides include receptor-binding domains.\",\n \"The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA.\",\n \"The polynucleotides may include all of the coding region of the cDNA or may represent a portion of the coding region of the cDNA.\",\n \"The present invention also provides genes corresponding to the cDNA sequences disclosed herein.\",\n \"The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein.\",\n \"Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.\",\n \"Further 5′ and 3′ sequence can be obtained using methods known in the art.\",\n \"For example, full length cDNA or genomic DNA that corresponds to any of the polynucleotides of SEQ ID NO: 1-30368 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any of the polynucleotides of SEQ ID NO: 1-30368 or a portion there of as a probe.\",\n \"Alternatively, the polynucleotides of SEQ ID NO: 1-30368 may be used as the basis for suitable primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.\",\n \"The nucleic acid sequences of the invention can be assembled from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases, such as dbEST, gbpri, and UniGene.\",\n \"The EST sequences can provide identifying sequence information, representative fragment or segment information, or novel segment information for the full-length gene.\",\n \"The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above.\",\n \"Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more typically at least about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%, 94%, and even more typically at least about 95%, 96%, 97%, 98%, 99%, sequence identity to a polynucleotide recited above.\",\n \"Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to any of the nucleotide sequences of SEQ ID NO: 1-30368, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides.\",\n \"Fragments of, e.g.\",\n \"15, 17, or 20 nucleotides or more that are selective for (i.e.\",\n \"specifically hybridize to any one of the polynucleotides of the invention) are contemplated.\",\n \"Probes capable of specifically hybridizing to a polynucleotide can differentiate polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate human genes from genes of other species, and are preferably based on unique nucleotide sequences.\",\n \"The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof.\",\n \"Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NO: 1-30368, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NO: 1-30368 with a sequence from another isolate of the same species.\",\n \"Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein.\",\n \"In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.\",\n \"The nearest neighbor or homology result for the nucleic acids of the present invention, including SEQ ID NO: 1-30368 can be obtained by searching a database using an algorithm or a program.\",\n \"Preferably, a BLAST which stands for Basic Local Alignment Search Tool is used to search for local sequence alignments (Altshul, S. F. J. Mol.\",\n \"Evol.\",\n \"36 290-300 (1993) and Altschul S. F. et al.\",\n \"J. Mol.\",\n \"Biol.\",\n \"21:403-410(1990)).\",\n \"Alternatively a FASTA version 3 search against Genpept, using Fastxy algorithm.\",\n \"Species homologs (or orthologs) of the disclosed polynucleotides and proteins are also provided by the present invention.\",\n \"Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.\",\n \"The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.\",\n \"The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids.\",\n \"These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide.\",\n \"There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation.\",\n \"Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature.\",\n \"These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions).\",\n \"Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site.\",\n \"Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous.\",\n \"Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.\",\n \"Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues.\",\n \"Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein.\",\n \"In a preferred method, polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis.\",\n \"This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed.\",\n \"In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183 (1983).\",\n \"A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res.\",\n \"10:6487-6500 (1982).\",\n \"PCR may also be used to create amino acid sequence variants of the novel nucleic acids.\",\n \"When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant.\",\n \"PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer.\",\n \"The product DNA fragments replace the corresponding region in the plasmid and this gives a polynucleotide encoding the desired amino acid variant.\",\n \"A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al.\",\n \"Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids.\",\n \"Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.\",\n \"Polynucleotides encoding preferred polypeptide truncations of the invention can be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.\",\n \"The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.\",\n \"The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA.\",\n \"Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides of the desired sequence identities.\",\n \"In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 1-30368, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells.\",\n \"Also included are the cDNA inserts of any of the clones identified herein.\",\n \"A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al.\",\n \"(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).\",\n \"Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art.\",\n \"Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.\",\n \"In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.\",\n \"Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.\",\n \"A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.\",\n \"The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-30368 or a fragment thereof or any other polynucleotides of the invention.\",\n \"In one embodiment, the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-30368 or a fragment thereof is inserted, in a forward or reverse orientation.\",\n \"In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF.\",\n \"Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention.\",\n \"The following vectors are provided by way of example.\",\n \"Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).\",\n \"Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).\",\n \"The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res.\",\n \"19, 44854490 (1991), in order to produce the protein recombinantly.\",\n \"Many suitable expression control sequences are known in the art.\",\n \"General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990).\",\n \"As defined herein “operably linked” means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.\",\n \"Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.\",\n \"Two appropriate vectors are pKK232-8 and pCM7.Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc.\",\n \"Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.\",\n \"Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.\",\n \"Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TP1gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.\",\n \"Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others.\",\n \"The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.\",\n \"Optionally, the heterologous sequence can encode a fusion protein including an amino terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.\",\n \"Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.\",\n \"The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.\",\n \"Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.\",\n \"As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).\",\n \"Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis., USA).\",\n \"These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.\",\n \"Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.\",\n \"Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.\",\n \"Polynucleotides of the invention can also be used to induce immune responses.\",\n \"For example, as described in Fan et al, Nat.\",\n \"Biotech.\",\n \"17:870-872 (1999), incorporated herein by reference, nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical administration of naked plasmid DNA or following injection, and preferably intramuscular injection of the DNA.\",\n \"The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.\",\n \"4.3 Antisense Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1-30368, or fragments, analogs or derivatives thereof.\",\n \"An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.\",\n \"In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only a portion thereof.\",\n \"Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a protein of any of SEQ ID NO: 30369-60736 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-30368 are additionally provided.\",\n \"In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence of the invention.\",\n \"The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.\",\n \"In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence of the invention.\",\n \"The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).\",\n \"Given the coding strand sequences encoding a nucleic acid disclosed herein (e.g., SEQ ID NO: 1-30368), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.\",\n \"The antisense nucleic acid molecule can be complementary to the entire coding region of a mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of a mRNA.\",\n \"For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a mRNA.\",\n \"An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.\",\n \"An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.\",\n \"For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.\",\n \"Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.\",\n \"Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).\",\n \"The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein according to the invention to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.\",\n \"The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.\",\n \"An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.\",\n \"Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.\",\n \"For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.\",\n \"The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.\",\n \"To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.\",\n \"In yet another embodiment, the antisense nucleic acid molecule of the invention is an-anomeric nucleic acid molecule.\",\n \"An-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual-units, the strands run parallel to each other (Gaultier et al.\",\n \"(1987) Nucleic Acids Res 15: 6625-6641).\",\n \"The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al.\",\n \"(1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al.\",\n \"(1987) FEBS Lett 215: 327-330).\",\n \"4.4 Ribozymes and PNA Moieties In still another embodiment, an antisense nucleic acid of the invention is a ribozyme.\",\n \"Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region.\",\n \"Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave a mRNA transcripts to thereby inhibit translation of a mRNA.\",\n \"A ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1-30368).\",\n \"For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an mRNA of SEQ ID NO: 1-30368 (see, e.g., Cech et al.\",\n \"U.S. Pat.\",\n \"No.\",\n \"4,987,071; and Cech et al.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,116,742).\",\n \"Alternatively, polynucleotides of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules.\",\n \"See, e.g., Bartel et al., (1993) Science 261:1411-1418.Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells.\",\n \"See generally, Helene.\",\n \"(1991) Anticancer Drug Des.\",\n \"6: 569-84; Helene.\",\n \"et al.\",\n \"(1992) Ann.\",\n \"N.Y. Acad.\",\n \"Sci.\",\n \"660:27-36; and Maher (1992) Bioassays 14: 807-15.In various embodiments, the nucleic acids of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.\",\n \"For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al.\",\n \"(1996) Bioorg Med Chem 4: 5-23).\",\n \"As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.\",\n \"The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.\",\n \"The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al.\",\n \"(1996) above; Perry-O'Keefe et al.\",\n \"(1996) PNAS 93: 14670-675.PNAs of the invention can be used in therapeutic and diagnostic applications.\",\n \"For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.\",\n \"PNAs of the invention can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymnes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B.\",\n \"(1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al.\",\n \"(1996), above; Perry-OKeefe (1996), above).\",\n \"In another embodiment, PNAs of the invention can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.\",\n \"For example, PNA-DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA.\",\n \"Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.\",\n \"PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above).\",\n \"The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al.\",\n \"(1996) Nucl Acids Res 24: 3357-63.For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag et al.\",\n \"(1989) Nucl Acid Res 17: 5973-88).\",\n \"PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al.\",\n \"(1996) above).\",\n \"Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment.\",\n \"See, Petersen et al.\",\n \"(1975) Bioorg Med Chem Lett 5: 1119-11124.In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"84:648-652; PCT Publication No.\",\n \"WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No.\",\n \"WO89/10134).\",\n \"In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm.\",\n \"Res.\",\n \"5:539-549).\",\n \"To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.\",\n \"4.5 Hosts The present invention further provides host cells genetically engineered to contain the polynucleotides of the invention.\",\n \"For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods.\",\n \"The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.\",\n \"Knowledge of nucleic acid sequences allows for modification of cells to permit, or increase, expression of endogenous polypeptide.\",\n \"Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the polypeptide at higher levels.\",\n \"The heterologous promoter is inserted in such a manner that it is operatively linked to the encoding sequences.\",\n \"See, for example, PCT International Publication No.\",\n \"WO94/12650, PCT International Publication No.\",\n \"WO92/20808, and PCT International Publication No.\",\n \"WO91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.\",\n \"If linked to the coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.\",\n \"The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.\",\n \"Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)).\",\n \"The host cells containing one of the polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF.\",\n \"Any host/vector system can be used to express one or more of the ORFs of the present invention.\",\n \"These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, 293 cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B. subtilis.\",\n \"The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level.\",\n \"Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters.\",\n \"Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.\",\n \"Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of which is hereby incorporated by reference.\",\n \"Various mammalian cell culture systems can also be employed to express recombinant protein.\",\n \"Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981).\",\n \"Other cell lines capable of expressing a compatible vector are, for example, the C127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.\",\n \"Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences.\",\n \"DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.\",\n \"Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps.\",\n \"Protein refolding steps can be used, as necessary, in completing configuration of the mature protein.\",\n \"Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.\",\n \"Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.\",\n \"Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or insects or in prokaryotes such as bacteria.\",\n \"Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.\",\n \"Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins.\",\n \"If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein.\",\n \"Such covalent attachments may be accomplished using known chemical or enzymatic methods.\",\n \"In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.\",\n \"As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.\",\n \"Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.\",\n \"Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.\",\n \"These sequence include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.\",\n \"The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.\",\n \"Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.\",\n \"Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.\",\n \"Here, the naturally occurring sequences are deleted and new sequences are added.\",\n \"In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome.\",\n \"The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative, selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.\",\n \"Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.\",\n \"The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.\",\n \"No.\",\n \"5,272,071 to Chappel; U.S. Pat.\",\n \"No.\",\n \"5,578,461 to Sherwin et al.\",\n \"; International Application No.\",\n \"PCT/US92/09627 (WO93/09222) by Selden et al.\",\n \"; and International Application No.\",\n \"PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.\",\n \"4.6 Polypeptides of the Invention The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences set forth as any one of SEQ ID NO: 30369-60736 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ID NO: 1-30368 or the corresponding full length or mature protein.\",\n \"Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ ID NO: 30369-60736 or (c) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions.\",\n \"The invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ ID NO: 30369-60736 or the corresponding full length or mature protein; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological activity.\",\n \"Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ ID NO: 30369-60736.Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention.\",\n \"Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer.\",\n \"Chem.\",\n \"Soc.\",\n \"114, 9245-9253 (1992), both of which are incorporated herein by reference.\",\n \"Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.\",\n \"The present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins.\",\n \"The protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences.\",\n \"The mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell.\",\n \"The sequence of the mature form of the protein is also determinable from the amino acid sequence of the full-length form.\",\n \"Where proteins of the present invention are membrane bound, soluble forms of the proteins are also provided.\",\n \"In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which they are expressed.\",\n \"Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.\",\n \"The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention.\",\n \"By “degenerate variant” is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence.\",\n \"Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.\",\n \"A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention.\",\n \"At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers.\",\n \"The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity.\",\n \"This technique is particularly useful in producing small peptides and fragments of larger polypeptides.\",\n \"Fragments are useful, for example, in generating antibodies against the native polypeptide.\",\n \"Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.\",\n \"The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein.\",\n \"As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level.\",\n \"One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.\",\n \"The invention also relates to methods for producing a polypeptide comprising growing a culture of host cells of the invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown.\",\n \"For example, the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide.\",\n \"The polypeptide can be recovered from the culture, conveniently from the culture medium, or from a lysate prepared from the host cells and further purified.\",\n \"Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein.\",\n \"In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein.\",\n \"One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention.\",\n \"These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography.\",\n \"See, e.g., Scopes, Protein Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in Molecular Biology.\",\n \"Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.\",\n \"The purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides.\",\n \"These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins.\",\n \"The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.\",\n \"In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.\",\n \"In addition, the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells.\",\n \"The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for SEQ ID NO: 30369-60736.The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.\",\n \"The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered.\",\n \"For example, modifications in the peptide or DNA sequence can be made by those skilled in the art using known techniques.\",\n \"Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.\",\n \"For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.\",\n \"Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat.\",\n \"No.\",\n \"4,518,584).\",\n \"Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.\",\n \"Regions of the protein that are important for the protein function can be determined by various methods known in the art including the alanine-scanning method which involved systematic substitution of single or strings of amino acids with alanine, followed by testing the resulting alanine-containing variant for biological activity.\",\n \"This type of analysis determines the importance of the substituted amino acid(s) in biological activity.\",\n \"Regions of the protein that are important for protein function may be determined by the eMATRIX program.\",\n \"Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and are useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein.\",\n \"Such modifications are encompassed by the present invention.\",\n \"The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.\",\n \"Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBat™ kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No.\",\n \"1555 (1987), incorporated herein by reference.\",\n \"As used herein, an insect cell capable of expressing a polynucleotide of the present invention is “transformed.” The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.\",\n \"The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.\",\n \"The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl™ or Cibacrom blue 3GA Sepharose™; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.\",\n \"Alternatively, the protein of the invention may also be expressed in a form that will facilitate purification.\",\n \"For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-5-transferase (GST) or thioredoxin (TRX), or as a His-tag.\",\n \"Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.\",\n \"), Pharmacia (Piscataway, N.J.) and Invitrogen, respectively.\",\n \"The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.\",\n \"One such epitope (“FLAG®”) is commercially available from Kodak (New Haven, Conn.).\",\n \"Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein.\",\n \"Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.\",\n \"The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an “isolated protein.” The polypeptides of the invention include analogs (variants).\",\n \"This embraces fragments, as well as peptides in which one or more amino acids has been deleted, inserted, or substituted.\",\n \"Also, analogs of the polypeptides of the invention embrace fusions of the polypeptides or modifications of the polypeptides of the invention, wherein the polypeptide or analog is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent.\",\n \"Such analogs may exhibit improved properties such as activity and/or stability.\",\n \"Examples of moieties which may be fused to the polypeptide or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to pancreatic cells, e.g., antibodies to pancreatic cells, antibodies to immune cells such as T-cells, monocytes, dendritic cells, granulocytes, etc., as well as receptor and ligands expressed on pancreatic or immune cells.\",\n \"Other moieties which may be fused to the polypeptide include therapeutic agents which are used for treatment, for example, immunosuppressive drugs such as cyclosporin, SK506, azathioprine, CD3 antibodies and steroids.\",\n \"Also, polypeptides may be fused to immune modulators, and other cytokines such as alpha or beta interferon.\",\n \"4.6.1 Determining Polypeptide and Polynucleotide Identity and Similarity Preferred identity and/or similarity are designed to give the largest match between the sequences tested.\",\n \"Methods to determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et al., J. Molec.\",\n \"Biol.\",\n \"215:403410 (1990), PSI-BLAST (Altschul S. F. et al., Nucleic Acids Res.\",\n \"vol.\",\n \"25, pp.\",\n \"3389-3402, herein incorporated by reference), eMatrix software (Wu et al., J. Comp.\",\n \"Biol., Vol.\",\n \"6, pp.\",\n \"219-235 (1999), herein incorporated by reference), eMotif software (Nevill-Manning et al, ISMB-97, Vol.\",\n \"4, pp.\",\n \"202-209, herein incorporated by reference), pFam software (Sonnhammer et al., Nucleic Acids Res., Vol.\",\n \"26(1), pp.\",\n \"320-322 (1998), herein incorporated by reference) and the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol.\",\n \"Biol, 157, pp.\",\n \"105-31 (1982), incorporated herein by reference).\",\n \"The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al.\",\n \"NCB NLM NIH Bethesda, Md.\",\n \"20894; Altschul, S., et al., J. Mol.\",\n \"Biol.\",\n \"215:403-410 (1990).\",\n \"4.7 Chimeric and Fusion Proteins The invention also provides chimeric or fusion proteins.\",\n \"As used herein, a “chimeric protein” or “fusion protein” comprises a polypeptide of the invention operatively linked to another polypeptide.\",\n \"Within a fusion protein the polypeptide according to the invention can correspond to all or a portion of a protein according to the invention.\",\n \"In one embodiment, a fusion protein comprises at least one biologically active portion of a protein according to the invention.\",\n \"In another embodiment, a fusion protein comprises at least two biologically active portions of a protein according to the invention.\",\n \"Within the fusion protein, the term “operatively linked” is intended to indicate that the polypeptide according to the invention and the other polypeptide are fused in-frame to each other.\",\n \"The polypeptide can be fused to the N-terminus or C-terminus.\",\n \"For example, in one embodiment a fusion protein comprises a polypeptide according to the invention operably linked to the extracellular domain of a second protein.\",\n \"In another embodiment, the fusion protein is a GST-fusion protein in which the polypeptide sequences of the invention are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences.\",\n \"In another embodiment, the fusion protein is an immunoglobulin fusion protein in which the polypeptide sequences according to the invention comprises one or more domains are fused to sequences derived from a member of the immunoglobulin protein family.\",\n \"The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand and a protein of the invention on the surface of a cell, to thereby suppress signal transduction in vivo.\",\n \"The immunoglobulin fusion proteins can be used to affect the bioavailability of a cognate ligand.\",\n \"Inhibition of the ligand/protein interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e,g., cancer as well as modulating (e.g.\",\n \"promoting or inhibiting) cell survival.\",\n \"Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction of a polypeptide of the invention with a ligand.\",\n \"A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques.\",\n \"For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.\",\n \"In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.\",\n \"Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al.\",\n \"(eds.)\",\n \"CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).\",\n \"Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).\",\n \"A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein of the invention.\",\n \"4.8 Gene Therapy Mutations in the polynucleotides of the invention gene may result in loss of normal function of the encoded protein.\",\n \"The invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention.\",\n \"Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments).\",\n \"See, for example, Anderson, Nature, supplement to vol.\",\n \"392, no.\",\n \"6679, pp.\",\n \"25-20 (1998).\",\n \"For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).\",\n \"Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression).\",\n \"Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.\",\n \"Treated cells can then be introduced in vivo for therapeutic purposes.\",\n \"Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states.\",\n \"It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.\",\n \"Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art.\",\n \"Further, the polypeptides of the present invention can be inhibited by using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific.\",\n \"The present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.\",\n \"These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.\",\n \"Knowledge of DNA sequences provided by the invention allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide.\",\n \"Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels.\",\n \"The heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences.\",\n \"See, for example, PCT International Publication No.\",\n \"WO 94/12650, PCT International Publication No.\",\n \"WO 92/20808, and PCT International Publication No.\",\n \"WO 91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.\",\n \"If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co-amplificationof the desired protein coding sequences in the cells.\",\n \"In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.\",\n \"As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.\",\n \"Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.\",\n \"Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.\",\n \"These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.\",\n \"The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.\",\n \"Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.\",\n \"Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.\",\n \"Here, the naturally occurring sequences are deleted and new sequences are added.\",\n \"In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome.\",\n \"The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.\",\n \"Markers useful for this purpose include the Herpes Sinplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphonbosyl-transferase (gpt) gene.\",\n \"The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.\",\n \"No.\",\n \"5,272,071 to Chappel; U.S. Pat.\",\n \"No.\",\n \"5,578,461 to Sherwin et al.\",\n \"; International Application No.\",\n \"PCT/US92/09627 (WO93/09222) by Selden et al.\",\n \"; and International Application No.\",\n \"PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.\",\n \"4.9 Transgenic Animals In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)].\",\n \"Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.\",\n \"Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.\",\n \"Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.\",\n \"No.\",\n \"5,557,032, incorporated herein by reference.\",\n \"Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.\",\n \"Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.\",\n \"Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.\",\n \"No.\",\n \"5,489,743 and PCT Publication No.\",\n \"WO94/28122, incorporated herein by reference.\",\n \"Transgenic animals can be prepared wherein all or part of a promoter of the polynucleotides of the invention is either activated or inactivated to alter the level of expression of the polypeptides of the invention.\",\n \"Inactivation can be carried out using homologous recombination methods described above.\",\n \"Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.\",\n \"The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.\",\n \"The polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express polypeptides of the invention or that express a variant polypeptide.\",\n \"Such animals are useful as models for studying the in vivo activities of polypeptide as well as for studying modulators of the polypeptides of the invention.\",\n \"In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)).\",\n \"Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.\",\n \"Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.\",\n \"Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.\",\n \"No.\",\n \"5,557,032, incorporated herein by reference.\",\n \"Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.\",\n \"Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.\",\n \"Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.\",\n \"No.\",\n \"5,489,743 and PCT Publication No.\",\n \"WO94/28122, incorporated herein by reference.\",\n \"Transgenic animals can be prepared wherein all or part of the polynucleotides of the invention promoter is either activated or inactivated to alter the level of expression of the polypeptides of the invention.\",\n \"Inactivation can be carried out using homologous recombination methods described above.\",\n \"Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.\",\n \"The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.\",\n \"4.10 Uses and Biological Activity The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified herein.\",\n \"Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).\",\n \"The mechanism underlying the particular condition or pathology will dictate whether the polypeptides of the invention, the polynucleotides of the invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment.\",\n \"Thus, “therapeutic compositions of the invention” include compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or polypeptides of the invention (including full length protein, mature protein and truncations or domains thereof), or compounds and other substances that modulate the overall activity of the target gene products, either at the level of target gene/protein expression or target protein activity.\",\n \"Such modulators include polypeptides, analogs, (variants), including fragments and fusion proteins, antibodies and other binding proteins; chemical compounds that directly or indirectly activate or inhibit the polypeptides of the invention (identified, e.g., via drug screening assays as described herein); antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies or other binding partners that specifically recognize one or more epitopes of the polypeptides of the invention.\",\n \"The polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.\",\n \"4.10.1 Research uses and Utilities The polynucleotides provided by the present invention can be used by the research community for various purposes.\",\n \"The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a “gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response.\",\n \"Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.\",\n \"The polypeptides provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.\",\n \"Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.\",\n \"Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.\",\n \"Methods for performing the uses listed above are well known to those skilled in the art.\",\n \"References disclosing such methods include without limitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.4.10.2 Nutritional Uses Polynucleotides and polypeptides of the present invention can also be used as nutritional sources or supplements.\",\n \"Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.\",\n \"In such cases the polypeptide or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.\",\n \"In the case of microorganisms, the polypeptide or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.\",\n \"4.10.3 Cytokine and Cell Proliferation/Differentiation Activity A polypeptide of the present invention may exhibit activity relating to cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.\",\n \"A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.\",\n \"Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.\",\n \"The activity of therapeutic compositions of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco.\",\n \"Therapeutic compositions of the invention can be used in the following: Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.\",\n \"137:3494-3500, 1986; Bertagnolli et al., J. Immunol.\",\n \"145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., 1.Immunol.\",\n \"149:3778-3783, 1992; Bowman et al., I. Immunol.\",\n \"152:1756-1761, 1994.Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology.\",\n \"J. E. e.a.\",\n \"Coligan eds.\",\n \"Vol 1 pp.\",\n \"3.12.1-3.12.14, John Wiley and Sons, Toronto.\",\n \"1994; and Measurement of mouse and human interleukin-γ, Schreiber, R. D. In Current Protocols in Immunology.\",\n \"J. E. e.a Coligan eds.\",\n \"Vol 1 pp.\",\n \"6.8.1-6.8.8, John Wiley and Sons, Toronto.\",\n \"1994.Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology.\",\n \"J. E. e.a Coligan eds.\",\n \"Vol 1 pp.\",\n \"6.3.1-6.3.12, John Wiley and Sons, Toronto.\",\n \"1991; deVries et al., J. Exp.\",\n \"Med.\",\n \"173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6-Nordan, R. In Current Protocols in Immunology.\",\n \"J. E. Coligan eds.\",\n \"Vol 1 pp.\",\n \"6.6.1-6.6.5, John Wiley and Sons, Toronto.\",\n \"1991; Smith et al., Proc.\",\n \"Natl.\",\n \"Aced.\",\n \"Sci.\",\n \"U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J.\",\n \"In Current Protocols in Immunology.\",\n \"J. E. Coligan eds.\",\n \"Vol 1 pp.\",\n \"6.15.1 John Wiley and Sons, Toronto.\",\n \"1991; Measurement of mouse and human Interleukin 9—Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J.\",\n \"In Current Protocols in Immunology.\",\n \"J. E. Coligan eds.\",\n \"Vol 1 pp.\",\n \"6.13.1, John Wiley and Sons, Toronto.\",\n \"1991.Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 77:6091-6095, 1980; Weinberger et al., Eur.\",\n \"J. Immun.\",\n \"11:405-411, 1981; Takai et al., J. Immunol.\",\n \"137:3494-3500, 1986; Takai et al., J. Immunol.\",\n \"140:508-512, 1988.4.10.4 Stem Cell Growth Factor Activity A polypeptide of the present invention may exhibit stem cell growth factor activity and be involved in the proliferation, differentiation and survival of pluripotent and totipotent stem cells including primordial germ cells, embryonic stem cells, hematopoietic stem cells and/or germ line stem cells.\",\n \"Administration of the polypeptide of the invention to stem cells in vivo or ex vivo is expected to maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering damaged or diseased tissues, transplantation, manufacture of bio-pharmaceuticals and the development of bio-sensors.\",\n \"The ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.\",\n \"It is contemplated that multiple different exogenous growth factors and/or cytokines may be administered in combination with the polypeptide of the invention to achieve the desired effect, including any of the growth factors listed herein, other stem cell maintenance factors, and specifically including stem cell factor (SCF), leukemia inhibitory factor (LIF), Flt-3 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatory protein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).\",\n \"Since totipotent stem cells can give rise to virtually any mature cell type, expansion of these cells in culture will facilitate the production of large quantities of mature cells.\",\n \"Techniques for culturing stem cells are known in the art and administration of polypeptides of the invention, optionally with other growth factors and/or cytokines, is expected to enhance the survival and proliferation of the stem cell populations.\",\n \"This can be accomplished by direct administration of the polypeptide of the invention to the culture medium.\",\n \"Alternatively, stroma cells transfected with a polynucleotide that encodes for the polypeptide of the invention can be used as a feeder layer for the stem cell populations in culture or in vivo.\",\n \"Stromal support cells for feeder layers may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see U.S. Pat.\",\n \"No.\",\n \"5,690,926).\",\n \"Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention.\",\n \"This will allow for generation of undifferentiated totipotential/pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types.\",\n \"These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential mRNA to create cDNA libraries and templates for polymerase chain reaction experiments.\",\n \"These studies would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.\",\n \"Expansion and maintenance of totipotent stem cell populations will be useful in the treatment of many pathological conditions.\",\n \"For example, polypeptides of the present invention may be used to manipulate stem cells in culture to give rise to neuroepithelial cells that can be used to augment or replace cells damaged by illness, autoimmune disease, accidental damage or genetic disorders.\",\n \"The polypeptide of the invention may be useful for inducing the proliferation of neural cells and for the regeneration of nerve and brain tissue, i.e.\",\n \"for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders which involve degeneration, death or trauma to neural cells or nerve tissue.\",\n \"In addition, the expanded stem cell populations can also be genetically altered for gene therapy purposes and to decrease host rejection of replacement tissues after grafting or implantation.\",\n \"Expression of the polypeptide of the invention and its effect on stem cells can also be manipulated to achieve controlled differentiation of the stem cells into more differentiated cell types.\",\n \"A broadly applicable method of obtaining pure populations of a specific differentiated cell type from undifferentiated stem cell populations involves the use of a cell-type specific promoter driving a selectable marker.\",\n \"The selectable marker allows only cells of the desired type to survive.\",\n \"For example, stem cells can be induced to differentiate into cardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin.\",\n \"Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In: Principles of Tissue Engineering eds.\",\n \"Lanza et al., Academic Press (1997)).\",\n \"Alternatively, directed differentiation of stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.\",\n \"In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity.\",\n \"Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson et al.\",\n \"Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci, U.S.A., 92: 7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines.\",\n \"The ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi-solid support e.g.\",\n \"as described by Bernstein et al., Blood, 77: 2316-2321 (1991).\",\n \"4.10.5 Hematopoiesis Regulating Activity A polypeptide of the present invention may be involved in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell disorders.\",\n \"Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.\",\n \"in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (ie., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.\",\n \"Therapeutic compositions of the invention can be used in the following: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.\",\n \"Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al.\",\n \"Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells.\",\n \"R. I. Freshney, et al.\",\n \"eds.\",\n \"Vol pp.\",\n \"265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A.\",\n \"In Culture of Hematopoietic Cells.\",\n \"R. I. Freshney, et al.\",\n \"eds.\",\n \"Vol pp.\",\n \"23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of Hematopoietic Cells.\",\n \"R. I. Freshney, et al.\",\n \"eds.\",\n \"Vol pp.\",\n \"1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells.\",\n \"R. I. Freshney, et al.\",\n \"eds.\",\n \"Vol pp.\",\n \"163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J.\",\n \"In Culture of Hematopoietic Cells.\",\n \"R. I. Freshney, et al.\",\n \"eds.\",\n \"Vol pp.\",\n \"139-162, Wiley-Liss, Inc., New York, N.Y. 1994.4.10.6 Tissue Growth Activity A polypeptide of the present invention also may be involved in bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of burns, incisions and ulcers.\",\n \"A polypeptide of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.\",\n \"Compositions of a polypeptide, antibody, binding partner, or other modulator of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints.\",\n \"De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.\",\n \"A polypeptide of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of bone-forming cells.\",\n \"Treatment of osteoporosis, osteoarthritis, bone degenerative disorders, or periodontal disease, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.)\",\n \"mediated by inflammatory processes may also be possible using the composition of the invention.\",\n \"Another category of tissue regeneration activity that may involve the polypeptide of the present invention is tendon/ligament formation.\",\n \"Induction of tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.\",\n \"Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.\",\n \"De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.\",\n \"The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.\",\n \"The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.\",\n \"The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.\",\n \"The compositions of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e.\",\n \"for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue.\",\n \"More specifically, a composition may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome.\",\n \"Further conditions that may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke.\",\n \"Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a composition of the invention.\",\n \"Compositions of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.\",\n \"Compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues.\",\n \"Part of the desired effects may be by inhibition or modulation of fibrotic scarring may allow normal tissue to regenerate.\",\n \"A polypeptide of the present invention may also exhibit angiogenic activity.\",\n \"A composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.\",\n \"A composition of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.\",\n \"Therapeutic compositions of the invention can be used in the following: Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No.\",\n \"WO95/16035 (bone, cartilage, tendon); International Patent Publication No.\",\n \"WO95/05846 (nerve, neuronal); International Patent Publication No.\",\n \"WO91/07491 (skin, endothelium).\",\n \"Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps.\",\n \"71-112 (Maibach, H. I. and Rovee, D. T., eds.\",\n \"), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.\",\n \"Invest.\",\n \"Dermatol 71:382-84 (1978).\",\n \"4.10.7 Immune Stimulating or Suppressing Activity A polypeptide of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.\",\n \"A polynucleotide of the invention can encode a polypeptide exhibiting such activities.\",\n \"A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.\",\n \"These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.\",\n \"More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp.\",\n \"and various fungal infections such as candidiasis.\",\n \"Of course, in this regard, proteins of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.\",\n \"Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.\",\n \"Such a protein (or antagonists thereof, including antibodies) of the present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems.\",\n \"Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein (or antagonists thereof) of the present invention.\",\n \"The therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig slin sensitization test (Vohr et al., Arch.\",\n \"Toxocol.\",\n \"73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol.\",\n \"Environ.\",\n \"Health 53: 563-79).\",\n \"Using the proteins of the invention it may also be possible to modulate immune responses, in a number of ways.\",\n \"Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.\",\n \"The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.\",\n \"Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.\",\n \"Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased.\",\n \"Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.\",\n \"Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).\",\n \"For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation.\",\n \"Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.\",\n \"The administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.\",\n \"Moreover, a lack of costinulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.\",\n \"Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.\",\n \"To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.\",\n \"The efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.\",\n \"Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4I g fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci USA, 89:11102-11105 (1992).\",\n \"In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.\",\n \"846-847) can be used to determine the effect of therapeutic compositions of the invention on the development of that disease.\",\n \"Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.\",\n \"Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self-tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.\",\n \"Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.\",\n \"Administration of reagents which block stimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process.\",\n \"Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.\",\n \"The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases.\",\n \"Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.\",\n \"840-856).\",\n \"Upregulation of an antigen function (e.g., a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy.\",\n \"Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response.\",\n \"For example, enhancing an immune response may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis.\",\n \"Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.\",\n \"Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.\",\n \"The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.\",\n \"A polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells.\",\n \"In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class 1 alpha chain protein and P2 microglobulin protein or an MHC class II alpha chain protein and an MHC class II beta chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.\",\n \"Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell.\",\n \"Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.\",\n \"Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.\",\n \"The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 78:2488-2492, 1981; Herrmnann et al., J. Immunol.\",\n \"128:1968-1974, 1982; Handa et al., J. Immunol.\",\n \"135:1564-1572, 1985; Takai et al., I. Immunol.\",\n \"137:3494-3500, 1986; Takai et al., J. Immunol.\",\n \"140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol.\",\n \"153:3079-3092, 1994.Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol.\",\n \"144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology.\",\n \"J. E. e.a.\",\n \"Coligan eds.\",\n \"Vol 1 pp.\",\n \"3.8.1-3.8.16, John Wiley and Sons, Toronto.\",\n \"1994.Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.\",\n \"137:3494-3500, 1986; Takai et al., J. Immunol.\",\n \"140:508-512, 1988; Bertagnolli et al., J. Immunol.\",\n \"149:3778-3783, 1992.Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol.\",\n \"134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260,1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:40374045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc.\",\n \"Nat.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 88:7548-7551, 1991.4.10.8 Activin/Inhibin Activity A polypeptide of the present invention may also exhibit activin- or inhibin-related activities.\",\n \"A polynucleotide of the invention may encode a polypeptide exhibiting such characteristics.\",\n \"Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH).\",\n \"Thus, a polypeptide of the present invention, alone or in heterodirners with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals.\",\n \"Administration of sufficient amounts of other inhibins can induce infertility in these mammals.\",\n \"Alternatively, the polypeptide of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary.\",\n \"See, for example, U.S. Pat.\",\n \"No.\",\n \"4,798,885.A polypeptide of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as, but not limited to, cows, sheep and pigs.\",\n \"The activity of a polypeptide of the invention may, among other means, be measured by the following methods.\",\n \"Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 83:3091-3095, 1986.4.10.9 Chemotactic/Chemokinetic Activity A polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.\",\n \"A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.\",\n \"Chemotactic and chemolinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action.\",\n \"Chemotactic or chemokinetic compositions (e.g.\",\n \"proteins, antibodies, binding partners, or modulators of the invention) provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections.\",\n \"For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.\",\n \"A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.\",\n \"Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells.\",\n \"Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.\",\n \"Therapeutic compositions of the invention can be used in the following: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.\",\n \"Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al.\",\n \"J. Clin.\",\n \"Invest.\",\n \"95:1370-1376, 1995; Lind et al.\",\n \"APMIS 103:140-146, 1995; Muller et al Eur.\",\n \"J. Immunol.\",\n \"25:1744-1748; Gruber et al.\",\n \"J. of Immunol.\",\n \"152:5860-5867, 1994; Johnston et al.\",\n \"J. of Immunol.\",\n \"153:1762-1768, 1994.4.10.10 Hemostatic and Thrombolytic Activity A polypeptide of the invention may also be involved in hemostatis or thrombolysis or thrombosis.\",\n \"A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.\",\n \"Compositions may be usefull in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.\",\n \"A composition of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).\",\n \"Therapeutic compositions of the invention can be used in the following: Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin.\",\n \"Pharmacol.\",\n \"26:131-140, 1986; Burdick et al., Thrombosis Res.\",\n \"45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79(1991); Schaub, Prostaglandins 35:467-474,1988.4.10.11 Cancer Diagnosis and Therapy Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis.\",\n \"Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer.\",\n \"For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy.\",\n \"Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition.\",\n \"Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.\",\n \"Cancer treatments promote tumor regression by inhibiting tumor cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumor growth) and/or prohibiting metastasis by reducing tumor cell motility or invasiveness.\",\n \"Therapeutic compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Karposi's sarcoma.\",\n \"Polypeptides, polynucleotides, or modulators of polypeptides of the invention (including inhibitors and stimulators of the biological activity of the polypeptide of the invention) may be administered to treat cancer.\",\n \"Therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g.\",\n \"reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.\",\n \"The composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail.\",\n \"An anti-cancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery.\",\n \"The use of anti-cancer cocktails as a cancer treatment is routine.\",\n \"Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.\",\n \"In addition, therapeutic compositions of the invention may be used for prophylactic treatment of cancer.\",\n \"There are hereditary conditions and/or environmental situations (e.g.\",\n \"exposure to carcinogens) known in the art that predispose an individual to developing cancers.\",\n \"Under these circumstances, it may be beneficial to treat these individuals with therapeutically effective doses of the polypeptide of the invention to reduce the risk of developing cancers.\",\n \"In vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment.\",\n \"These in vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, N.Y. Ch 18 and Ch 21), tumor systems in nude mice as described in Giovanella et al., J. Natl.\",\n \"Can.\",\n \"Inst., 52: 921-30 (1974), mobility and invasive potential of tumor cells in Boyden Chamber assays as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays such as induction of vascularization of the chick chorioallantoic membrane or induction of vascular endothelial cell migration as described in Ribatta et al., Intl.\",\n \"J. Dev.\",\n \"Biol., 40: 1189-97 (1999) and Li et al., Clin.\",\n \"Exp.\",\n \"Metastasis, 17:423-9 (1999), respectively.\",\n \"Suitable tumor cells lines are available, e.g.\",\n \"from American Type Tissue Culture Collection catalogs.\",\n \"4.10.12 RECEPTOR/LIGAND ACTIVITY A polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions.\",\n \"A polynucleotide of the invention can encode a polypeptide exhibiting such characteristics.\",\n \"Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses.\",\n \"Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.\",\n \"A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.\",\n \"The activity of a polypeptide of the invention may, among other means, be measured by the following methods: Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 84:6864-6868, 1987; Bierer et al., J. Exp.\",\n \"Med.\",\n \"168:1145-1156, 1988; Rosenstein et al., J. Exp.\",\n \"Med.\",\n \"169:149-160 1989; Stoltenborg et al., J. Immunol.\",\n \"Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.By way of example, the polypeptides of the invention may be used as a receptor for a ligand(s) thereby transmitting the biological activity of that ligand(s).\",\n \"Ligands may be identified through binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays, or other methods known in the art.\",\n \"Studies characterizing drugs or proteins as agonist or antagonist or partial agonists or a partial antagonist require the use of other proteins as competing ligands.\",\n \"The polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, colorimetric molecules or toxin molecules by conventional methods.\",\n \"(“Guide to Protein Purification” Murray P. Deutscher (ed) Methods in Enzymology Vol.\",\n \"182 (1990) Academic Press, Inc. San Diego).\",\n \"Examples of radioisotopes include, but are not limited to, tritium and carbon-14.Examples of colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules.\",\n \"Examples of toxins include, but are not limited, to ricin.\",\n \"4.10.13 DRUG SCREENING This invention is particularly useful for screening chemical compounds by using the novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques.\",\n \"The polypeptides or fragments employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.\",\n \"One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof.\",\n \"Drugs are screened against such transformed cells in competitive binding assays.\",\n \"Such cells, either in viable or fixed form, can be used for standard binding assays.\",\n \"One may measure, for example, the formation of complexes between polypeptides of the invention or fragments and the agent being tested or examine the diminution in complex formation between the novel polypeptides and an appropriate cell line, which are well known in the art.\",\n \"Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include (1) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.\",\n \"Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.\",\n \"The sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves.\",\n \"Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof.\",\n \"For a review, see Science 282:63-68 (1998).\",\n \"Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods.\",\n \"Of particular interest are peptide and oligonucleotide combinatorial libraries.\",\n \"Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.\",\n \"For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr.\",\n \"Opin.\",\n \"Biotechnol.\",\n \"8:701-707 (1997).\",\n \"For reviews and examples of peptidomimetic libraries, see Al-Obeidi et al., Mol.\",\n \"Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol, 1(1): 114-19 (1997); Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996) (alkylated dipeptides).\",\n \"Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to bind a polypeptide of the invention.\",\n \"The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.\",\n \"In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.\",\n \"The binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes.\",\n \"The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention.\",\n \"Alternatively, the binding molecules may be complexed with imaging agents for targeting and imaging purposes.\",\n \"4.10.14 ASSAY FOR RECEPTOR ACTIVITY The invention also provides methods to detect specific binding of a polypeptide e.g.\",\n \"a ligand or a receptor.\",\n \"The art provides numerous assays particularly useful for identifying previously unknown binding partners for receptor polypeptides of the invention.\",\n \"For example, expression cloning using mammalian or bacterial cells, or dihybrid screening assays can be used to identify polynucleotides encoding binding partners.\",\n \"As another example, affinity chromatography with the appropriate immobilized polypeptide of the invention can be used to isolate polypeptides that recognize and bind polypeptides of the invention.\",\n \"There are a number of different libraries used for the identification of compounds, and in particular small molecules, that modulate (i.e., increase or decrease) biological activity of a polypeptide of the invention.\",\n \"Ligands for receptor polypeptides of the invention can also be identified by adding exogenous ligands, or cocktails of ligands to two cells populations that are genetically identical except for the expression of the receptor of the invention: one cell population expresses the receptor of the invention whereas the other does not.\",\n \"The responses of the two cell populations to the addition of ligands(s) are then compared.\",\n \"Alternatively, an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s).\",\n \"As still another example, BIAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.\",\n \"The role of downstream intracellular signaling molecules in the signaling cascade of the polypeptide of the invention can be determined.\",\n \"For example, a chimeric protein in which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of a protein, whose ligand has been identified, is produced in a host cell.\",\n \"The cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor.\",\n \"Known downstream proteins involved in intracellular signaling can then be assayed for expected modifications i.e.\",\n \"phosphorylation.\",\n \"Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.\",\n \"4.10.15 Anti-Inflammtory Activity Compositions of the present invention may also exhibit anti-inflammatory activity.\",\n \"The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.\",\n \"Compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1.Compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.\",\n \"Compositions of this invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.\",\n \"4.10.16 LEUKEMIAS Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention.\",\n \"Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.\",\n \"B. Lippincott Co., Philadelphia).\",\n \"4.10.17 NERVOUS SYSTEM DISORDERS Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.\",\n \"Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (iii) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis; (iv) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis; (v) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (vi) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (vii) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.\",\n \"Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons.\",\n \"For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful according to the invention: (i) increased survival time of neurons in culture; (ii) increased sprouting of neurons in culture or in vivo; (iii) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (iv) decreased symptoms of neuron dysfunction in vivo.\",\n \"Such effects may be measured by any method known in the art.\",\n \"In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al.\",\n \"(1990, J. Neurosci.\",\n \"10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al.\",\n \"(1980, Exp.\",\n \"Neurol.\",\n \"70:65-82) or Brown et al.\",\n \"(1981, Ann.\",\n \"Rev.\",\n \"Neurosci.\",\n \"4:17-42); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.\",\n \"In specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).\",\n \"4.10.18 Other Activities A polypeptide of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fingi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.\",\n \"4.10.19 Identification of Polymorphisms The demonstration of polymorphisms makes possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment.\",\n \"Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately.\",\n \"For example, the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.\",\n \"Polymorphisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymorphism in the DNA.\",\n \"For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced.\",\n \"Alternatively, the DNA may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymorphism is extended with one or more labeled nucleotides).\",\n \"In addition, traditional restriction fragment length polymorphism analysis (using restriction enzymes that provide differential digestion of the genomic DNA depending on the presence or absence of the polymorphism) may be performed.\",\n \"Arrays with nucleotide sequences of the present invention can be used to detect polymorphisms.\",\n \"The array can comprise modified nucleotide sequences of the present invention in order to detect the nucleotide sequences of the present invention.\",\n \"In the alternative, any one of the nucleotide sequences of the present invention can be placed on the array to detect changes from those sequences.\",\n \"Alternatively a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.\",\n \"4.10.20 Arthritis and Inflammation The immunosuppressive effects of the compositions of the invention against rheumatoid arthritis are determined in an experimental animal model system.\",\n \"The experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et al., 1963, Int.\",\n \"Arch.\",\n \"Allergy Appl.\",\n \"Immunol., 23:129.Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA).\",\n \"The route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture.\",\n \"The polypeptide is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg.\",\n \"The control consists of administering PBS only.\",\n \"The procedure for testing the effects of the test compound would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24.At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall arthritis score may be obtained as described by J. Holoskitz above.\",\n \"An analysis of the data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.\",\n \"4.11 Therapeutic Methods The compositions (including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides) of the invention have numerous applications in a variety of therapeutic methods.\",\n \"Examples of therapeutic applications include, but are not limited to, those exemplified herein.\",\n \"4.11.1 Example One embodiment of the invention is the administration of an effective amount of the polypeptides or other composition of the invention to individuals affected by a disease or disorder that can be modulated by regulating the peptides of the invention.\",\n \"While the mode of administration is not particularly important, parenteral administration is preferred.\",\n \"An exemplary mode of administration is to deliver an intravenous bolus.\",\n \"The dosage of the polypeptides or other composition of the invention will normally be determined by the prescribing physician.\",\n \"It is to be expected that the dosage will vary according to the age, weight, condition and response of the individual patient.\",\n \"Typically, the amount of polypeptide administered per dose will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight, with the preferred dose being about 0.1 μg/kg to 10 mg/kg of patient body weight.\",\n \"For parenteral administration, polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle.\",\n \"Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin.\",\n \"The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient.\",\n \"The preparation of such solutions is within the skill of the art.\",\n \"4.12 Pharmaceutical Formulations and Routes of Administration A protein or other composition of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources and including antibodies and other binding partners of the polypeptides of the invention) may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders.\",\n \"Such a composition may optionally contain (in addition to protein or other active ingredient and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.\",\n \"The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).\",\n \"The characteristics of the carrier will depend on the route of administration.\",\n \"The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, ThF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.\",\n \"In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question.\",\n \"These agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), as well as cytokines described herein.\",\n \"The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or other active ingredient or complement its activity or use in treatment.\",\n \"Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein or other active ingredient of the invention, or to minimize side effects.\",\n \"Conversely, protein or other active ingredient of the present invention may be included in formulations of the particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent (such as IL-1Ra, IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents).\",\n \"A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.\",\n \"As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.\",\n \"As an alternative to being included in a pharmaceutical composition of the invention including a first protein, a second protein or a therapeutic agent may be concurrently administered with the first protein (e.g., at the same time, or at differing times provided that therapeutic concentrations of the combination of agents is achieved at the treatment site).\",\n \"Techniques for formulation and administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.\",\n \"A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.\",\n \"When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone.\",\n \"When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.\",\n \"In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated.\",\n \"Protein or other active ingredient of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.\",\n \"When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein or other active ingredient of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially.\",\n \"If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.\",\n \"4.12.1 Routes of Administration Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.\",\n \"Administration of protein or other active ingredient of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.\",\n \"Intravenous administration to the patient is preferred.\",\n \"Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthritic joints or in fibrotic tissue, often in a depot or sustained release formulation.\",\n \"In order to prevent the scarring process frequently occurring as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops.\",\n \"Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue.\",\n \"The liposomes will be targeted to and taken up selectively by the afflicted tissue.\",\n \"The polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action.\",\n \"The determination of a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art.\",\n \"Preferably for wound treatment, one administers the therapeutic compound directly to the site.\",\n \"Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models.\",\n \"Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.\",\n \"4.12.2 Compositions/Formulations Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.\",\n \"These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.\",\n \"Proper formulation is dependent upon the route of administration chosen.\",\n \"When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.\",\n \"When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.\",\n \"The tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient of the present invention.\",\n \"When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.\",\n \"The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.\",\n \"When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and preferably from about 1 to 50% protein or other active ingredient of the present invention.\",\n \"When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.\",\n \"The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.\",\n \"A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.\",\n \"The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.\",\n \"For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.\",\n \"Such penetrants are generally known in the art.\",\n \"For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.\",\n \"Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.\",\n \"Pharmaceutical preparations for oral use can be obtained from a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.\",\n \"Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).\",\n \"If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.\",\n \"Dragee cores are provided with suitable coatings.\",\n \"For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.\",\n \"Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.\",\n \"Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.\",\n \"The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.\",\n \"In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.\",\n \"In addition, stabilizers may be added.\",\n \"All formulations for oral administration should be in dosages suitable for such administration.\",\n \"For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.\",\n \"For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.\",\n \"In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.\",\n \"Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.\",\n \"The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.\",\n \"Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.\",\n \"The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.\",\n \"Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.\",\n \"Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.\",\n \"Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.\",\n \"Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.\",\n \"Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.\",\n \"Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.\",\n \"The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.\",\n \"In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.\",\n \"Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.\",\n \"Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.\",\n \"A pharmaceutical carrier for the hydrophobic compounds of the invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.\",\n \"The co-solvent system may be the VPD co-solvent system.\",\n \"VPD is a solution of 3%/o w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.\",\n \"The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution.\",\n \"This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.\",\n \"Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.\",\n \"Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g.\",\n \"polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.\",\n \"Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed.\",\n \"Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.\",\n \"Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.\",\n \"Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.\",\n \"Various types of sustained-release materials have been established and are well known by those skilled in the art.\",\n \"Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.\",\n \"Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed.\",\n \"The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients.\",\n \"Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.\",\n \"Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions.\",\n \"Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.\",\n \"The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) or other active ingredient(s) of present invention along with protein or peptide antigens.\",\n \"The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.\",\n \"B-lymphocytes will respond to antigen through their surface immunoglobulin receptor.\",\n \"T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.\",\n \"MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.\",\n \"The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.\",\n \"Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.\",\n \"The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.\",\n \"Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like.\",\n \"Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat.\",\n \"Nos.\",\n \"4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.\",\n \"The amount of protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone.\",\n \"Ultimately, the attending physician will decide the amount of protein or other active ingredient of the present invention with which to treat each individual patient.\",\n \"Initially, the attending physician will administer low doses of protein or other active ingredient of the present invention and observe the patient's response.\",\n \"Larger doses of protein or other active ingredient of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.\",\n \"It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein or other active ingredient of the present invention per kg body weight.\",\n \"For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.\",\n \"When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.\",\n \"Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.\",\n \"Topical administration may be suitable for wound healing and tissue repair.\",\n \"Therapeutically useful agents other than a protein or other active ingredient of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.\",\n \"Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing or other active ingredient-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.\",\n \"Such matrices may be formed of materials presently in use for other implanted medical applications.\",\n \"The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties.\",\n \"The particular application of the compositions will define the appropriate formulation.\",\n \"Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.\",\n \"Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen.\",\n \"Further matrices are comprised of pure proteins or extracellular matrix components.\",\n \"Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.\",\n \"Matrices may be comprised of combinations of any of the above-mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate.\",\n \"The biocermics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.\",\n \"Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns.\",\n \"In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.\",\n \"A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).\",\n \"Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).\",\n \"The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.\",\n \"In further compositions, proteins or other active ingredients of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.\",\n \"These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF).\",\n \"The therapeutic compositions are also presently valuable for veterinary applications.\",\n \"Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins or other active ingredients of the present invention.\",\n \"The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.\",\n \"The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.\",\n \"For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage.\",\n \"Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.\",\n \"Polynucleotides of the present invention can also be used for gene therapy.\",\n \"Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject.\",\n \"Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).\",\n \"Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.\",\n \"Treated cells can then be introduced in vivo for therapeutic purposes.\",\n \"4.12.3 Effective Dosage Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose.\",\n \"More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated.\",\n \"Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.\",\n \"For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays.\",\n \"For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans.\",\n \"For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the protein's biological activity).\",\n \"Such information can be used to more accurately determine useful doses in humans.\",\n \"A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient.\",\n \"Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).\",\n \"The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50.Compounds which exhibit high therapeutic indices are preferred.\",\n \"The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.\",\n \"The dosage of such compounds lies preferably within a range of circulating concentrations that include the EDso with little or no toxicity.\",\n \"The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.\",\n \"The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.\",\n \"See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.\",\n \"1 p.1.Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC).\",\n \"The MEC will vary for each compound but can be estimated from in vitro data.\",\n \"Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration.\",\n \"However, HPLC assays or bioassays can be used to determine plasma concentrations.\",\n \"Dosage intervals can also be determined using MEC value.\",\n \"Compounds should be administered using a regimen that maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.\",\n \"In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.\",\n \"An exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 μg/kg to 25 mg/kg of patient body weight daily, varying in adults and children.\",\n \"Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.\",\n \"The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.\",\n \"4.12.4 Packaging The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.\",\n \"The pack may, for example, comprise metal or plastic foil, such as a blister pack.\",\n \"The pack or dispenser device may be accompanied by instructions for administration.\",\n \"Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.\",\n \"4.13 Antibodies Also included in the invention are antibodies to proteins, or fragments of proteins of the invention.\",\n \"The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen.\",\n \"Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab, and F(ab′)2 fragments, and an Fab expression library.\",\n \"In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.\",\n \"Certain classes have subclasses as well, such as IgG1, IgG2, and others.\",\n \"Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.\",\n \"Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.\",\n \"An isolated related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.\",\n \"The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens.\",\n \"An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, (for example the amino acid sequence shown in SEQ ID NO: 30369), and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.\",\n \"Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.\",\n \"Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.\",\n \"In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region on the surface of the protein of the invention that is located on the surface of the protein, e.g., a hydrophilic region.\",\n \"A hydrophobicity analysis of the human related protein sequence will indicate which regions of a related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.\",\n \"As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation.\",\n \"See, e.g., Hopp and Woods, 1981, Proc.\",\n \"Nat.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol.\",\n \"Biol.\",\n \"157: 105-142, each of which is incorporated herein by reference in its entirety.\",\n \"Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.\",\n \"A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.\",\n \"Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference).\",\n \"Some of these antibodies are discussed below.\",\n \"5.13.1 Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing.\",\n \"An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.\",\n \"Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.\",\n \"Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.\",\n \"The preparation can further include an adjuvant.\",\n \"Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.\",\n \"), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.\",\n \"Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).\",\n \"The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum.\",\n \"Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography.\",\n \"Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol.\",\n \"14, No.\",\n \"8 (Apr.\",\n \"17, 2000), pp.\",\n \"25-28).\",\n \"5.13.2 Monoclonal Antibodies The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product.\",\n \"In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.\",\n \"MAbs thus contain an antigen-binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.\",\n \"Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).\",\n \"In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.\",\n \"Alternatively, the lymphocytes can be immunized in vitro.\",\n \"The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.\",\n \"Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.\",\n \"The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.\",\n \"59-103).\",\n \"Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.\",\n \"Usually, rat or mouse myeloma cell lines are employed.\",\n \"The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.\",\n \"For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.\",\n \"Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.\",\n \"More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.\",\n \"Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.\",\n \"51-63).\",\n \"The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.\",\n \"Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).\",\n \"Such techniques and assays are known in the art.\",\n \"The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal.\",\n \"Biochem.\",\n \"107:220 (1980).\",\n \"Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.\",\n \"After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods.\",\n \"Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.\",\n \"Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.\",\n \"The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.\",\n \"The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat.\",\n \"No.\",\n \"4,816,567.DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).\",\n \"The hybridoma cells of the invention serve as a preferred source of such DNA.\",\n \"Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.\",\n \"The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat.\",\n \"No.\",\n \"4,816,567; Morrison, Nature 368 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.\",\n \"Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.\",\n \"5.13.2 Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies.\",\n \"These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.\",\n \"Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.\",\n \"Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.\",\n \"(See also U.S. Pat.\",\n \"No.\",\n \"5,225,539.)\",\n \"In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.\",\n \"Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.\",\n \"In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.\",\n \"The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr.\",\n \"Op.\",\n \"Struct.\",\n \"Biol., 2:593-596 (1992)).\",\n \"5.13.3 Human Antibodies Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes.\",\n \"Such antibodies are termed “human antibodies”, or “fully human antibodies” herein.\",\n \"Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.\",\n \"77-96).\",\n \"Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983.Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.\",\n \"77-96).\",\n \"In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol.\",\n \"Biol., 227:381 (1991); Marks et al., J. Mol.\",\n \"Biol., 222:581 (1991)).\",\n \"Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.\",\n \"Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.\",\n \"This approach is described, for example, in U.S. Pat.\",\n \"Nos.\",\n \"5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.\",\n \"(Bio/Technology 10, 779-783 (1992)); Lonberg et al.\",\n \"(Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern.\",\n \"Rev.\",\n \"Immunol.\",\n \"13 65-93 (1995)).\",\n \"Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.\",\n \"(See PCT publication WO94/02602).\",\n \"The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.\",\n \"The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments.\",\n \"An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.\",\n \"The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096.This animal produces B cells which secrete fully human immunoglobulins.\",\n \"The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.\",\n \"Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.\",\n \"An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Pat.\",\n \"No.\",\n \"5,939,598.It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.\",\n \"A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat.\",\n \"No.\",\n \"5,916,771.It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.\",\n \"The hybrid cell expresses an antibody containing the heavy chain and the light chain.\",\n \"In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.5.13.4 Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Pat.\",\n \"No.\",\n \"4,946,778).\",\n \"In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.\",\n \"Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab′)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab′)2fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) FV fragments.\",\n \"5.13.5 Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.\",\n \"In the present case, one of the binding specificities is for an antigenic protein of the invention.\",\n \"The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.\",\n \"Methods for making bispecific antibodies are known in the art.\",\n \"Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)).\",\n \"Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure.\",\n \"The purification of the correct molecule is usually accomplished by affinity chromatography steps.\",\n \"Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences.\",\n \"The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.\",\n \"It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions.\",\n \"DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism.\",\n \"For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).\",\n \"According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.\",\n \"The preferred interface comprises at least a part of the CH3 region of an antibody constant domain.\",\n \"In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g.\",\n \"tyrosine or tryptophan).\",\n \"Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.\",\n \"alanine or threonine).\",\n \"This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.\",\n \"Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g.\",\n \"F(ab′)2 bispecific antibodies).\",\n \"Techniques for generating bispecific antibodies from antibody fragments have been described in the literature.\",\n \"For example, bispecific antibodies can be prepared using chemical linkage.\",\n \"Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments.\",\n \"These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.\",\n \"The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.\",\n \"One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody.\",\n \"The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.\",\n \"Additionally, Fab′ fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies.\",\n \"Shalaby et al., J. Exp.\",\n \"Med.\",\n \"175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule.\",\n \"Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.\",\n \"The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.\",\n \"Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described.\",\n \"For example, bispecific antibodies have been produced using leucine zippers.\",\n \"Kostelny et al., J. Immunol.\",\n \"148(5):1547-1553 (1992).\",\n \"The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion.\",\n \"The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.\",\n \"This method can also be utilized for the production of antibody homodimers.\",\n \"The “diabody” technology described by Hollinger et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.\",\n \"The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain.\",\n \"Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.\",\n \"Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) diners has also been reported.\",\n \"See, Gruber et al., J. Immunol.\",\n \"152:5368 (1994).\",\n \"Antibodies with more than two valencies are contemplated.\",\n \"For example, trispecific antibodies can be prepared.\",\n \"Tutt et al., J. Immunol.\",\n \"147:60 (1991).\",\n \"Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.\",\n \"Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.\",\n \"CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.\",\n \"Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.\",\n \"These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.\",\n \"Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (° F.).\",\n \"5.13.6 Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.\",\n \"Heteroconjugate antibodies are composed of two covalently joined antibodies.\",\n \"Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat.\",\n \"No.\",\n \"4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089).\",\n \"It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.\",\n \"For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond.\",\n \"Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat.\",\n \"No.\",\n \"4,676,980.5.13.7 Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer.\",\n \"For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.\",\n \"The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).\",\n \"See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.\",\n \"Immunol., 148: 2918-2922 (1992).\",\n \"Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al.\",\n \"Cancer Research, 53: 2560-2565 (1993).\",\n \"Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities.\",\n \"See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).\",\n \"5.13.8 Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).\",\n \"Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above.\",\n \"Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.\",\n \"A variety of radionuclides are available for the production of radioconjugated antibodies.\",\n \"Examples include 212Bi, 131I, 90Y, 90Y, and 186Re.\",\n \"Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).\",\n \"For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).\",\n \"Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.\",\n \"See WO94/11026.In another embodiment, the antibody can be conjugated to a “receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.\",\n \"4.14 Computer Readable Sequences In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media.\",\n \"As used herein, “computer readable media” refers to any medium which can be read and accessed directly by a computer.\",\n \"Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention.\",\n \"As used herein, “recorded” refers to a process for storing information on computer readable medium.\",\n \"A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.\",\n \"A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention.\",\n \"The choice of the data storage structure will generally be based on the means chosen to access the stored information.\",\n \"In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium.\",\n \"The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.\",\n \"A skilled artisan can readily adapt any number of data processor structuring formats (e.g.\",\n \"text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.\",\n \"By providing any of the nucleotide sequences SEQ ID NO: 1-30368 or a representative fragment thereof; or a nucleotide sequence at least 95% identical to any of the nucleotide sequences of SEQ ID NO: 1-30368 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes.\",\n \"Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium.\",\n \"The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol.\",\n \"Biol.\",\n \"215:403-410 (1990)) and BLAZE (Brutlag et al., Comp.\",\n \"Chem.\",\n \"17:203-207 (1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) within a nucleic acid sequence.\",\n \"Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.\",\n \"As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention.\",\n \"The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.\",\n \"A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention.\",\n \"As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means.\",\n \"As used herein, “data storage means” refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.\",\n \"As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means.\",\n \"Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif.\",\n \"A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention.\",\n \"Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASIN and BLASTA (NPOLYPEPTIDEIA).\",\n \"A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems.\",\n \"As used herein, a “target sequence” can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids.\",\n \"A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database.\",\n \"The most preferred sequence length of a target sequence is from about 10 to 300 amino acids, more preferably from about 30 to 100 nucleotide residues.\",\n \"However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.\",\n \"As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.\",\n \"There are a variety of target motifs known in the art.\",\n \"Protein target motifs include, but are not limited to, enzyme active sites and signal sequences.\",\n \"Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).\",\n \"4.15 Triple Helix Formation In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA.\",\n \"Polynucleotides suitable for use in these methods are preferably 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.\",\n \"Acids Res.\",\n \"6:3073 (1979); Cooney et al., Science 15241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Olnno, J. Neurochem.\",\n \"56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).\",\n \"Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.\",\n \"Both techniques have been demonstrated to be effective in model systems.\",\n \"Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.\",\n \"4.16 Diagnostic Assays and Kits The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label.\",\n \"In general, methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample.\",\n \"Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample.\",\n \"In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample.\",\n \"In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of the nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample.\",\n \"Conditions for incubating a nucleic acid probe or antibody with a test sample vary.\",\n \"Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay.\",\n \"One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention.\",\n \"Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol.\",\n \"1 (1982), Vol.\",\n \"2 (1983), Vol.\",\n \"3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).\",\n \"The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine.\",\n \"The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed.\",\n \"Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.\",\n \"In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.\",\n \"Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.\",\n \"In detail, a compartment kit includes any kit in which reagents are contained in separate containers.\",\n \"Such containers include small glass containers, plastic containers or strips of plastic or paper.\",\n \"Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.\",\n \"Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.\",\n \"), and containers which contain the reagents used to detect the bound antibody or probe.\",\n \"Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.\",\n \"One skilled in the art will readily recognize that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.\",\n \"4.17 Medical Imaging The novel polypeptides and binding partners of the invention are useful in medical imaging of sites expressing the molecules of the invention (e.g., where the polypeptide of the invention is involved in the immune response, for imaging sites of inflammation or infection).\",\n \"See, e.g., Kunkel et al., U.S. Pat.\",\n \"No.\",\n \"5,413,778.Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.\",\n \"4.18 Screening Assays Using the isolated proteins and polynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any of the nucleotide sequences set forth in SEQ ID NO: 1-30368, or bind to a specific domain of the polypeptide encoded by the nucleic acid.\",\n \"In detail, said method comprises the steps of: (a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and (b) determining whether the agent binds to said protein or said nucleic acid.\",\n \"In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.\",\n \"Likewise, in general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.\",\n \"Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified.\",\n \"Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound).\",\n \"Alternatively, compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound).\",\n \"Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.\",\n \"The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents.\",\n \"The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.\",\n \"For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention.\",\n \"Alternatively, agents may be rationally selected or designed.\",\n \"As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the particular protein.\",\n \"For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like, capable of binding to a specific peptide sequence, in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide, W.H.\",\n \"Freeman, N.Y. (1992), pp.\",\n \"289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.\",\n \"In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention.\",\n \"As described above, such agents can be randomly screened or rationally designed/selected.\",\n \"Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control.\",\n \"One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA.\",\n \"Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.\",\n \"Agents suitable for use in these methods preferably contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.\",\n \"Acids Res.\",\n \"6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem.\",\n \"56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).\",\n \"Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.\",\n \"Both techniques have been demonstrated to be effective in model systems.\",\n \"Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.\",\n \"Agents that bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent.\",\n \"Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.\",\n \"4.19 Use of Nucleic Acids as Probes Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences.\",\n \"The hybridization probes of the subject invention may be derived from any of the nucleotide sequences SEQ ID NO: 1-30368.Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from of any of the nucleotide sequences SEQ ID NO: 1-30368 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.\",\n \"Any suitable hybridization technique can be employed, such as, for example, in situ hybridization.\",\n \"PCR as described in U.S. Pat.\",\n \"Nos.\",\n \"4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences.\",\n \"Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both.\",\n \"The probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.\",\n \"Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA probes.\",\n \"Such vectors are known in the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides.\",\n \"The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences.\",\n \"The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques.\",\n \"These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like.\",\n \"The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y. Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data.\",\n \"Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981f).\",\n \"Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease.\",\n \"The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.\",\n \"4.20 Preparation of Support Bound Oligonucleotides Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.\",\n \"Support bound oligonucleotides may be prepared by any of the methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon.\",\n \"One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers.\",\n \"Immobilization can be achieved using passive adsorption (Inouye & Hondo, (1990) J. Clin.\",\n \"Microbiol.\",\n \"28(6) 1469-72); using UV light (Nagata et al., 1985; Dahlen et al., 1987; Morrissey& Collins, (1989) Mol.\",\n \"Cell Probes 3(2) 189-207) or by covalent binding of base modified DNA (Keller et al., 1988; 1989); all references being specifically incorporated herein.\",\n \"Another strategy that may be employed is the use of the strong biotin-streptavidin interaction as a linker.\",\n \"For example, Broude et al.\",\n \"(1994) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 91(8) 3072-6, describe the use of biotinylated probes, although these are duplex probes, that are immobilized on streptavidin-coated magnetic beads.\",\n \"Streptavidin-coated beads may be purchased from Dynal, Oslo.\",\n \"Of course, this same linking chemistry is applicableto coating any surface with streptavidin Biotinylated probes may be purchased from various sources, such as, e.g., Operon Technologies (Alameda, Calif.).\",\n \"Nunc Laboratories (Naperville, Ill.) is also selling suitable material that could be used.\",\n \"Nunc Laboratories have developed a method by which DNA can be covalently bound to the microwell surface termed Covalink NH.\",\n \"CovaLink NH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridge-heads for further covalent coupling.\",\n \"CovaLink Modules may be purchased from Nunc Laboratories.\",\n \"DNA molecules may be bound to CovaLink exclusively at the 5′-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA (Rasmussen et al., (1991) Anal.\",\n \"Biochem.\",\n \"198(1) 138-42).\",\n \"The use of CovaLink NH strips for covalent binding of DNA molecules at the 5′-end has been described (Rasmussen et al., (1991).\",\n \"In this technology, a phosphoramidate bond is employed (Chu et al., (1983) Nucleic Acids Res.\",\n \"1 (8) 6513-29).\",\n \"This is beneficial as immobilization using only a single covalent bond is preferred.\",\n \"The phosphoramidate bond joins the DNA to the CovaLink NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm.\",\n \"To link an oligonucleotide to CovaLink NH via an phosphoramidate bond, the oligonucleotide terminus must have a 5′-end phosphate group.\",\n \"It is, perhaps, even possible for biotin to be covalently bound to CovaLink and then streptavidin used to bind the probes.\",\n \"More specifically, the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min.\",\n \"at 95° C. and cooling on ice for 10 min.\",\n \"Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm7), is then added to a final concentration of 10 mM 1-MeIm7.A ss DNA solution is then dispensed into CovaLink NH strips (75 ul/well) standing on ice.\",\n \"Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIm7, is made fresh and 25 ul added per well.\",\n \"The strips are incubated for 5 hours at 50° C. After incubation the strips are washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3 times, then they are soaked with washing solution for 5 min., and finally they are washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50° C.).\",\n \"It is contemplated that a further suitable method for use with the present invention is that described in PCT Patent Application WO 90/03382 (Southern & Maskos), incorporated herein by reference.\",\n \"This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3′-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups carried by the support.\",\n \"The oligonucleotide is then synthesized on the supported nucleoside and protecting groups removed from the synthetic oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support.\",\n \"Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphorate.\",\n \"An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed.\",\n \"For example, addressable laser-activated photodeprotection may be employed in the chemical synthesis of oligonucleotides directly on a glass surface, as described by Fodor et al.\",\n \"(1991) Science 251(4995) 767-73, incorporated herein by reference.\",\n \"Probes may also be immobilized on nylon supports as described by Van Ness et al.\",\n \"(1991) Nucleic Acids Res.\",\n \"19(12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier (1988) Anal.\",\n \"Biochem.\",\n \"169(1) 104-8; all references being specifically incorporated herein.\",\n \"To link an oligonucleotide to a nylon support, as described by Van Ness et at (1991), requires activation of the nylon surface via alkylation and selective activation of the 5′-amine of oligonucleotides with cyanuric chloride.\",\n \"One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described by Pease et al., (1994) PNAS USA 91(11) 5022-6, incorporated herein by reference).\",\n \"These authors used current photolithographic techniques to generate arrays of immobilized oligonucleotide probes (DNA chips).\",\n \"These methods, in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5′-protected N-acyl-deoxynucleoside phosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies.\",\n \"A matrix of 256 spatially defined oligonucleotide probes may be generated in this manner.\",\n \"4.21 Preparation of Nucleic Acid Fragments The nucleic acids may be obtained from any appropriate source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC inserts, and RNA, including mRNA without any amplification steps.\",\n \"For example, Sambrook et al.\",\n \"(1989) describes three protocols for the isolation ofhigh molecular weight DNA from mammalian cells (p. 9.14-9.23).\",\n \"DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and/or prepared directly from genomic DNA or cDNA by PCR or other amplification methods.\",\n \"Samples may be prepared or dispensed in multiwell plates.\",\n \"About 100-1000 ng of DNA samples may be prepared in 2-500 ml of final volume.\",\n \"The nucleic acids would then be fragmented by any of the methods known to those of skill in the art including, for example, using restriction enzymes as described at 9.24-9.28 of Sambrook et al.\",\n \"(1989), shearing by ultrasound and NaOH treatment.\",\n \"Low pressure shearing is also appropriate, as described by Schriefer et at (1.990) Nucleic Acids Res.\",\n \"18(24) 7455-6, incorporated herein by reference).\",\n \"In this method, DNA samples are passed through a small French pressure cell at a variety of low to intermediate pressures.\",\n \"A lever device allows controlled application of low to intermediate pressures to the cell.\",\n \"The results of these studies indicate that low-pressure shearing is a useful alternative to sonic and enzymatic DNA fragmentation methods.\",\n \"One particularly suitable way for fragmenting DNA is contemplated to be that using the two base recognition endonuclease, CviJI, described by Fitzgerald et al.\",\n \"(1992) Nucleic Acids Res.\",\n \"20(14) 3753-62.These authors described an approach for the rapid fragmentation and fractionation of DNA into particular sizes that they contemplated to be suitable for shotgun cloning and sequencing.\",\n \"The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends.\",\n \"A typical reaction conditions, which alter the specificity of this enzyme (CviJI**), yield a quasi-random distribution of DNA fragments form the small molecule pUC19 (2688 base pairs).\",\n \"Fitzgerald et al.\",\n \"(1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI** digest ofpUCl9 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z minus M13 cloning vector.\",\n \"Sequence analysis of 76 clones showed that CviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation.\",\n \"As reported in the literature, advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed.\",\n \"Irrespective of the manner in which the nucleic acid fragments are obtained or prepared, it is important to denature the DNA to give single stranded pieces available for hybridization.\",\n \"This is achieved by incubating the DNA solution for 2-5 minutes at 80-90° C. The solution is then cooled quickly to 2° C. to prevent renaturation of the DNA fragments before they are contacted with the chip.\",\n \"Phosphate groups must also be removed from genomic DNA by methods known in the art.\",\n \"4.22 Preparation of DNA Arrays Arrays may be prepared by spotting DNA samples on a support such as a nylon membrane.\",\n \"Spotting may be performed by using arrays of metal pins (the positions of which correspond to an array of wells in a microtiter plate) to repeated by transfer of about 20 nl of a DNA solution to a nylon membrane.\",\n \"By offset printing, a density of dots higher than the density of the wells is achieved.\",\n \"One to 25 dots may be accommodated in 1 mm2, depending on the type of label used.\",\n \"By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed.\",\n \"Samples in one subarray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones.\",\n \"Each of the subarrays may represent replica spotting of the same samples.\",\n \"In one example, a selected gene segment may be amplified from 64 patients.\",\n \"For each patient, the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample).\",\n \"A plate for each of the 64 patients is prepared.\",\n \"By using a 96-pin device, all samples may be spotted on one 8×12 cm membrane.\",\n \"Subarrays may contain 64 samples, one from each patient.\",\n \"Where the 96 subarrays are identical, the dot span may be 1 mm2 and there may be a 1 mm space between subarrays.\",\n \"Another approach is to use membranes or plates (available from NUNC, Naperville, Ill.) which may be partitioned by physical spacers e.g.\",\n \"a plastic grid molded over the membrane, the grid being similar to the sort of membrane applied to the bottom of multiwell plates, or hydrophobic strips.\",\n \"A fixed physical spacer is not preferred for imaging by exposure to flat phosphor-storage screens or x-ray films.\",\n \"The present invention is illustrated in the following examples.\",\n \"Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention.\",\n \"Accordingly, it is intended that the broader aspects of the present invention not be limited to the disclosure of the following examples.\",\n \"The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention.\",\n \"Indeed, numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the present preferred embodiments.\",\n \"Consequently, the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims.\",\n \"All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.\",\n \"5.0 EXAMPLES 5.1 Example 1 Novel Nucleic Acid Sequences Obtained From Various Libraries A plurality of novel nucleic acids were obtained from cDNA libraries prepared from various human tissues and in some cases isolated from a genomic library derived from human chromosome using standard PCR, SBH sequence signature analysis and Sanger sequencing techniques.\",\n \"The inserts of the library were amplified with PCR using primers specific for the vector sequences which flank the inserts.\",\n \"Clones from cDNA libraries were spotted on nylon membrane filters and screened with oligonucleotide probes (e.g., 7-mers) to obtain signature sequences.\",\n \"The clones were clustered into groups of similar or identical sequences.\",\n \"Representative clones were selected for sequencing.\",\n \"In some cases, the 5′ sequence of the amplified inserts was then deduced using a typical Sanger sequencing protocol.\",\n \"PCR products were purified and subjected to fluorescent dye terminator cycle sequencing.\",\n \"Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer to obtain the novel nucleic acid sequences.\",\n \"In some cases RACE (Rapid Amplification of cDNA Ends) was performed to further extend the sequence in the 5′ direction.\",\n \"5.2 Example 2 Novel Contigs The novel contigs of the invention were assembled from sequences that were obtained from a cDNA library by methods described in Example 1 above, and in some cases sequences obtained from one or more public databases.\",\n \"The sequences for the resulting nucleic acid contigs are designated as SEQ ID NO: 1-30368 and are provided in the attached Sequence Listing.\",\n \"The contigs were assembled using an EST sequence as a seed.\",\n \"Then a recursive algorithm was used to extend the seed EST into an extended assemblage, by pulling additional sequences from different databases (i.e., Hyseq's database containing EST sequences, dbEST version 115, gb pri 115, and UniGene version 103, and exons from public domain genomic sequences predicted by GenScan) that belong to this assemblage.\",\n \"The algorithm terminated when there was no additional sequences from the above databases that would extend the assemblage.\",\n \"Further, the inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST score greater than 300 and percent identity greater than 95%.\",\n \"The novel predicted polypeptides (including proteins) encoded by the novel polynucleotides (SEQ ID NO: 1-30368) of the present invention are incorporated in the attached Sequence Listing.\",\n \"A subset the predicted polypeptide sequences contain an unknown amino acid, a stop codon, a possible nucleotide deletion or a possible nucleotide insertion.\",\n \"These sequences have been shown in their entirety with the special characters in Table 2.Table 2 also shows the corresponding start and stop nucleotide locations to each of SEQ ID NO: 1-30368.Table 2 also indicates the method by which the polypeptide was predicted.\",\n \"Method A refers to a polypeptide obtained by using a software program called FASTY (available from http://fastabioch.virginia.edu) which selects a polypeptide based on a comparison of the translated novel polynucleotide to known polynucleotides (W. R Pearson, Methods in Enzymology, 183:63-98 (1990), herein incorporated by reference).\",\n \"Method B refers to a polypeptide obtained by using a software program called GenScan for human/vertebrate sequences (available from Stanford University, Office of Technology Licensing) that predicts the polypeptide based on a probabilistic model of gene structure/compositional properties (C. Burge and S. Karlin, J. Mol.\",\n \"Biol., 268:78-94(1997), incorporated herein by reference).\",\n \"Method C refers to a polypeptide obtained by using a Hyseq proprietary software program that translates the novel polynucleotide and its complementary strand into six possible amino acid sequences (forward and reverse frames) and chooses the polypeptide with the longest open reading frame.\",\n \"The nearest neighbor results for SEQ ID NO: 1-30368 were obtained by a BLASTP version 2.0al 19 MP-WashU search against Genpept release 121 and Geneseq release 200103 (Derwent), using BLAST algorithm.\",\n \"The nearest neighbor result showed the closest homologue for SEQ ID NO: 1-30368.The nearest neighbor results for SEQ ID NO: 1-30368 are incorporated in the attached Sequence Listing.\",\n \"Using eMatrix software package (Stanford University, Stanford, Calif.) (Wu et al., J. Comp.\",\n \"Biol., Vol.\",\n \"6 pp.\",\n \"219-235 (1999) herein incorporated by reference), all the sequences were examined to determine whether they had identifiable signature regions.\",\n \"The attached Sequence Listing provodes the results obtained by eMatrix analysis for each polypeptide as follows: the signature region found in the indicated polypeptide sequences, the description of the signature, the eMatrix p-value(s) and the position(s) of the signature within the polypeptide sequence.\",\n \"Using the pFam software program (Sonnhammer et al., Nucleic Acids Res., Vol.\",\n \"26(1) pp.\",\n \"320-322 (1998) herein incorporated by reference) all the polypeptide sequences were examined for domains with homology to certain peptide domains.\",\n \"The attached Sequence Listing provides the results obtained by PFAM analysis for each peptide, namely: the name of the domain found, the description, the p-value and the pFam score for the identified domain within the sequence.\",\n \"Tables 1 and 2 follow.\",\n \"Table I shows the various tissue sources of SEQ ID NO: 1-30368.Table 2 shows the start and stop nucleotides for the translated amino acid sequence for which each assemblage encodes.\",\n \"Table 2 also provides a correlation between the amino acid sequences set forth in the Sequence Listing, the nucleotide sequences set forth in the Sequence Listing and the SEQ ID NO: in U.S. Ser.\",\n \"No.\",\n \"09/540,217\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10450763"}}},{"rowIdx":283,"cells":{"text":{"kind":"list like","value":[["Novel means for the diagnosis and therapy of ctcl","A means for the diagnosis and therapy of T lymphomas, in particular Cutaneous T Cell Lymphomas (“CTCL”) are provided.","Tumor markers which are universal for CTCL are described.","More particularly, a molecule, termed SC5 by the inventors, an allelic form of p140, and biological applications of SC5 and p140 molecules, notably in the diagnosis and therapy of CTCL are described."],["1 A monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.","2 A hybridoma deposited as deposit number I-2575 at the C.N.C.M.","3 An isolated protein obtainable by: (i) collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), and stimulating them with PHA at 1 microgram/ml, (ii) lysing the cells by incubation in a lysis buffer containing Triton X-100 at 1%, (iii) recovering from the lysate the compound onto which the mAb of claim 1 binds under conditions enabling this mAb to perform reactions of the antigen-antibody type.","4 An isolated protein obtainable by recovering the isolated protein compound of claim 3 under non reducing conditions.","5 An isolated cDNA obtainable by: collecting cells selected from the group consisting of total blood cells and PBL5 incubating the collected cell population with a CD3 activator at 1 microgram per ml, extracting and purifying the whole mRNA population from said cells, synthesizing every complementary cDNA, operably cloning each cDNA so that expression of this cDNA in this clone is under appropriate clone culture conditions, and cultivating every clone accordingly, selecting those clones which express a compound onto which the monoclonal antibody of claim 1 binds when placed under conditions suitable for this monoclonal antibody to perform reactions of the antigen-antibody type, and/or of which lysate would give an immuno-precipitation reaction with this mAb, optionally, amplifying those clones that have been thus selected, and recovering the inserted cDNA from the selected clones.","6 An isolated mRNA obtainable by selection among the mRNA population of cells selected from the group consisting of total blood cells and PBL, of a mRNA which is complementary to a cDNA of claim 5.7 An isolated genomic DNA encoding an isolated mRNA of claim 6.8 An engineered cell in which a cDNA according to claim 5, has been transfected.","9 An isolated protein encoded by an isolated cDNA according to claim 5.10 A bank of polypeptide compounds, obtainable by cleavage of a protein according to claim 3, with an enzyme selected from the group consisting of V8 protease and alpha chymotrypsine.","11 An isolated polypeptide compound obtainable by: collecting PBL cells or total blood cells, incubating the collected cells with a CD3 activator at 1 microgram par ml, labeling the cells with a polypeptide-specific label, lysing the cells in a lysis buffer comprising Trition X-100 at 1%, and submitting the lysate to an immuno-precipitation reaction with the mAb of claim 1, and recovering from the digested immuno-precipitate any compound onto which bear a label.","12 A monoclonal antibody obtainable by: (i) immunizing an animal against a protein according to claim 3, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein or to the polypeptide compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells maizy at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with We a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.","for binding to a protein according to claim 3, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.","13 A fragment of a monoclonal antibody according to claim 1, selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3.14 An isolated compound, comprising a fragment according to claim 13.15 The isolated compound according to claim 14, wherein it is a humanized antibody.","16 The isolated compound according to claim 14, wherein it further comprises at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3 fragments of anti-CD4 antibodies.","17 An isolated protein of SEQ ID No.","2.18 The isolated DNA encoding an isolated protein according to claim 17.19 An isolated DNA of SEQ No.","1.20 A polypeptidic vector comprising a monoclonal antibody according to claim 1, or a fragment thereof selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2 and CDR3.21 A polypeptidic vector for use in the treatment of CTCL, wherein said vector comprises a p140 binding compound.","22 The polypeptidic vector according to claim 20, further comprising an element selected from the group consisting of tumoral toxins and radioelements.","23 The polypeptidic vector according to claim 20, further comprising an enzyme capable of transforming an anti-mitotic pro-drug into an active drug form.","24 A medical kit comprising a polypeptidic vector according to claim 23 and an antimitotic pro-drug.","25 A medicament comprising, as an active principle, an element selected from the group consisting of a monoclonal antibody according to claim 1, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody according to claim 1.26 A method for manufacturing an anti-CTCL medicament comprising including a p140 binding compound in said medicament.","27 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a protein according to claim 3 in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.","28 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a protein according to claim 3, in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.","29 A method for CTCL diagnosis, comprising measuring the percentage of CD4+ cells expressing an element selected from the group consisting of the proteins according to claim 3, in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.","30 A method for CTCL diagnosis, comprising determining the presence of CD4+ cells expressing p140 in a biological sample which comprises potential CTCL cells, a CTCL-positive diagnosis being made when such a presence is significantly detected.","31 A method according to claim 27, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody according to claim 1 and the DNA encoding SEQ ID No.","3 or SEQ ID No.","4.32 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises an element selected from the group consisting of the monoclonal antibodies according to claim 1, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody according to claim 1.33 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a protein according to claim 3, or to a p140 molecule, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.","34 The isolated protein of claim 4, wherein said non reducing conditions are of chromatography by affinity with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.","35 The isolated cDNA of claim 5, wherein said CD3 activator is PHA at 1 microgram per ml.","36 An engineered cell in which a mRNA according to claim 6 has been transfected.","37 An engineered cell in which a DNA according to claim 7 has been transfected.","38 An isolated protein encoded by an isolated mRNA according to claim 6.39 An isolated protein encoded by an isolated cDNA according to claim 7.40 A bank of polypeptide compounds, obtainable by cleavage of a protein according to claim 4, with an enzyme selected from the group consisting of V8 protease and alpha chymotrypsine.","41 A bank of polypeptide compounds, obtainable by cleavage of a protein according to claim 9, with an enzyme selected from the group consisting of V8 protease and alpha chymotrypsine.","42 A monoclonal antibody obtainable by: (i) immunizing an animal against a protein according to claim 4, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.","for binding to a protein according to claim 4, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.","43 A monoclonal antibody obtainable by: (i) immunizing an animal against a protein according to claim 9, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.","for binding to a protein according to claim 9, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.","44 A monoclonal antibody obtainable by: (i) immunizing an animal against a polypeptide compound according to claim 11, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the polypeptide compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.","for binding to a polypeptide according to claim 11, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.","45 A fragment of a monoclonal antibody according to claim 12, selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3.46 The isolated compound according to claim 15, wherein it further comprises at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3 fragments of anti-CD4 antibodies.","47 The polypeptidic vector according to claim 20, wherein said fragment is the Fab or F(ab′)2 fragment.","48 A polypeptidic vector comprising an isolated compound according to claim 15, optionally including at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3 fragments of anti-CD4 antibodies.","49 The polypeptidic vector according to claim 21, further comprising an element selected from the group consisting of tumoral toxins and radioelements.","50 The polypeptidic vector according to claim 21, further comprising an enzyme capable of transforming an anti-mitotic pro-drug into an active drug form 51 The polypeptidic vector according to claim 23, wherein said enzyme capable of transforming an anti-mitotic pro-drug into an active drug form is a carboxypeptidase.","52 The polypeptidic vector according to claim 50, wherein said enzyme capable of transforming an anti-mitotic pro-drug into an active drug form is a carboxypeptidase.","53 The medical kit according to claim 24, wherein said antimitotic pro-drug is phenol mustard pro-drug.","54 A medicament comprising, as an active principle, an element selected from the group consisting of a monoclonal antibody according to claim 12, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.","55 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a protein according to claim 4 in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.","56 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a protein according to claim 9 in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.","57 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a member selected from the group consisting of the polypeptide compounds of claim 11 and the p140 molecules in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.","58 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a protein according to claim 4, in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.","59 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a protein according to claim 9, in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.","60 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a member selected from the group consisting of the polypeptide compounds of claim 11 and the p140 molecules in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.","61 A method for CTCL diagnosis comprising measuring the percentage of CD4+ cells expressing a protein according to claim 4 in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.","62 A method for CTCL diagnosis comprising measuring the percentage of CD4+ cells expressing a protein according to claim 9 in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.","63 A method for CTCL diagnosis comprising measuring the percentage of CD4+ cells expressing the polypeptide compounds of claim 11 in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.","64 A method according to claim 28, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.","65 A method according to claim 29, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.","66 A method according to claim 30, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody, and the DNA encoding SEQ ID No.","3 or SEQ ID No.","4.67 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises an element selected from the group consisting of the monoclonal antibodies according to claim 12, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.","68 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises at least one polypeptide compound selected from the bank of polypeptide compounds according to claim 10.69 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises an element selected from the group consisting the cDNA according to claim 5, an mRNA complementary to said cDNA, a p140 binding compound, a p140 DNA, an isolated DNA encoding an isolated protein of SEQ ID NO: 2, an isolated DNA of SEQ ID NO: 1, the DNA encoding SEQ ID No.","3 and the DNA encoding SEQ ID No.","4.70 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a protein according to claim 4, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.","71 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a protein according to claim 9, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.","72 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a polypeptide compound according to claim 10, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.","73 The method according to claim 33, wherein said p140 molecule is of SEQ ID No.","2 or SEQ ID No.","4."],["<SOH> BACKGROUND OF THE INVENTION <EOH>CTCL is a group of T lymphomas which primarily involve the skin.","The CTCL group namely comprises transformed Mycosis Fungoides (abbreviated into transformed MF), Sézary Syndrome (abbreviated into SS), Lymphomatoide Papulosis (abbreviated into LP), and CD30+ lymphomas.","Transformed MF is characterized by skin invasion of clonally-derived malignant T lymphocytes that phenotypically resemble mature T helper cells.","LP and CD30+ lymphomas also develop in the skin.","More aggressive forms of CTCL develop when the malignant cells become non-epidermotropic, and are associated with extra-cutaneous involvement.","SS is a more aggressive form of CTCL that is characterized by a clonal expansion of CD4+/CD45RO+ T cells and the appearance of these malignant T cells in the blood.","Most CTCL are CD4+ CTCL, but some rare CD8+ CTCL cases exist.","The biology of CTCL disease remains poorly understood, as it is difficult to identify the malignant cells, due to the lack of specific cell surface markers.","In cutaneous lesions, it is therefore difficult to distinguish CTCL cells from reactive infiltrating (non-tumoral) T lymphocytes.","Diagnosis of T lymphomas such as CTCL is at present time mainly based on cytological and histological observations of the presence or absence of tumoral cells in a sample collected from a suspected body area (observation of histopathological aspect on skin biopsis and/or presence of SS cells in the blood, via detection of cells showing a cerebriform nucleus).","Such a diagnosis method is not fully reliable, notably at the early stages of the transformation of skin lymphocytes into malignant lymphocytes.","Such a diagnosis method does also not enable to stage the disease.","Technically speaking, it is also time-consuming.","Today therapy of CTCL is tentatively achieved by induction of tumoral cell apoptosis via non specific chemotherapy.","There is therefore still a need for more accurate and appropriate solutions to the problem of the diagnosis and therapy of diseases involving the proliferation of malignant T cells, such as CTCL."],["<SOH> SUMMARY OF THE INVENTION <EOH>In view of this prior art situation, the inventors now provide with two molecules which give solutions to the problem of CTCL diagnostic and therapy: one has been termed SC5 by the inventors, and the other one is the p140 molecule (p140 is also referred to as KIR3DL2).","The SC5 molecule of the invention appears as a biochemically and functionally new protein, of which apparent molecular weight is of 96 kD under reducing conditions.","It has been isolated as the antigen of a monoclonal antibody that has been produced by the inventors.","The hybridoma producing this monoclonal antibody has been deposited on Oct. 30, 2000 at the C.N.C.M.","(C.N.C.M.","Institut Pasteur; 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France) in accordance with the Budapest Treaty (C.N.C.M.","deposit number: I-2575).","When expressed at the cell surface, and made to aggregate, SC5 molecules act as inhibitory receptors for cell activity and proliferation.","The second marker of the invention, i.e.","p140, was already known to be an inhibitory receptor, but has been previously described only on sub-groups of NK cells and of CD3+ CD8+ cells from healthy humans.","The inventors now demonstrate that p140 is expressed at the surface of tumoral T cells such as CTCL cells.","They further demonstrate that p140 is expressed at the surface of malignant CD4+ T cells, such as CD4+ CTCL cells, whereas those other receptors that are usually observed at the surface of NK cells (such as p58.1, p58.2, p70 KIRs, CD94/NKG2A) are not found at the surface of malignant CD4+ T cells.","Of further note is that p140 has not been observed by the inventors at the surface of CD4+ T cells collected from patients suffering from non-tumoral dermatological diseases such as inflammatory skin diseases (e.g.","lupus, lichen), or toxic epidermal necrolysis.","At the surface of tumoral CD4+ T cells, two allelic forms of p140 have been identified by the inventors: allelic form KIR3D clone 24 (SEQ ID No.1), and allelic form p140 clone 1.1 (SEQ ID No.3).","Allelic form KIR3D clone 24 (SEQ ID No.","1) is a new polynucleotide encoding a new protein (SEQ ID No.2): KIR3D clone 24 (SEQ ID No.","1) displays five differences when compared to the previously described p140 clone 1.1 DNA sequence (SEQ ID No.","3), resulting in four amino acid substitutions in the mature protein (SEQ ID No.","2) when compared to p140 clone 1.1.mature protein (SEQ ID No.","4).","The inventors demonstrate that the SC5 and p140 both share the same technical following features: SC5 and p140 both are membranar differentiation antigens which are characteristic of malignant T cells, and notably of malignant CD4+ T cells, whichever form of CD4+ CTCL is concerned, there are malignant CD4+ T cells which express SC5 or p140 at their surface (usually both SC5 and p140 are expressed): SC5 as well as p140 indeed cover the whole range of CD4+ CTCL, and notably the Sézary Syndrome (abbreviated into “SS”), transformed Mycosis Fungoides (abbreviated into “transformed MF”), Lymphomatoide Papulosis (abbreviated onto “LP”), and CD30+ lymphomas, there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that a CD4+ CTCL diagnosis based on the analysis of the presence of SC5 or p140 at the surface of CD4+ cells collected from the suspected body area (e.g.","sample of skin erythroderma when transformed MF is suspected, or sample of peripheral blood when a more aggressive CTCL form, such as SS, is suspected) has a reliability of more than 90%, preferably of more than of 95%, most preferably of 100% (among those patients who were tested up to now, the reliability is of 100%): indeed, according to the invention, it can be concluded that a CD4+ T cell is tumoral as soon as there are p140 molecules detected at the surface of these CD4+ T cells, or as soon as a percentage of SC5+ CD4+ T cells higher than the average standard level is measured (the average standard level is in the 1-15% range, generally in the 5-10% range), and there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that the percentage of CD4+ SC5+ T cells, as well as of CD4+ p140+ T cells, that is measured in a sample of peripheral blood collected from a patient for whom a Sézary Syndrome (SS) is suspected, substantially corresponds to the percentage of malignant SS cells that are actually present in the peripheral blood of this patient (within a ±10% range for SC5+ CD4+ cells, within a ±5% range for p140+ CD4+ cells): the SC5 and p140 markers of the invention therefore share the common particular technical feature of enabling to assess the staging of a SS.","To the best of the inventors' knowledge, SC5 and p140 are the first molecules which are described as having these common technical features.","The invention thus provides with the first CTCL universal markers.","No prior art product was known to be such a CTCL universal marker.","The closest prior art product in this respect would be CD30 of which presence at the surface of malignant CD4+ T cells directs to the conclusion that the patient has a particular form of CD4+ CTCL which is referred to in the art as CD30+ lymphoma.","CD30 is therefore a CTCL marker, but its reliability is limited to a particular form of CTCL (CD30+ lymphomas), and CD30 does not cover every form of CD4+ CTCL: for CD4+ CTCL such as SS, transformed MF, or LP, there does not necessarily exist a malignant CD4+ T cell which would express CD30 at its surface.","CD30 uses in CTCL diagnosis and therapy is thus restricted to a particular form of CTCL, whereas the markers of the invention advantageously cover the whole range of CD4+ CTCL.","In addition to the technical features shared with p140, SC5 has the further characteristic of being a positive indicator of proliferation and/or functional activity (e.g.","cytokine profile) of non-tumoral T cells.","The presence of SC5 at the surface of T cells is indeed positively linked with the activation status of non-tumoral T cells, such as normal (CD4+ and CD8+) T cells or virus-infected CD4+ T cells.","SC5 is thus also a useful novel means for the diagnostic and therapy of T cell viral infections such as HIV-infection, and of inflammatory diseases such as those of the auto-immune type (e.g.","rhumatoid arthritis such as spondyloarthropathis, or skin immune mediated diseases such as psoriasis, eczema, atopic dermatitis).","SC5 is also a useful target for modulating graft-host reactions: the activation of SC5 transduces an inhibitory signal that can be used to inhibit the reactions that effector recipient cells may develop against a graft.","The invention also provides with products capable of binding to the SC5 new molecule, and in particular with monoclonal antibodies directed against SC5 (anti-SC5 mAbs).","It is also provided with anti-SC5 monoclonal antibodies which are capable of modulating the proliferation and/or the functional activity of T cells, and notably of tumoral T cells such as malignant CTCL cells.","In particular, the monoclonal antibody produced by hybridoma I-2575 is capable of inhibiting the proliferation and/or functional activity of T cells, and according to a particularly useful feature, is capable of inhibiting the proliferation and/or activity of malignant T cells such as malignant CTCL cells (see examples below).","Compounds binding to SC5, but not aggregating them will prevent SC5 inhibitory signal transduction, or will prevent compounds to reach SC5+ cells (target masking).","More generally, compounds binding to SC5 or p140 may, according to the invention, be used as complement recruiting agents, as ADCC stimulators, or as vectors for therapeutic agents so as to prevent, palliate, treat T lymphomas, such as CTCL.","The invention therefore also relates to the diagnostic and therapeutic uses of SC5- or p140-binding compounds such as anti-SC5 and/or anti-p140 monoclonal antibodies, and of products directly-derived therefrom (e.g.","humanized monoclonal antibodies, or monoclonal antibodies with a double CD4-p140/SC5 specificity)."],["FIELD OF THE INVENTION The present application relates to novel means for the diagnosis and therapy of T lymphomas, and more particularly of Cutaneous T Cell Lymphomas (abbreviated into CTCL).","The invention indeed provides novel tumor markers which are universal for CTCL, and describes biotechnological and medical uses thereof.","BACKGROUND OF THE INVENTION CTCL is a group of T lymphomas which primarily involve the skin.","The CTCL group namely comprises transformed Mycosis Fungoides (abbreviated into transformed MF), Sézary Syndrome (abbreviated into SS), Lymphomatoide Papulosis (abbreviated into LP), and CD30+ lymphomas.","Transformed MF is characterized by skin invasion of clonally-derived malignant T lymphocytes that phenotypically resemble mature T helper cells.","LP and CD30+ lymphomas also develop in the skin.","More aggressive forms of CTCL develop when the malignant cells become non-epidermotropic, and are associated with extra-cutaneous involvement.","SS is a more aggressive form of CTCL that is characterized by a clonal expansion of CD4+/CD45RO+ T cells and the appearance of these malignant T cells in the blood.","Most CTCL are CD4+ CTCL, but some rare CD8+ CTCL cases exist.","The biology of CTCL disease remains poorly understood, as it is difficult to identify the malignant cells, due to the lack of specific cell surface markers.","In cutaneous lesions, it is therefore difficult to distinguish CTCL cells from reactive infiltrating (non-tumoral) T lymphocytes.","Diagnosis of T lymphomas such as CTCL is at present time mainly based on cytological and histological observations of the presence or absence of tumoral cells in a sample collected from a suspected body area (observation of histopathological aspect on skin biopsis and/or presence of SS cells in the blood, via detection of cells showing a cerebriform nucleus).","Such a diagnosis method is not fully reliable, notably at the early stages of the transformation of skin lymphocytes into malignant lymphocytes.","Such a diagnosis method does also not enable to stage the disease.","Technically speaking, it is also time-consuming.","Today therapy of CTCL is tentatively achieved by induction of tumoral cell apoptosis via non specific chemotherapy.","There is therefore still a need for more accurate and appropriate solutions to the problem of the diagnosis and therapy of diseases involving the proliferation of malignant T cells, such as CTCL.","SUMMARY OF THE INVENTION In view of this prior art situation, the inventors now provide with two molecules which give solutions to the problem of CTCL diagnostic and therapy: one has been termed SC5 by the inventors, and the other one is the p140 molecule (p140 is also referred to as KIR3DL2).","The SC5 molecule of the invention appears as a biochemically and functionally new protein, of which apparent molecular weight is of 96 kD under reducing conditions.","It has been isolated as the antigen of a monoclonal antibody that has been produced by the inventors.","The hybridoma producing this monoclonal antibody has been deposited on Oct. 30, 2000 at the C.N.C.M.","(C.N.C.M.","Institut Pasteur; 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France) in accordance with the Budapest Treaty (C.N.C.M.","deposit number: I-2575).","When expressed at the cell surface, and made to aggregate, SC5 molecules act as inhibitory receptors for cell activity and proliferation.","The second marker of the invention, i.e.","p140, was already known to be an inhibitory receptor, but has been previously described only on sub-groups of NK cells and of CD3+ CD8+ cells from healthy humans.","The inventors now demonstrate that p140 is expressed at the surface of tumoral T cells such as CTCL cells.","They further demonstrate that p140 is expressed at the surface of malignant CD4+ T cells, such as CD4+ CTCL cells, whereas those other receptors that are usually observed at the surface of NK cells (such as p58.1, p58.2, p70 KIRs, CD94/NKG2A) are not found at the surface of malignant CD4+ T cells.","Of further note is that p140 has not been observed by the inventors at the surface of CD4+ T cells collected from patients suffering from non-tumoral dermatological diseases such as inflammatory skin diseases (e.g.","lupus, lichen), or toxic epidermal necrolysis.","At the surface of tumoral CD4+ T cells, two allelic forms of p140 have been identified by the inventors: allelic form KIR3D clone 24 (SEQ ID No.1), and allelic form p140 clone 1.1 (SEQ ID No.3).","Allelic form KIR3D clone 24 (SEQ ID No.","1) is a new polynucleotide encoding a new protein (SEQ ID No.2): KIR3D clone 24 (SEQ ID No.","1) displays five differences when compared to the previously described p140 clone 1.1 DNA sequence (SEQ ID No.","3), resulting in four amino acid substitutions in the mature protein (SEQ ID No.","2) when compared to p140 clone 1.1.mature protein (SEQ ID No.","4).","The inventors demonstrate that the SC5 and p140 both share the same technical following features: SC5 and p140 both are membranar differentiation antigens which are characteristic of malignant T cells, and notably of malignant CD4+ T cells, whichever form of CD4+ CTCL is concerned, there are malignant CD4+ T cells which express SC5 or p140 at their surface (usually both SC5 and p140 are expressed): SC5 as well as p140 indeed cover the whole range of CD4+ CTCL, and notably the Sézary Syndrome (abbreviated into “SS”), transformed Mycosis Fungoides (abbreviated into “transformed MF”), Lymphomatoide Papulosis (abbreviated onto “LP”), and CD30+ lymphomas, there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that a CD4+ CTCL diagnosis based on the analysis of the presence of SC5 or p140 at the surface of CD4+ cells collected from the suspected body area (e.g.","sample of skin erythroderma when transformed MF is suspected, or sample of peripheral blood when a more aggressive CTCL form, such as SS, is suspected) has a reliability of more than 90%, preferably of more than of 95%, most preferably of 100% (among those patients who were tested up to now, the reliability is of 100%): indeed, according to the invention, it can be concluded that a CD4+ T cell is tumoral as soon as there are p140 molecules detected at the surface of these CD4+ T cells, or as soon as a percentage of SC5+ CD4+ T cells higher than the average standard level is measured (the average standard level is in the 1-15% range, generally in the 5-10% range), and there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that the percentage of CD4+ SC5+ T cells, as well as of CD4+ p140+ T cells, that is measured in a sample of peripheral blood collected from a patient for whom a Sézary Syndrome (SS) is suspected, substantially corresponds to the percentage of malignant SS cells that are actually present in the peripheral blood of this patient (within a ±10% range for SC5+ CD4+ cells, within a ±5% range for p140+ CD4+ cells): the SC5 and p140 markers of the invention therefore share the common particular technical feature of enabling to assess the staging of a SS.","To the best of the inventors' knowledge, SC5 and p140 are the first molecules which are described as having these common technical features.","The invention thus provides with the first CTCL universal markers.","No prior art product was known to be such a CTCL universal marker.","The closest prior art product in this respect would be CD30 of which presence at the surface of malignant CD4+ T cells directs to the conclusion that the patient has a particular form of CD4+ CTCL which is referred to in the art as CD30+ lymphoma.","CD30 is therefore a CTCL marker, but its reliability is limited to a particular form of CTCL (CD30+ lymphomas), and CD30 does not cover every form of CD4+ CTCL: for CD4+ CTCL such as SS, transformed MF, or LP, there does not necessarily exist a malignant CD4+ T cell which would express CD30 at its surface.","CD30 uses in CTCL diagnosis and therapy is thus restricted to a particular form of CTCL, whereas the markers of the invention advantageously cover the whole range of CD4+ CTCL.","In addition to the technical features shared with p140, SC5 has the further characteristic of being a positive indicator of proliferation and/or functional activity (e.g.","cytokine profile) of non-tumoral T cells.","The presence of SC5 at the surface of T cells is indeed positively linked with the activation status of non-tumoral T cells, such as normal (CD4+ and CD8+) T cells or virus-infected CD4+ T cells.","SC5 is thus also a useful novel means for the diagnostic and therapy of T cell viral infections such as HIV-infection, and of inflammatory diseases such as those of the auto-immune type (e.g.","rhumatoid arthritis such as spondyloarthropathis, or skin immune mediated diseases such as psoriasis, eczema, atopic dermatitis).","SC5 is also a useful target for modulating graft-host reactions: the activation of SC5 transduces an inhibitory signal that can be used to inhibit the reactions that effector recipient cells may develop against a graft.","The invention also provides with products capable of binding to the SC5 new molecule, and in particular with monoclonal antibodies directed against SC5 (anti-SC5 mAbs).","It is also provided with anti-SC5 monoclonal antibodies which are capable of modulating the proliferation and/or the functional activity of T cells, and notably of tumoral T cells such as malignant CTCL cells.","In particular, the monoclonal antibody produced by hybridoma I-2575 is capable of inhibiting the proliferation and/or functional activity of T cells, and according to a particularly useful feature, is capable of inhibiting the proliferation and/or activity of malignant T cells such as malignant CTCL cells (see examples below).","Compounds binding to SC5, but not aggregating them will prevent SC5 inhibitory signal transduction, or will prevent compounds to reach SC5+ cells (target masking).","More generally, compounds binding to SC5 or p140 may, according to the invention, be used as complement recruiting agents, as ADCC stimulators, or as vectors for therapeutic agents so as to prevent, palliate, treat T lymphomas, such as CTCL.","The invention therefore also relates to the diagnostic and therapeutic uses of SC5- or p140-binding compounds such as anti-SC5 and/or anti-p140 monoclonal antibodies, and of products directly-derived therefrom (e.g.","humanized monoclonal antibodies, or monoclonal antibodies with a double CD4-p140/SC5 specificity).","DETAILED DESCRIPTION OF THE INVENTION The invention thus provides with an hybridoma deposited on Oct. 30, 2000 as deposit number I-2575 at the C.N.C.M.","in accordance with the Budapest Treaty (C.N.C.M.","Collection Nationale de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, F-75724 Paris Cedex 15, France), and with a method for preparing an anti-SC5 monoclonal antibody which comprises cultivating hybridoma I-2575 under conditions suitable for the metabolism of the hybridoma cells (e.g.","culture for three days at 37° C., with 5% CO2 in a humid atmosphere at 2.106 cells par ml of appropriate culture medium such as RPMI 1640 medium Gibco BRL Cat.","No21875-034, with 10% Fetal Calf Serum, penicillin 100 U/ml and streptomycin 100 microgram/ml).","The invention therefore encompasses the monoclonal antibody (mAb) which is produced by hybridoma I-2575, or which is obtainable from the culture supernatant of this deposited hybridoma.","This mAb is a pentameric anti-SC5 IgM.","It is a useful tool for the diagnosis and therapy of an inappropriate or undesired T cell proliferation and/or functional activity (cytokine profile), of an inappropriate or undesired IL-2 production.","Its inhibits the proliferation and/or functional activity of normal T cells, of virus-infected T cells (e.g.","HIV-infected CD4+ T cells), and of malignant T cells, such as notably malignant CTCL cells.","The monoclonal antibody produced by hybridoma I-2575 enables the isolation of a is protein, termed SC5 by the inventors.","SC5 protein appears as a biochemically and functionally new molecule, with an apparent molecular weight of 96 kD under reducing conditions.","It has been located in and on numerous PBL cells: normal T cells (both from CD4+ and CD8+ subsets), and in particular CD3+ T cells, CD45RO+ T cells, but also normal NK cells, B cells, granulocytes and macrophages.","In addition, SC5 has been located in the cytoplasm of cells from other lineages and other species such as NIH/3T3 (ATCC CRL-1658) and CHO-K1 (ATCC CRL-9618).","In resting normal PBL cells, SC5 is mainly intracellularly located, but CD3 stimulation induces the transfer of SC5 to the cell membrane.","Remarkably, SC5 has also been observed by the inventors on malignant T cells, such as malignant CTCL cells, and in particular malignant CD4+ CTCL cells, where they are mainly located at the cell surface (without any ex vivo CD3 stimulation).","As a further remarkable feature, SC5 surface expression has been observed for all the CTCL patients who were tested until now (see the examples below).","Another further remarkable feature of the invention is that the percentage of CD4+ T cells which co-express SC5 is statistically higher in patients developing tumoral CD4+ T cells than in healthy patients.","The percentage of SC5+ CD4+ cells in CD4+ CTCL patients is significantly higher than the percentage observed in healthy patients (average standard percentage of SC5+ CD4+ cells is in the 1-15% range, generally in the 5-10% range).","In the particular CD4+ CTCL case of Sézary Syndrome (SS), the percentage of CD4+ cells which co-express SC5 is furthermore statistically closely correlated to the percentage of malignant CTCL cells in the PBL of the CTCL patient: the percentage of SC5+ CD4+ T cells is substantially equal to the percentage of malignant CTCL cells (within ±10% of this percentage).","SC5 protein is therefore a new molecule whose transmembrane expression at the cell surface is linked to an activation and/or proliferation status for non-tumoral T cells (such as virus-infected cells, e.g.","HIV-infected cells, and such as normal CD4+ or CD8+ T cells), and to a malignant status for non-activated T cells.","According to a remarkable feature, it enables a CD4+ CTCL-positive diagnosis with a confidence of more than 90% (actually of 100% of those humans who have been tested up-to-date).","A further outstanding feature of the invention is that the SC5 new molecule is capable.","of acting as an inhibitory receptor for cell proliferation and/or functional activity when expressed at the cell surface: SC5 aggregation induces a decrease in the proliferation and activity of the cell expressing it.","For example, the anti-SC5 monoclonal antibody produced by hybridoma I-2575 (which is a pentameric Ig) inhibits the anti-CD3 induced proliferation of SC5+ cells such as normal CD3+ T cells collected from a human or an animal, or normal CD3+ T cell clones, and also inhibits the anti-CD3 induced in vitro proliferation of CTCL cells collected from a CTCL patient, and the in vivo natural proliferation of human CTCL cells in an animal model.","This mAb also binds HIV-infected CD4+ T cells, and is therefore useful as an active principle in an anti-HIV drug intended for killing infected cells, and therefore reducing HIV propagation.","Several procedures enabling the isolation of the SC5 protein are available to the skilled person to whom a monoclonal antibody of the invention has been made available.","Standard procedures comprise recovering the SC5 molecules from a cell lysate by affinity chromatography, or by electrophoresis under reducing conditions.","All human or animal cells which the monoclonal antibody produced by hybridoma I-2575 recognizes as an antigen, or of which lysate gives an immuno-precipitation reaction with this monoclonal antibody, are appropriate cell sources for isolating the SC5 molecule.","Such cells notably include normal cells (collected from a human or an animal, or cell clones) such as T cells, CD3+ T cells, CD45RO+ T cells, B cells, NK cells, macrophages, granulocytes.","Appropriate cells also include tumoral cells such as malignant T cells, e.g.","CD4+ CTCL cells collected from a CTCL patient, or tumoral clones such as the CTCL clone HUT78 (ATCC TIB-161).","Due to the wide range of appropriate cell sources, it will be understood that rough total blood cell or PBL may be directly used as a SC5 source without any further purification.","Normal cells enables the isolation of both the intracellular form and the transmembrane receptor form of SC5.When it is desired to isolate the SC5 receptor form, or a portion thereof, it may thus be preferable to use a source of SC5+ cells (i.e.","cells expressing SC5 at their surface).","Such a source include normal cells as above-mentioned but that have been CD3-stimulated so that SC5 expression increases at the cell surface (see examples below for CD3 stimulation procedures).","When total blood cells or PBL are used as a source of SC5+ cells, CD3 activation can be induced by incubation with a polyclonal activator such as PHA (obtainable from Wellcome) at a concentration of about 1 microgram per ml, because total blood cells or PBL also contain T cells and mononuclear cells.","When a more homogeneous normal cell population is used as a source of SC5+ cells (e.g.","only SC5+ NK cells), then CD3 activation is preferably performed by incubation with immobilized anti-CD3 antibodies, or alternatively with both soluble anti-CD3 antibodies and polyclonal activator such as PHA.","Optionally, a culture step may be performed before and/or after CD3 activation so as to increase cell amount (e.g.","culture for 3 days in RPMI 10% fetal calf serum at 37° C., 95% humidity, and 5% CO2).","When it is desired to isolate SC5 in its intracellular form, then CD3 stimulation may be omitted.","Tumoral cells such as CTCL cells, and notably malignant transformed MF or SS cells, enable the isolation of SC5 molecules in their receptor form, without any required ex vivo CD3 activation: CTCL cells already express SC5 in a transmembranar location.","The invention therefore encompasses any isolated protein obtainable by: (i) collecting cells selected from the group consisting of T cells, CD3+ T cells, CD45RO+ T cells, B cells, NIK cells, macrophages, granulocytes, and stimulating the collected cells with immobilized anti-CD3 antibodies, or with both soluble anti-CD3 antibodies and PHA at 1 micrograimn/ml, or collecting malignant CTCL cells such as malignant CD4+ CTCL cells, or collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), and stimulating the collected cells with PHA at about 1 microgram per ml, (ii) lysing the cells under conditions enabling the dissociation of multimeric polypeptidic complexes, for example by incubation in a drastic lysis buffer containing Triton X-100 at 1%, (iii) recovering from the lysate the protein compound onto which the monoclonal antibody produced by hybridoma I-2575 binds under conditions enabling this monoclonal antibody to perform reactions of the antigen-antibody type (i.e.","the compound which is recognized as an antigen by this mAb).","The aminoacid sequence of the recovered protein compound can be obtained by any standard technique, such as mass spectrophotometry.","As above-indicated, the cells mentioned in step i. are activated cells, and therefore enables the isolation of SC5 in the form it has when it is a transmembrane receptor.","If the SC5 source used in step i. consists of resting normal T cells (without ex vivo stimulation), the the protein compound thus isolated will be SC5 in its cytoplamsic form.","When the cells selected in step i. are activated or malignant cells, lysis in step ii.","therefore may be performed either on whole cells, or on the membranar fraction of these cells.","The lysis step itself can be achieved by any technique enabling the dissociation of multimeric polypeptidic complexes from each others.","For example, cells (or their membrane fraction) may be incubated in a lysis buffer containing a drastic detergent such as Triton X-100 at about 1% for 1 hour at 4° C.; this enables the dissociation of the SC5 protein from the other cell components.","If a mild detergent such as digitonin 1% or Bridg58 1% is used, then SC5 won't dissociate from those compounds which are naturally associated with it; the use of a mild detergent in the above step ii.","therefore enables the isolation of SC5, and the further isolation of the SC5 transducers and effectors.","The invention therefore also encompasses any isolated compound obtainable by such processes.","The lysate obtained from step ii.","is then incubated with the monoclonal antibody produced by hybridoma I-2575 under conditions appropriate for this mAb to perform reactions of the antigen-antibody type, such as coating the antibodies into microwells containing the cell lysate (e.g.","2 microgrammes of the monoclonal antibody produced by hybridoma I-2575 for a lysate of 107 cells) and incubating the monoclonal antibodies with the cell lysate for two hours at 4° C. The step of recovering the compound onto which the monoclonal antibody produced by hybridoma I-2575 binds (step iii.)","can be achieved by any technique available the skilled person.","Recovering under non reducing conditions enables the isolation of SC5 in its native conformation, and will be preferred when it is desired to retain the native biological properties of SC5.Appropriate techniques for recovering SC5 protein under non reducing conditions namely include chromatography by affinity, and, for example: percolation of the lysate through a column in which the monoclonal antibody (mAb) produced by hybridoma I-2575 has been immobilized (e.g.","by coupling this mAb onto agar beads and placing these beads inside said column); preferably, the lysate is mixed with a buffer which has a.physiological pH, e.g.","at a pH comprised between 6 and 8, such as 7, before percolation so as to make the percolation easier without substantially altering the conformation of the compounds contained in the lysate, rinsing the column with a buffer at the same physiological pH, and recovering the compound which has bound to the immobilized mAb, e.g.","by elution with a buffer which disrupts the binding of the compound onto the mAb without substantially modifying the native conformation of the compound.","Such an elution buffer usually has a slightly acidic pH (usually comprised between 2 and 5, e.g.","a pH of 3).","For the preparation of a matrix which has an optimal orientation of antibody molecules, and which thereby enables antigen binding at a high efficiency, and for a one-step purification procedure for the isolation of membrane proteins, see e.g.","Schneider et al.","1982 (J. Biol.","Chem.","257: 10766-10769) of which content is herein incorporated by reference.","The protein compound thus recovered is the isolated SC5 protein in its native form.","Alternatively, step iii.","under non reducing conditions can be achieved by placing the lysate obtained from step ii.","for immuno-precipitation with the monoclonal produced by hybridoma I-2575, and the immuno-precipitate thus obtained can be percolated through a column comprising antibodies capable of binding mice IgM.","The protein compound which has bound onto the monoclonal antibody produced by hybridoma I-2575 is then recovered by elution similarly to what has been above described.","The protein compound thus recovered is also the isolated SC5 protein in its native form.","Another standard way for recovering the SC5 protein (step iii.)","is to proceed through electrophoresis under reducing conditions.","This notably includes incubating the monoclonal antibody produced by hybridoma I-2575 with the lysate obtained from step ii.","under conditions enabling this mAb to perform reactions of the antigen-antibody type, collecting the immuno-precipitate thus formed, separating the compounds contained in this immuno-precipitate under reducing conditions, and recovering the protein compound which has an apparent molecular weight of 96 kD.","An alternative way to localize on the electrophoresis gel or membrane the desired SC5 protein (and to subsequently isolate it from the gel or membrane) is to proceed with a labeling step prior to lysis so that surface polypeptidic components of the cells are labeled (e.g.","125 iode or fluorescent labeling with e.g.","sulfo-NHS-LC-biotin), and to recover from the immuno-precipitate those compounds which bear the label.","It has to be noted that if the labeling step is performed after lysis, then both the intra-cellular form and the trans-membranar receptor form of SC5 will be isolated (in this case, as above-indicated, non-activated T cells may be used as a SC5 source).","Because denaturing conditions are used, the protein compound thus recovered is in a non native form: it still has the native amino acid sequence, but its native biological properties have been substantially lost.","It is however the SC5 protein in its non-native conformation that is observed (and has to be searched for) when the biological sample to be tested has been treated under denaturing conditions (such as e.g.","paraffin sections of skin sample).","The SC5 protein in its non-native form may therefore be used for the production of monoclonal antibodies directed against it, such monoclonal antibodies being then useful for SC5 detection on denaturated samples.","If the SC5 protein in its native conformation is desired, portions of at least 10 amino acids of the denaturated protein may then be sequenced (e.g.","by mass spectrophotometry), oligonucleotides deduced from the sequenced portions may be synthetized, and then used as probes to screen a cDNA library obtained from SC5+ cells.","The cDNA thus selected may the be produced and cloned into a cell under conditions enabling the production, and preferably the excretion, of the cDNA-encoded product by culture of the clone (e.g.","via insertion of the SC5-encoding cDNA in a baculo virus vector and transformation of insect cells—e.g.","Sf9 cells—, or via the vaccina virus and EBV cells, see Rindis Bacher 1995, J. Biol.","Chem.","270(23): 14220-14228).","The invention therefore encompasses any isolated protein of which sequence is obtainable by: collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), stimulating the collected cells with PHA at 1 microgram/ml, labeling the stimulated cells with a polypeptide-specific label such as biotin, lysing the labeled cells by incubation in a lysis buffer containing Triton X-100 at 1%, submitting the lysate to immuno-precipitation with the monoclonal antibody produced by hybridoma I-2575, recovering from the immuno-precipitate the protein compound which bears the label, synthesing the cDNA which encodes the protein sequence thus obtained, operably transfecting insect cells with this cDNA, recovering the protein produced by the transfected cells, e.g.","with the monoclonal antibody produced by hybridoma I-2575, or obtainable by: collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), stimulating the collected cells with PHA at 1 microgram/ml, lysing the labeled cells by incubation in a lysis buffer containing Triton X-100 at 1%, submitting the lysate to immuno-precipitation with the monoclonal antibody produced by hybridoma I-2575, recovering from the immuno-precipitate the protein compound which has an apparent molecular weight of 96 kD under reducing conditions, synthesing the cDNA which encodes the protein sequence thus obtained, operably transfecting insect cells with this cDNA, the protein compound produced by the transfected insect cells, e.g.","with the monoclonal antibody produced by hybridoma I-2575.The invention thus encompasses any isolated protein compound obtainable by any of the above-mentioned processes, as well as any amino acid sequence of such an isolated protein compound, and any isolated protein of which sequence comprises such a sequence.","Falls within the scope of the invention any solid support onto which a protein compound or protein of the invention has been placed.","Such solid supports notably include sepharose beads.","The invention also encompasses any isolated DNA (or cDNA) of which sequence codes for an isolated protein of the invention.","It also relates to engineered cells transfected by such a DNA, to engineered cells which excrete an isolated protein of the invention, and to isolated protein obtainable by isolation from the culture medium of such engineered cells.","The invention notably encompasses any cDNA obtainable by: collecting a cell population which consists of, or comprises cells onto which the monoclonal antibody produced by hybridoma I-2575 binds (i.e.","onto which this mAb recognizes an antigen), or cells of which lysate would give an immuno-precipitation reaction with this monoclonal antibody, e.g.","collecting cells selected from.","the group consisting of total blood cells and PBL, if the sole transmembranar form of SC5 is desired: stimulating the CD3 pathway if said cell population mainly comprises or consists of non-activated cells (e.g.","resting non-tumoral cells) so as to increase SC5 expression at the cell surface, e.g.","by incubating the collected cell population with a CD3 activator (for example a polyclonal activator such as PHA at 1 microgram par ml when the collected cell population is PBL or total blood, or immobilized anti-CD3 antibodies, or both a polyclonal activator such as PHA and soluble anti-CD3 antibodies when the collected cell population is more homogeneous such as a cell population mainly comprising, or consisting of CD3+ T cells, or of CD45RO+ cells, or of NK cells, or of macrophages, or of granulocytes); if the collected cell population consists of, or mainly comprises activated cells (such as CD3-activated non-tumoral cells, or such as tumoral cells such as CD4+ CTCL cells), then CD3 stimulation is not necessary because SC5 location is already mainly transmembranar in these activated cells; if it is desired to isolate both the intracellular form and the trans-membranar form of SC5, then non-activated cells are preferred and CD3 activation is omitted, extracting and purifying the whole mRNA population from said cell population (commercial kits are available for mRNA extraction and purification, e.g.","a polydT columns), synthetising every complementary cDNA (with a reverse transcriptase, e.g.","such as described in Seed B. and Arrufo A.","1987, Proc.","Natl.","Acad.","Sci.","USA 84: 3365-3369, of which content is herewith incorporated by reference), operably cloning each cDNA so that expression of this cDNA in this clone is possible under appropriate clone culture conditions, and cultivating every clone accordingly, selecting those clones which express a compound onto which the monoclonal antibody produced by hybridoma I-2575 binds when placed under conditions suitable for this monoclonal antibody to perform reactions of the antigen-antibody type, and/or those clones of which lysate would give an immuno-precipitation reaction with the monoclonal antibody produced by hybridoma I-2575 will be selected.","optionally amplifying those clones that have been thus selected, recovering the inserted cDNA from the selected clones.","The cDNA thus recovered is an SC5 cDNA of the invention, it can be sequenced using standard DNA sequencing techniques.","Operably cloning each cDNA so that expression of this cDNA in this clone is possible under appropriate clone culture conditions may be achieved by any conventional cloning procedure available to the skilled person.","An example of cloning procedure comprises inserting each cDNA in a transfection vector under the control of a promoter suitable for the expression of this cDNA in the host cell, transfecting the host cells with this transfection vector, and cultivating the transfected host cells under conditions appropriate to their metabolism.","Appropriate transfection vectors and host cells comprise virus such as Baculo virus and host cells such as insect cells (Sf9 cells), or vaccinia virus as a vector and EBV cells as host cells (see Rindis Bacher reference supra).","Such a cDNA can be used for cell transfection, and for SC5 production from the clones thus obtained.","It may also be used as probe for identifying or isolating the native SC5 mRNA.","The invention therefore also encompasses any isolated mRNA obtainable by selection among the above-mentioned mRNA population, of a mRNA which is complementary to a cDNA of the invention (e.g.","via selection of a mRNA which hybridizes to the above-mentioned cDNA under stringent conditions such as described in Freeman G. J. et al.","1992, J. Immunol.","149: 3745).","Are particularly encompassed any isolated mRNA obtainable by using an isolated cDNA of the invention as a probe so as to recover from the whole mRNA population of said cell population, the mRNA which is complementary to this extracted cDNA.","Alternatively, the one of ordinary skill in the art may directly sequence the cDNA that is recovered from the selected clones, and deduce from this sequence the sequence of the mRNA.","The invention also encompasses any DNA encoding an isolated mRNA according to the invention.","It particularly encompasses any genomic DNA obtainable by searching a genomic bank (such as www.ucsc.edu) for a genomic DNA matching with the above-mentioned SC5 cDNA.","The invention also encompasses any engineered cell in which a cDNA, a mRNA or a DNA of the invention has been transfected.","Are also encompassed SC5-specific probes and primers.","Such probes and primers can be obtained by any technique available to the skilled person (e.g.","selecting, as candidate sequences, portions of SC5 sequence that may be unique to SC5, comparing these candidate sequences on DNA/RNA banks with the sequences of cell receptors other than SC5, and selecting those candidate sequences which target a SC5 sequence that appears as unique after said comparison, and/or a SC5 sequence of which length appears as unique after said comparison).","Any solid support onto which is placed a cDNA, a mRNA or a genomic DNA of the invention falls within the scope of the present invention.","Such solid supports notably include DNA chips, or DNA microspheres.","The invention also encompasses any isolated protein encoded by an isolated cDNA, mRNA, or DNA according to the invention.","The invention further encompasses any isolated portion of a protein of the invention, provided that this portion is still characteristic of CTCL cells by comparison with normal resting cells.","It particularly encompasses any portion that is recognized by the monoclonal antibody produced by the hybridoma I-2575 at the C.N.C.M.","It also encompasses any isolated portion of a protein compound of the invention, provided that this portion is capable of inducing a modulation (inhibition or stimulation) of the CD3-induced proliferation of a normal T cell, and/or is capable of inducing a modulation of the in vivo proliferation of a malignant T cell such as a CTCL cell, and/or is capable of inducing a modulation of the IL-2 production of a normal T cell or of a malignant T cell, when said portion is expressed by said cell and contacted with the mAb produced by hybridoma I-2575.It particularly encompasses any isolated portion of a protein compound of the invention, provided that this portion is a part of a molecule that is expressed in a trans-membrane location by malignant CTCL cells and that is expressed in the cytoplasm compartment by non-tumoral resting T cells.","The invention notably provides with a bank of polypeptidic fragments which is obtainable by enzymatic cleavage of the SC5 protein of the invention.","Preferred enzymes comprises proteolytic enzymes such as those serine endopeptidases which cleave at the level of Tyr, Phe and Trp (e.g.","alpha-chymotrypsin), and also those enzymes which cleaves at the C-terminus of glutamic acid and aspartic acid (e.g.","V8 protease); see e.g.","Shesberadaran and Payne 1988, Proc.","Natl.","Acad.","USA 85:1-5, of which content is herein incorporated by reference.","Such a bank of SC5 polypeptidic fragments is of particular use in analysis such as mass spectrophotometry analysis: such analysis use the characteristic separation of such a polypeptidic bank, and give a (polypeptide) profile that is characteristic of SC5, and that can be used for SC5 detection.","Such a use and such a profile is encompassed by the present application.","The invention particularly encompasses the extra-cellular, trans-membranar and intra-cytoplasmic portions of any SC5 protein compound of the invention.","Determining the extra-cellular, trans-membranar and intra-cytoplasmic portions of a receptor molecule may be achieved by the skilled person according to any appropriate technique.","They may be identified on the whole molecule by identification of the region which contains glycosylation sites (extra-cellular portion), of the region that is hydrophobic (trans-membrane portion), and of the region that is hydrophylic (intra-cytoplasmic region), and they may then be isolated either by cleavage or synthesis.","An alternative way to isolate the extra-cellular portion of a protein compound of the invention is to proceed as above-described for the isolation of the whole protein compound, but with an additional step of enzymatic digestion so as to cleave from the whole molecule its extra-cellular portion.","Appropriate enzymes for such a cleavage includes enzymes such as V8 protease or alpha-chymotrypsin as above-mentioned.","The invention thus encompasses any isolated polypeptide compound obtainable by: collecting a SC5 cell source as above-described, such as e.g.","PBL or total blood cells, if it is desired to isolate a polypeptide compound in the form it has when it is naturally expressed at the cell surface, and if the collected SC5 cell source is, or mainly comprises normal resting cells, then stimulating the CD3 pathway, e.g.","via incubation with an appropriate CD3 activator (e.g.","incubation with PHA at 1 microgramme per ml when said SC5 source is total blood cells or PBL), labeling the cells with a polypeptide-specific label, such as biotin (surface labeling on whole cells), lysing the SC5 cell source (such as PBL or total blood cells) so as to dissociate multimeric polypeptidic complexes (and thereby recovering SC5 dissociated from the transducers and effectors naturally associated therewith) e.g.","by incubating the cells in a lysis buffer comprising a drastic detergent such as Triton X-100 at 1%, and submitting the lysate to an enzymatic digestion enabling the cleavage of SC5 into portions, e.g.","by incubating the lysate with a proteolytic enzyme such as an enzyme selected from the group consisting of V8 protease or alpha-chemotrypsin, submitting the lysate to immuno-precipitation with the monoclonal antibody produced by hybridoma I-2575, and recovering from the immuno-precipitate a polypeptide compound which bears a label.","Such recovered compounds are SC5 portions which either are the complete SC5 extra-cellular portion, or are part of such a complete SC5 extra-cellular portion, or comprise such a SC5 extra-cellular portion.","The set of polypeptide compounds that are thus obtainable constitute a bank of SC5 “extra-cellular” portions.","Such a bank, and its medical uses, such as e.g.","its use for the diagnosis of a T-related disease (e.g.","via mass spectrophotometry analysis), falls within the scope of the present invention.","The person of ordinary skill in the art will note that lysis and enzymatic digestion may be performed in a single step.","Labeling on whole cells is performed prior to cell lysis so as to identify SC5 portions which are extra-cellular portions, or which are part of such extra-cellular portions, or which comprise such portions.","The invention also encompasses any isolated compound which comprises a portion of a protein of the invention.","The mAb of the invention also enables the identification of the SC5 epitope onto which this mAb binds.","Conventional techniques such as directed mutagenesis are appropriate (see e.g.","Chang H. C. et al.","1989, J. Exp.","Med.","169: 2073-2083, of which content is herein incorporated by reference).","The proteins of the invention and their portions according to the invention, and notably their extra-cellular portions, enable the production of monoclonal antibodies directed against them.","An example of such monoclonal antibodies is produced by hybridoma I-2575.The monoclonal antibodies of the invention are particularly useful for the detection of SC5, for the diagnosis of T lymphomas such as CTCL, and are also useful as modulators of an undesired or inappropriate proliferation and/or activity of T cells, such as normal CD4+ and CD8+ T cells, virus-infected T cells such as HIV-infected T cells, T lymphomas such as CTCL.","The invention therefore encompasses any monoclonal antibody obtainable by: (i) immunizing an animal against a protein of the invention or against a portion thereof as above-defined, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them for them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein or protein portion that as been used as an immunogen in step i., and which has at least one property selected from the group consisting of the property of: binding resting non-tumoral T cells mainly in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells mainly at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of malignant T cells such as CD4+ CTCL cells, modulating the proliferation of malignant T cells such as CD4+ CTCL cells in an animal, and preferably in a human, competing with a monoclonal antibody of the invention, such as the monoclonal antibody produced by hybridoma I-2575, for binding to a protein of the invention, or to a portion thereof as above-defined, and in particular with the extra-cellular portion of such a protein, (iv) selecting and cloning hybridomas producing the desired antibody, (v) recovering the antibody from the supernatant above said clones.","Production of hybridomas may be achieved using any conventional technique.","This includes removing the spleen from said animal, making a suspension of spleen cells, fusing the spleen cells with myeloma cells in the presence of a fusion promoter, diluting and cultivating the fused cells in separate wells in a medium which will not support unfused cells.","The invention also encompasses any ascite isolated from said immunized animal.","For step iii., T and malignant T cells can be collected from a human using standard procedures.","Alternatively, clones can be used such as the CD4+ CTCL HUT 28 cell line (ATCC TIB-161).","In the case of CD4+ tumors, collection is made from the body part which will display the CD4+ tumoral cells (PBL for SS, cutaneous erythroderma for transformed MF, etc.).","The above-mentioned properties can be easily assessed by the skilled person using common knowledge in the art.","Examples of appropriate experimental conditions for assessing the properties mentioned in step iii.","can be found in the below examples.","The person of ordinary skill in the art will adjust them to the particular cells, or concentrations used.","Modulation of cell proliferation herein refers to stimulation as well as inhibition of this proliferation.","When CTCL cells are used, inhibition of proliferation is preferred in perspective of therapeutic application.","The mAbs of the invention are all anti-SC5 mAbs which have either diagnostic or therapeutic applications, or both.","For certain applications, it may be desired to immobilize one or several of these mAb onto a support, e.g.","for protein purification purposes, or for exerting the modulation property of which they are capable.","The present application also encompasses any support onto which a mAb of the invention has been immobilized.","Such supports notably include those appropriate for columns of chromatography by affinity (e.g.","agar beads).","The present application is also directed to any fragment of a mAb of the invention (anti-SC5 fragments) selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, CDR3.It is directed to any compound, and namely to any antibody, comprising such a fragment.","It notably relates to any humanized antibody comprising at least one of such fragments.","Techniques for the production of humanized antibodies have been widely described in the prior art (see e.g.","Farah et al.","1998, Crit.","Rev.","Eucaryote Gene Exp.","Vol.","8 pp 321-345, of which content is incorporated herein by reference).","In particular, it relates to any compound, and notably to any antibody and humanized antibody, comprising at least one anti-SC5 fragment as herein defined and also at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, CDR3 (see e.g.","WO 94/13804 “Multivalent and multispecific binding proteins, their manufacture and use”, Inventors: Holliger et al.","Applicants: Cambridge Antibody Technology Ltd and Medical Research Council; see also Merchant et al.","“An efficient route to human bi-specific IgG” Nat.","Biotechnol.","1998 vol.","16 pp 677-681).","As previously indicated, the invention provides with two molecules linked by a common general inventive concept: SC5 and p140.The SC5 is a novel molecule fully herein described.","The p140 molecule has been described in the prior art as an inhibitory receptor which is expressed by sub-groups of NK cells and of CD3+ CD8+ cells from healthy (non-cancerous) patients.","The present invention now demonstrates that p140 is expressed by malignant T cells, and notably by CTCL cells, and provides with a novel p140 allelic form (clone 24 in the below example 3; SEQ ID No.","1) encoding a novel protein (SEQ ID No.","2).","The cDNA and amino acid sequences of this novel allelic form are as follow: SEQ ID No.1: CATGTCGCTCACTGTCGTCAGCATGGCGTGCGTTGGGTTCTTCTTGCTGC AGGGGGCCTGGCCACTCATGGGTGGTCAGGACAAACCCTTCCTGTCTGCC CGGCGCAGCACTGTGGTGCCTCAAGGAGGACACGTGGCTCTTCAGTGTCA CTATCGTCGTGGGTTTAACAATTTCATGCTGTACAAAGAAGACAGAAGCC ACGTTCCCATCTTCCACGGCAGAATATTCCAGGAGAGCTTGATCATGGGC CCTGTGACCCCAGCACATGCAGGGACCTACAGATGTCGGGGTTCACGCCC ACACTCCCTCACTGGGTGGTCGGCACCCAGCAACCCCCTGGTGATCATGG TCACAGGAAACCACAGAAAACCTTCCCTCCTGGCCCACCCAGGGACCCTG CTGAAATCAGGAGAGACAGTCATCGTGCAATGTTGGTCAGATGTCATGTT TGAGCACTTCTTTCTGCACAGAGAGGGGATCTCTGAGGACCCCTCACGCC TCGTTGGACAGATCCATGATGGGGTCTCCAAGGCCAACTTCTCCATCGGT CCCTTGATGCCTGTCCTTGCAGGAACCTACAGATGTTATGGTTCTGTTCC TCACTCCCCCTATCAGTTGTCAGCTCCCAGTGACCCCCTGGACATCGTGA TCACAGGTCTATATGAGAAACCTTCTCTCTCAGCCCAGCCGGGCCCCACG GTTCAGGCAGGAGAGAACGTGACCTTGTCCTGTAGCTCCTGGAGCTCCTA TGACATCTACCATCTGTCCAGGGAAGGGGAGGCCCATGAACGTAGGCTCC GTGCAGTGCCGAAGGTCAACAGAACATTCCAGGCAGACTTTCCTCTGGGG CCTGCCACCCACGGAGGGACCTACAGATGCTTCGGCTCTTTCCGTGCCCT GCCCTGCGTGTGGTCAAACTCAAGTGACCCACTGCTTGTTTCTGTCACAG GAAACCCTTCAAGTAGTTGGCCTTCACCCACAGAACCAAGCTCCAAATCT GGTATCTGCAGACAGGTGCATGTTCTGATTGGGACGTCAGTGGTCATCTT CCTCTTCATCCTCCTCCTCTTCTTTCTCCTTTATCGCTGGTGCTCCAACA AAAAGAATGGTGCTGTAATGGACCAAGAGCCTGCGGGGGACAGAACAGTG AATAGGCAGGACTCTGATGAACAAGACCGTCAGGAGGTGACGTACGCACA GTTGGATCACTGCGTTTTCATACAGAGAAAAATCAGTCGCCCTTCTCAGA GGGCCAAGAGACCCCCAACAGATACCAGCGTGTACACGGAACTTCCAAAT GCTGAGCCCAGATCCAAAGTTGTCTCCTGCCCACGAGCACCACAGTCAGG TCTTGAGGGGGTTTTCTAGGGAGACAACAGCCCTGTCTCAAAACC SEQ ID No.2: pLMGGQDKPF LSARPSTVVP QGGHVALQCH YRRGFNNFML YKEDRSHVPI FHGRIFQESF IMGPVTPAHA GTYRCRGSRP HSLTGWSAPS NPLVIMVTGN HRKPSLLAHP GTLLKSGETV ILQCWSDVMF EHFFLHREGI SEDPSRLVGQ IHDGVSKANF SIGPLMPVLA GTYRCYGSVP HSPYQLSAPS ˜DPLDIVITGL YEKPSLSAQP GPTVQAGENV TLSCSSWSSY DIYHLSREGE AHERRLRAVP KVNRTFQADF PLGPATHGGT YRCFGSFRAL PCVWSNSSDP LLVSVTGNPS SSWPSPTEPS SKSGICRHLH VLIGTSVVIF LFILLLFFLL YRWCSNKKNA AVMDQEPAGD RTVNRQDSDE QDPQEVTYAQ LDHCVFIQRK ISRPSQRPKT PPTDTSVYTE LPNAEPRSKV VSCPRAPQSG LEGVF The DNA and amino acid sequences of the previously described p140 allelic form (clone 1.1) are as follow: SEQ ID No.3: CATGTCGCTCACGGTCGTCAGCATGGCGTGCGTTGGGTTCTTCTTGCTGCA GGGGGCCTGGCCACTCATGGGTGGTCAGGACAAACCCTTCCTGTCTGCCCG GCCCAGCACTGTGGTGCCTCGAGGAGGACACGTGGCTCTTCAGTGTCACTA TCGTCGTGGGTTTAACAATTTCATGCTGTACAAAGAAGACAGAAGCGACGT TCCCATCTTCCACGGCAGAATATTCCAGGAGAGCTTCATCATGGGCCCTGT GACCCCAGCACATGCAGGGACCTACAGATGTCGGGGTTCACGCCCACACT CCCTCACTGGGTGGTCGGGACCCAGCAACCCCGTGGTGATCATGGTCACAG GAAACCACAGAAAACCTTCCCTCCTGGCCCACCCAGGGCCCCTGCTGAAA TCACGAGAGACAGTCATGCTGCAATGTTGGTCAGATGTCATGTTTGAGCAC TTCTTTCTGCACAGAGAGGGGATCTCTGAGGACCCCTCACGCCTCGTTGGA CAGATCCATGATGGGGTCTCCAAGGCCAACTTCTCCATCGGTGGCTTGATG CCTGTGCTTGCAGGAACGTACAGATGTTATGGTTCTGTTCCTCACTCCGGC TATCAGTTGTCAGCTCGCAGTGACCCCCTCGACATCGTGATCACAGGTCTA TATGAGAAACCTTCTCTCTCAGCGCAGCGGGGCCCCACGGTTCAGGCAGGA GAGAACGTGACCTTGTCCTGTAGCTCCTGGAGCTCCTATGACATCTACCAT CTGTCCAGGGAAGGGGAGGCCCATGAACGTAGGCTCCGTGCAGTGCCGAA GGTCAACAGAACATTCCAGGCAGACTTTCCTCTGGGCCCTGCCACCCACGG AGGGACCTACAGATGCTTCGGCTCTTTGCGTGCCCTGCCCTGCGTGTGGTC AAACTCAAGTGACCCACTGCTTGTTTCTGTCACAGGAAACCCTTCAAGTAG TTGGCCTTCACCCACAGAACCAAGCTCCAAATCTGGTATCTGCAGACACCT GCATGTTCTGATTGGGACCTCAGTGGTCATCTTCCTCTTCATCCTCCTCCT CTTCTTTCTCCTTTATCGCTGGTGCTCCAACAAAAAGAATGCTGCTGTAAT GGACCAAGAGCCTGCGGGGGACAGAACAGTGAATAGGCAGGACTCTGATG AACAAGACCCTCAGGAGGTGACGTACGCACAGTTGGATCACTGCGTTTTC ATACAGAGAAAAATCAGTCGCCCTTCTCAGAGGCCCAAGACACCCCTAAC AGATACCAGCGTGTACACGGAACTTCCAAATGCTGAGCCCAGATCCAAAG TTGTCTCCTGCCCACGAGCACCACAGTCAGGTCTTGAGGGGGTTTTCTAGG GAGACAACAGCCCTGTCTCAAAACC SEQ ID No.4: pLMGGQDKPF LSARPSTVVP RGGHVALQCH YRRGFNNFML YKEDRSHVPI FHGRIFQESF IMGPVTPAHA GTYRCRGSRP HSLTGWSAPS NPVVIMVTGN HRKPSLLAHP GPLLKSGETV ILQCWSDVMF EHFFLHREGI SEDPSRLVGQ IHDGVSKANF SIGPLMPVLA GTYRCYGSVP HSPYQLSAPS DPLDIVITGL YEKPSLSAQP GPTVQAGENV TLSCSSWSSY DIYHLSREGE AHERRLRAVP KVNRTFQADF PLGPATHGGT YRCFGSFRAL PCVWSNSSDP LLVSVTGNPS SSWPSPTEPS SKSGICRHLH VLIGTSVVIF LFILLLFFLL YRWCSNKKNA AVMDQEPAGD RTVNRQDSDE QDPQBVTYAQ LDHCVFIQRK ISRPSQRPKT PLTDTSVYTE LPNAEPRSKV VSCPRAPQSG LEGVF SEQ ID No.","1 differs from SEQ ID No.","3 in five locations.","The resulting SEQ ID No.2 differs from the p140 disclosed in prior art (SEQ ID No.","4) by four amino acid substitutions (shown in bold and underlined characters): R instead of Q in position 20, V instead of L in position 92, P instead of T in position 102, and L instead of P in position 401 (see FIG.","11).","The present application encompasses any isolated compound of which sequence is, or comprises SEQ ID No.","1 and/or SEQ ID No.","2.It also encompasses any mRNA that is complementary to SEQ ID No.","1, and any genomic DNA coding for SEQ ID No.","2.Appropriate techniques for producing p140 proteins are available to the skilled persons.","They notably include the production of cells which have been engineered so as to produce p140 proteins in their culture medium.","An example of such techniques comprises inserting SEQ ID No.","1 or No.","3 in a baculo virus vector, operably transfecting an insect cell such as Sf9 cell line with this vector, recovering the p140 protein produced in the culture medium (e.g.","by percolation of the culture medium is through a column—e.g.","a Sephadex column—which protein compounds as a function of their molecular weight, and isolating the 140 kD eluate).","Compounds capable of binding to p140 under conditions of the physiological type (in vivo conditions, or in vitro conditions mimicking the in vivo ones) have been previously described, and may be obtained by the person of ordinary skill in the art by any appropriate available technique.","By “p140 binding compound”, it is herein meant any compound which is capable under said conditions of the physiological type to recognize a p140 molecule as an antigen (SEQ ID No.","2 and/or SEQ ID No.","4) when it is expressed by a cell in a transmembrane receptor location (i.e.","recognition of the SEQ ID No.","2 and/or No.","4 portion that is above the lipid bi-layer of said cell).","Such p140 binding compound notably comprise HLA-A11 and HLA-A3 molecules (natural p140 ligands), and notably fusion proteins such as Fc-HLA-A11 and Fc-HLA-A3 fusion proteins.","p140 binding compounds also comprise p140 antiserum (obtainable by immunizing an animal such as a rabbit against p140, and by recovering the antiserum thus produced—an additional step of immuno-purification may be performed so as to increase p140 concentration in the antiserum, e.g.","via percolation through a p140 column).","Other p140 binding compounds comprise monoclonal antibodies directed to p140 SEQ ID No.","4, such as e.g.","AZ158, Q66, Q241 described in Pende et al.","1996 (J. Exp.","Med.","184: 505-518).","Such anti-SEQ ID No.","4 mAbs may also recognize the new SEQ ID No.","2 as an antigen.","Any available technique for mAb production is appropriate for production of anti-p140 mAbs.","An example of such a technique comprise the steps of: (i) immunizing an animal against p140, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them for them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the p140 molecule that as been used as an immunogen in step i., and which is also capable of binding resting non-malignant T cells mainly in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells mainly at their cell surface, (iv) selecting and cloning hybridomas producing the desired antibody, (v) recovering the antibody from the supernatant above said clones.","Preferred p140 binding compounds do also comprise an anti-CD4 entity (e.g.","bi-specific mAb).","Such p140 binding compounds are useful for p140 detection on malignant T cells, such as CTCL cells, in particular CD4+ CTCL.","They are thus useful for CD4+ cancer detection.","They are also useful as polypeptidic vectors, as described below.","According to another aspect of the invention, there are provided polypeptidic vectors comprising at least one element selected from the group consisting of the anti-SC5 mAbs of the invention and the anti-SC5 mAb fragments of the invention.","Such polypeptidic vectors are useful for molecules, such as active principle molecules, to reach activated T cells, or their vicinity (e.g.","activated T cells observed in inflammatory diseases, or in virus-infected T cells, or malignant T cells such as malignant CTCL cells, and malignant CD4+ CTCL cells in particular).","It also relates to polypeptidic vectors comprising a p140 binding compound such as a mAb directed against SEQ ID No.","2 and/or SEQ ID No.","4 extra-cellular portion.","Such polypeptidic vectors are useful for delivering molecules to, or in the vicinity of malignant T cells, such as notably CTCL cells, and malignant CD4+ CTCL cells in particular.","Molecules which may be usefully delivered to malignant T cells, such as CTCL cells notably comprise molecules capable of inducing cell death or apoptosis, such as a radio-element or a toxin.","Such molecules also comprise any enzyme capable of transforming an antimititotic pro-drug into an active drug form, such as a carboxypeptidase.","A polypeptidic vector carrying such an enzyme is then advantageously used in combination with an anti-mitotic pro-drug (such as phenol mustard pro-drug).","The invention therefore also encompasses a medical kit comprising such a vector and such an anti-mitotic pro-drug.","Molecules which may be usefully delivered in the vicinity of malignant T cells such as CTCL cells notably comprise molecules capable of stimulating the immune functions of cells which are in the vicinity-of malignant T cells in an effort to induce an anti-tumor effect from these neighboring cells towards said malignant T cells.","Examples of such molecules notably comprise cytokines such as interferon gamma, and interleukine such as IL-2 (see e.g.","Xu et al.","2000, Cancer Research 60: 4475-4484).","When a polypeptide vector of the invention is intended for targeting CD4+ cells such as CD4+ CTCL cells, then this vector advantageously further comprises an anti-CD4 entity.","The invention also encompasses any pharmaceutical composition comprising such polypeptidic vectors, and in particular any medicament comprising such polypeptidic vectors.","Such medicaments are useful in the control of an inappropriate or undesired T cell proliferation and/or functional activity (including undesired IL-2 production).","They are particularly useful for the prevention, palliation, relieve or therapy of an inappropriate immune activity, and notably for the prevention or inhibition of a T lymphoma such as CTCL and in particular CD4+ CTCL.","The use of such vectors for the manufacture of a drug intended for the prevention, palliation, relieve or therapy of an inappropriate T cell proliferation and/or activity, and notably for the prevention or inhibition of a CTCL proliferation is thus also an object of the present application.","A compound which binds to SC5 under conditions of the physiological type may be used as a SC5 ligand agonist so as to stimulate the activation of SC5, the SC5 molecule being then regarded as a cell inhibitory receptor.","Such anti-SC5 compounds namely comprise anti-SC5 antisera, the anti-SC5 mAb of the invention, the Fab, F(ab′)2, fragments thereof, the humanized mAbs derived therefrom.","When CD4+ cells are more particularly concerned, said anti-SC5 compound advantageously further comprises an anti-CD4 entity.","Anti-SC5 compounds are particularly useful for modulating an inappropriate or undesired proliferation and/or functional activity of SC5+ cells.","They are thus useful for modulating the proliferation of T cells, CD45RO+ cells, CD3+ cells, CD4+ T cells, CD8+ T cells, or of virus-infected T cells (e.g.","HIV-infected CD4+ T cells), or of malignant T cells such as CTCL cells and in particular CD4+ CTCL cells.","They are also useful for modulating IL-2 production from T cells.","When the mAb produced by the hybridoma deposited as deposit number I-2575 at the C.N.C.M.","(which is a pentameric IgM) is used, this modulation goes in favor of an inhibition.","It is therefore particularly appropriate for inhibition of T lymphomas such as CTCL.","The application encompasses any drug comprising, as an active principle, at least one anti-SC5 compound.","Such a drug may be intended for the prevention, palliation, therapy of T lymphomas, of CTCL, of T infections such as viral (e.g.","HIV) infections, of inflammatory diseases such as those of the auto-immune type (e.g.","rhumatoid arthritis—e.g.","spondyloarthropathis—or skin immune mediated diseases such as psoriasis, eczema, atopic dermatitis).","Such a drug may also be intended for graft improvement (inhibition of graft rejection).","The invention also encompasses the use of such anti-SC5 compounds as complement recruiting agents, or as ADCC activators or stimulators.","The p140 binding compounds as defined above, may also be used as complement recruiting agents, or as ADCC stimulators or activators in the prevention, palliation, relieve or treatment of T lymphomas, such as CTCL and in particular CD4+ CTCL.","The invention therefore encompasses the use of such p140 binding compounds in the manufacture of an anti-T lymphoma drug, of an anti-CTCL drug, of an anti-CD4+ CTCL drugs, and also encompasses such drugs.","The invention also provides with a method for the assessment of the development level of a CTCL (staging): it enables to evaluate the proportion (e.g.","percentage) of malignant CD4+ CTCL cells present within a certain body compartment of a patient.","According to this method, the proportion of CD4+ cells expressing at their surface an element selected from the group consisting of the SC5 proteins of the invention, the SC5 portions of the invention (notably, those extracellular portions), and the p140 molecules is measured in a biological sample collected from said body compartment.","The proportion of CD4+ CTCL cells that are actually present in said body compartment can be considered as substantially equal to said measured proportion.","It usually falls within a ±10% range around this measured proportion.","The invention also provides with a method for CTCL diagnosis, wherein the percentage of T cells expressing an element selected from the group consisting of the SC5 proteins of the invention, the SC5 portions of the invention (notably, those extracellular portions), and the p140 molecules is measured in a biological sample collected from a patient, and is compared to the average percentage observed in non-CTCL humans (preferably in healthy humans), and in that a CTCL-positive diagnosis is decided when said measured percentage is significantly higher than said average percentage.","This CTCL diagnosis method particularly applies to those CTCL which are CD4+; in this case the measured T cell percentage is preferably a CD4+ percentage.","The average standard proportion of CD4+ T cells expressing SC5 is generally in between 1-15%, usually in between 5-10% among healthy humans.","When the measured percentage of SC5+ CD4+ T cells is higher than such an average percentage, then a CD4+ CTCL positive diagnosis can be concluded.","Of note is that, as far as SC5 protein or portions thereof are concerned, the methods of the invention more generally enables to evaluate the proportion of CD4+ activated T cells that are present in said body compartment.","This may e.g.","be useful for assessing whether a graft induces rejection reactions, and for the diagnosis and follow-up of inflammatory diseases such as those of the auto-immune type (e.g.","rhumatoid arthritis such as spondyloarthropathis, skin immune mediated disease such as psoriasis, eczema, atopic dermatitis).","Said biological sample may then be intra-synovial fluid.","This may be also useful for the diagnosis and monitoring of non-CTCL T lymphomas (e.g.","CD4+ ALL, or CD4+ T-LL).","The CTCL assessing/diagnostic methods of the invention are highly efficient for all kinds of CD4+ CTCL, and notably for transformed MF, for SS, for LP, as well as for CD30+ lymphomas.","Said biological sample may e.g.","then be a skin sample collected from an erythroderma (e.g; suspicion of transformed MF), and/or a PBL or total blood sample (in order to evaluate whether a transformed MF has evolved into a more aggressive form such as SS).","When the element detected at the surface of said CD4+ cells is a SC5 compound, then a (preliminary or ulterior) step of cytological observation (search for tumor-alike cytology) is the preferably performed to conclude with a confidence of more than 90% to an actual CD4+ CTCL.","When p140 is detected at the surface of said CD4+ cells, then no additional step is required: the simple fact of detecting p140 at the surface of CD4+ T cells directs to a positive diagnosis of CD4+ CTCL.","The methods of the invention can be implemented with any appropriate element, and notably with an element selected from the group consisting of the anti-SC5 monoclonal antibodies of the invention, the Fab, F(ab′)2 fragments thereof, the humanized mAb derived therefrom, the SC5 “extra-cellular” bank of the invention (as above-defined), the SC5 cDNA, mRNA, genomic DNA of the invention, the p140 binding compounds as herein defined, the p140 DNA, the DNA comprising SEQ ID No.","1 or SEQ ID No.","3 or encoding SEQ ID No.","2 or SEQ ID No.","4.Such an element is then used as a detection means for SC5 or p140 cell surface expression.","Preferably, said detection means also comprises an anti-CD4 entity so as to detect SC5+ /p140+ CD4+ cells in a one-step procedure.","When cDNA/mRNA material is used as a detection means, then CD4+ cells have to be lysed so as to detect those SC5 or p140 hybridizing mRNA which are actually present in said cells (detection of the transcripts).","Such cDNA/mRNA detection means may advantageously be placed onto or into a solid support such as a DNA chip or a DNA microsphere.","When mAb material or mAb-derived material is used as a detection means, then surface expression of SC5 or p140 is detected.","Any appropriate antibody-antigen technique is convenient.","Examples notably comprise flow cytometry analysis.","The invention also encompasses a method for the identification of a compound which is useful in the palliation, prevention, relieve, therapy of an undesired or inappropriate T cell activity, such as notably a proliferation of malignant T cells such as CTCL cells and in particular CD4+ CTCL cells.","This method is characterized in that it comprises the detection of a compound that is capable of binding to a SC5 protein as above-defined, or to a SC5 polypeptide compound as above-defined, or to a p140 molecule (such as SEQ ID No.","2 and/or SEQ ID No.","4).","Such compounds can be used as polypeptidic vectors as above-described.","Those compounds which, in addition to binding to SC5 molecules, are also capable of aggregating these SC5 molecules at the cell surface, are preferred for stimulating SC5 inhibitory functions.","An easy way to implement this identification method is to proceed with SC5 and/or p140 molecules immobilized onto a solid support such as protein A Sepharose CL-4B.","Candidates compounds for implementation of the method notably comprise sera immunized against SC5 or p140.The invention is illustrated by the examples, which are in no way intended to restrict the invention to the particular embodiments described below.","The person of ordinary skill in the art will contemplate alternative embodiments, and such alternative embodiments derived from common knowledge in the art are within the scope of the invention.","The skilled person will in particular appreciate that various standard immuno-techniques, and various standard molecular biology techniques are available to him (see e.g.","“Antibodies: a laboratory manual” Ed Harlow, David lane, Cold Spring Harbor Laboratory 1988; Maniatis 1982, “Molecular cloning: a laboratory manual”, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory; “Current protocols in Immunology” protocols on CD-ROM, John Wiley, West Sussex, England; “Immunological techniques made easy”, edited by Olivier Cochet et at.","; John Wiley, West Sussex, England; and the publication specifically referred to in the present application).","The content of such prior art documents is herein incorporated by reference.","It is also to be understood that experiments described herein in full details for one particular form of CD4+ CTCL can be directly transposed by the skilled person to any other form of CD4+ CTCL.","In these examples, reference is made to the following figures: FIGS.","1A, 1B and 1C illustrate cell membrane expression of SC5 molecule on PBLs from normal individual.","PBMC from a normal individual were stained with anti-SC5 mAb (ascites ({fraction (1/200)}) followed by FITC-conjugated isotype specific goat anti-mouse IgM second reagent and one of the following PE-conjugated mAbs: anti-CD3 (FIG.","1A), anti-CD56 (FIG.","1C) and anti-CD45RO (FIG.","1B).","The percentage of double-stained lymphocyte-gated cells is shown in the upper right quadrant (3.3 for FIG.","1A; 5.1 for FIG.","1B; 0.6 for FIG.","1C).","This experiment is representative for the 10 donors studied.","FIG.","2 illustrates cell membrane expression of SC5 molecule during activation of PBLs.","PBMC were stimulated with 1 μg/mL PHA and the kinetics of SC5 and CD69 expression were studied in parallel on the CD3+ cells.","FIG.","3 illustrates intracellular localization of SC5 molecule in PBL.","Anti-SC5, anti-CD3ζ chain mAb and anti-BY55 isotype-matched mAb were used for staining permeabilized and non-permeabilized gated lymphocytes from a normal donor.","The shaded histograms represent the labeling obtained with the indicated mAbs compared to an irrelevant control mAb.","FIG.","4 illustrates biochemical analysis of SC5 molecule.","DS6 T cell clone was surface labeled with biotin and 1% Triton X-100 lysates were immunoprecipitated, using anti SC5 mAb.","The samples were analyzed by SDS-10%-PAGE under reducing conditions.","Anti-SC5 mAb was used at two concentrations {fraction (1/200)} (lanes 2) and {fraction (1/500)} (lane 3).","The negative control samples were precipitated with goat anti-mouse Ig alone (lane 1) and isotype-matched irrelevant mAb (lane 4).","The molecular weight markers (kD), are indicated on the left.","FIGS.","5A, 5B, 5C, 5D illkustrate that anti-SC5 mAb modulates the anti-CD3-induced proliferation and cytokine secretion of normal T lymphocytes whithout affecting their cytotoxic activity: FIG.","5A: PBL or T cell clones LSO and GDS.3 were stimulated with immobilized anti-CD3 mAb (1 μg of mAb coated per well) or with IL-2 (50 IU/ml) in the presence of anti-SC5 mAb (1:200 final dilution of ascites) or an isotype-matched irrelevant mAb.","Results shown are representative of at least three separate experiments and are expressed as mean cpm±SD of triplicate wells.","FIG.","5B: GDS.3 CD4+ T cell clone was stimulated with the indicated concentrations of pre-coated anti-CD3 mAb in the presence of different concentrations of anti-SC5 mAb.","FIG.","5C: IL-2 secretion in the supernatant of anti-CD3-stimulated PBL, in the presence of anti-SC5 or control mAb, was assessed by measuring the proliferation of an IL-2 dependant T cell clone in the presence of: medium alone (a), supernatant from PBL/anti-CD3+ control mAb (b), supernatant from PBL/anti-CD3+ anti-SC5 mAb (c).","Data represent the mean values±SD of triplicate determinations of one out of three representative experiments.","FIG.","5D: JF1, a cytotoxic CD8+ cell clone was incubated with anti-SC5 mAb ({fraction (1/200)} final dilution of ascites) or with an isotype matched control mAb before the coculture with the FcγR+ murine tumor cells P815 at an E/T ratio of 5/1.Target cells were preincubated with the indicated final concentrations of anti-CD3 mAb.","Results are expressed as mean of triplicate wells.","FIGS.","6A, 6B illustrate cell membrane expression of SC5 molecule on PBL from CTCL patients.","Cells from peripheral blood of 8 patients with Sézary syndrome, 3 patients with Mycosis fungoides and 8 healthy donors were analysed by two-color fluorescence for the expression of SC5 molecule by CD4+ lymphocytes.","FIG.","6A: co-expression of SC5 and CD4 molecules in PBL from a patient with Sézary syndrome in comparison to a normal individual.","FIG.","6B: histograms showing the mean values of SC5 expression in the 3 indicated groups.","Statistically significant differences between SS patients and 2 other groups are marked, (Mann-Witney U test, p<0.001) FIGS.","7A, 7B, 7C illustrate surface membrane expression on malignant cell from Sézary syndrome patient.","FIG.","7A: two-color immunofluorescence analysis of PBMC from SS patient with a circulating TCRVβ22+ malignant T cell clone.","Cells were stained with anti-SC5 mAb followed by PE-conjugated isotype specific goat anti-mouse IgM and FITC-conjugated anti-TCRVP22 mAb.","FIG.","7B: anti-SC5-mAb labeled weakly (shaded histogram) the TCRVβ22-positive malignant cells.","FIG.","7C: cells from the same patient stained with PE-conjugated anti-CD69 mAb and FITC-conjugated anti-TCRVβ22 mAb.","FIG.","8 illustrates the modulation of anti-CD3-induced proliferation of CTCL malignant cell line by anti-SC5 mAb.","Pno cell line was stimulated with IL-7 (10 ng/mL), PMA alone (2 ng/mL), immobilized anti-CD3 mAb or a combination of PMA/anti-CD3 mAb.","SC5 mAb or an isotype-matched irrelevant mAb (1:200 final dilution of ascites) was added at the start of cultures.","Results shown are representative of four separate experiments and are expressed as mean cpm±SD of triplicate wells.","FIGS.","9A, 9B illustrate the detection of p140/KIR molecules on CTCL cell lines (FIG.","9A) and on fresh tumor lymphocytes (FIG.","9B).","Double immunostaining flow cytometric analysis was performed as described in Bagot et al.","1998 (Blood 91:4331-434 1) and in Poszepczynska et al.","2000 (Blood 96: 1056-1063).","FIG.","10 illustrates the biochemical analysis of p140 molecules: the NK clone AM61 (p140+/p70−), derived from a healthy donor, and the cell lines Pno and Cou-L were surface labeled with biotin and immunoprecipitated with anti-p140 and anti-CD8 mAbs.","Samples were treated (+) or not (−) with N-glycosidase F and analyzed in an 8% (left panel) or 11% (right panel) SDS-PAGE under reducing conditions.","Sepharose Protein-A alone represents the negative control.","Molecular weight markers (kD) are indicated on the right.","FIG.","11 shows the amino acid sequence alignment of KIR3D cl.","24 (SEQ ID No.","2) and cl.","1.1 (SEQ ID No.","4) encoded proteins.","Amino acids corresponding to the signal peptide are in small case letters, while transmembrane region is underlined in the consensus sequence.","Amino acids identical to the consensus sequence are indicated by dots.","EXAMPLE 1 SC5 Isolation, SC5 Biochemical and Functional Characterization, Production of Anti-SC5 mAbs T lymphocyte immune responses are regulated by functional cell surface molecules providing positive signals that lead to the expansion of antigen-specific clones, and negative signals which prevent excessive stimulation or responsiveness to self antigens.","The activation of resting T lymphocytes requires two independent signals.","The signal provided by the engagement of T-cell receptor (TCR) must be accompanied by a second positive signal in order to result in optimal T cell response.","CD28 is considered as the major co-stimulatory receptor inducing T lymphocyte activation and IL-2 synthesis and preventing cell-death.","Recent studies demonstrated that CD3/TCR stimulation leads to a redistribution of the detergent-insoluble glycolipid-enriched membrane fraction (DIG) or raft, which results in the aggregation of TCR/CD3 and DIG-associated signal-transducing molecules.","CD28 was reported to enhance raft redistribution to the site of TCR engagement.","Further, a series of other molecules may provide co-stimulatory signals to T cells, acting at different time points, affecting particular subsets or promoting distinct effector functions.","Negative regulation of T lymphocyte responses can be mediated by CD152-induced signals or by apoptosis through the members of TNF receptor superfamily.","Recently, a novel type of negative regulation has been reported for subsets of NK cells and CD8+ T lymphocytes (Lan998, Annu.","Rev.","Immunol.","16: 359-393).","The immunoglobulin-like (KIRs) or C-type lectin structures responsible for these signals prevent unwanted effector functions such as activation of CTL activity or cytokine production.","We now describe a novel 96 kD transmembrane receptor, SC5, which delineates a minor subset of peripheral blood lymphocytes in normal individuals.","Anti-SC5 mAb stained mainly CD45RO+ lymphocytes, from both CD8 and CD4 subsets, as well as NK lineage cells.","We found that SC5 was predominantly located in the intracellular compartment of resting T lymphocytes and its surface membrane expression increased rapidly after cell activation.","SC5 molecule engagement inhibited the anti-CD3 mAb-induced proliferation of resting T lymphocytes or T cell clones, whereas it had no effect on the cytokine-induced proliferation of these cells.","At the same time, the effector cytotoxic activity mediated by CD8+ T cell clones was not altered by anti-SC5 mAb.","Further on, we observed that the percentage of SC5+CD4+ circulating lymphocytes was significantly increased in Sézary syndrome (SS) patients as compared to peripheral blood lymphocytes of normal individuals.","Importantly, we were able to clearly demonstrate that Sézary cells express SC5 molecules.","In addition, we found that ligation of SC5 molecules in a cutaneous T cell lymphoma (CTCL) cell line resulted in a strong inhibition of the malignant cell proliferation induced by anti-CD3 mAbs.","Thus, SC5 molecule expression can serve to detect the presence of circulating malignant CD4+ cells in SS patients.","Moreover, the identification of a cell surface molecule providing negative signals upon ligation constitutes a new tool to investigate the mechanisms of pathological T cell growth and could be used to prevent it.","Material and Methods Production of Anti-CS mAb SC5 mAb was obtained by immunizing 6 wk old Balb/c mice with the NK cell line Ytindi, using an established protocol (David et al.","1990; J. Immunol.","144: 1-6).","Hybridoma supernatants were screened for selective reactivity with the immunizing cell line and a CTCL cell line termed Pno (Poszcepczynska et al.","2000; Blood 96: 1056-1063) by indirect immunofluorescence and flow cytometry.","Pno cell line derives from CTCL cells from a patient which had a CD3+ Vβ22+ CD4+ CD8αα+ CD25-phenotype, and which proliferates in response to IL-7 and to anti-CD3 mAb stimulation.","Any CTCL cell line is appropriate for this screening.","Examples of other CTCL cell lines include the HUT78 cell line (ATCC TIB-161).","The reactive supernatants were further tested for their reactivity with peripheral blood lymphocytes (PBL).","A hybridoma supernatant was selected for its reactivity with both tumoral cell lines and with a minor PBL sub-population.","The hybridoma was cloned twice and cloned hybridomas were passaged into pristane-primed Balb/c mice to produce ascites.","The antibody isotype was determined as IgM and termed anti-SC5 mAb.","The ascites was dialysed against PBS, sterilized by ultrafiltration and further utilized at final dilution {fraction (1/200)}.","This hybridoma has been deposited on Oct. 30, 2000 at the C.N.C.M.","(Collection Nationale de Microorganismes, Institut Pasteur, 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France) in accordance with the Budapest Treaty; the C.N.C.M.","deposit number is I-2575.In this example, “anti-SC5 mAb” refers to the mAb produced by hybridoma I-2575.Patients After informed consent and approval by an ethic committee (CCPPRB, Hôpital Henri Mondor, Creteil, France), we obtained blood samples from eleven patients with CTCL.","Eight patients had a Sézary syndrome with 10 to 80% circulating CD3+, CD4+, CD8-Sézary cells, whereas three patients had a transformed mycosis fungoides (MF) and presented with disseminated skin tumors with a CD3+, CD4+, CD8-phenotype.","All patients were not previously treated with chemotherapy.","Cells and Cell Lines Peripheral blood mononuclear cells (PBMC) were isolated by the technique of Ficoll-Isopaque (Pharmacia fine Chemicals, Piscataway, N.J.) density gradient centrifuigation.","Human T cell clones GDS.3 (CD3+TCRαβ+CD4+CD8−), DS6 (CD3+TCRγδ+CD4−CD8−), LSO (CD3+TCRγδ+CD4−CD8−) and JF1 (CD3+TCRαβ+CD4−CD8+), described in Bensussan et al.","1984 (J. Exp.","Med.","159: 947-952) and Yssel et al 1984.(J.","Immunol.","Methods 72: 219), were fed each 8 days with irradiated allogenic PBMC in culture medium consisting of RPMI 1640 (GIBCO, Paisley, UK), 2 mmol/L L-glutamine, penicillin (100 U/ml), streptomycin (100 mg/ml), 10% heat-inactivated human serum, 50 IU/ml recombinant interleukin-2 (rIL-2, Sanofi Synthélabo, Labège, France) and 1 μg/ml phytohemagglutinin (PHA), (Wellcome, Beckenham, UK).","Functional studies were performed at day 7 after the feeding.","Standard human leukemic cell lines were mycoplasma-free and maintained in logarithmic growth in complete RPMI medium supplemented with 10% fetal calf serum (FCS) and antibiotics.","The human CTCL cell clone, Pno, was established from peripheral blood of a Sézary patient and maintained in culture as previously described (Poszcepczynska et al.","2000; see supra).","Pno cell line has a CD3+Vβ22+CD4+CD8αα+CD25-phenotype and proliferates in response to IL-7 and to anti-CD3 mAb stimulation.","Other CTCL cell lines include the HUT78 cell line (ATCC TIB-161).","Monoclonal Antibodies and Flow Cytometry Studies Indirect immuno-fluorescent staining was performed with hybridoma supernatants or ascites fluid using FITC-conjugated goat anti-mouse Ig from Caltag laboratories (San Francisco, Calif.).","For two-color analysis the immuno-fluorescent staining was performed by incubating 3×105 cells with anti-SC5 mAb for 30 min at 4° C. Cells were then washed and incubated with FITC-conjugated goat anti-mouse IgM (Caltag laboratories) followed by a second PE-, ECD- or TRI-conjugated specific mAb of IgG isotype.","Stained cells were analyzed using a single argon flow cytometer analyser (Epics XL, Bekman-Coulter, Miami, Fla.) as described in Schiavon V et al.","1999; Tissue Antigens 53: 23-32.For intracellular labeling, cells were fixed in PBS 4% p-formaldehyde for 20 min at 4° C., washed, and then permeabilized with staining buffer containing 0.1% saponin.","Conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD45RO, anti-CD69, and anti-TCRVβ22 mAbs were purchased from Immunotech (Marseille, France).","Other mAbs are available from Beckman Coulter (see Cell Analysis 2000 catalogue): anti-CD3 (PE) Cell Analysis 2000 catalogue reference IM1282; purified anti-CD3 Cell Analysis 2000 catalogue reference IM0178; Cell Analysis 2000 catalogue reference anti-CD4 IM0449; anti-CD8 Cell Analysis 2000 catalogue reference IM0452; anti-CD45RO Cell Analysis 2000 catalogue reference IM1307; anti-CD69 Cell Analysis 2000 catalogue reference IM1943; anti-CD94 Cell Analysis 2000 catalogue reference IM2276; anti-CD158a Cell Analysis 2000 catalogue reference IM2277; anti-CD158b Cell Analysis 2000 catalogue reference IM2278; anti-NKG2a Cell Analysis 2000 catalogue reference IM3291; anti-BY55 Cell Analysis 2000 catalogue reference IM2745.Immunoprecipitation of Biotinylated Surface Molecules.","2×107 cells were washed twice in PBS and surface-labeled with Sulfo-NHS-LC biotin (Pierce Europe, Interchim, Montlucon, France) (2 mg/mL in PBS) for 20 min at 4° C. After quenching for 20 min with RPMI 1640, cells were washed twice in PBS and re-suspended in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM Na Vanadate, 10 mM NaF, 1 mM PMSF, 1 μg/mL aprotinin, and 1 μg/mL leupeptin) for 1 h at 4° C. Postnuclear supernatant was then incubated for 2 h at 4° C. in a 96-well plate (Maxisorp Nunc immuno-plate) pre-coated with goat anti-mouse IgG+IgM (Caltag laboratories) followed by anti-SC5 mAb or anti-TCRβ (anti-TCRβ C305 isotype matched mAb, Cell Analysis 2000 catalogue reference IM1466; anti-TCRVbeta22, Cell Analysis 2000 catalogue reference IM2051; anti-TCRzeta, Cell Analysis 2000 catalogue reference IM3169; all from Beckman Coulter).","Immuno-precipitates were washed 4 times with washing buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM Na Vanadate, 10 mM NaF, 1 mM PMSF) and precipitated proteins were subjected to SDS-PAGE.","Western blot analysis was performed using streptavidin-peroxidase (Immunotech) and the ECL detection system according to manufacturers' recommendations (Amersham Pharmacia, Orsay, France).","Proliferation Assays For lymphocyte activation, PBMC were cultured in medium containing 15% human serum in the presence of 1 μg/ml PHA (Wellcome, Beckenham, UK).","For proliferation assays, 5×104 cells were cultured in triplicates in 96-well round-bottomed plates (Greiner, Nürtingen, Germany) in a final volume of 0.2 ml culture medium.","When needed, the plates were pre-coated with anti-CD3 mAb at the indicated concentrations as previously described (Poszcepczynska et al.","2000, see supra).","For stimulation of CTCL cell line Pno, pre-coated anti-CD3 mAb was used in combination with 2 ng/ml phorbol 12 β-myristate 13 α-acetate (PMA, Sigma Biochemicals).","Cells were cultured for 4 days and were pulsed with 1 μCi of 3H[TdR] during the last 8-16 h of culture.","3H[TdR] incorporation was measured in a liquid scintillation counter (Topcount; Packard Instrument Co, Meriden, Conn.).","For the determination of IL-2 production, 105 PBL were stimulated with immobilized anti-CD3 mAb in the presence of anti-SC5 or control mAb.","After 32 hours of culture, 100 μL of supernatant was added to 105 cells from an IL-2-dependent T cell clone.","CD3-Induced Redirected Cytotoxicity Assays The redirected cytotoxicity assay was carried out as described in Le Cleach et al.","2000, Clin.","Exp.","Immunol.","119: 225-230.The target murine mastocytoma P815 tumor cells were loaded with 100 μl 51Cr (2.5 mCi/mL) for 90 min at 37° C., washed and incubated with anti-CD3 mAb for 15 min at room temperature.","Effector cells were likewise pre-incubated with anti-SC5 mAb (1:200 final dilution of ascites) and added to target cells in ratio 5:1, in a final volume of 150 μl in 96 well V-bottomed microtiter plates for 4 h. After centrifugation 100 μl aliquots were counted in a gamma-counter to determine 51Cr release.","The spontaneous release was always less than 20% from the maximum release (target cells with 1% NP40).","Percentage of specific 51Cr release was calculated as described in David et al.","1987, J. Immunol.","138: 2831-2836.Statistical Analysis Statistical analysis of the results was performed with Statistica software vers.","5.0 (StatSoft, Los Angeles, Calif.).","Significant differences between SC5 expression in patients and control group were evaluated by the Mann-Whitney U test, and the correlation between the percentage of SC5+CD4+ cells and the percentage of malignant cells in patients peripheral blood was evaluated by the Spearman Rank Order Correlations test.","Results The results herein reported have been obtained with the CTCL cell line Pno, and with CTCL cells collected from patients.","Equivalent experiments and results can be performed and obtained with any CTCL cell line, such as e.g.","HUT 78 (ATCC TIB-161).","Expression of SC5 Molecules in Normal Resting and Activated Peripheral Blood Lymphocytes Monoclonal antibodies raised against the functional tumor cell line Ytindi were analyzed for their simultaneous reactivity with the CTCL cell line Pno (Poszcepczynska et al.","2000, see supra) and normal peripheral blood T cells.","The SC5-specific mAb, of IgM isotype, stained both the immunizing and CTCL cell lines.","Among peripheral blood lymphocytes, anti-SC5 mAb delineated a small sub-population (see FIGS.","1A, 1B, 1C).","The SC5 mAb reactive molecule was expressed by a variable percentage of CD3+ T cells, never exceeding 20% (mean=10.5%, SD±5.6).","Both CD4+ and CD8+ lymphocytes were stained by anti-SC5 mAb.","A sub-population of peripheral blood CD56+ NK cells was also reactive with anti-SC5 mAb, though with more important inter-individual variations (mean=19.4%, SD±11.4); see the below Table 1.TABLE 1 expression of SC5 in peripheral blood lymphocyte sub-populations Cell population N tested mean (%) SD Total Ly 15 14.1 6.8 CD3+ Ly 10 10.5 5.6 CD4+ Ly 8 9.4 5.1 CD8+ Ly 8 14.8 5.1 CD56+ Ly 10 19.4 11.4 A representative example of SC5 co-expression with CD3 or CD56 molecules on gated lymphocytes from PBL of normal donors is shown in FIG.","1A.","It should be noted that most SC5+ lymphocytes belonged to the activation/memory pool, as demonstrated in FIG.","1B.","Over 70% of the SC5-positive cells co-expressed CD45RO.","As SC5 molecule expression was detected in all IL-2 dependent T lymphocyte clones tested, we verified whether its cell surface expression was induced during T lymphocyte activation.","We found that stimulation of PBL with PHA promptly increased the expression of SC5 on CD3+ lymphocytes.","Both the percentage of SC5+ T cells and the level of SC5 expression rose very rapidly after stimulation (see FIG.","2).","As early as 4 hours after stimulation with PHA, the percentage of SC5+CD3+ cells increased two to threefold and reached a maximum after 24 to 48 hours.","In the course of 5 to 7 days of in vitro stimulation SC5 expression returned to the basal level.","In parallel we studied the expression of the very early activation antigen CD69 and we observed similar kinetic profiles of CD69 and SC5 on CD3+ lymphocytes.","It should be noted, that unlike CD69 molecules, low levels of SC5 were already present on circulating PBL before stimulation (FIG.","2) Activation-induced antigen expression results either from de novo protein synthesis or cell surface export of preexisting intracellular molecules.","Therefore, we studied the expression of SC5 in non-activated permeabilized PBL.","As shown in FIG.","3, anti-SC5 mAb stained over 90% of permeabilized lymphocytes vs. 16% of the same non-permeabilized cells.","The specificity of anti-SC5 mAb staining was confirmed by using the isotype-matched mAb BY55 which detects a GPI-anchored cell surface structure expressed by a subset of circulating lymphocytes (see Agrawal et al.","1999, J. Immunol.","162: 1223-1226).","A comparable percentage of BY55+ cells was found in permeabilized and non-permeabilized lymphocytes.","As a positive control for the detection of an intracellular epitope, we used an anti-CD3ζ chain mAb which only labeled permeabilized T lymphocytes.","Thus, the surface expression of SC5 is dynamically regulated by the activation status of cells and its induction during T cell activation is due to the enhanced transport of the molecule from an intracellular pool to the surface membrane.","Biochemical Characterization of SC5 Antigen In order to characterize the molecular weight of the structure identified by anti-SC5 mAb, an IL-2 dependent TCRγδ+ clone, termed DS6, was surface labeled with biotin and the cell lysates were immun-precipitated with either anti-SC5 mAb or an isotype-matched anti-TCRβ mAb (as negative control).","Anti-SC5 mAb recognized a single molecule with apparent molecular weight of 96 kD under reducing conditions (see FIG.","4).","As tumor cell lines (the immunizing Ytindi cells) are often characterized by aberrant expression of carbohydrate epitopes (20, 22), we studied the reactivity of anti-SC5 mAb with the SC5+ NK cell line NK3.3 and with the CTCL cell line HUT78 (ATCC TIB-161) after treatment with neuraminidase or sodium periodate in concentrations destroying known sialylated epitopes (digestion of CD75 epitope on Raji cells was used as a positive control).","Expression of SC5 was not affected by neuraminidase or sodium periodate treatment.","Thus, we excluded a possible carbohydrate nature of the epitope.","Inhibition of Tile Anti-CD3 mAb-Induced T Lymphocyte Proliferation by Anti-SC5 mAb In order to examine the possible function of SC5 molecule in normal T cells, we studied the effect of anti-SC5 mAb alone or during the proliferative responses induced by immobilized anti-CD3 mAb.","We found that anti-SC5 mAb alone or in combination with PMA did not induce the proliferation of peripheral blood T lymphocytes.","Interestingly, when soluble anti-SC5 mAb was present together with immobilized anti-CD3 mAb, a significant inhibition of lymphocyte proliferation was observed.","The inhibition varied between 33 and 66% at day 4 depending on the donor as compared to the effect of an isotype-matched irrelevant control mAb (see FIG.","5A, upper panel).","As SC5 molecule is expressed on monocytes, it was important to determine whether the inhibitory effect was monocyte-independent.","We studied the proliferation of two T cell clones, GDS.3 and LSO, stimulated by immobilized anti-CD3 mAb in the presence of anti-SC5 mAb or an isotype control mAb.","The results obtained with both T cell clones indicated that engagement of SC5 molecules resulted in an inhibition of the proliferation to anti-CD3 mAb (see FIG.","5A, lower panel).","It should be noted that the inhibitory effect of anti-SC5 mAb on T cell proliferation depended on its concentration, as well as on the concentration of the agonistic anti-CD3 mAb.","The inhibition obtained with anti-SC5 mAb was significant only when T cells were stimulated with optimally diluted anti-CD3 mAb (see FIG.","5B).","Such inhibitory behaviour was also reported for anti-KIRs mAbs (Cambiaggi et al.","1999, Blood 94: 2396-2402).","The decrease of T cell proliferation following SC5 engagement could be due to a direct induction of cell death, a perturbation of the IL-2R expression, or the inhibition of cytokine synthesis.","We found that anti-SC5 mAb did not inhibit the IL-2-dependent proliferation of PBL or T cell clones (FIG.","5A).","Furthermore, the addition of rIL-2 to T cells pre-incubated with anti-SC5 mAb (ascites diluted to {fraction (1/200)}) in combination with anti-CD3 for 48 h restored their proliferation.","This would not be the case if the specific mAb inhibited IL-2R expression or caused cell death.","Finally, in order to prove that SC5 triggering may inhibit cytokine production, we compared the amount of IL-2 secreted by anti CD3-activated PBL in the presence or absence of anti-SC5 mAb, using an IL-2 dependant T cell line.","The quantity of IL-2 produced in the presence of anti-SC5 mAb represented 40 to 65% of the control values obtained with an isotype-matched antibody (see FIG.","5C).","Thus, the simultaneous engagement of SC5 and CD3 molecules in T cells results in the decrease of cytokine production.","We next studied the effect of anti-SC5 mnAb on T cell effector cytotoxic activity using an anti-CD3 mAb redirected killing assay against the FcgR+ murine cell line P815.FIG.","5D shows a representative experiment performed with a cytotoxic T cell clone at an E/T ratio of 5/1.The CD8+ T cell clone JF1, expressing high amount of SC5 molecules, was induced to kill murine target cells following anti-CD3 mAb is stimulation.","The simultaneous targeting of SC5 by its specific mAb did not influence JF1 cytotoxic activity, regardless of the E/T ratio or the concentration of stimulating anti-CD3 mAb.","Expression of SC5 Molecules on Malignant Lymphocytes Obtainedfrom Patients with Sézary Syndrome As we initially screened anti-SC5 mAb for its reactivity with a CTCL cell line, we further studied the expression of SC5 molecule in the periphcral blood lymphocytes isolated from 8 patients with Sézary syndrome.","Two-color flow cytometry analysis showed that expression of SC5 was significantly increased in the CD4+ subset (see representative results obtained with one patient in FIG.","6A).","On the average, 35.5% of the CD4+ lymphocytes in SS patient blood expressed SC5 as compared to 9.4% in healthy controls (p<0.001), (FIG.","6B).","In the 8 SS patients studied, the expression of SC5 on CD4+ cells correlated with the percentage of circulating malignant cells detected by cytomorphology (r=0.91, p=0.0012).","It should be noted that in these patients the percentage of total CD4+ cells (mean=88%, SD±8.8) was higher than that of Sézary cells (mean=26%, SD±12.8).","We also studied SC5 molecule expression in peripheral blood samples from 3 MF patients without blood involvement.","No significant difference in the levels of SC5 expression on peripheral blood CD4+ cells was observed between MF patients and healthy donors (FIG.","6B).","Still, an increased percentage of SC5+CD4+ was detected in the skin samples from two of these patients, that we were able to to test (26% and 19% respectively).","In one SS patient we had previously demonstrated that the circulating malignant cells were clonal lymphocytes with a CD4+TCRVβ22+ phenotype (Poszcepczynska et al.","2000, see supra).","Here we demonstrated that SC5 was co-expressed on the TCRVβ22+ cells, i.e.—by the malignant cell population (FIG.","7A).","It should be noted that the profile of SC5-mAb binding to TCRVβ22+ cells corresponded to a weak homogeneous expression on the whole malignant clonal cell population (FIG.","7B).","Interestingly, the malignant TCRVβ22+ cells did not co-express common activation markers as CD69 (FIG.","7C), CD25 or CD30 as previously described (Poszcepczynska et al.","2000, see supra).","Thus, the increased levels of SC5 in SS PBL were mostly due to the expression of the antigen on circulating malignant T cells.","Inhibition of the Anti-CD3mAb-Induced CTCL Cell Line Proliferation by Anti-SC5 mAb We next investigated whether SC5 molecules were able to provide a negative signal in malignant CTCL cells.","We used the long-term cultured Pno CTCL tumor T cell line which strongly proliferates in the presence of IL-7.As previously reported that Pno cell line expresses functional T cell receptors, since immobilized anti-CD3 mAbs or the combination of soluble anti-CD3 mAbs and PMA induced significantly their proliferation (Poszcepczynska et al.","2000, see supra).","We examined the effect of SC5 molecule engagement on the proliferation of Pno cells.","FIG.","8 shows that anti-SC5 mAb strongly inhibited the anti-CD3 mAb-induced proliferation of the CTCL cell line.","In contrast, PMA alone (2 ng/mL) produced a low level of proliferation which was not significantly affected by anti-SC5.Notably, anti-SC5 mAb had no effect on the IL-7-dependent proliferation, since Pno cells incubated with IL-7 in the presence of anti-SC5 mAb proliferated as vigorously as in the presence of the isotype control mAb.","Thus, we concluded that SC5 antigen is functional in CTCL malignant cells and may specifically and profoundly inhibit their proliferation triggered through the TCR/CD3 signaling pathway.","A CTCL tumor was then successfully grafted onto transgenic mice having a substantial deficiency in functionally active NK cells and T lymphocytes (SCID mice) as described in Charley et al.","(1990) “Establishment of a human cutaneous T cell lymphoma in CB-17 SCID mice” J.","Invest.","Dermatol.","94: 381-384 (see also U.S. Pat.","No.","5,530,179 Cornelius P. Terhorst and Baoping Wang for obtention of SCID mice).","Anti-SC5 mAb was intraveneously or intracutaneously administered at different times to half of the mice at various concentrations of purified mAb per gram of mouse, whereas the other half received the same amount of purified CD56 (isotype IgM) as a control.","PBL and cutaneous cells were collected for standard cytological observations of infiltrating large T cells, and the percentage of CTCL cells was assessed for each mouse of the experiment.","This procedure is an example of procedure that enables the is skilled person to check that anti-SC5 compounds, such as the mAb produced by the hybridoma deposited as deposit number I-2575 at the C.N.C.M., are capable of inhibiting the in vivo proliferation of human CTCL.","Discussion A number of receptors with dynamic surface expression mediate T cell interactions with other immune cells or cytokines and, consequently modulate T cell immune responses.","In this study, we identified a novel 96 kD functional molecule expressed by a minor subset of PBL whose surface expression is induced rapidly during in vitro T-cell activation.","SC5 is distinct from the established molecules with similar molecular weight and/or T cell modulatory functions.","First, SC5 was induced very early upon T cell activation, with important increase of its surface expression detected after 4 h. A similar kinetics has been observed for the very early activation antigen CD69, which is detected on the cell surface between 2 and 4 h after stimulation.","Most established T-cell specific inducible receptors such as 4-1BB, OX-40 LIGHT or CD152 (CTLA-4) appear on T-cell surface between 10 to 24 h following stimulation.","Further, SC5 expression is not T cell restricted.","The antigen was detected on sub-populations of NK and B lymphocytes, monocytes and granulocytes.","Such a wide distribution pattern is typical of certain killer cell Ig-like receptors (KIRs), as the recently described AIRM1 and IRp60, which are found on NK cells, T and B lymphocytes, myeloid and other antigen-presenting cells.","The family of immunoglobulin—like transcript (ILT) inhibitory receptors has also a pan-leukocyte type of expression, the individual receptors being detected on different NK, T, B and myeloid sub-populations.","Moreover, ILT4 has a molecular weight very close to SC5.However, neither the expression of AIRM1 and IRp60, nor of ILTRs depends on the activation status of cells.","The prompt induction of SC5 molecule at the surface of activated T cells followed by its down-regulation may result from trafficking of intracellular molecules to the cell membrane and vice versa, as we detected SC5 in most non-activated T lymphocytes after cell permeabilization.","Such a regulatory mechanism has been already reported for another T cell receptor, CD152.Further on, a heterogeneous family of lysosome associated membrane proteins (LAMP) exists, including molecules involved in endocytosis, lysosomal trafficking and secretion from intracellular granules and that may be induced on the surface of monocytes, neutrophiles, NK, B or T cells upon activation.","Additional studies on the intracellular localization and transport of SC5 would precise its proper role in T cell activation and functions.","Our studies rcvealed that triggering of SC5 by its specific mAb inhibited the anti-CD3 mAb-induced T cell proliferation.","This inhibitory effect required an optimal anti-CD3 mAb stimulation, which is necessary to induce maximal SC5 surface expression in PBL (see FIG.","5B).","A 1:200 final dilution of anti-SC5 ascites significantly inhibits the proliferation induced by anti-CD3 mAb at 2 microgram par well and this effect of inhibition increases with lower anti-CD3 mAb concentrations −1 microgram par well; 0.5 microgram per well—A 1:400 final dilution of anti-SC5 ascites do not significantly inhibits the proliferation induced by anti-CD3 mAb at 2 microgram per well, but inhibition is significant when a lower concentration of anti-CD3 is used −1 microgram per well; 0.5 microgram per well—The person of ordinary skill in the art can proceed with such adjustments by standard interpolation from the particular results described herein, or from his own trials.","At the same time, the inhibitory.","threshold posed by SC5 engagement could be bypassed by excessive anti-CD3 mAb stimulation.","It should be noted that the inhibitory functional effects obtained by ligation of SC5 molecules were observed in an APC-independent system.","Further experiments are needed to determine whether SC5 stimulation may have different effects on the proliferation of APC or allo-stimulated T cells.","Recently, the negative regulation of immune responses has been extensively investigated, in particular in T lymphocytes and NK cells.","The best studied T cell specific inhibitory receptor, CD152 (CTLA-4), is a typical activation-induced antigen, functionally coupled to CD28 co-stimulatory receptor.","Indeed, CD152 may overcome CD28 positive signals at low antigenic concentrations and prevent immune response.","Alternatively, it may downregulate and terminate T-cell proliferation induced through CD28 by affecting IL-2R expression and IL-2 synthesis.","Similarly to CD152, SC5 is an intracellular molecule whose surface expression is regulated by the activation status of cells.","Both receptors mediate their effects by affecting IL-2 synthesis.","The identification of SC5 specific ligands and signaling pathways will precise the temporal and spatial correlation between these negative T-cell regulators.","In consideration to its wide cell distribution, SC5 functional effects are probably not restricted to T cells.","In several aspects, SC5 is comparable to NK cell inhibitory receptors, equally expressed by T cells and other lymphocytes sub-populations.","Both KIRs and CD94/NKG2 receptors were shown to inhibit antigen-specific and superantigen-driven activation of TCRαβ and TCRγδ T cells, including cytotoxicity, proliferation and cytokine secretion.","The KIR-induced inhibition depends on the activation status of T cells and the potency of T cell stimulation and may be bypassed under specific in vivo conditions.","However, SC5 is definitely distinct from KIRs, since we demonstrated that it selectively inhibited the proliferation of anti-CD3 mAb stimulated T cells without affecting their effector cytotoxic functions.","An important characteristic of SC5 molecule is its significantly increased expression in peripheral blood lymphocytes of patients with SS in comparison to healthy subjects.","SS is characterized by the generalization of a primary epidermotropic malignant T cell clone with mature phenotype.","Actually, none of the established T cell functional receptors conclusively identifies peripheral blood SS tumor cells.","MF/SS has been considered as the expansion of a T cell subset with a particular phenotype, CD4+CD7−, corresponding to a normally existing T memory subset.","However, a recent study demonstrated that the CD4+CD7− population does not always represent the dominant T cell clone in SS patients (Dummer et al.","1999, Arch.","Dermatol.","Res.","291: 307-311).","SS is regarded as a proliferation of specifically activated T cells although the nature of the stimulus is not known (Fargnoli et al.","1997, Leukemia 11: 1338-1346).","For this reason the distinct expression of activation induced antigens on SS and normal T cells is of special interest.","Established activation antigens such as CD69, CD25, CD30 are not consistently detected on Sézary cells, as confirmed by our own investigations.","Expression of CD25 rather correlates with the progression of the disease and the transformation of MF/SS cells into a large cell variant in a minority of the cases.","An increased rate of expression of T cell activation molecules as HLA-DR, CD25 or CD71 in SS has been primarily associated with reactive tumor-infiltrating T cells.","A 78 kD very late activation antigen, BE2, was reported to be specifically increased in SS peripheral blood, while not detected on normal T cells.","In fact, BE2 was not consistently detected in all SS patients studied, a comparatively low percentage of peripheral blood cells in relation to the reported percentage of malignant cells were BE2-positive, and BE2 was equally detected in patients without clinical evidence of blood involvement.","In contrast, SC5 was detected in 8/8 SS cases studied.","Although the percentage of malignant cells determined by cytomorphology varied considerably from patient to patient (10-80%), the expression of SC5 on CD4+ cells correlated with this percentage.","Finally, no elevated expression of SC5 on CD4+ cells was detected in MF patients without blood involvement.","Further, we demonstrated that Sézary cells identified by the expression of the clonotypic TCRVβ chain co-expressed SC5 molecule, confirming that increased levels of SC5 in the peripheral blood of SS patients were due to its expression on the malignant T cell clone.","A very important observation was that SC5 triggering on Sézary cells induced down-modulation of the CD3-mediated proliferation of tumor cells, analogous to the inhibition observed in normal T cells.","It has been already demonstrated that malignant CTCL lymphocytes preserve certain functional properties as they may be activated through basic signaling pathways although producing aberrant profiles of cytokines.","We now demonstrate that an inhibitory pathway is preserved in CTCL tumor cells, that may permit to counterbalance their proliferation.","Notably, the inhibitory effect observed after anti-SC5 mAb binding on anti-CD3 mAb-stimulated CTCL Pno cells was much more important than in normal T cells.","Anti-SC5 mAb does not seem to directly mediate cell death of neither normal nor Sézary T cells, since it does not modulate the rIL-induced proliferation.","Moreover, T-cells that had been cultivated in the presence of anti-SC5 mAb can be re-stimulated by addition of rIL-2.At the same time, we observed a diminished IL-2 production by anti-SC5 mAb-treated T cells.","which should account at least partially for the inhibition of their proliferation.","Inhibitory effects of functional receptors on the proliferation of malignant cells have been already observed.","It has been recently reported the inhibition of the proliferation of normal and leukemic myeloid cells mediated by the homologous NK cell sialoadhesin receptor AIRMI and CD33 myeloid-specific antigen (Vitale et al.","1999, Proc.","Natl.","Acad.","Sci.","USA 96: 15091-15096).","Unlike SC5, these receptors inhibited proliferation in the presence of growvth factors and most probably by induction of apoptosis.","Interestingly, another T cell activation antigen, CD30, expressed by a subset of normal CD45RO+ T cells, is likely to mediate the regression of primary CTCL other than MF/SS.","This effect is related to the natural ligand of CD30 (CD30L) which is co-located within the cytoplasm of malignant cells and may induce cytolytic cell death independently of Fas/FasL system (Mori et al.","1999, Blood 94: 3077-3083).","In this aspect, the identification of SC5 ligand will enlighten the mechanism of its inhibitory action.","In conclusion, we have identified an early activation antigen which is distinct, by phenotypic, biochemical and functional criteria, from the established T lymphocyte receptors.","As it was underlined, SC5 molecule is expressed by a sub-population of the post-activation/memory peripheral blood T cell subset.","Likewise, continuous expression of KIRs is detected on minor T cell clones, and this expression seems to depend on TCR-occupancy by specific antigens.","It was recently proposed that such clones might be auto-reactive and maintained tolerant by KIRs signaling.","We may speculate that SC5 surface expression is also preserved on auto-reactive T cells, suggesting that Sézary syndrome tumor cells possibly originate from such clones.","Importantly, the expression of SC5 on CTCL malignant cells will serve to better define their origin and the nature of the signal driving mature T cells to uncontrolled proliferation.","In addition, the inhibitory effect of anti-SC5 mAb on tumor T cells constitutes a novel therapeutic approach in patients with advanced CTCL.","Furthermore, expression of SC5 has also been observed at the surface of CD8+ CTCL, such as CD8+ transformed MF.","EXAMPLE 2 P140 is Expressed by CD4+ CTCL Cells, and Identification of a Novel P140 Allelic Form Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of lymphomas primarily involving the skin.","Mycosis fungoides (MF) is characterized by skin invasion of clonally-derived malignant CD4+ T lymphocytes that phenotypically resemble mature T helper cells.","A more aggressive form of CTCL develops when the is malignant cells become non-epidermotropic and is associated with extra-cutaneous involvement.","Sézary syndrome (SS) is a more aggressive form of CTCL that is characterized by a clonal expansion of CD4+/CD45RO+ T cells and the appearance of these malignant T cells in the blood.","The biology of the disease remains poorly understood, as it is difficult to identify the malignant cell, due to the lack of specific cell surface markers.","Thus, in cutaneous lesions it is difficult to distinguish CD4+ CTCL cells from reactive infiltrating CD4+ T lymphocytes.","Bagot et al.","previously described a unique CD4+ T cell line derived from CTCL lesions (Bagot et al.","1998, Blood 91: 4331-4341); this cell line and the in vivo tumor cells expressed an identical size of the complementarily-determining region 3 of TCR-Vβ transcripts.","More recently, Poszepczynska et al.","(Blood 2000, 96: 1056-1063) functionally characterized an IL-7-dependent CD4+CD8αα+ tumor T cell line isolated from the blood of another patient with a cutaneous erythrodermic CTCL.","This tumor T-cell line was identical to the major circulating T cell populations, as demonstrated by the expression of TCR-Vβ22 and the identity of the TCRβ-VDJ sequences.","Here, we show that these two different CTCL lines express the p140/KIR inhibitory receptor for HLA-A alleles.","Importantly, this receptor is detected on freshly isolated tumor cells derived from the same patients.","Moreover, the p140/KIR was also co-expressed by a major subset of CD4+ lymphocytes in seven other patients with SS, and by tumor skin CD4+ cells in two additional patients with advanced MF.","The p140/KIR is detected in normal individuals on a minor NK cell-subset and on rare peripheral blood CD3+CD8+ cells.","Moreover, T cells obtained from skin in other dermatological diseases such as inflammatory skin diseases and toxic epidermal necrolysis did not express this receptor (Le Cleach et al.","2000, Clin.","Exp.","Immunol.","119: 225-230).","Thus, our present findings demonstrate that the p140/KIR represents a suitable marker on CD4+ cells for the identification of CTCL.","Materials and Methods Patients After informed consent and approval by an ethic committee, we obtained skin and blood samples from eleven patients with a CTCL.","Eight patients had a Sézary syndrome with 10 to 45% circulating Sézary cells in the blood.","In seven cases, the phenotype of tumor cells was CD3+,CD4+,CD8−.","In one case, the phenotype was CD3+,CD4+,CD8αα+ (patient Pno).","Three patients had a transformed mycosis fungoides (Lez, Cou, Bic) and presented with disseminated skin tumors with a CD3+,CD4+,CD8− phenotype.","All patients were not previously treated with chemotherapy.","Isolation of Tumoral Lymphocytes Fresh CTCL tumor cells were obtained from tumor fragments mechanically dispersed into single-cell suspensions (Bagot et al.","1998, Blood 91: 4331-4341).","The mononuclear cells were then washed and frozen in human serum plus 10% dimethyl sulfoxide for later use.","For Sézary syndrome patients, the mononuclear blood cells were isolated by the technique of Ficoll-Isopaque (Pharmacia fine Chemicals, Piscataway, N.J.).","Long Term Culture of Tumor Cell Lines We established the long term culture of Pno cell line (TCRVβ22+,CD3+, CD4+,CD8αα+,MHC class I+,MHC class II−) in vitro from the peripheral blood of the patient as previously described (Bagot et al.","1998, Blood 91: 4331-4341).","We demonstrated that both the malignant clone circulating in the patient blood and the derived cultured T-cell line were identical for their cell surface phenotype and for their size and sequence of the TCRβ VDJ region (Poszepczynska et al.","2000, cf.","supra).","Other sources of SS cell lines may be found at the ATCC (e.g.","HUT 78 ATCC TIB-161).","The Cou-L cell line (TCRVβ13+) was cultured in vitro with rIL-2 for more than three years.","It corresponds to a subelone of the CD4+ Cou-LS CTCL line previously described (Bagot et al.","1998, cf supra).","The Cou-L cell line, the original TCRVβ13+, CD4+ Cou-LS CTCL, and the tumor cells freshly isolated from the skin shared the same size and sequence of the TCRβ VDJ region (Bagot et al.","1998, cf.","supra).","Monoclonal Antibodies (mAbs) and Flow Cytometry Studies One- and two-color immunofluorescence analysis was performed as previously described (Bagot et al.","1998, cf.","supra).","Anti-CD3 (Cell Analysis 2000 catalogue reference 6604629), anti-CD4 (Cell Analysis 2000 catalogue reference 6602138), anti-MHC Class I (Cell Analysis 2000 catalogue reference IM0107) and II (Cell Analysis 2000 catalogue reference IM0108) are obtainable from Beckman Coulter (see Cell Analysis 2000 catalogue).","The anti-TCRVβ13+ mAb was purchased from Bioadvance (Emerainville, France, catalogue reference TCR 1738), and the anti-TCRVβ22+ mAb was obtained from Beckman-Coulter (Marseille, France, Cell Analysis 2000 catalogue reference IM 1484/IM1365).","The anti-CD8 αβ 2ST8.5H7 mAb is obtainable from Beckman Coulter (Cell Analysis 2000 catalogue reference IM 2014.As anti-p140 mAbs, Q66 (IgM, anti-p140) and AZ158 (IgG2a, recognizing both p70/NKB1 and p140) which have been described in Pende et al.","1996 (J. Exp.","Med.","184: 505-518) have been used; however any anti-140 binding compound is appropriate (e.g.","anti-p140 antiserum, Fc-HLA-A11, Fc-HLA-A3 fuision proteins).","Z27 (IgG1, anti-p70/NKB1), EB6 (IgG1, anti-p58.1/S50.1), GL183 (IgG1, anti-p58.2/p50.2), XA185 (IgG1, anti-CD94), Z199 and Z270 (IgG2b and IgG2a respectively, anti-NKG2A), and B9.4 (IgG2b, anti-CD8) mAbs are all obtainable from Beckman Coulter (see Cell Analysis 2000 catalogue references IM2748, IM2277, IM2278, IM1610, IM2750, IM0102).","Goat anti-mouse IgG FITC and goat anti-mouse IgM PE are also obtainable from Beckman Coulter (see Cell Analysis Catalogue references IM08 and IM0555).","Biochemical Characterization 20×106 cells were incubated (15′ at 20° C.) in 1 ml PBS pH8 containing 250 μg of EZ-LINK SULFO-NHS-LC-LC-BIOTIN (Pierce, Rockford, Ill.) and washed three times in Washing Buffer (10 mM TRIS pH 8, 0.14 M NaCl).","Cells were then lysed in 1% NP-40 and immuno-precipitated with Sepharose-PA (Pharmacia Biotech Inc. Piscataway, N.J.)-coupled AZ158 (IgG2a, antip70/140) or B9.4 ([gG2b, anti-CD8) mAbs.","Samples were analyzed by discontinuous SDS-PAGE either undigested or digested with N-glycosidase F (Boehringer Mannheim, GmbH, Germany) and transferred to Immobilon P (Millipore Corp, Bedform, Mass.).","After staining with Neutravidin (Pierce) the Renaissance Chemiluminescence Kit (NEN, Boston, Mass.)","was used for detection.","NK cell clones were obtained by limiting dilution as described in Pende et al.","1996 (J. Exp.","Med.","184: 505-518).","RT-PCR Analysis Total RNA was extracted from CTCL cell lines Pno and Cou-LCD8 αα using RNA-Clean System (AGS GmbH, Heidelberg, Germany).","cDNA synthesis was performed using oligodT priming.","Primers used for cDNA amplification of the complete ORF (open reading frame) of KIR displaying three Ig-like domains (1395 bp) were the following: 5′CATGT(CT)GCTCA(CT)GGTCGTC (Ig3 UP; SEQ ID No.","5) and 5′ GGTTTTGAGACAGGGCTG (Ig3 DOWN; SEQ ID No.","6).","Amplification was performed for 30 cycles (30 sec.","at 94° C., 30 sec.","at 55° C., 30 sec.","at 72° C.), followed by a 7 min.","incubation at 72° C., utilizing AmpliTAQ (Perkin Elmer-Applied Biosystems, Foster City, Calif.).","PCR products were sub-cloned into pcDNA3.1/V5-His-TOPO vector (Invitrogen, Carlsbad, Calif.).","DNA sequencing was performed using d-Rhodamine Terminator Cycle Sequencing Kit and a 377 Applied Biosystems Automatic Sequencer (Perkin Elmer-Applied Biosystems).","Transient Transfections COS-7 cells were transfected with pcDNA3.1 TOPO-KIR3D cl.24 or with pCR3-cl.1.1 (Pende et al.","1996, J. Exp.","Med.","184: 505-518) utilizing Fugene 6 (Roche, Monza Italy).","Briefly, cells were seeded at 5×105/plate; 24 hr later they were incubated with 6 μg plasmid and 10 μl of Fugene-6 reagent in DMEM/10% FCS.","After 48 or 72 hrs, transfected cells were used for cytofluorimetric analysis.","Cell transfectants were stained with Q66 and AZ158 mAbs, followed by a phycoerythrin-conjugated goat antibody to mouse IgG2a or IgM and analyzed by flow cytometry using a FACSort (Becton Dickinson, San Jose, Calif.).","Results CTCL Cell Lines are Stained by mAbs to the p140/KIR Two long-term CTCL tumor lines Pno (labeled with anti-TCR-Vβ22 mAb) (Poszepczynska et al.","2000, cf supra) and Cou-L (labeled with anti-CD3 mAb) (Bagot et al.","1998, cf.","supra) were analyzed for reactivity with different anti-KIR mAbs.","We found that both cell lines were reactive with mAbs Q66 (see FIG.","9A) and AZ158, both recognizing the p140/KIR.","In contrast, these cell lines did not express other inhibitory receptors specific for HLA class I molecules, including p58.1, p58.2, p70 KIRs and the CD94/NKG2A lectin-like receptor (Poszepczynska et al.","2000, cf supra).","Tumor T Lymph Ocytes Freshly Isolated from CTCL Patients are Stained by Anti-p140/KIR mAbs To determine whether p140/KIR was expressed by freshly isolated tumor cells, we tested the reactivity of Q66 mAb with uncultured tumor cells isolated from the blood in SS patient Pno and from tumoral skin fragments of MF patient Cou.","We found that the majority of tumor cells were stained by this mAb (FIG.","9B).","In particular, we observed that most TCRVβ22+ tumor lymphocytes isolated from the blood of patient Pno were reactive with Q66 antibody, and that most of the TCRVβ13+ tumor lymphocytes isolated from the skin of patient Cou were stained by the same antibody (FIG.","9B).","Next, we studied the phenotype of tumor T lymphocytes from the blood of seven additional patients with a Sézary syndrome, with malignant cells representing 10 to 45% of circulating CD4+ lymphocytes, and tumor T lymphocytes isolated from skin tumors of two other patients with a MF.","Remarkably, all patients tested exhibited a significant population co-expressing CD4 and p140/KIR (see Table 2 below).","TABLE 2 Anti-Q66 mAb stained CD4+ lymphocytes isolated from the skin of patients with transformed mycosis fungoides and from blood of patients with Sezary syndrome.","Percentage of positive cells with Patient anti- anti-CD4 mAb + samples CD4 mAb anti-Q66 mAb MF Lez 65 29 Bic 53 44 SS Bri 85 35 Bar 90 18 Att 95 35 Ros 98 15 Can 78 36 Pet 45 9 Riv 98 19 It should be noted that all Q66+ cells were included in the CD4+ cell population.","Thus, the expression of p140/KIR, which in normal individuals is restricted to subsets of lymphocytes from the NK and CD8+ populations, appears to be a characteristic of CTCL tumor CD4+ T lymphocytes, both in the skin and in the blood.","As control, skin T lymphocytes derived from another dermatological disease, toxic epidermal necrolysis, which were shown to contain small percentages of various KIR expressing T lymphocytes (Le Cleach et al.","2000, Clin.","Exp.","Immunol.","119: 225-230), failed to express p140/KIR.","Molecular Characterization of the p140 Receptor Expressed by CTCL.","The Pno and Cou-L cell lines were surface labeled with biotin and cell lysates were immuno-precipitated with an anti-p140 (AZ158 mAb).","As shown in FIG.","10, this mAb immuno-precipitated from a NK clone and from the Pno and Cou-L cell lines a molecule with a molecular mass of approximately 70 kD under reducing conditions.","Treatment with N-glycosidase revealed a protein backbone of approximately 50 kD with a slightly higher mobility for Pno and Cou-L compared to NK clone.","These data suggested that the p140 inhibitory receptor expressed by these CTCL tumor cell lines could be almost similar to that previously detected on normal NK cells (Pende et al.","1996, J. Exp.","Med.","184: 505-518).","Next, we determined whether the cDNA encoding the molecule recognized by Q66 and AZ158 mAbs on Pno and Cou-L CTCL cell lines corresponded to one of the already described cDNA encoding p140/KIR (SEQ ID No.","3).","To this end, RT-PCR was performed on RNA derived from these cell lines using a set of primers able to amplify all cDNA encoding KIR with three Ig-like domains.","From the Pno cell line, we isolated a full-length cDNA, termed KIR3D cl.24 (SEQ ID No.","1).","Comparison of its nucleotide sequence with DNA sequences coding for all KIR characterized by three extra-cellular Ig-like domains revealed that KIR3D cl.24 represents a novel allelic form of p140 receptor.","In particular, its nucleotide sequence displays five differences compared to the previously described cl.","1.1 cDNA (Pende et al.","1996, J. Exp.","Med.","184: 505-518), resulting in four amino acid substitutions in the mature protein (see FIG.","11; SEQ ID No.","2: clone 24 protein; SEQ ID No.","4: clone 1.1 protein).","Three of the four substitutions are found in the extra-cellular Ig-like domain (pos.","20, 92 and 111 of the mature protein), whereas the other is located in the cytoplasmic region (pos.401).","RT-PCR performed on Cou-L CTCL cell line revealed two different allelic fonns of p140/KIR one corresponding to cl.","1.1 cDNA and the other identical to KIR3D cl.24 (isolated from Pno cell line).","The cDNA derived from the CTCL cell lines were then transiently transfected in COS-7 cells.","As expected, all cell transfectants were brightly stained by Q66 and AZ158 mAb, while they were unreactive with p70/KIR-specific Z27 mAb used as a negative control.","Finally, RT-PCR was performed on RNA extracted from PBL derived from three SS patients (including patient Pno) and from skin-derived T cells of one additional MF patients.","Also in these samples one or another allelic isoform of p140/KIR described above could be identified.","The same results were obtained on malignant CTCL cells collected from patients.","Discussion Skin lesions in CTCL contain a heterogeneous lymphocytic infiltrate composed of malignant T cells, which are most often CD4+, and non-neoplastic tumor infiltrating T lymphocytes.","We previously reported CD4+ cytotoxic tumor infiltrating lymphocytes specifically directed against autologous malignant CTCL CD4+ cell line.","However, as no tumor restricted cell surface structure, besides the clonotypic TCR expressed by tumor cells, has been identified on CTCL, it is difficult using standard methods to distinguish malignant from non-malignant reactive CD4+ lymphocytes.","In the present study, we report for the first time that tumor cells from MF and SS patients express the p140/KIR.","This receptor has been identified in both skin and blood tumor cells from CTCL patients as well as in two long-term culture CTCL lines.","Two color fluorescence analysis indicated that p140/KIR expression is restricted to T cells characterized by a given TCRβ-VDJ previously identified on tumor cells.","Thus, p140/KIR allows rapid identification of tumor cells from tumor reactive cells among the T lymphocyte CD4+ population.","This could be particularly useful in SS patients, in which the tumor T cell clone is not always easily identified within the peripheral blood CD4+ lymphocyte population.","Moreover, during the course of the disease or after treatment, it is crucial to assess whether an increase of the CD4+ population is due to an expansion of the tumor cell population or of reactive T lymphocytes.","Since the anti-p140/KIR mAbs appear to recognize tumor cells in all CTCL patients analyzed, they may be considered as a suitable tool for the direct identification of CTCLs.","In addition, the use of p140/KIR co-expression on CD4+ CTCL cells provides a unique tool to distinguish malignant cells from normal cells in every patient.","This is in contrast to TCR determinations which is unique to an individual patient.","Moreover, the p140/KIR represents a possible target for the development of novel specific immuno-therapy of CTCL.","Previous studies indicated that this receptor was able to generate inhibitory signals upon recognition of HLA.A3 and HLA.A11 alleles.","It is of note, however, that p140/KIR expression in the various patients analyzed appears apparently independent from their own HLA Class I haplotype.","Nevertheless, the actual role of p140/KIR in the patho-physiology of CTCL is an important feature that remains to be studied by taking into account the potential role of this receptor in the tolerance to self.","In conclusion, the present study demonstrates for the first time the expression of the p140/KIR in CD4+ CTCL cells and the isolation of a novel allelic form of this receptor in tumor cells.","This finding is an important new issue, both for the patho-physiology and for the clinical management of CTCL patients.","Furthermore, p140 expression has also been observed at the surface of CD8+ CTCL such as CD8+ transformed MF.","EXAMPLE 3 Production of Anti-CTCL Drugs The person of ordinary skill in the art is enabled to produce anti-CTCL drugs pursuant to the following outlines, given for illustration purposes: use of anti-SC5 or anti-p140 mAb with a cytokine such as Interferon gamma or IL-2 for stimulating the immune system of cells in the vicinity of CTCL cells, use of these anti-SC5 and anti-p140 mAb as vectors for delivering anti-CTCL ingredients onto CTCL cells; examples of anti-CTCL ingredients comprise standard compounds used in chimiotherapy such as radioelements, toxines.","mAb vectors for radioelements can be produced following standard techniques applied to an anti-SC5 mAb of the invention (see e.g.","Press O W et al.","“Phase two trial of iode131-B1 (anti-CD20) antibody therapy with autologous stem cell transplantation for relapsed B cell lymphoma”, Lancet 1995 vol.","346 pp336-340, of which content is herein incorporated by reference).","mAb vectors for toxine can be produced following standard techniques applied to an anti-SC5 mAb of the invention (see e.g.","Ghetie U et al.","“Large scale preparation of an immunoconjuguate constructed with human recombinant CD4 and deglycosylated ricin A chain”, J. Immunol.","Methods 1990, vol.","126 pp 135-141, of which content is herein incorporated by reference).","combined use of an antimitotic pro-drug and of anti-SC5 mAb and/or anti-p140 mAb vectors delivering an enzyme capable of transforming an antimitotic pro-drug to the active drug form (mitotic agent in a functional form).","Anti-SC5 mAb and anti-p140 mAb vectors delivering an enzyme capable of transforming an antimitotic pro-drug to the active drug form can be produced following standard techniques applied to an anti-SC5 mAb of the invention and to an anti-p140 mAb (see e.g.","the ADEPT technique, for Antibody Directed Enzymre Prodrug Therapy, described in Plakey T C et al.","“Anti-tumor effects of an antibody-carboxypeptidase G2 conjuguate in combination with phenol mustard prodrugs”, British J.","Cancer 1995 vol.","72 pp 1083-1088, of which content is herein incorporated by reference).","use of anti-SC5 or anti-p140 mAb as ligand agonists (SC5 stimulation or p140 stimulation) use of anti-SC5 or anti-p140 mAb as complement recruiting agent For the above-mentioned uses, mAbs with a double anti-SC5/anti-CD4 or anti-p140/anti-CD4 specificity are preferred.","use of anti-SC5 or anti-p140 (no CD4+ specificity needed) as activators of ADCC (Antibody Dependent Cell Cytotoxicity): polynuclear cells, macrophages, NK cells bear Fc receptors which are activated by the Fc portion of the anti-SC5 and anti-p140 mAb, these mAb therefore enable the ADCC activation in the vicinity of CTCL cells.","Abbreviations: APC: Antigen Presenting Cell, CTCL: cutaneous T cell lymphoma, DIG: detergent-insoluble glycolipid-enriched fraction, ILT: immunoglobulin-like transcript, KIR: killer cell immunoglobulin receptor, mAb: monoclonal antibody, MF: mycosis fungoides, PBL: peripheral blood lymphocytes, PBMC: peripheral blood mononuclear cells, PHA: phytohemagglutinin, PMA: phorbo112 beta-myristate13 alpha-acetate, SS: Sézary syndrome, TCR: T cell receptor"]],"string":"[\n [\n \"Novel means for the diagnosis and therapy of ctcl\",\n \"A means for the diagnosis and therapy of T lymphomas, in particular Cutaneous T Cell Lymphomas (“CTCL”) are provided.\",\n \"Tumor markers which are universal for CTCL are described.\",\n \"More particularly, a molecule, termed SC5 by the inventors, an allelic form of p140, and biological applications of SC5 and p140 molecules, notably in the diagnosis and therapy of CTCL are described.\"\n ],\n [\n \"1 A monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.\",\n \"2 A hybridoma deposited as deposit number I-2575 at the C.N.C.M.\",\n \"3 An isolated protein obtainable by: (i) collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), and stimulating them with PHA at 1 microgram/ml, (ii) lysing the cells by incubation in a lysis buffer containing Triton X-100 at 1%, (iii) recovering from the lysate the compound onto which the mAb of claim 1 binds under conditions enabling this mAb to perform reactions of the antigen-antibody type.\",\n \"4 An isolated protein obtainable by recovering the isolated protein compound of claim 3 under non reducing conditions.\",\n \"5 An isolated cDNA obtainable by: collecting cells selected from the group consisting of total blood cells and PBL5 incubating the collected cell population with a CD3 activator at 1 microgram per ml, extracting and purifying the whole mRNA population from said cells, synthesizing every complementary cDNA, operably cloning each cDNA so that expression of this cDNA in this clone is under appropriate clone culture conditions, and cultivating every clone accordingly, selecting those clones which express a compound onto which the monoclonal antibody of claim 1 binds when placed under conditions suitable for this monoclonal antibody to perform reactions of the antigen-antibody type, and/or of which lysate would give an immuno-precipitation reaction with this mAb, optionally, amplifying those clones that have been thus selected, and recovering the inserted cDNA from the selected clones.\",\n \"6 An isolated mRNA obtainable by selection among the mRNA population of cells selected from the group consisting of total blood cells and PBL, of a mRNA which is complementary to a cDNA of claim 5.7 An isolated genomic DNA encoding an isolated mRNA of claim 6.8 An engineered cell in which a cDNA according to claim 5, has been transfected.\",\n \"9 An isolated protein encoded by an isolated cDNA according to claim 5.10 A bank of polypeptide compounds, obtainable by cleavage of a protein according to claim 3, with an enzyme selected from the group consisting of V8 protease and alpha chymotrypsine.\",\n \"11 An isolated polypeptide compound obtainable by: collecting PBL cells or total blood cells, incubating the collected cells with a CD3 activator at 1 microgram par ml, labeling the cells with a polypeptide-specific label, lysing the cells in a lysis buffer comprising Trition X-100 at 1%, and submitting the lysate to an immuno-precipitation reaction with the mAb of claim 1, and recovering from the digested immuno-precipitate any compound onto which bear a label.\",\n \"12 A monoclonal antibody obtainable by: (i) immunizing an animal against a protein according to claim 3, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein or to the polypeptide compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells maizy at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with We a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.\",\n \"for binding to a protein according to claim 3, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.\",\n \"13 A fragment of a monoclonal antibody according to claim 1, selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3.14 An isolated compound, comprising a fragment according to claim 13.15 The isolated compound according to claim 14, wherein it is a humanized antibody.\",\n \"16 The isolated compound according to claim 14, wherein it further comprises at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3 fragments of anti-CD4 antibodies.\",\n \"17 An isolated protein of SEQ ID No.\",\n \"2.18 The isolated DNA encoding an isolated protein according to claim 17.19 An isolated DNA of SEQ No.\",\n \"1.20 A polypeptidic vector comprising a monoclonal antibody according to claim 1, or a fragment thereof selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2 and CDR3.21 A polypeptidic vector for use in the treatment of CTCL, wherein said vector comprises a p140 binding compound.\",\n \"22 The polypeptidic vector according to claim 20, further comprising an element selected from the group consisting of tumoral toxins and radioelements.\",\n \"23 The polypeptidic vector according to claim 20, further comprising an enzyme capable of transforming an anti-mitotic pro-drug into an active drug form.\",\n \"24 A medical kit comprising a polypeptidic vector according to claim 23 and an antimitotic pro-drug.\",\n \"25 A medicament comprising, as an active principle, an element selected from the group consisting of a monoclonal antibody according to claim 1, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody according to claim 1.26 A method for manufacturing an anti-CTCL medicament comprising including a p140 binding compound in said medicament.\",\n \"27 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a protein according to claim 3 in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.\",\n \"28 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a protein according to claim 3, in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.\",\n \"29 A method for CTCL diagnosis, comprising measuring the percentage of CD4+ cells expressing an element selected from the group consisting of the proteins according to claim 3, in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.\",\n \"30 A method for CTCL diagnosis, comprising determining the presence of CD4+ cells expressing p140 in a biological sample which comprises potential CTCL cells, a CTCL-positive diagnosis being made when such a presence is significantly detected.\",\n \"31 A method according to claim 27, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody according to claim 1 and the DNA encoding SEQ ID No.\",\n \"3 or SEQ ID No.\",\n \"4.32 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises an element selected from the group consisting of the monoclonal antibodies according to claim 1, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody according to claim 1.33 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a protein according to claim 3, or to a p140 molecule, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.\",\n \"34 The isolated protein of claim 4, wherein said non reducing conditions are of chromatography by affinity with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.\",\n \"35 The isolated cDNA of claim 5, wherein said CD3 activator is PHA at 1 microgram per ml.\",\n \"36 An engineered cell in which a mRNA according to claim 6 has been transfected.\",\n \"37 An engineered cell in which a DNA according to claim 7 has been transfected.\",\n \"38 An isolated protein encoded by an isolated mRNA according to claim 6.39 An isolated protein encoded by an isolated cDNA according to claim 7.40 A bank of polypeptide compounds, obtainable by cleavage of a protein according to claim 4, with an enzyme selected from the group consisting of V8 protease and alpha chymotrypsine.\",\n \"41 A bank of polypeptide compounds, obtainable by cleavage of a protein according to claim 9, with an enzyme selected from the group consisting of V8 protease and alpha chymotrypsine.\",\n \"42 A monoclonal antibody obtainable by: (i) immunizing an animal against a protein according to claim 4, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.\",\n \"for binding to a protein according to claim 4, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.\",\n \"43 A monoclonal antibody obtainable by: (i) immunizing an animal against a protein according to claim 9, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.\",\n \"for binding to a protein according to claim 9, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.\",\n \"44 A monoclonal antibody obtainable by: (i) immunizing an animal against a polypeptide compound according to claim 11, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the polypeptide compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.\",\n \"for binding to a polypeptide according to claim 11, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.\",\n \"45 A fragment of a monoclonal antibody according to claim 12, selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3.46 The isolated compound according to claim 15, wherein it further comprises at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3 fragments of anti-CD4 antibodies.\",\n \"47 The polypeptidic vector according to claim 20, wherein said fragment is the Fab or F(ab′)2 fragment.\",\n \"48 A polypeptidic vector comprising an isolated compound according to claim 15, optionally including at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3 fragments of anti-CD4 antibodies.\",\n \"49 The polypeptidic vector according to claim 21, further comprising an element selected from the group consisting of tumoral toxins and radioelements.\",\n \"50 The polypeptidic vector according to claim 21, further comprising an enzyme capable of transforming an anti-mitotic pro-drug into an active drug form 51 The polypeptidic vector according to claim 23, wherein said enzyme capable of transforming an anti-mitotic pro-drug into an active drug form is a carboxypeptidase.\",\n \"52 The polypeptidic vector according to claim 50, wherein said enzyme capable of transforming an anti-mitotic pro-drug into an active drug form is a carboxypeptidase.\",\n \"53 The medical kit according to claim 24, wherein said antimitotic pro-drug is phenol mustard pro-drug.\",\n \"54 A medicament comprising, as an active principle, an element selected from the group consisting of a monoclonal antibody according to claim 12, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.\",\n \"55 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a protein according to claim 4 in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.\",\n \"56 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a protein according to claim 9 in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.\",\n \"57 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a member selected from the group consisting of the polypeptide compounds of claim 11 and the p140 molecules in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.\",\n \"58 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a protein according to claim 4, in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.\",\n \"59 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a protein according to claim 9, in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.\",\n \"60 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a member selected from the group consisting of the polypeptide compounds of claim 11 and the p140 molecules in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.\",\n \"61 A method for CTCL diagnosis comprising measuring the percentage of CD4+ cells expressing a protein according to claim 4 in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.\",\n \"62 A method for CTCL diagnosis comprising measuring the percentage of CD4+ cells expressing a protein according to claim 9 in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.\",\n \"63 A method for CTCL diagnosis comprising measuring the percentage of CD4+ cells expressing the polypeptide compounds of claim 11 in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.\",\n \"64 A method according to claim 28, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.\",\n \"65 A method according to claim 29, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.\",\n \"66 A method according to claim 30, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody, and the DNA encoding SEQ ID No.\",\n \"3 or SEQ ID No.\",\n \"4.67 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises an element selected from the group consisting of the monoclonal antibodies according to claim 12, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.\",\n \"68 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises at least one polypeptide compound selected from the bank of polypeptide compounds according to claim 10.69 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises an element selected from the group consisting the cDNA according to claim 5, an mRNA complementary to said cDNA, a p140 binding compound, a p140 DNA, an isolated DNA encoding an isolated protein of SEQ ID NO: 2, an isolated DNA of SEQ ID NO: 1, the DNA encoding SEQ ID No.\",\n \"3 and the DNA encoding SEQ ID No.\",\n \"4.70 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a protein according to claim 4, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.\",\n \"71 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a protein according to claim 9, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.\",\n \"72 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a polypeptide compound according to claim 10, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.\",\n \"73 The method according to claim 33, wherein said p140 molecule is of SEQ ID No.\",\n \"2 or SEQ ID No.\",\n \"4.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>CTCL is a group of T lymphomas which primarily involve the skin.\",\n \"The CTCL group namely comprises transformed Mycosis Fungoides (abbreviated into transformed MF), Sézary Syndrome (abbreviated into SS), Lymphomatoide Papulosis (abbreviated into LP), and CD30+ lymphomas.\",\n \"Transformed MF is characterized by skin invasion of clonally-derived malignant T lymphocytes that phenotypically resemble mature T helper cells.\",\n \"LP and CD30+ lymphomas also develop in the skin.\",\n \"More aggressive forms of CTCL develop when the malignant cells become non-epidermotropic, and are associated with extra-cutaneous involvement.\",\n \"SS is a more aggressive form of CTCL that is characterized by a clonal expansion of CD4+/CD45RO+ T cells and the appearance of these malignant T cells in the blood.\",\n \"Most CTCL are CD4+ CTCL, but some rare CD8+ CTCL cases exist.\",\n \"The biology of CTCL disease remains poorly understood, as it is difficult to identify the malignant cells, due to the lack of specific cell surface markers.\",\n \"In cutaneous lesions, it is therefore difficult to distinguish CTCL cells from reactive infiltrating (non-tumoral) T lymphocytes.\",\n \"Diagnosis of T lymphomas such as CTCL is at present time mainly based on cytological and histological observations of the presence or absence of tumoral cells in a sample collected from a suspected body area (observation of histopathological aspect on skin biopsis and/or presence of SS cells in the blood, via detection of cells showing a cerebriform nucleus).\",\n \"Such a diagnosis method is not fully reliable, notably at the early stages of the transformation of skin lymphocytes into malignant lymphocytes.\",\n \"Such a diagnosis method does also not enable to stage the disease.\",\n \"Technically speaking, it is also time-consuming.\",\n \"Today therapy of CTCL is tentatively achieved by induction of tumoral cell apoptosis via non specific chemotherapy.\",\n \"There is therefore still a need for more accurate and appropriate solutions to the problem of the diagnosis and therapy of diseases involving the proliferation of malignant T cells, such as CTCL.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>In view of this prior art situation, the inventors now provide with two molecules which give solutions to the problem of CTCL diagnostic and therapy: one has been termed SC5 by the inventors, and the other one is the p140 molecule (p140 is also referred to as KIR3DL2).\",\n \"The SC5 molecule of the invention appears as a biochemically and functionally new protein, of which apparent molecular weight is of 96 kD under reducing conditions.\",\n \"It has been isolated as the antigen of a monoclonal antibody that has been produced by the inventors.\",\n \"The hybridoma producing this monoclonal antibody has been deposited on Oct. 30, 2000 at the C.N.C.M.\",\n \"(C.N.C.M.\",\n \"Institut Pasteur; 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France) in accordance with the Budapest Treaty (C.N.C.M.\",\n \"deposit number: I-2575).\",\n \"When expressed at the cell surface, and made to aggregate, SC5 molecules act as inhibitory receptors for cell activity and proliferation.\",\n \"The second marker of the invention, i.e.\",\n \"p140, was already known to be an inhibitory receptor, but has been previously described only on sub-groups of NK cells and of CD3+ CD8+ cells from healthy humans.\",\n \"The inventors now demonstrate that p140 is expressed at the surface of tumoral T cells such as CTCL cells.\",\n \"They further demonstrate that p140 is expressed at the surface of malignant CD4+ T cells, such as CD4+ CTCL cells, whereas those other receptors that are usually observed at the surface of NK cells (such as p58.1, p58.2, p70 KIRs, CD94/NKG2A) are not found at the surface of malignant CD4+ T cells.\",\n \"Of further note is that p140 has not been observed by the inventors at the surface of CD4+ T cells collected from patients suffering from non-tumoral dermatological diseases such as inflammatory skin diseases (e.g.\",\n \"lupus, lichen), or toxic epidermal necrolysis.\",\n \"At the surface of tumoral CD4+ T cells, two allelic forms of p140 have been identified by the inventors: allelic form KIR3D clone 24 (SEQ ID No.1), and allelic form p140 clone 1.1 (SEQ ID No.3).\",\n \"Allelic form KIR3D clone 24 (SEQ ID No.\",\n \"1) is a new polynucleotide encoding a new protein (SEQ ID No.2): KIR3D clone 24 (SEQ ID No.\",\n \"1) displays five differences when compared to the previously described p140 clone 1.1 DNA sequence (SEQ ID No.\",\n \"3), resulting in four amino acid substitutions in the mature protein (SEQ ID No.\",\n \"2) when compared to p140 clone 1.1.mature protein (SEQ ID No.\",\n \"4).\",\n \"The inventors demonstrate that the SC5 and p140 both share the same technical following features: SC5 and p140 both are membranar differentiation antigens which are characteristic of malignant T cells, and notably of malignant CD4+ T cells, whichever form of CD4+ CTCL is concerned, there are malignant CD4+ T cells which express SC5 or p140 at their surface (usually both SC5 and p140 are expressed): SC5 as well as p140 indeed cover the whole range of CD4+ CTCL, and notably the Sézary Syndrome (abbreviated into “SS”), transformed Mycosis Fungoides (abbreviated into “transformed MF”), Lymphomatoide Papulosis (abbreviated onto “LP”), and CD30+ lymphomas, there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that a CD4+ CTCL diagnosis based on the analysis of the presence of SC5 or p140 at the surface of CD4+ cells collected from the suspected body area (e.g.\",\n \"sample of skin erythroderma when transformed MF is suspected, or sample of peripheral blood when a more aggressive CTCL form, such as SS, is suspected) has a reliability of more than 90%, preferably of more than of 95%, most preferably of 100% (among those patients who were tested up to now, the reliability is of 100%): indeed, according to the invention, it can be concluded that a CD4+ T cell is tumoral as soon as there are p140 molecules detected at the surface of these CD4+ T cells, or as soon as a percentage of SC5+ CD4+ T cells higher than the average standard level is measured (the average standard level is in the 1-15% range, generally in the 5-10% range), and there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that the percentage of CD4+ SC5+ T cells, as well as of CD4+ p140+ T cells, that is measured in a sample of peripheral blood collected from a patient for whom a Sézary Syndrome (SS) is suspected, substantially corresponds to the percentage of malignant SS cells that are actually present in the peripheral blood of this patient (within a ±10% range for SC5+ CD4+ cells, within a ±5% range for p140+ CD4+ cells): the SC5 and p140 markers of the invention therefore share the common particular technical feature of enabling to assess the staging of a SS.\",\n \"To the best of the inventors' knowledge, SC5 and p140 are the first molecules which are described as having these common technical features.\",\n \"The invention thus provides with the first CTCL universal markers.\",\n \"No prior art product was known to be such a CTCL universal marker.\",\n \"The closest prior art product in this respect would be CD30 of which presence at the surface of malignant CD4+ T cells directs to the conclusion that the patient has a particular form of CD4+ CTCL which is referred to in the art as CD30+ lymphoma.\",\n \"CD30 is therefore a CTCL marker, but its reliability is limited to a particular form of CTCL (CD30+ lymphomas), and CD30 does not cover every form of CD4+ CTCL: for CD4+ CTCL such as SS, transformed MF, or LP, there does not necessarily exist a malignant CD4+ T cell which would express CD30 at its surface.\",\n \"CD30 uses in CTCL diagnosis and therapy is thus restricted to a particular form of CTCL, whereas the markers of the invention advantageously cover the whole range of CD4+ CTCL.\",\n \"In addition to the technical features shared with p140, SC5 has the further characteristic of being a positive indicator of proliferation and/or functional activity (e.g.\",\n \"cytokine profile) of non-tumoral T cells.\",\n \"The presence of SC5 at the surface of T cells is indeed positively linked with the activation status of non-tumoral T cells, such as normal (CD4+ and CD8+) T cells or virus-infected CD4+ T cells.\",\n \"SC5 is thus also a useful novel means for the diagnostic and therapy of T cell viral infections such as HIV-infection, and of inflammatory diseases such as those of the auto-immune type (e.g.\",\n \"rhumatoid arthritis such as spondyloarthropathis, or skin immune mediated diseases such as psoriasis, eczema, atopic dermatitis).\",\n \"SC5 is also a useful target for modulating graft-host reactions: the activation of SC5 transduces an inhibitory signal that can be used to inhibit the reactions that effector recipient cells may develop against a graft.\",\n \"The invention also provides with products capable of binding to the SC5 new molecule, and in particular with monoclonal antibodies directed against SC5 (anti-SC5 mAbs).\",\n \"It is also provided with anti-SC5 monoclonal antibodies which are capable of modulating the proliferation and/or the functional activity of T cells, and notably of tumoral T cells such as malignant CTCL cells.\",\n \"In particular, the monoclonal antibody produced by hybridoma I-2575 is capable of inhibiting the proliferation and/or functional activity of T cells, and according to a particularly useful feature, is capable of inhibiting the proliferation and/or activity of malignant T cells such as malignant CTCL cells (see examples below).\",\n \"Compounds binding to SC5, but not aggregating them will prevent SC5 inhibitory signal transduction, or will prevent compounds to reach SC5+ cells (target masking).\",\n \"More generally, compounds binding to SC5 or p140 may, according to the invention, be used as complement recruiting agents, as ADCC stimulators, or as vectors for therapeutic agents so as to prevent, palliate, treat T lymphomas, such as CTCL.\",\n \"The invention therefore also relates to the diagnostic and therapeutic uses of SC5- or p140-binding compounds such as anti-SC5 and/or anti-p140 monoclonal antibodies, and of products directly-derived therefrom (e.g.\",\n \"humanized monoclonal antibodies, or monoclonal antibodies with a double CD4-p140/SC5 specificity).\"\n ],\n [\n \"FIELD OF THE INVENTION The present application relates to novel means for the diagnosis and therapy of T lymphomas, and more particularly of Cutaneous T Cell Lymphomas (abbreviated into CTCL).\",\n \"The invention indeed provides novel tumor markers which are universal for CTCL, and describes biotechnological and medical uses thereof.\",\n \"BACKGROUND OF THE INVENTION CTCL is a group of T lymphomas which primarily involve the skin.\",\n \"The CTCL group namely comprises transformed Mycosis Fungoides (abbreviated into transformed MF), Sézary Syndrome (abbreviated into SS), Lymphomatoide Papulosis (abbreviated into LP), and CD30+ lymphomas.\",\n \"Transformed MF is characterized by skin invasion of clonally-derived malignant T lymphocytes that phenotypically resemble mature T helper cells.\",\n \"LP and CD30+ lymphomas also develop in the skin.\",\n \"More aggressive forms of CTCL develop when the malignant cells become non-epidermotropic, and are associated with extra-cutaneous involvement.\",\n \"SS is a more aggressive form of CTCL that is characterized by a clonal expansion of CD4+/CD45RO+ T cells and the appearance of these malignant T cells in the blood.\",\n \"Most CTCL are CD4+ CTCL, but some rare CD8+ CTCL cases exist.\",\n \"The biology of CTCL disease remains poorly understood, as it is difficult to identify the malignant cells, due to the lack of specific cell surface markers.\",\n \"In cutaneous lesions, it is therefore difficult to distinguish CTCL cells from reactive infiltrating (non-tumoral) T lymphocytes.\",\n \"Diagnosis of T lymphomas such as CTCL is at present time mainly based on cytological and histological observations of the presence or absence of tumoral cells in a sample collected from a suspected body area (observation of histopathological aspect on skin biopsis and/or presence of SS cells in the blood, via detection of cells showing a cerebriform nucleus).\",\n \"Such a diagnosis method is not fully reliable, notably at the early stages of the transformation of skin lymphocytes into malignant lymphocytes.\",\n \"Such a diagnosis method does also not enable to stage the disease.\",\n \"Technically speaking, it is also time-consuming.\",\n \"Today therapy of CTCL is tentatively achieved by induction of tumoral cell apoptosis via non specific chemotherapy.\",\n \"There is therefore still a need for more accurate and appropriate solutions to the problem of the diagnosis and therapy of diseases involving the proliferation of malignant T cells, such as CTCL.\",\n \"SUMMARY OF THE INVENTION In view of this prior art situation, the inventors now provide with two molecules which give solutions to the problem of CTCL diagnostic and therapy: one has been termed SC5 by the inventors, and the other one is the p140 molecule (p140 is also referred to as KIR3DL2).\",\n \"The SC5 molecule of the invention appears as a biochemically and functionally new protein, of which apparent molecular weight is of 96 kD under reducing conditions.\",\n \"It has been isolated as the antigen of a monoclonal antibody that has been produced by the inventors.\",\n \"The hybridoma producing this monoclonal antibody has been deposited on Oct. 30, 2000 at the C.N.C.M.\",\n \"(C.N.C.M.\",\n \"Institut Pasteur; 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France) in accordance with the Budapest Treaty (C.N.C.M.\",\n \"deposit number: I-2575).\",\n \"When expressed at the cell surface, and made to aggregate, SC5 molecules act as inhibitory receptors for cell activity and proliferation.\",\n \"The second marker of the invention, i.e.\",\n \"p140, was already known to be an inhibitory receptor, but has been previously described only on sub-groups of NK cells and of CD3+ CD8+ cells from healthy humans.\",\n \"The inventors now demonstrate that p140 is expressed at the surface of tumoral T cells such as CTCL cells.\",\n \"They further demonstrate that p140 is expressed at the surface of malignant CD4+ T cells, such as CD4+ CTCL cells, whereas those other receptors that are usually observed at the surface of NK cells (such as p58.1, p58.2, p70 KIRs, CD94/NKG2A) are not found at the surface of malignant CD4+ T cells.\",\n \"Of further note is that p140 has not been observed by the inventors at the surface of CD4+ T cells collected from patients suffering from non-tumoral dermatological diseases such as inflammatory skin diseases (e.g.\",\n \"lupus, lichen), or toxic epidermal necrolysis.\",\n \"At the surface of tumoral CD4+ T cells, two allelic forms of p140 have been identified by the inventors: allelic form KIR3D clone 24 (SEQ ID No.1), and allelic form p140 clone 1.1 (SEQ ID No.3).\",\n \"Allelic form KIR3D clone 24 (SEQ ID No.\",\n \"1) is a new polynucleotide encoding a new protein (SEQ ID No.2): KIR3D clone 24 (SEQ ID No.\",\n \"1) displays five differences when compared to the previously described p140 clone 1.1 DNA sequence (SEQ ID No.\",\n \"3), resulting in four amino acid substitutions in the mature protein (SEQ ID No.\",\n \"2) when compared to p140 clone 1.1.mature protein (SEQ ID No.\",\n \"4).\",\n \"The inventors demonstrate that the SC5 and p140 both share the same technical following features: SC5 and p140 both are membranar differentiation antigens which are characteristic of malignant T cells, and notably of malignant CD4+ T cells, whichever form of CD4+ CTCL is concerned, there are malignant CD4+ T cells which express SC5 or p140 at their surface (usually both SC5 and p140 are expressed): SC5 as well as p140 indeed cover the whole range of CD4+ CTCL, and notably the Sézary Syndrome (abbreviated into “SS”), transformed Mycosis Fungoides (abbreviated into “transformed MF”), Lymphomatoide Papulosis (abbreviated onto “LP”), and CD30+ lymphomas, there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that a CD4+ CTCL diagnosis based on the analysis of the presence of SC5 or p140 at the surface of CD4+ cells collected from the suspected body area (e.g.\",\n \"sample of skin erythroderma when transformed MF is suspected, or sample of peripheral blood when a more aggressive CTCL form, such as SS, is suspected) has a reliability of more than 90%, preferably of more than of 95%, most preferably of 100% (among those patients who were tested up to now, the reliability is of 100%): indeed, according to the invention, it can be concluded that a CD4+ T cell is tumoral as soon as there are p140 molecules detected at the surface of these CD4+ T cells, or as soon as a percentage of SC5+ CD4+ T cells higher than the average standard level is measured (the average standard level is in the 1-15% range, generally in the 5-10% range), and there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that the percentage of CD4+ SC5+ T cells, as well as of CD4+ p140+ T cells, that is measured in a sample of peripheral blood collected from a patient for whom a Sézary Syndrome (SS) is suspected, substantially corresponds to the percentage of malignant SS cells that are actually present in the peripheral blood of this patient (within a ±10% range for SC5+ CD4+ cells, within a ±5% range for p140+ CD4+ cells): the SC5 and p140 markers of the invention therefore share the common particular technical feature of enabling to assess the staging of a SS.\",\n \"To the best of the inventors' knowledge, SC5 and p140 are the first molecules which are described as having these common technical features.\",\n \"The invention thus provides with the first CTCL universal markers.\",\n \"No prior art product was known to be such a CTCL universal marker.\",\n \"The closest prior art product in this respect would be CD30 of which presence at the surface of malignant CD4+ T cells directs to the conclusion that the patient has a particular form of CD4+ CTCL which is referred to in the art as CD30+ lymphoma.\",\n \"CD30 is therefore a CTCL marker, but its reliability is limited to a particular form of CTCL (CD30+ lymphomas), and CD30 does not cover every form of CD4+ CTCL: for CD4+ CTCL such as SS, transformed MF, or LP, there does not necessarily exist a malignant CD4+ T cell which would express CD30 at its surface.\",\n \"CD30 uses in CTCL diagnosis and therapy is thus restricted to a particular form of CTCL, whereas the markers of the invention advantageously cover the whole range of CD4+ CTCL.\",\n \"In addition to the technical features shared with p140, SC5 has the further characteristic of being a positive indicator of proliferation and/or functional activity (e.g.\",\n \"cytokine profile) of non-tumoral T cells.\",\n \"The presence of SC5 at the surface of T cells is indeed positively linked with the activation status of non-tumoral T cells, such as normal (CD4+ and CD8+) T cells or virus-infected CD4+ T cells.\",\n \"SC5 is thus also a useful novel means for the diagnostic and therapy of T cell viral infections such as HIV-infection, and of inflammatory diseases such as those of the auto-immune type (e.g.\",\n \"rhumatoid arthritis such as spondyloarthropathis, or skin immune mediated diseases such as psoriasis, eczema, atopic dermatitis).\",\n \"SC5 is also a useful target for modulating graft-host reactions: the activation of SC5 transduces an inhibitory signal that can be used to inhibit the reactions that effector recipient cells may develop against a graft.\",\n \"The invention also provides with products capable of binding to the SC5 new molecule, and in particular with monoclonal antibodies directed against SC5 (anti-SC5 mAbs).\",\n \"It is also provided with anti-SC5 monoclonal antibodies which are capable of modulating the proliferation and/or the functional activity of T cells, and notably of tumoral T cells such as malignant CTCL cells.\",\n \"In particular, the monoclonal antibody produced by hybridoma I-2575 is capable of inhibiting the proliferation and/or functional activity of T cells, and according to a particularly useful feature, is capable of inhibiting the proliferation and/or activity of malignant T cells such as malignant CTCL cells (see examples below).\",\n \"Compounds binding to SC5, but not aggregating them will prevent SC5 inhibitory signal transduction, or will prevent compounds to reach SC5+ cells (target masking).\",\n \"More generally, compounds binding to SC5 or p140 may, according to the invention, be used as complement recruiting agents, as ADCC stimulators, or as vectors for therapeutic agents so as to prevent, palliate, treat T lymphomas, such as CTCL.\",\n \"The invention therefore also relates to the diagnostic and therapeutic uses of SC5- or p140-binding compounds such as anti-SC5 and/or anti-p140 monoclonal antibodies, and of products directly-derived therefrom (e.g.\",\n \"humanized monoclonal antibodies, or monoclonal antibodies with a double CD4-p140/SC5 specificity).\",\n \"DETAILED DESCRIPTION OF THE INVENTION The invention thus provides with an hybridoma deposited on Oct. 30, 2000 as deposit number I-2575 at the C.N.C.M.\",\n \"in accordance with the Budapest Treaty (C.N.C.M.\",\n \"Collection Nationale de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, F-75724 Paris Cedex 15, France), and with a method for preparing an anti-SC5 monoclonal antibody which comprises cultivating hybridoma I-2575 under conditions suitable for the metabolism of the hybridoma cells (e.g.\",\n \"culture for three days at 37° C., with 5% CO2 in a humid atmosphere at 2.106 cells par ml of appropriate culture medium such as RPMI 1640 medium Gibco BRL Cat.\",\n \"No21875-034, with 10% Fetal Calf Serum, penicillin 100 U/ml and streptomycin 100 microgram/ml).\",\n \"The invention therefore encompasses the monoclonal antibody (mAb) which is produced by hybridoma I-2575, or which is obtainable from the culture supernatant of this deposited hybridoma.\",\n \"This mAb is a pentameric anti-SC5 IgM.\",\n \"It is a useful tool for the diagnosis and therapy of an inappropriate or undesired T cell proliferation and/or functional activity (cytokine profile), of an inappropriate or undesired IL-2 production.\",\n \"Its inhibits the proliferation and/or functional activity of normal T cells, of virus-infected T cells (e.g.\",\n \"HIV-infected CD4+ T cells), and of malignant T cells, such as notably malignant CTCL cells.\",\n \"The monoclonal antibody produced by hybridoma I-2575 enables the isolation of a is protein, termed SC5 by the inventors.\",\n \"SC5 protein appears as a biochemically and functionally new molecule, with an apparent molecular weight of 96 kD under reducing conditions.\",\n \"It has been located in and on numerous PBL cells: normal T cells (both from CD4+ and CD8+ subsets), and in particular CD3+ T cells, CD45RO+ T cells, but also normal NK cells, B cells, granulocytes and macrophages.\",\n \"In addition, SC5 has been located in the cytoplasm of cells from other lineages and other species such as NIH/3T3 (ATCC CRL-1658) and CHO-K1 (ATCC CRL-9618).\",\n \"In resting normal PBL cells, SC5 is mainly intracellularly located, but CD3 stimulation induces the transfer of SC5 to the cell membrane.\",\n \"Remarkably, SC5 has also been observed by the inventors on malignant T cells, such as malignant CTCL cells, and in particular malignant CD4+ CTCL cells, where they are mainly located at the cell surface (without any ex vivo CD3 stimulation).\",\n \"As a further remarkable feature, SC5 surface expression has been observed for all the CTCL patients who were tested until now (see the examples below).\",\n \"Another further remarkable feature of the invention is that the percentage of CD4+ T cells which co-express SC5 is statistically higher in patients developing tumoral CD4+ T cells than in healthy patients.\",\n \"The percentage of SC5+ CD4+ cells in CD4+ CTCL patients is significantly higher than the percentage observed in healthy patients (average standard percentage of SC5+ CD4+ cells is in the 1-15% range, generally in the 5-10% range).\",\n \"In the particular CD4+ CTCL case of Sézary Syndrome (SS), the percentage of CD4+ cells which co-express SC5 is furthermore statistically closely correlated to the percentage of malignant CTCL cells in the PBL of the CTCL patient: the percentage of SC5+ CD4+ T cells is substantially equal to the percentage of malignant CTCL cells (within ±10% of this percentage).\",\n \"SC5 protein is therefore a new molecule whose transmembrane expression at the cell surface is linked to an activation and/or proliferation status for non-tumoral T cells (such as virus-infected cells, e.g.\",\n \"HIV-infected cells, and such as normal CD4+ or CD8+ T cells), and to a malignant status for non-activated T cells.\",\n \"According to a remarkable feature, it enables a CD4+ CTCL-positive diagnosis with a confidence of more than 90% (actually of 100% of those humans who have been tested up-to-date).\",\n \"A further outstanding feature of the invention is that the SC5 new molecule is capable.\",\n \"of acting as an inhibitory receptor for cell proliferation and/or functional activity when expressed at the cell surface: SC5 aggregation induces a decrease in the proliferation and activity of the cell expressing it.\",\n \"For example, the anti-SC5 monoclonal antibody produced by hybridoma I-2575 (which is a pentameric Ig) inhibits the anti-CD3 induced proliferation of SC5+ cells such as normal CD3+ T cells collected from a human or an animal, or normal CD3+ T cell clones, and also inhibits the anti-CD3 induced in vitro proliferation of CTCL cells collected from a CTCL patient, and the in vivo natural proliferation of human CTCL cells in an animal model.\",\n \"This mAb also binds HIV-infected CD4+ T cells, and is therefore useful as an active principle in an anti-HIV drug intended for killing infected cells, and therefore reducing HIV propagation.\",\n \"Several procedures enabling the isolation of the SC5 protein are available to the skilled person to whom a monoclonal antibody of the invention has been made available.\",\n \"Standard procedures comprise recovering the SC5 molecules from a cell lysate by affinity chromatography, or by electrophoresis under reducing conditions.\",\n \"All human or animal cells which the monoclonal antibody produced by hybridoma I-2575 recognizes as an antigen, or of which lysate gives an immuno-precipitation reaction with this monoclonal antibody, are appropriate cell sources for isolating the SC5 molecule.\",\n \"Such cells notably include normal cells (collected from a human or an animal, or cell clones) such as T cells, CD3+ T cells, CD45RO+ T cells, B cells, NK cells, macrophages, granulocytes.\",\n \"Appropriate cells also include tumoral cells such as malignant T cells, e.g.\",\n \"CD4+ CTCL cells collected from a CTCL patient, or tumoral clones such as the CTCL clone HUT78 (ATCC TIB-161).\",\n \"Due to the wide range of appropriate cell sources, it will be understood that rough total blood cell or PBL may be directly used as a SC5 source without any further purification.\",\n \"Normal cells enables the isolation of both the intracellular form and the transmembrane receptor form of SC5.When it is desired to isolate the SC5 receptor form, or a portion thereof, it may thus be preferable to use a source of SC5+ cells (i.e.\",\n \"cells expressing SC5 at their surface).\",\n \"Such a source include normal cells as above-mentioned but that have been CD3-stimulated so that SC5 expression increases at the cell surface (see examples below for CD3 stimulation procedures).\",\n \"When total blood cells or PBL are used as a source of SC5+ cells, CD3 activation can be induced by incubation with a polyclonal activator such as PHA (obtainable from Wellcome) at a concentration of about 1 microgram per ml, because total blood cells or PBL also contain T cells and mononuclear cells.\",\n \"When a more homogeneous normal cell population is used as a source of SC5+ cells (e.g.\",\n \"only SC5+ NK cells), then CD3 activation is preferably performed by incubation with immobilized anti-CD3 antibodies, or alternatively with both soluble anti-CD3 antibodies and polyclonal activator such as PHA.\",\n \"Optionally, a culture step may be performed before and/or after CD3 activation so as to increase cell amount (e.g.\",\n \"culture for 3 days in RPMI 10% fetal calf serum at 37° C., 95% humidity, and 5% CO2).\",\n \"When it is desired to isolate SC5 in its intracellular form, then CD3 stimulation may be omitted.\",\n \"Tumoral cells such as CTCL cells, and notably malignant transformed MF or SS cells, enable the isolation of SC5 molecules in their receptor form, without any required ex vivo CD3 activation: CTCL cells already express SC5 in a transmembranar location.\",\n \"The invention therefore encompasses any isolated protein obtainable by: (i) collecting cells selected from the group consisting of T cells, CD3+ T cells, CD45RO+ T cells, B cells, NIK cells, macrophages, granulocytes, and stimulating the collected cells with immobilized anti-CD3 antibodies, or with both soluble anti-CD3 antibodies and PHA at 1 micrograimn/ml, or collecting malignant CTCL cells such as malignant CD4+ CTCL cells, or collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), and stimulating the collected cells with PHA at about 1 microgram per ml, (ii) lysing the cells under conditions enabling the dissociation of multimeric polypeptidic complexes, for example by incubation in a drastic lysis buffer containing Triton X-100 at 1%, (iii) recovering from the lysate the protein compound onto which the monoclonal antibody produced by hybridoma I-2575 binds under conditions enabling this monoclonal antibody to perform reactions of the antigen-antibody type (i.e.\",\n \"the compound which is recognized as an antigen by this mAb).\",\n \"The aminoacid sequence of the recovered protein compound can be obtained by any standard technique, such as mass spectrophotometry.\",\n \"As above-indicated, the cells mentioned in step i. are activated cells, and therefore enables the isolation of SC5 in the form it has when it is a transmembrane receptor.\",\n \"If the SC5 source used in step i. consists of resting normal T cells (without ex vivo stimulation), the the protein compound thus isolated will be SC5 in its cytoplamsic form.\",\n \"When the cells selected in step i. are activated or malignant cells, lysis in step ii.\",\n \"therefore may be performed either on whole cells, or on the membranar fraction of these cells.\",\n \"The lysis step itself can be achieved by any technique enabling the dissociation of multimeric polypeptidic complexes from each others.\",\n \"For example, cells (or their membrane fraction) may be incubated in a lysis buffer containing a drastic detergent such as Triton X-100 at about 1% for 1 hour at 4° C.; this enables the dissociation of the SC5 protein from the other cell components.\",\n \"If a mild detergent such as digitonin 1% or Bridg58 1% is used, then SC5 won't dissociate from those compounds which are naturally associated with it; the use of a mild detergent in the above step ii.\",\n \"therefore enables the isolation of SC5, and the further isolation of the SC5 transducers and effectors.\",\n \"The invention therefore also encompasses any isolated compound obtainable by such processes.\",\n \"The lysate obtained from step ii.\",\n \"is then incubated with the monoclonal antibody produced by hybridoma I-2575 under conditions appropriate for this mAb to perform reactions of the antigen-antibody type, such as coating the antibodies into microwells containing the cell lysate (e.g.\",\n \"2 microgrammes of the monoclonal antibody produced by hybridoma I-2575 for a lysate of 107 cells) and incubating the monoclonal antibodies with the cell lysate for two hours at 4° C. The step of recovering the compound onto which the monoclonal antibody produced by hybridoma I-2575 binds (step iii.)\",\n \"can be achieved by any technique available the skilled person.\",\n \"Recovering under non reducing conditions enables the isolation of SC5 in its native conformation, and will be preferred when it is desired to retain the native biological properties of SC5.Appropriate techniques for recovering SC5 protein under non reducing conditions namely include chromatography by affinity, and, for example: percolation of the lysate through a column in which the monoclonal antibody (mAb) produced by hybridoma I-2575 has been immobilized (e.g.\",\n \"by coupling this mAb onto agar beads and placing these beads inside said column); preferably, the lysate is mixed with a buffer which has a.physiological pH, e.g.\",\n \"at a pH comprised between 6 and 8, such as 7, before percolation so as to make the percolation easier without substantially altering the conformation of the compounds contained in the lysate, rinsing the column with a buffer at the same physiological pH, and recovering the compound which has bound to the immobilized mAb, e.g.\",\n \"by elution with a buffer which disrupts the binding of the compound onto the mAb without substantially modifying the native conformation of the compound.\",\n \"Such an elution buffer usually has a slightly acidic pH (usually comprised between 2 and 5, e.g.\",\n \"a pH of 3).\",\n \"For the preparation of a matrix which has an optimal orientation of antibody molecules, and which thereby enables antigen binding at a high efficiency, and for a one-step purification procedure for the isolation of membrane proteins, see e.g.\",\n \"Schneider et al.\",\n \"1982 (J. Biol.\",\n \"Chem.\",\n \"257: 10766-10769) of which content is herein incorporated by reference.\",\n \"The protein compound thus recovered is the isolated SC5 protein in its native form.\",\n \"Alternatively, step iii.\",\n \"under non reducing conditions can be achieved by placing the lysate obtained from step ii.\",\n \"for immuno-precipitation with the monoclonal produced by hybridoma I-2575, and the immuno-precipitate thus obtained can be percolated through a column comprising antibodies capable of binding mice IgM.\",\n \"The protein compound which has bound onto the monoclonal antibody produced by hybridoma I-2575 is then recovered by elution similarly to what has been above described.\",\n \"The protein compound thus recovered is also the isolated SC5 protein in its native form.\",\n \"Another standard way for recovering the SC5 protein (step iii.)\",\n \"is to proceed through electrophoresis under reducing conditions.\",\n \"This notably includes incubating the monoclonal antibody produced by hybridoma I-2575 with the lysate obtained from step ii.\",\n \"under conditions enabling this mAb to perform reactions of the antigen-antibody type, collecting the immuno-precipitate thus formed, separating the compounds contained in this immuno-precipitate under reducing conditions, and recovering the protein compound which has an apparent molecular weight of 96 kD.\",\n \"An alternative way to localize on the electrophoresis gel or membrane the desired SC5 protein (and to subsequently isolate it from the gel or membrane) is to proceed with a labeling step prior to lysis so that surface polypeptidic components of the cells are labeled (e.g.\",\n \"125 iode or fluorescent labeling with e.g.\",\n \"sulfo-NHS-LC-biotin), and to recover from the immuno-precipitate those compounds which bear the label.\",\n \"It has to be noted that if the labeling step is performed after lysis, then both the intra-cellular form and the trans-membranar receptor form of SC5 will be isolated (in this case, as above-indicated, non-activated T cells may be used as a SC5 source).\",\n \"Because denaturing conditions are used, the protein compound thus recovered is in a non native form: it still has the native amino acid sequence, but its native biological properties have been substantially lost.\",\n \"It is however the SC5 protein in its non-native conformation that is observed (and has to be searched for) when the biological sample to be tested has been treated under denaturing conditions (such as e.g.\",\n \"paraffin sections of skin sample).\",\n \"The SC5 protein in its non-native form may therefore be used for the production of monoclonal antibodies directed against it, such monoclonal antibodies being then useful for SC5 detection on denaturated samples.\",\n \"If the SC5 protein in its native conformation is desired, portions of at least 10 amino acids of the denaturated protein may then be sequenced (e.g.\",\n \"by mass spectrophotometry), oligonucleotides deduced from the sequenced portions may be synthetized, and then used as probes to screen a cDNA library obtained from SC5+ cells.\",\n \"The cDNA thus selected may the be produced and cloned into a cell under conditions enabling the production, and preferably the excretion, of the cDNA-encoded product by culture of the clone (e.g.\",\n \"via insertion of the SC5-encoding cDNA in a baculo virus vector and transformation of insect cells—e.g.\",\n \"Sf9 cells—, or via the vaccina virus and EBV cells, see Rindis Bacher 1995, J. Biol.\",\n \"Chem.\",\n \"270(23): 14220-14228).\",\n \"The invention therefore encompasses any isolated protein of which sequence is obtainable by: collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), stimulating the collected cells with PHA at 1 microgram/ml, labeling the stimulated cells with a polypeptide-specific label such as biotin, lysing the labeled cells by incubation in a lysis buffer containing Triton X-100 at 1%, submitting the lysate to immuno-precipitation with the monoclonal antibody produced by hybridoma I-2575, recovering from the immuno-precipitate the protein compound which bears the label, synthesing the cDNA which encodes the protein sequence thus obtained, operably transfecting insect cells with this cDNA, recovering the protein produced by the transfected cells, e.g.\",\n \"with the monoclonal antibody produced by hybridoma I-2575, or obtainable by: collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), stimulating the collected cells with PHA at 1 microgram/ml, lysing the labeled cells by incubation in a lysis buffer containing Triton X-100 at 1%, submitting the lysate to immuno-precipitation with the monoclonal antibody produced by hybridoma I-2575, recovering from the immuno-precipitate the protein compound which has an apparent molecular weight of 96 kD under reducing conditions, synthesing the cDNA which encodes the protein sequence thus obtained, operably transfecting insect cells with this cDNA, the protein compound produced by the transfected insect cells, e.g.\",\n \"with the monoclonal antibody produced by hybridoma I-2575.The invention thus encompasses any isolated protein compound obtainable by any of the above-mentioned processes, as well as any amino acid sequence of such an isolated protein compound, and any isolated protein of which sequence comprises such a sequence.\",\n \"Falls within the scope of the invention any solid support onto which a protein compound or protein of the invention has been placed.\",\n \"Such solid supports notably include sepharose beads.\",\n \"The invention also encompasses any isolated DNA (or cDNA) of which sequence codes for an isolated protein of the invention.\",\n \"It also relates to engineered cells transfected by such a DNA, to engineered cells which excrete an isolated protein of the invention, and to isolated protein obtainable by isolation from the culture medium of such engineered cells.\",\n \"The invention notably encompasses any cDNA obtainable by: collecting a cell population which consists of, or comprises cells onto which the monoclonal antibody produced by hybridoma I-2575 binds (i.e.\",\n \"onto which this mAb recognizes an antigen), or cells of which lysate would give an immuno-precipitation reaction with this monoclonal antibody, e.g.\",\n \"collecting cells selected from.\",\n \"the group consisting of total blood cells and PBL, if the sole transmembranar form of SC5 is desired: stimulating the CD3 pathway if said cell population mainly comprises or consists of non-activated cells (e.g.\",\n \"resting non-tumoral cells) so as to increase SC5 expression at the cell surface, e.g.\",\n \"by incubating the collected cell population with a CD3 activator (for example a polyclonal activator such as PHA at 1 microgram par ml when the collected cell population is PBL or total blood, or immobilized anti-CD3 antibodies, or both a polyclonal activator such as PHA and soluble anti-CD3 antibodies when the collected cell population is more homogeneous such as a cell population mainly comprising, or consisting of CD3+ T cells, or of CD45RO+ cells, or of NK cells, or of macrophages, or of granulocytes); if the collected cell population consists of, or mainly comprises activated cells (such as CD3-activated non-tumoral cells, or such as tumoral cells such as CD4+ CTCL cells), then CD3 stimulation is not necessary because SC5 location is already mainly transmembranar in these activated cells; if it is desired to isolate both the intracellular form and the trans-membranar form of SC5, then non-activated cells are preferred and CD3 activation is omitted, extracting and purifying the whole mRNA population from said cell population (commercial kits are available for mRNA extraction and purification, e.g.\",\n \"a polydT columns), synthetising every complementary cDNA (with a reverse transcriptase, e.g.\",\n \"such as described in Seed B. and Arrufo A.\",\n \"1987, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 84: 3365-3369, of which content is herewith incorporated by reference), operably cloning each cDNA so that expression of this cDNA in this clone is possible under appropriate clone culture conditions, and cultivating every clone accordingly, selecting those clones which express a compound onto which the monoclonal antibody produced by hybridoma I-2575 binds when placed under conditions suitable for this monoclonal antibody to perform reactions of the antigen-antibody type, and/or those clones of which lysate would give an immuno-precipitation reaction with the monoclonal antibody produced by hybridoma I-2575 will be selected.\",\n \"optionally amplifying those clones that have been thus selected, recovering the inserted cDNA from the selected clones.\",\n \"The cDNA thus recovered is an SC5 cDNA of the invention, it can be sequenced using standard DNA sequencing techniques.\",\n \"Operably cloning each cDNA so that expression of this cDNA in this clone is possible under appropriate clone culture conditions may be achieved by any conventional cloning procedure available to the skilled person.\",\n \"An example of cloning procedure comprises inserting each cDNA in a transfection vector under the control of a promoter suitable for the expression of this cDNA in the host cell, transfecting the host cells with this transfection vector, and cultivating the transfected host cells under conditions appropriate to their metabolism.\",\n \"Appropriate transfection vectors and host cells comprise virus such as Baculo virus and host cells such as insect cells (Sf9 cells), or vaccinia virus as a vector and EBV cells as host cells (see Rindis Bacher reference supra).\",\n \"Such a cDNA can be used for cell transfection, and for SC5 production from the clones thus obtained.\",\n \"It may also be used as probe for identifying or isolating the native SC5 mRNA.\",\n \"The invention therefore also encompasses any isolated mRNA obtainable by selection among the above-mentioned mRNA population, of a mRNA which is complementary to a cDNA of the invention (e.g.\",\n \"via selection of a mRNA which hybridizes to the above-mentioned cDNA under stringent conditions such as described in Freeman G. J. et al.\",\n \"1992, J. Immunol.\",\n \"149: 3745).\",\n \"Are particularly encompassed any isolated mRNA obtainable by using an isolated cDNA of the invention as a probe so as to recover from the whole mRNA population of said cell population, the mRNA which is complementary to this extracted cDNA.\",\n \"Alternatively, the one of ordinary skill in the art may directly sequence the cDNA that is recovered from the selected clones, and deduce from this sequence the sequence of the mRNA.\",\n \"The invention also encompasses any DNA encoding an isolated mRNA according to the invention.\",\n \"It particularly encompasses any genomic DNA obtainable by searching a genomic bank (such as www.ucsc.edu) for a genomic DNA matching with the above-mentioned SC5 cDNA.\",\n \"The invention also encompasses any engineered cell in which a cDNA, a mRNA or a DNA of the invention has been transfected.\",\n \"Are also encompassed SC5-specific probes and primers.\",\n \"Such probes and primers can be obtained by any technique available to the skilled person (e.g.\",\n \"selecting, as candidate sequences, portions of SC5 sequence that may be unique to SC5, comparing these candidate sequences on DNA/RNA banks with the sequences of cell receptors other than SC5, and selecting those candidate sequences which target a SC5 sequence that appears as unique after said comparison, and/or a SC5 sequence of which length appears as unique after said comparison).\",\n \"Any solid support onto which is placed a cDNA, a mRNA or a genomic DNA of the invention falls within the scope of the present invention.\",\n \"Such solid supports notably include DNA chips, or DNA microspheres.\",\n \"The invention also encompasses any isolated protein encoded by an isolated cDNA, mRNA, or DNA according to the invention.\",\n \"The invention further encompasses any isolated portion of a protein of the invention, provided that this portion is still characteristic of CTCL cells by comparison with normal resting cells.\",\n \"It particularly encompasses any portion that is recognized by the monoclonal antibody produced by the hybridoma I-2575 at the C.N.C.M.\",\n \"It also encompasses any isolated portion of a protein compound of the invention, provided that this portion is capable of inducing a modulation (inhibition or stimulation) of the CD3-induced proliferation of a normal T cell, and/or is capable of inducing a modulation of the in vivo proliferation of a malignant T cell such as a CTCL cell, and/or is capable of inducing a modulation of the IL-2 production of a normal T cell or of a malignant T cell, when said portion is expressed by said cell and contacted with the mAb produced by hybridoma I-2575.It particularly encompasses any isolated portion of a protein compound of the invention, provided that this portion is a part of a molecule that is expressed in a trans-membrane location by malignant CTCL cells and that is expressed in the cytoplasm compartment by non-tumoral resting T cells.\",\n \"The invention notably provides with a bank of polypeptidic fragments which is obtainable by enzymatic cleavage of the SC5 protein of the invention.\",\n \"Preferred enzymes comprises proteolytic enzymes such as those serine endopeptidases which cleave at the level of Tyr, Phe and Trp (e.g.\",\n \"alpha-chymotrypsin), and also those enzymes which cleaves at the C-terminus of glutamic acid and aspartic acid (e.g.\",\n \"V8 protease); see e.g.\",\n \"Shesberadaran and Payne 1988, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"USA 85:1-5, of which content is herein incorporated by reference.\",\n \"Such a bank of SC5 polypeptidic fragments is of particular use in analysis such as mass spectrophotometry analysis: such analysis use the characteristic separation of such a polypeptidic bank, and give a (polypeptide) profile that is characteristic of SC5, and that can be used for SC5 detection.\",\n \"Such a use and such a profile is encompassed by the present application.\",\n \"The invention particularly encompasses the extra-cellular, trans-membranar and intra-cytoplasmic portions of any SC5 protein compound of the invention.\",\n \"Determining the extra-cellular, trans-membranar and intra-cytoplasmic portions of a receptor molecule may be achieved by the skilled person according to any appropriate technique.\",\n \"They may be identified on the whole molecule by identification of the region which contains glycosylation sites (extra-cellular portion), of the region that is hydrophobic (trans-membrane portion), and of the region that is hydrophylic (intra-cytoplasmic region), and they may then be isolated either by cleavage or synthesis.\",\n \"An alternative way to isolate the extra-cellular portion of a protein compound of the invention is to proceed as above-described for the isolation of the whole protein compound, but with an additional step of enzymatic digestion so as to cleave from the whole molecule its extra-cellular portion.\",\n \"Appropriate enzymes for such a cleavage includes enzymes such as V8 protease or alpha-chymotrypsin as above-mentioned.\",\n \"The invention thus encompasses any isolated polypeptide compound obtainable by: collecting a SC5 cell source as above-described, such as e.g.\",\n \"PBL or total blood cells, if it is desired to isolate a polypeptide compound in the form it has when it is naturally expressed at the cell surface, and if the collected SC5 cell source is, or mainly comprises normal resting cells, then stimulating the CD3 pathway, e.g.\",\n \"via incubation with an appropriate CD3 activator (e.g.\",\n \"incubation with PHA at 1 microgramme per ml when said SC5 source is total blood cells or PBL), labeling the cells with a polypeptide-specific label, such as biotin (surface labeling on whole cells), lysing the SC5 cell source (such as PBL or total blood cells) so as to dissociate multimeric polypeptidic complexes (and thereby recovering SC5 dissociated from the transducers and effectors naturally associated therewith) e.g.\",\n \"by incubating the cells in a lysis buffer comprising a drastic detergent such as Triton X-100 at 1%, and submitting the lysate to an enzymatic digestion enabling the cleavage of SC5 into portions, e.g.\",\n \"by incubating the lysate with a proteolytic enzyme such as an enzyme selected from the group consisting of V8 protease or alpha-chemotrypsin, submitting the lysate to immuno-precipitation with the monoclonal antibody produced by hybridoma I-2575, and recovering from the immuno-precipitate a polypeptide compound which bears a label.\",\n \"Such recovered compounds are SC5 portions which either are the complete SC5 extra-cellular portion, or are part of such a complete SC5 extra-cellular portion, or comprise such a SC5 extra-cellular portion.\",\n \"The set of polypeptide compounds that are thus obtainable constitute a bank of SC5 “extra-cellular” portions.\",\n \"Such a bank, and its medical uses, such as e.g.\",\n \"its use for the diagnosis of a T-related disease (e.g.\",\n \"via mass spectrophotometry analysis), falls within the scope of the present invention.\",\n \"The person of ordinary skill in the art will note that lysis and enzymatic digestion may be performed in a single step.\",\n \"Labeling on whole cells is performed prior to cell lysis so as to identify SC5 portions which are extra-cellular portions, or which are part of such extra-cellular portions, or which comprise such portions.\",\n \"The invention also encompasses any isolated compound which comprises a portion of a protein of the invention.\",\n \"The mAb of the invention also enables the identification of the SC5 epitope onto which this mAb binds.\",\n \"Conventional techniques such as directed mutagenesis are appropriate (see e.g.\",\n \"Chang H. C. et al.\",\n \"1989, J. Exp.\",\n \"Med.\",\n \"169: 2073-2083, of which content is herein incorporated by reference).\",\n \"The proteins of the invention and their portions according to the invention, and notably their extra-cellular portions, enable the production of monoclonal antibodies directed against them.\",\n \"An example of such monoclonal antibodies is produced by hybridoma I-2575.The monoclonal antibodies of the invention are particularly useful for the detection of SC5, for the diagnosis of T lymphomas such as CTCL, and are also useful as modulators of an undesired or inappropriate proliferation and/or activity of T cells, such as normal CD4+ and CD8+ T cells, virus-infected T cells such as HIV-infected T cells, T lymphomas such as CTCL.\",\n \"The invention therefore encompasses any monoclonal antibody obtainable by: (i) immunizing an animal against a protein of the invention or against a portion thereof as above-defined, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them for them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein or protein portion that as been used as an immunogen in step i., and which has at least one property selected from the group consisting of the property of: binding resting non-tumoral T cells mainly in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells mainly at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of malignant T cells such as CD4+ CTCL cells, modulating the proliferation of malignant T cells such as CD4+ CTCL cells in an animal, and preferably in a human, competing with a monoclonal antibody of the invention, such as the monoclonal antibody produced by hybridoma I-2575, for binding to a protein of the invention, or to a portion thereof as above-defined, and in particular with the extra-cellular portion of such a protein, (iv) selecting and cloning hybridomas producing the desired antibody, (v) recovering the antibody from the supernatant above said clones.\",\n \"Production of hybridomas may be achieved using any conventional technique.\",\n \"This includes removing the spleen from said animal, making a suspension of spleen cells, fusing the spleen cells with myeloma cells in the presence of a fusion promoter, diluting and cultivating the fused cells in separate wells in a medium which will not support unfused cells.\",\n \"The invention also encompasses any ascite isolated from said immunized animal.\",\n \"For step iii., T and malignant T cells can be collected from a human using standard procedures.\",\n \"Alternatively, clones can be used such as the CD4+ CTCL HUT 28 cell line (ATCC TIB-161).\",\n \"In the case of CD4+ tumors, collection is made from the body part which will display the CD4+ tumoral cells (PBL for SS, cutaneous erythroderma for transformed MF, etc.).\",\n \"The above-mentioned properties can be easily assessed by the skilled person using common knowledge in the art.\",\n \"Examples of appropriate experimental conditions for assessing the properties mentioned in step iii.\",\n \"can be found in the below examples.\",\n \"The person of ordinary skill in the art will adjust them to the particular cells, or concentrations used.\",\n \"Modulation of cell proliferation herein refers to stimulation as well as inhibition of this proliferation.\",\n \"When CTCL cells are used, inhibition of proliferation is preferred in perspective of therapeutic application.\",\n \"The mAbs of the invention are all anti-SC5 mAbs which have either diagnostic or therapeutic applications, or both.\",\n \"For certain applications, it may be desired to immobilize one or several of these mAb onto a support, e.g.\",\n \"for protein purification purposes, or for exerting the modulation property of which they are capable.\",\n \"The present application also encompasses any support onto which a mAb of the invention has been immobilized.\",\n \"Such supports notably include those appropriate for columns of chromatography by affinity (e.g.\",\n \"agar beads).\",\n \"The present application is also directed to any fragment of a mAb of the invention (anti-SC5 fragments) selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, CDR3.It is directed to any compound, and namely to any antibody, comprising such a fragment.\",\n \"It notably relates to any humanized antibody comprising at least one of such fragments.\",\n \"Techniques for the production of humanized antibodies have been widely described in the prior art (see e.g.\",\n \"Farah et al.\",\n \"1998, Crit.\",\n \"Rev.\",\n \"Eucaryote Gene Exp.\",\n \"Vol.\",\n \"8 pp 321-345, of which content is incorporated herein by reference).\",\n \"In particular, it relates to any compound, and notably to any antibody and humanized antibody, comprising at least one anti-SC5 fragment as herein defined and also at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, CDR3 (see e.g.\",\n \"WO 94/13804 “Multivalent and multispecific binding proteins, their manufacture and use”, Inventors: Holliger et al.\",\n \"Applicants: Cambridge Antibody Technology Ltd and Medical Research Council; see also Merchant et al.\",\n \"“An efficient route to human bi-specific IgG” Nat.\",\n \"Biotechnol.\",\n \"1998 vol.\",\n \"16 pp 677-681).\",\n \"As previously indicated, the invention provides with two molecules linked by a common general inventive concept: SC5 and p140.The SC5 is a novel molecule fully herein described.\",\n \"The p140 molecule has been described in the prior art as an inhibitory receptor which is expressed by sub-groups of NK cells and of CD3+ CD8+ cells from healthy (non-cancerous) patients.\",\n \"The present invention now demonstrates that p140 is expressed by malignant T cells, and notably by CTCL cells, and provides with a novel p140 allelic form (clone 24 in the below example 3; SEQ ID No.\",\n \"1) encoding a novel protein (SEQ ID No.\",\n \"2).\",\n \"The cDNA and amino acid sequences of this novel allelic form are as follow: SEQ ID No.1: CATGTCGCTCACTGTCGTCAGCATGGCGTGCGTTGGGTTCTTCTTGCTGC AGGGGGCCTGGCCACTCATGGGTGGTCAGGACAAACCCTTCCTGTCTGCC CGGCGCAGCACTGTGGTGCCTCAAGGAGGACACGTGGCTCTTCAGTGTCA CTATCGTCGTGGGTTTAACAATTTCATGCTGTACAAAGAAGACAGAAGCC ACGTTCCCATCTTCCACGGCAGAATATTCCAGGAGAGCTTGATCATGGGC CCTGTGACCCCAGCACATGCAGGGACCTACAGATGTCGGGGTTCACGCCC ACACTCCCTCACTGGGTGGTCGGCACCCAGCAACCCCCTGGTGATCATGG TCACAGGAAACCACAGAAAACCTTCCCTCCTGGCCCACCCAGGGACCCTG CTGAAATCAGGAGAGACAGTCATCGTGCAATGTTGGTCAGATGTCATGTT TGAGCACTTCTTTCTGCACAGAGAGGGGATCTCTGAGGACCCCTCACGCC TCGTTGGACAGATCCATGATGGGGTCTCCAAGGCCAACTTCTCCATCGGT CCCTTGATGCCTGTCCTTGCAGGAACCTACAGATGTTATGGTTCTGTTCC TCACTCCCCCTATCAGTTGTCAGCTCCCAGTGACCCCCTGGACATCGTGA TCACAGGTCTATATGAGAAACCTTCTCTCTCAGCCCAGCCGGGCCCCACG GTTCAGGCAGGAGAGAACGTGACCTTGTCCTGTAGCTCCTGGAGCTCCTA TGACATCTACCATCTGTCCAGGGAAGGGGAGGCCCATGAACGTAGGCTCC GTGCAGTGCCGAAGGTCAACAGAACATTCCAGGCAGACTTTCCTCTGGGG CCTGCCACCCACGGAGGGACCTACAGATGCTTCGGCTCTTTCCGTGCCCT GCCCTGCGTGTGGTCAAACTCAAGTGACCCACTGCTTGTTTCTGTCACAG GAAACCCTTCAAGTAGTTGGCCTTCACCCACAGAACCAAGCTCCAAATCT GGTATCTGCAGACAGGTGCATGTTCTGATTGGGACGTCAGTGGTCATCTT CCTCTTCATCCTCCTCCTCTTCTTTCTCCTTTATCGCTGGTGCTCCAACA AAAAGAATGGTGCTGTAATGGACCAAGAGCCTGCGGGGGACAGAACAGTG AATAGGCAGGACTCTGATGAACAAGACCGTCAGGAGGTGACGTACGCACA GTTGGATCACTGCGTTTTCATACAGAGAAAAATCAGTCGCCCTTCTCAGA GGGCCAAGAGACCCCCAACAGATACCAGCGTGTACACGGAACTTCCAAAT GCTGAGCCCAGATCCAAAGTTGTCTCCTGCCCACGAGCACCACAGTCAGG TCTTGAGGGGGTTTTCTAGGGAGACAACAGCCCTGTCTCAAAACC SEQ ID No.2: pLMGGQDKPF LSARPSTVVP QGGHVALQCH YRRGFNNFML YKEDRSHVPI FHGRIFQESF IMGPVTPAHA GTYRCRGSRP HSLTGWSAPS NPLVIMVTGN HRKPSLLAHP GTLLKSGETV ILQCWSDVMF EHFFLHREGI SEDPSRLVGQ IHDGVSKANF SIGPLMPVLA GTYRCYGSVP HSPYQLSAPS ˜DPLDIVITGL YEKPSLSAQP GPTVQAGENV TLSCSSWSSY DIYHLSREGE AHERRLRAVP KVNRTFQADF PLGPATHGGT YRCFGSFRAL PCVWSNSSDP LLVSVTGNPS SSWPSPTEPS SKSGICRHLH VLIGTSVVIF LFILLLFFLL YRWCSNKKNA AVMDQEPAGD RTVNRQDSDE QDPQEVTYAQ LDHCVFIQRK ISRPSQRPKT PPTDTSVYTE LPNAEPRSKV VSCPRAPQSG LEGVF The DNA and amino acid sequences of the previously described p140 allelic form (clone 1.1) are as follow: SEQ ID No.3: CATGTCGCTCACGGTCGTCAGCATGGCGTGCGTTGGGTTCTTCTTGCTGCA GGGGGCCTGGCCACTCATGGGTGGTCAGGACAAACCCTTCCTGTCTGCCCG GCCCAGCACTGTGGTGCCTCGAGGAGGACACGTGGCTCTTCAGTGTCACTA TCGTCGTGGGTTTAACAATTTCATGCTGTACAAAGAAGACAGAAGCGACGT TCCCATCTTCCACGGCAGAATATTCCAGGAGAGCTTCATCATGGGCCCTGT GACCCCAGCACATGCAGGGACCTACAGATGTCGGGGTTCACGCCCACACT CCCTCACTGGGTGGTCGGGACCCAGCAACCCCGTGGTGATCATGGTCACAG GAAACCACAGAAAACCTTCCCTCCTGGCCCACCCAGGGCCCCTGCTGAAA TCACGAGAGACAGTCATGCTGCAATGTTGGTCAGATGTCATGTTTGAGCAC TTCTTTCTGCACAGAGAGGGGATCTCTGAGGACCCCTCACGCCTCGTTGGA CAGATCCATGATGGGGTCTCCAAGGCCAACTTCTCCATCGGTGGCTTGATG CCTGTGCTTGCAGGAACGTACAGATGTTATGGTTCTGTTCCTCACTCCGGC TATCAGTTGTCAGCTCGCAGTGACCCCCTCGACATCGTGATCACAGGTCTA TATGAGAAACCTTCTCTCTCAGCGCAGCGGGGCCCCACGGTTCAGGCAGGA GAGAACGTGACCTTGTCCTGTAGCTCCTGGAGCTCCTATGACATCTACCAT CTGTCCAGGGAAGGGGAGGCCCATGAACGTAGGCTCCGTGCAGTGCCGAA GGTCAACAGAACATTCCAGGCAGACTTTCCTCTGGGCCCTGCCACCCACGG AGGGACCTACAGATGCTTCGGCTCTTTGCGTGCCCTGCCCTGCGTGTGGTC AAACTCAAGTGACCCACTGCTTGTTTCTGTCACAGGAAACCCTTCAAGTAG TTGGCCTTCACCCACAGAACCAAGCTCCAAATCTGGTATCTGCAGACACCT GCATGTTCTGATTGGGACCTCAGTGGTCATCTTCCTCTTCATCCTCCTCCT CTTCTTTCTCCTTTATCGCTGGTGCTCCAACAAAAAGAATGCTGCTGTAAT GGACCAAGAGCCTGCGGGGGACAGAACAGTGAATAGGCAGGACTCTGATG AACAAGACCCTCAGGAGGTGACGTACGCACAGTTGGATCACTGCGTTTTC ATACAGAGAAAAATCAGTCGCCCTTCTCAGAGGCCCAAGACACCCCTAAC AGATACCAGCGTGTACACGGAACTTCCAAATGCTGAGCCCAGATCCAAAG TTGTCTCCTGCCCACGAGCACCACAGTCAGGTCTTGAGGGGGTTTTCTAGG GAGACAACAGCCCTGTCTCAAAACC SEQ ID No.4: pLMGGQDKPF LSARPSTVVP RGGHVALQCH YRRGFNNFML YKEDRSHVPI FHGRIFQESF IMGPVTPAHA GTYRCRGSRP HSLTGWSAPS NPVVIMVTGN HRKPSLLAHP GPLLKSGETV ILQCWSDVMF EHFFLHREGI SEDPSRLVGQ IHDGVSKANF SIGPLMPVLA GTYRCYGSVP HSPYQLSAPS DPLDIVITGL YEKPSLSAQP GPTVQAGENV TLSCSSWSSY DIYHLSREGE AHERRLRAVP KVNRTFQADF PLGPATHGGT YRCFGSFRAL PCVWSNSSDP LLVSVTGNPS SSWPSPTEPS SKSGICRHLH VLIGTSVVIF LFILLLFFLL YRWCSNKKNA AVMDQEPAGD RTVNRQDSDE QDPQBVTYAQ LDHCVFIQRK ISRPSQRPKT PLTDTSVYTE LPNAEPRSKV VSCPRAPQSG LEGVF SEQ ID No.\",\n \"1 differs from SEQ ID No.\",\n \"3 in five locations.\",\n \"The resulting SEQ ID No.2 differs from the p140 disclosed in prior art (SEQ ID No.\",\n \"4) by four amino acid substitutions (shown in bold and underlined characters): R instead of Q in position 20, V instead of L in position 92, P instead of T in position 102, and L instead of P in position 401 (see FIG.\",\n \"11).\",\n \"The present application encompasses any isolated compound of which sequence is, or comprises SEQ ID No.\",\n \"1 and/or SEQ ID No.\",\n \"2.It also encompasses any mRNA that is complementary to SEQ ID No.\",\n \"1, and any genomic DNA coding for SEQ ID No.\",\n \"2.Appropriate techniques for producing p140 proteins are available to the skilled persons.\",\n \"They notably include the production of cells which have been engineered so as to produce p140 proteins in their culture medium.\",\n \"An example of such techniques comprises inserting SEQ ID No.\",\n \"1 or No.\",\n \"3 in a baculo virus vector, operably transfecting an insect cell such as Sf9 cell line with this vector, recovering the p140 protein produced in the culture medium (e.g.\",\n \"by percolation of the culture medium is through a column—e.g.\",\n \"a Sephadex column—which protein compounds as a function of their molecular weight, and isolating the 140 kD eluate).\",\n \"Compounds capable of binding to p140 under conditions of the physiological type (in vivo conditions, or in vitro conditions mimicking the in vivo ones) have been previously described, and may be obtained by the person of ordinary skill in the art by any appropriate available technique.\",\n \"By “p140 binding compound”, it is herein meant any compound which is capable under said conditions of the physiological type to recognize a p140 molecule as an antigen (SEQ ID No.\",\n \"2 and/or SEQ ID No.\",\n \"4) when it is expressed by a cell in a transmembrane receptor location (i.e.\",\n \"recognition of the SEQ ID No.\",\n \"2 and/or No.\",\n \"4 portion that is above the lipid bi-layer of said cell).\",\n \"Such p140 binding compound notably comprise HLA-A11 and HLA-A3 molecules (natural p140 ligands), and notably fusion proteins such as Fc-HLA-A11 and Fc-HLA-A3 fusion proteins.\",\n \"p140 binding compounds also comprise p140 antiserum (obtainable by immunizing an animal such as a rabbit against p140, and by recovering the antiserum thus produced—an additional step of immuno-purification may be performed so as to increase p140 concentration in the antiserum, e.g.\",\n \"via percolation through a p140 column).\",\n \"Other p140 binding compounds comprise monoclonal antibodies directed to p140 SEQ ID No.\",\n \"4, such as e.g.\",\n \"AZ158, Q66, Q241 described in Pende et al.\",\n \"1996 (J. Exp.\",\n \"Med.\",\n \"184: 505-518).\",\n \"Such anti-SEQ ID No.\",\n \"4 mAbs may also recognize the new SEQ ID No.\",\n \"2 as an antigen.\",\n \"Any available technique for mAb production is appropriate for production of anti-p140 mAbs.\",\n \"An example of such a technique comprise the steps of: (i) immunizing an animal against p140, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them for them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the p140 molecule that as been used as an immunogen in step i., and which is also capable of binding resting non-malignant T cells mainly in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells mainly at their cell surface, (iv) selecting and cloning hybridomas producing the desired antibody, (v) recovering the antibody from the supernatant above said clones.\",\n \"Preferred p140 binding compounds do also comprise an anti-CD4 entity (e.g.\",\n \"bi-specific mAb).\",\n \"Such p140 binding compounds are useful for p140 detection on malignant T cells, such as CTCL cells, in particular CD4+ CTCL.\",\n \"They are thus useful for CD4+ cancer detection.\",\n \"They are also useful as polypeptidic vectors, as described below.\",\n \"According to another aspect of the invention, there are provided polypeptidic vectors comprising at least one element selected from the group consisting of the anti-SC5 mAbs of the invention and the anti-SC5 mAb fragments of the invention.\",\n \"Such polypeptidic vectors are useful for molecules, such as active principle molecules, to reach activated T cells, or their vicinity (e.g.\",\n \"activated T cells observed in inflammatory diseases, or in virus-infected T cells, or malignant T cells such as malignant CTCL cells, and malignant CD4+ CTCL cells in particular).\",\n \"It also relates to polypeptidic vectors comprising a p140 binding compound such as a mAb directed against SEQ ID No.\",\n \"2 and/or SEQ ID No.\",\n \"4 extra-cellular portion.\",\n \"Such polypeptidic vectors are useful for delivering molecules to, or in the vicinity of malignant T cells, such as notably CTCL cells, and malignant CD4+ CTCL cells in particular.\",\n \"Molecules which may be usefully delivered to malignant T cells, such as CTCL cells notably comprise molecules capable of inducing cell death or apoptosis, such as a radio-element or a toxin.\",\n \"Such molecules also comprise any enzyme capable of transforming an antimititotic pro-drug into an active drug form, such as a carboxypeptidase.\",\n \"A polypeptidic vector carrying such an enzyme is then advantageously used in combination with an anti-mitotic pro-drug (such as phenol mustard pro-drug).\",\n \"The invention therefore also encompasses a medical kit comprising such a vector and such an anti-mitotic pro-drug.\",\n \"Molecules which may be usefully delivered in the vicinity of malignant T cells such as CTCL cells notably comprise molecules capable of stimulating the immune functions of cells which are in the vicinity-of malignant T cells in an effort to induce an anti-tumor effect from these neighboring cells towards said malignant T cells.\",\n \"Examples of such molecules notably comprise cytokines such as interferon gamma, and interleukine such as IL-2 (see e.g.\",\n \"Xu et al.\",\n \"2000, Cancer Research 60: 4475-4484).\",\n \"When a polypeptide vector of the invention is intended for targeting CD4+ cells such as CD4+ CTCL cells, then this vector advantageously further comprises an anti-CD4 entity.\",\n \"The invention also encompasses any pharmaceutical composition comprising such polypeptidic vectors, and in particular any medicament comprising such polypeptidic vectors.\",\n \"Such medicaments are useful in the control of an inappropriate or undesired T cell proliferation and/or functional activity (including undesired IL-2 production).\",\n \"They are particularly useful for the prevention, palliation, relieve or therapy of an inappropriate immune activity, and notably for the prevention or inhibition of a T lymphoma such as CTCL and in particular CD4+ CTCL.\",\n \"The use of such vectors for the manufacture of a drug intended for the prevention, palliation, relieve or therapy of an inappropriate T cell proliferation and/or activity, and notably for the prevention or inhibition of a CTCL proliferation is thus also an object of the present application.\",\n \"A compound which binds to SC5 under conditions of the physiological type may be used as a SC5 ligand agonist so as to stimulate the activation of SC5, the SC5 molecule being then regarded as a cell inhibitory receptor.\",\n \"Such anti-SC5 compounds namely comprise anti-SC5 antisera, the anti-SC5 mAb of the invention, the Fab, F(ab′)2, fragments thereof, the humanized mAbs derived therefrom.\",\n \"When CD4+ cells are more particularly concerned, said anti-SC5 compound advantageously further comprises an anti-CD4 entity.\",\n \"Anti-SC5 compounds are particularly useful for modulating an inappropriate or undesired proliferation and/or functional activity of SC5+ cells.\",\n \"They are thus useful for modulating the proliferation of T cells, CD45RO+ cells, CD3+ cells, CD4+ T cells, CD8+ T cells, or of virus-infected T cells (e.g.\",\n \"HIV-infected CD4+ T cells), or of malignant T cells such as CTCL cells and in particular CD4+ CTCL cells.\",\n \"They are also useful for modulating IL-2 production from T cells.\",\n \"When the mAb produced by the hybridoma deposited as deposit number I-2575 at the C.N.C.M.\",\n \"(which is a pentameric IgM) is used, this modulation goes in favor of an inhibition.\",\n \"It is therefore particularly appropriate for inhibition of T lymphomas such as CTCL.\",\n \"The application encompasses any drug comprising, as an active principle, at least one anti-SC5 compound.\",\n \"Such a drug may be intended for the prevention, palliation, therapy of T lymphomas, of CTCL, of T infections such as viral (e.g.\",\n \"HIV) infections, of inflammatory diseases such as those of the auto-immune type (e.g.\",\n \"rhumatoid arthritis—e.g.\",\n \"spondyloarthropathis—or skin immune mediated diseases such as psoriasis, eczema, atopic dermatitis).\",\n \"Such a drug may also be intended for graft improvement (inhibition of graft rejection).\",\n \"The invention also encompasses the use of such anti-SC5 compounds as complement recruiting agents, or as ADCC activators or stimulators.\",\n \"The p140 binding compounds as defined above, may also be used as complement recruiting agents, or as ADCC stimulators or activators in the prevention, palliation, relieve or treatment of T lymphomas, such as CTCL and in particular CD4+ CTCL.\",\n \"The invention therefore encompasses the use of such p140 binding compounds in the manufacture of an anti-T lymphoma drug, of an anti-CTCL drug, of an anti-CD4+ CTCL drugs, and also encompasses such drugs.\",\n \"The invention also provides with a method for the assessment of the development level of a CTCL (staging): it enables to evaluate the proportion (e.g.\",\n \"percentage) of malignant CD4+ CTCL cells present within a certain body compartment of a patient.\",\n \"According to this method, the proportion of CD4+ cells expressing at their surface an element selected from the group consisting of the SC5 proteins of the invention, the SC5 portions of the invention (notably, those extracellular portions), and the p140 molecules is measured in a biological sample collected from said body compartment.\",\n \"The proportion of CD4+ CTCL cells that are actually present in said body compartment can be considered as substantially equal to said measured proportion.\",\n \"It usually falls within a ±10% range around this measured proportion.\",\n \"The invention also provides with a method for CTCL diagnosis, wherein the percentage of T cells expressing an element selected from the group consisting of the SC5 proteins of the invention, the SC5 portions of the invention (notably, those extracellular portions), and the p140 molecules is measured in a biological sample collected from a patient, and is compared to the average percentage observed in non-CTCL humans (preferably in healthy humans), and in that a CTCL-positive diagnosis is decided when said measured percentage is significantly higher than said average percentage.\",\n \"This CTCL diagnosis method particularly applies to those CTCL which are CD4+; in this case the measured T cell percentage is preferably a CD4+ percentage.\",\n \"The average standard proportion of CD4+ T cells expressing SC5 is generally in between 1-15%, usually in between 5-10% among healthy humans.\",\n \"When the measured percentage of SC5+ CD4+ T cells is higher than such an average percentage, then a CD4+ CTCL positive diagnosis can be concluded.\",\n \"Of note is that, as far as SC5 protein or portions thereof are concerned, the methods of the invention more generally enables to evaluate the proportion of CD4+ activated T cells that are present in said body compartment.\",\n \"This may e.g.\",\n \"be useful for assessing whether a graft induces rejection reactions, and for the diagnosis and follow-up of inflammatory diseases such as those of the auto-immune type (e.g.\",\n \"rhumatoid arthritis such as spondyloarthropathis, skin immune mediated disease such as psoriasis, eczema, atopic dermatitis).\",\n \"Said biological sample may then be intra-synovial fluid.\",\n \"This may be also useful for the diagnosis and monitoring of non-CTCL T lymphomas (e.g.\",\n \"CD4+ ALL, or CD4+ T-LL).\",\n \"The CTCL assessing/diagnostic methods of the invention are highly efficient for all kinds of CD4+ CTCL, and notably for transformed MF, for SS, for LP, as well as for CD30+ lymphomas.\",\n \"Said biological sample may e.g.\",\n \"then be a skin sample collected from an erythroderma (e.g; suspicion of transformed MF), and/or a PBL or total blood sample (in order to evaluate whether a transformed MF has evolved into a more aggressive form such as SS).\",\n \"When the element detected at the surface of said CD4+ cells is a SC5 compound, then a (preliminary or ulterior) step of cytological observation (search for tumor-alike cytology) is the preferably performed to conclude with a confidence of more than 90% to an actual CD4+ CTCL.\",\n \"When p140 is detected at the surface of said CD4+ cells, then no additional step is required: the simple fact of detecting p140 at the surface of CD4+ T cells directs to a positive diagnosis of CD4+ CTCL.\",\n \"The methods of the invention can be implemented with any appropriate element, and notably with an element selected from the group consisting of the anti-SC5 monoclonal antibodies of the invention, the Fab, F(ab′)2 fragments thereof, the humanized mAb derived therefrom, the SC5 “extra-cellular” bank of the invention (as above-defined), the SC5 cDNA, mRNA, genomic DNA of the invention, the p140 binding compounds as herein defined, the p140 DNA, the DNA comprising SEQ ID No.\",\n \"1 or SEQ ID No.\",\n \"3 or encoding SEQ ID No.\",\n \"2 or SEQ ID No.\",\n \"4.Such an element is then used as a detection means for SC5 or p140 cell surface expression.\",\n \"Preferably, said detection means also comprises an anti-CD4 entity so as to detect SC5+ /p140+ CD4+ cells in a one-step procedure.\",\n \"When cDNA/mRNA material is used as a detection means, then CD4+ cells have to be lysed so as to detect those SC5 or p140 hybridizing mRNA which are actually present in said cells (detection of the transcripts).\",\n \"Such cDNA/mRNA detection means may advantageously be placed onto or into a solid support such as a DNA chip or a DNA microsphere.\",\n \"When mAb material or mAb-derived material is used as a detection means, then surface expression of SC5 or p140 is detected.\",\n \"Any appropriate antibody-antigen technique is convenient.\",\n \"Examples notably comprise flow cytometry analysis.\",\n \"The invention also encompasses a method for the identification of a compound which is useful in the palliation, prevention, relieve, therapy of an undesired or inappropriate T cell activity, such as notably a proliferation of malignant T cells such as CTCL cells and in particular CD4+ CTCL cells.\",\n \"This method is characterized in that it comprises the detection of a compound that is capable of binding to a SC5 protein as above-defined, or to a SC5 polypeptide compound as above-defined, or to a p140 molecule (such as SEQ ID No.\",\n \"2 and/or SEQ ID No.\",\n \"4).\",\n \"Such compounds can be used as polypeptidic vectors as above-described.\",\n \"Those compounds which, in addition to binding to SC5 molecules, are also capable of aggregating these SC5 molecules at the cell surface, are preferred for stimulating SC5 inhibitory functions.\",\n \"An easy way to implement this identification method is to proceed with SC5 and/or p140 molecules immobilized onto a solid support such as protein A Sepharose CL-4B.\",\n \"Candidates compounds for implementation of the method notably comprise sera immunized against SC5 or p140.The invention is illustrated by the examples, which are in no way intended to restrict the invention to the particular embodiments described below.\",\n \"The person of ordinary skill in the art will contemplate alternative embodiments, and such alternative embodiments derived from common knowledge in the art are within the scope of the invention.\",\n \"The skilled person will in particular appreciate that various standard immuno-techniques, and various standard molecular biology techniques are available to him (see e.g.\",\n \"“Antibodies: a laboratory manual” Ed Harlow, David lane, Cold Spring Harbor Laboratory 1988; Maniatis 1982, “Molecular cloning: a laboratory manual”, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory; “Current protocols in Immunology” protocols on CD-ROM, John Wiley, West Sussex, England; “Immunological techniques made easy”, edited by Olivier Cochet et at.\",\n \"; John Wiley, West Sussex, England; and the publication specifically referred to in the present application).\",\n \"The content of such prior art documents is herein incorporated by reference.\",\n \"It is also to be understood that experiments described herein in full details for one particular form of CD4+ CTCL can be directly transposed by the skilled person to any other form of CD4+ CTCL.\",\n \"In these examples, reference is made to the following figures: FIGS.\",\n \"1A, 1B and 1C illustrate cell membrane expression of SC5 molecule on PBLs from normal individual.\",\n \"PBMC from a normal individual were stained with anti-SC5 mAb (ascites ({fraction (1/200)}) followed by FITC-conjugated isotype specific goat anti-mouse IgM second reagent and one of the following PE-conjugated mAbs: anti-CD3 (FIG.\",\n \"1A), anti-CD56 (FIG.\",\n \"1C) and anti-CD45RO (FIG.\",\n \"1B).\",\n \"The percentage of double-stained lymphocyte-gated cells is shown in the upper right quadrant (3.3 for FIG.\",\n \"1A; 5.1 for FIG.\",\n \"1B; 0.6 for FIG.\",\n \"1C).\",\n \"This experiment is representative for the 10 donors studied.\",\n \"FIG.\",\n \"2 illustrates cell membrane expression of SC5 molecule during activation of PBLs.\",\n \"PBMC were stimulated with 1 μg/mL PHA and the kinetics of SC5 and CD69 expression were studied in parallel on the CD3+ cells.\",\n \"FIG.\",\n \"3 illustrates intracellular localization of SC5 molecule in PBL.\",\n \"Anti-SC5, anti-CD3ζ chain mAb and anti-BY55 isotype-matched mAb were used for staining permeabilized and non-permeabilized gated lymphocytes from a normal donor.\",\n \"The shaded histograms represent the labeling obtained with the indicated mAbs compared to an irrelevant control mAb.\",\n \"FIG.\",\n \"4 illustrates biochemical analysis of SC5 molecule.\",\n \"DS6 T cell clone was surface labeled with biotin and 1% Triton X-100 lysates were immunoprecipitated, using anti SC5 mAb.\",\n \"The samples were analyzed by SDS-10%-PAGE under reducing conditions.\",\n \"Anti-SC5 mAb was used at two concentrations {fraction (1/200)} (lanes 2) and {fraction (1/500)} (lane 3).\",\n \"The negative control samples were precipitated with goat anti-mouse Ig alone (lane 1) and isotype-matched irrelevant mAb (lane 4).\",\n \"The molecular weight markers (kD), are indicated on the left.\",\n \"FIGS.\",\n \"5A, 5B, 5C, 5D illkustrate that anti-SC5 mAb modulates the anti-CD3-induced proliferation and cytokine secretion of normal T lymphocytes whithout affecting their cytotoxic activity: FIG.\",\n \"5A: PBL or T cell clones LSO and GDS.3 were stimulated with immobilized anti-CD3 mAb (1 μg of mAb coated per well) or with IL-2 (50 IU/ml) in the presence of anti-SC5 mAb (1:200 final dilution of ascites) or an isotype-matched irrelevant mAb.\",\n \"Results shown are representative of at least three separate experiments and are expressed as mean cpm±SD of triplicate wells.\",\n \"FIG.\",\n \"5B: GDS.3 CD4+ T cell clone was stimulated with the indicated concentrations of pre-coated anti-CD3 mAb in the presence of different concentrations of anti-SC5 mAb.\",\n \"FIG.\",\n \"5C: IL-2 secretion in the supernatant of anti-CD3-stimulated PBL, in the presence of anti-SC5 or control mAb, was assessed by measuring the proliferation of an IL-2 dependant T cell clone in the presence of: medium alone (a), supernatant from PBL/anti-CD3+ control mAb (b), supernatant from PBL/anti-CD3+ anti-SC5 mAb (c).\",\n \"Data represent the mean values±SD of triplicate determinations of one out of three representative experiments.\",\n \"FIG.\",\n \"5D: JF1, a cytotoxic CD8+ cell clone was incubated with anti-SC5 mAb ({fraction (1/200)} final dilution of ascites) or with an isotype matched control mAb before the coculture with the FcγR+ murine tumor cells P815 at an E/T ratio of 5/1.Target cells were preincubated with the indicated final concentrations of anti-CD3 mAb.\",\n \"Results are expressed as mean of triplicate wells.\",\n \"FIGS.\",\n \"6A, 6B illustrate cell membrane expression of SC5 molecule on PBL from CTCL patients.\",\n \"Cells from peripheral blood of 8 patients with Sézary syndrome, 3 patients with Mycosis fungoides and 8 healthy donors were analysed by two-color fluorescence for the expression of SC5 molecule by CD4+ lymphocytes.\",\n \"FIG.\",\n \"6A: co-expression of SC5 and CD4 molecules in PBL from a patient with Sézary syndrome in comparison to a normal individual.\",\n \"FIG.\",\n \"6B: histograms showing the mean values of SC5 expression in the 3 indicated groups.\",\n \"Statistically significant differences between SS patients and 2 other groups are marked, (Mann-Witney U test, p<0.001) FIGS.\",\n \"7A, 7B, 7C illustrate surface membrane expression on malignant cell from Sézary syndrome patient.\",\n \"FIG.\",\n \"7A: two-color immunofluorescence analysis of PBMC from SS patient with a circulating TCRVβ22+ malignant T cell clone.\",\n \"Cells were stained with anti-SC5 mAb followed by PE-conjugated isotype specific goat anti-mouse IgM and FITC-conjugated anti-TCRVP22 mAb.\",\n \"FIG.\",\n \"7B: anti-SC5-mAb labeled weakly (shaded histogram) the TCRVβ22-positive malignant cells.\",\n \"FIG.\",\n \"7C: cells from the same patient stained with PE-conjugated anti-CD69 mAb and FITC-conjugated anti-TCRVβ22 mAb.\",\n \"FIG.\",\n \"8 illustrates the modulation of anti-CD3-induced proliferation of CTCL malignant cell line by anti-SC5 mAb.\",\n \"Pno cell line was stimulated with IL-7 (10 ng/mL), PMA alone (2 ng/mL), immobilized anti-CD3 mAb or a combination of PMA/anti-CD3 mAb.\",\n \"SC5 mAb or an isotype-matched irrelevant mAb (1:200 final dilution of ascites) was added at the start of cultures.\",\n \"Results shown are representative of four separate experiments and are expressed as mean cpm±SD of triplicate wells.\",\n \"FIGS.\",\n \"9A, 9B illustrate the detection of p140/KIR molecules on CTCL cell lines (FIG.\",\n \"9A) and on fresh tumor lymphocytes (FIG.\",\n \"9B).\",\n \"Double immunostaining flow cytometric analysis was performed as described in Bagot et al.\",\n \"1998 (Blood 91:4331-434 1) and in Poszepczynska et al.\",\n \"2000 (Blood 96: 1056-1063).\",\n \"FIG.\",\n \"10 illustrates the biochemical analysis of p140 molecules: the NK clone AM61 (p140+/p70−), derived from a healthy donor, and the cell lines Pno and Cou-L were surface labeled with biotin and immunoprecipitated with anti-p140 and anti-CD8 mAbs.\",\n \"Samples were treated (+) or not (−) with N-glycosidase F and analyzed in an 8% (left panel) or 11% (right panel) SDS-PAGE under reducing conditions.\",\n \"Sepharose Protein-A alone represents the negative control.\",\n \"Molecular weight markers (kD) are indicated on the right.\",\n \"FIG.\",\n \"11 shows the amino acid sequence alignment of KIR3D cl.\",\n \"24 (SEQ ID No.\",\n \"2) and cl.\",\n \"1.1 (SEQ ID No.\",\n \"4) encoded proteins.\",\n \"Amino acids corresponding to the signal peptide are in small case letters, while transmembrane region is underlined in the consensus sequence.\",\n \"Amino acids identical to the consensus sequence are indicated by dots.\",\n \"EXAMPLE 1 SC5 Isolation, SC5 Biochemical and Functional Characterization, Production of Anti-SC5 mAbs T lymphocyte immune responses are regulated by functional cell surface molecules providing positive signals that lead to the expansion of antigen-specific clones, and negative signals which prevent excessive stimulation or responsiveness to self antigens.\",\n \"The activation of resting T lymphocytes requires two independent signals.\",\n \"The signal provided by the engagement of T-cell receptor (TCR) must be accompanied by a second positive signal in order to result in optimal T cell response.\",\n \"CD28 is considered as the major co-stimulatory receptor inducing T lymphocyte activation and IL-2 synthesis and preventing cell-death.\",\n \"Recent studies demonstrated that CD3/TCR stimulation leads to a redistribution of the detergent-insoluble glycolipid-enriched membrane fraction (DIG) or raft, which results in the aggregation of TCR/CD3 and DIG-associated signal-transducing molecules.\",\n \"CD28 was reported to enhance raft redistribution to the site of TCR engagement.\",\n \"Further, a series of other molecules may provide co-stimulatory signals to T cells, acting at different time points, affecting particular subsets or promoting distinct effector functions.\",\n \"Negative regulation of T lymphocyte responses can be mediated by CD152-induced signals or by apoptosis through the members of TNF receptor superfamily.\",\n \"Recently, a novel type of negative regulation has been reported for subsets of NK cells and CD8+ T lymphocytes (Lan998, Annu.\",\n \"Rev.\",\n \"Immunol.\",\n \"16: 359-393).\",\n \"The immunoglobulin-like (KIRs) or C-type lectin structures responsible for these signals prevent unwanted effector functions such as activation of CTL activity or cytokine production.\",\n \"We now describe a novel 96 kD transmembrane receptor, SC5, which delineates a minor subset of peripheral blood lymphocytes in normal individuals.\",\n \"Anti-SC5 mAb stained mainly CD45RO+ lymphocytes, from both CD8 and CD4 subsets, as well as NK lineage cells.\",\n \"We found that SC5 was predominantly located in the intracellular compartment of resting T lymphocytes and its surface membrane expression increased rapidly after cell activation.\",\n \"SC5 molecule engagement inhibited the anti-CD3 mAb-induced proliferation of resting T lymphocytes or T cell clones, whereas it had no effect on the cytokine-induced proliferation of these cells.\",\n \"At the same time, the effector cytotoxic activity mediated by CD8+ T cell clones was not altered by anti-SC5 mAb.\",\n \"Further on, we observed that the percentage of SC5+CD4+ circulating lymphocytes was significantly increased in Sézary syndrome (SS) patients as compared to peripheral blood lymphocytes of normal individuals.\",\n \"Importantly, we were able to clearly demonstrate that Sézary cells express SC5 molecules.\",\n \"In addition, we found that ligation of SC5 molecules in a cutaneous T cell lymphoma (CTCL) cell line resulted in a strong inhibition of the malignant cell proliferation induced by anti-CD3 mAbs.\",\n \"Thus, SC5 molecule expression can serve to detect the presence of circulating malignant CD4+ cells in SS patients.\",\n \"Moreover, the identification of a cell surface molecule providing negative signals upon ligation constitutes a new tool to investigate the mechanisms of pathological T cell growth and could be used to prevent it.\",\n \"Material and Methods Production of Anti-CS mAb SC5 mAb was obtained by immunizing 6 wk old Balb/c mice with the NK cell line Ytindi, using an established protocol (David et al.\",\n \"1990; J. Immunol.\",\n \"144: 1-6).\",\n \"Hybridoma supernatants were screened for selective reactivity with the immunizing cell line and a CTCL cell line termed Pno (Poszcepczynska et al.\",\n \"2000; Blood 96: 1056-1063) by indirect immunofluorescence and flow cytometry.\",\n \"Pno cell line derives from CTCL cells from a patient which had a CD3+ Vβ22+ CD4+ CD8αα+ CD25-phenotype, and which proliferates in response to IL-7 and to anti-CD3 mAb stimulation.\",\n \"Any CTCL cell line is appropriate for this screening.\",\n \"Examples of other CTCL cell lines include the HUT78 cell line (ATCC TIB-161).\",\n \"The reactive supernatants were further tested for their reactivity with peripheral blood lymphocytes (PBL).\",\n \"A hybridoma supernatant was selected for its reactivity with both tumoral cell lines and with a minor PBL sub-population.\",\n \"The hybridoma was cloned twice and cloned hybridomas were passaged into pristane-primed Balb/c mice to produce ascites.\",\n \"The antibody isotype was determined as IgM and termed anti-SC5 mAb.\",\n \"The ascites was dialysed against PBS, sterilized by ultrafiltration and further utilized at final dilution {fraction (1/200)}.\",\n \"This hybridoma has been deposited on Oct. 30, 2000 at the C.N.C.M.\",\n \"(Collection Nationale de Microorganismes, Institut Pasteur, 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France) in accordance with the Budapest Treaty; the C.N.C.M.\",\n \"deposit number is I-2575.In this example, “anti-SC5 mAb” refers to the mAb produced by hybridoma I-2575.Patients After informed consent and approval by an ethic committee (CCPPRB, Hôpital Henri Mondor, Creteil, France), we obtained blood samples from eleven patients with CTCL.\",\n \"Eight patients had a Sézary syndrome with 10 to 80% circulating CD3+, CD4+, CD8-Sézary cells, whereas three patients had a transformed mycosis fungoides (MF) and presented with disseminated skin tumors with a CD3+, CD4+, CD8-phenotype.\",\n \"All patients were not previously treated with chemotherapy.\",\n \"Cells and Cell Lines Peripheral blood mononuclear cells (PBMC) were isolated by the technique of Ficoll-Isopaque (Pharmacia fine Chemicals, Piscataway, N.J.) density gradient centrifuigation.\",\n \"Human T cell clones GDS.3 (CD3+TCRαβ+CD4+CD8−), DS6 (CD3+TCRγδ+CD4−CD8−), LSO (CD3+TCRγδ+CD4−CD8−) and JF1 (CD3+TCRαβ+CD4−CD8+), described in Bensussan et al.\",\n \"1984 (J. Exp.\",\n \"Med.\",\n \"159: 947-952) and Yssel et al 1984.(J.\",\n \"Immunol.\",\n \"Methods 72: 219), were fed each 8 days with irradiated allogenic PBMC in culture medium consisting of RPMI 1640 (GIBCO, Paisley, UK), 2 mmol/L L-glutamine, penicillin (100 U/ml), streptomycin (100 mg/ml), 10% heat-inactivated human serum, 50 IU/ml recombinant interleukin-2 (rIL-2, Sanofi Synthélabo, Labège, France) and 1 μg/ml phytohemagglutinin (PHA), (Wellcome, Beckenham, UK).\",\n \"Functional studies were performed at day 7 after the feeding.\",\n \"Standard human leukemic cell lines were mycoplasma-free and maintained in logarithmic growth in complete RPMI medium supplemented with 10% fetal calf serum (FCS) and antibiotics.\",\n \"The human CTCL cell clone, Pno, was established from peripheral blood of a Sézary patient and maintained in culture as previously described (Poszcepczynska et al.\",\n \"2000; see supra).\",\n \"Pno cell line has a CD3+Vβ22+CD4+CD8αα+CD25-phenotype and proliferates in response to IL-7 and to anti-CD3 mAb stimulation.\",\n \"Other CTCL cell lines include the HUT78 cell line (ATCC TIB-161).\",\n \"Monoclonal Antibodies and Flow Cytometry Studies Indirect immuno-fluorescent staining was performed with hybridoma supernatants or ascites fluid using FITC-conjugated goat anti-mouse Ig from Caltag laboratories (San Francisco, Calif.).\",\n \"For two-color analysis the immuno-fluorescent staining was performed by incubating 3×105 cells with anti-SC5 mAb for 30 min at 4° C. Cells were then washed and incubated with FITC-conjugated goat anti-mouse IgM (Caltag laboratories) followed by a second PE-, ECD- or TRI-conjugated specific mAb of IgG isotype.\",\n \"Stained cells were analyzed using a single argon flow cytometer analyser (Epics XL, Bekman-Coulter, Miami, Fla.) as described in Schiavon V et al.\",\n \"1999; Tissue Antigens 53: 23-32.For intracellular labeling, cells were fixed in PBS 4% p-formaldehyde for 20 min at 4° C., washed, and then permeabilized with staining buffer containing 0.1% saponin.\",\n \"Conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD45RO, anti-CD69, and anti-TCRVβ22 mAbs were purchased from Immunotech (Marseille, France).\",\n \"Other mAbs are available from Beckman Coulter (see Cell Analysis 2000 catalogue): anti-CD3 (PE) Cell Analysis 2000 catalogue reference IM1282; purified anti-CD3 Cell Analysis 2000 catalogue reference IM0178; Cell Analysis 2000 catalogue reference anti-CD4 IM0449; anti-CD8 Cell Analysis 2000 catalogue reference IM0452; anti-CD45RO Cell Analysis 2000 catalogue reference IM1307; anti-CD69 Cell Analysis 2000 catalogue reference IM1943; anti-CD94 Cell Analysis 2000 catalogue reference IM2276; anti-CD158a Cell Analysis 2000 catalogue reference IM2277; anti-CD158b Cell Analysis 2000 catalogue reference IM2278; anti-NKG2a Cell Analysis 2000 catalogue reference IM3291; anti-BY55 Cell Analysis 2000 catalogue reference IM2745.Immunoprecipitation of Biotinylated Surface Molecules.\",\n \"2×107 cells were washed twice in PBS and surface-labeled with Sulfo-NHS-LC biotin (Pierce Europe, Interchim, Montlucon, France) (2 mg/mL in PBS) for 20 min at 4° C. After quenching for 20 min with RPMI 1640, cells were washed twice in PBS and re-suspended in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM Na Vanadate, 10 mM NaF, 1 mM PMSF, 1 μg/mL aprotinin, and 1 μg/mL leupeptin) for 1 h at 4° C. Postnuclear supernatant was then incubated for 2 h at 4° C. in a 96-well plate (Maxisorp Nunc immuno-plate) pre-coated with goat anti-mouse IgG+IgM (Caltag laboratories) followed by anti-SC5 mAb or anti-TCRβ (anti-TCRβ C305 isotype matched mAb, Cell Analysis 2000 catalogue reference IM1466; anti-TCRVbeta22, Cell Analysis 2000 catalogue reference IM2051; anti-TCRzeta, Cell Analysis 2000 catalogue reference IM3169; all from Beckman Coulter).\",\n \"Immuno-precipitates were washed 4 times with washing buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM Na Vanadate, 10 mM NaF, 1 mM PMSF) and precipitated proteins were subjected to SDS-PAGE.\",\n \"Western blot analysis was performed using streptavidin-peroxidase (Immunotech) and the ECL detection system according to manufacturers' recommendations (Amersham Pharmacia, Orsay, France).\",\n \"Proliferation Assays For lymphocyte activation, PBMC were cultured in medium containing 15% human serum in the presence of 1 μg/ml PHA (Wellcome, Beckenham, UK).\",\n \"For proliferation assays, 5×104 cells were cultured in triplicates in 96-well round-bottomed plates (Greiner, Nürtingen, Germany) in a final volume of 0.2 ml culture medium.\",\n \"When needed, the plates were pre-coated with anti-CD3 mAb at the indicated concentrations as previously described (Poszcepczynska et al.\",\n \"2000, see supra).\",\n \"For stimulation of CTCL cell line Pno, pre-coated anti-CD3 mAb was used in combination with 2 ng/ml phorbol 12 β-myristate 13 α-acetate (PMA, Sigma Biochemicals).\",\n \"Cells were cultured for 4 days and were pulsed with 1 μCi of 3H[TdR] during the last 8-16 h of culture.\",\n \"3H[TdR] incorporation was measured in a liquid scintillation counter (Topcount; Packard Instrument Co, Meriden, Conn.).\",\n \"For the determination of IL-2 production, 105 PBL were stimulated with immobilized anti-CD3 mAb in the presence of anti-SC5 or control mAb.\",\n \"After 32 hours of culture, 100 μL of supernatant was added to 105 cells from an IL-2-dependent T cell clone.\",\n \"CD3-Induced Redirected Cytotoxicity Assays The redirected cytotoxicity assay was carried out as described in Le Cleach et al.\",\n \"2000, Clin.\",\n \"Exp.\",\n \"Immunol.\",\n \"119: 225-230.The target murine mastocytoma P815 tumor cells were loaded with 100 μl 51Cr (2.5 mCi/mL) for 90 min at 37° C., washed and incubated with anti-CD3 mAb for 15 min at room temperature.\",\n \"Effector cells were likewise pre-incubated with anti-SC5 mAb (1:200 final dilution of ascites) and added to target cells in ratio 5:1, in a final volume of 150 μl in 96 well V-bottomed microtiter plates for 4 h. After centrifugation 100 μl aliquots were counted in a gamma-counter to determine 51Cr release.\",\n \"The spontaneous release was always less than 20% from the maximum release (target cells with 1% NP40).\",\n \"Percentage of specific 51Cr release was calculated as described in David et al.\",\n \"1987, J. Immunol.\",\n \"138: 2831-2836.Statistical Analysis Statistical analysis of the results was performed with Statistica software vers.\",\n \"5.0 (StatSoft, Los Angeles, Calif.).\",\n \"Significant differences between SC5 expression in patients and control group were evaluated by the Mann-Whitney U test, and the correlation between the percentage of SC5+CD4+ cells and the percentage of malignant cells in patients peripheral blood was evaluated by the Spearman Rank Order Correlations test.\",\n \"Results The results herein reported have been obtained with the CTCL cell line Pno, and with CTCL cells collected from patients.\",\n \"Equivalent experiments and results can be performed and obtained with any CTCL cell line, such as e.g.\",\n \"HUT 78 (ATCC TIB-161).\",\n \"Expression of SC5 Molecules in Normal Resting and Activated Peripheral Blood Lymphocytes Monoclonal antibodies raised against the functional tumor cell line Ytindi were analyzed for their simultaneous reactivity with the CTCL cell line Pno (Poszcepczynska et al.\",\n \"2000, see supra) and normal peripheral blood T cells.\",\n \"The SC5-specific mAb, of IgM isotype, stained both the immunizing and CTCL cell lines.\",\n \"Among peripheral blood lymphocytes, anti-SC5 mAb delineated a small sub-population (see FIGS.\",\n \"1A, 1B, 1C).\",\n \"The SC5 mAb reactive molecule was expressed by a variable percentage of CD3+ T cells, never exceeding 20% (mean=10.5%, SD±5.6).\",\n \"Both CD4+ and CD8+ lymphocytes were stained by anti-SC5 mAb.\",\n \"A sub-population of peripheral blood CD56+ NK cells was also reactive with anti-SC5 mAb, though with more important inter-individual variations (mean=19.4%, SD±11.4); see the below Table 1.TABLE 1 expression of SC5 in peripheral blood lymphocyte sub-populations Cell population N tested mean (%) SD Total Ly 15 14.1 6.8 CD3+ Ly 10 10.5 5.6 CD4+ Ly 8 9.4 5.1 CD8+ Ly 8 14.8 5.1 CD56+ Ly 10 19.4 11.4 A representative example of SC5 co-expression with CD3 or CD56 molecules on gated lymphocytes from PBL of normal donors is shown in FIG.\",\n \"1A.\",\n \"It should be noted that most SC5+ lymphocytes belonged to the activation/memory pool, as demonstrated in FIG.\",\n \"1B.\",\n \"Over 70% of the SC5-positive cells co-expressed CD45RO.\",\n \"As SC5 molecule expression was detected in all IL-2 dependent T lymphocyte clones tested, we verified whether its cell surface expression was induced during T lymphocyte activation.\",\n \"We found that stimulation of PBL with PHA promptly increased the expression of SC5 on CD3+ lymphocytes.\",\n \"Both the percentage of SC5+ T cells and the level of SC5 expression rose very rapidly after stimulation (see FIG.\",\n \"2).\",\n \"As early as 4 hours after stimulation with PHA, the percentage of SC5+CD3+ cells increased two to threefold and reached a maximum after 24 to 48 hours.\",\n \"In the course of 5 to 7 days of in vitro stimulation SC5 expression returned to the basal level.\",\n \"In parallel we studied the expression of the very early activation antigen CD69 and we observed similar kinetic profiles of CD69 and SC5 on CD3+ lymphocytes.\",\n \"It should be noted, that unlike CD69 molecules, low levels of SC5 were already present on circulating PBL before stimulation (FIG.\",\n \"2) Activation-induced antigen expression results either from de novo protein synthesis or cell surface export of preexisting intracellular molecules.\",\n \"Therefore, we studied the expression of SC5 in non-activated permeabilized PBL.\",\n \"As shown in FIG.\",\n \"3, anti-SC5 mAb stained over 90% of permeabilized lymphocytes vs. 16% of the same non-permeabilized cells.\",\n \"The specificity of anti-SC5 mAb staining was confirmed by using the isotype-matched mAb BY55 which detects a GPI-anchored cell surface structure expressed by a subset of circulating lymphocytes (see Agrawal et al.\",\n \"1999, J. Immunol.\",\n \"162: 1223-1226).\",\n \"A comparable percentage of BY55+ cells was found in permeabilized and non-permeabilized lymphocytes.\",\n \"As a positive control for the detection of an intracellular epitope, we used an anti-CD3ζ chain mAb which only labeled permeabilized T lymphocytes.\",\n \"Thus, the surface expression of SC5 is dynamically regulated by the activation status of cells and its induction during T cell activation is due to the enhanced transport of the molecule from an intracellular pool to the surface membrane.\",\n \"Biochemical Characterization of SC5 Antigen In order to characterize the molecular weight of the structure identified by anti-SC5 mAb, an IL-2 dependent TCRγδ+ clone, termed DS6, was surface labeled with biotin and the cell lysates were immun-precipitated with either anti-SC5 mAb or an isotype-matched anti-TCRβ mAb (as negative control).\",\n \"Anti-SC5 mAb recognized a single molecule with apparent molecular weight of 96 kD under reducing conditions (see FIG.\",\n \"4).\",\n \"As tumor cell lines (the immunizing Ytindi cells) are often characterized by aberrant expression of carbohydrate epitopes (20, 22), we studied the reactivity of anti-SC5 mAb with the SC5+ NK cell line NK3.3 and with the CTCL cell line HUT78 (ATCC TIB-161) after treatment with neuraminidase or sodium periodate in concentrations destroying known sialylated epitopes (digestion of CD75 epitope on Raji cells was used as a positive control).\",\n \"Expression of SC5 was not affected by neuraminidase or sodium periodate treatment.\",\n \"Thus, we excluded a possible carbohydrate nature of the epitope.\",\n \"Inhibition of Tile Anti-CD3 mAb-Induced T Lymphocyte Proliferation by Anti-SC5 mAb In order to examine the possible function of SC5 molecule in normal T cells, we studied the effect of anti-SC5 mAb alone or during the proliferative responses induced by immobilized anti-CD3 mAb.\",\n \"We found that anti-SC5 mAb alone or in combination with PMA did not induce the proliferation of peripheral blood T lymphocytes.\",\n \"Interestingly, when soluble anti-SC5 mAb was present together with immobilized anti-CD3 mAb, a significant inhibition of lymphocyte proliferation was observed.\",\n \"The inhibition varied between 33 and 66% at day 4 depending on the donor as compared to the effect of an isotype-matched irrelevant control mAb (see FIG.\",\n \"5A, upper panel).\",\n \"As SC5 molecule is expressed on monocytes, it was important to determine whether the inhibitory effect was monocyte-independent.\",\n \"We studied the proliferation of two T cell clones, GDS.3 and LSO, stimulated by immobilized anti-CD3 mAb in the presence of anti-SC5 mAb or an isotype control mAb.\",\n \"The results obtained with both T cell clones indicated that engagement of SC5 molecules resulted in an inhibition of the proliferation to anti-CD3 mAb (see FIG.\",\n \"5A, lower panel).\",\n \"It should be noted that the inhibitory effect of anti-SC5 mAb on T cell proliferation depended on its concentration, as well as on the concentration of the agonistic anti-CD3 mAb.\",\n \"The inhibition obtained with anti-SC5 mAb was significant only when T cells were stimulated with optimally diluted anti-CD3 mAb (see FIG.\",\n \"5B).\",\n \"Such inhibitory behaviour was also reported for anti-KIRs mAbs (Cambiaggi et al.\",\n \"1999, Blood 94: 2396-2402).\",\n \"The decrease of T cell proliferation following SC5 engagement could be due to a direct induction of cell death, a perturbation of the IL-2R expression, or the inhibition of cytokine synthesis.\",\n \"We found that anti-SC5 mAb did not inhibit the IL-2-dependent proliferation of PBL or T cell clones (FIG.\",\n \"5A).\",\n \"Furthermore, the addition of rIL-2 to T cells pre-incubated with anti-SC5 mAb (ascites diluted to {fraction (1/200)}) in combination with anti-CD3 for 48 h restored their proliferation.\",\n \"This would not be the case if the specific mAb inhibited IL-2R expression or caused cell death.\",\n \"Finally, in order to prove that SC5 triggering may inhibit cytokine production, we compared the amount of IL-2 secreted by anti CD3-activated PBL in the presence or absence of anti-SC5 mAb, using an IL-2 dependant T cell line.\",\n \"The quantity of IL-2 produced in the presence of anti-SC5 mAb represented 40 to 65% of the control values obtained with an isotype-matched antibody (see FIG.\",\n \"5C).\",\n \"Thus, the simultaneous engagement of SC5 and CD3 molecules in T cells results in the decrease of cytokine production.\",\n \"We next studied the effect of anti-SC5 mnAb on T cell effector cytotoxic activity using an anti-CD3 mAb redirected killing assay against the FcgR+ murine cell line P815.FIG.\",\n \"5D shows a representative experiment performed with a cytotoxic T cell clone at an E/T ratio of 5/1.The CD8+ T cell clone JF1, expressing high amount of SC5 molecules, was induced to kill murine target cells following anti-CD3 mAb is stimulation.\",\n \"The simultaneous targeting of SC5 by its specific mAb did not influence JF1 cytotoxic activity, regardless of the E/T ratio or the concentration of stimulating anti-CD3 mAb.\",\n \"Expression of SC5 Molecules on Malignant Lymphocytes Obtainedfrom Patients with Sézary Syndrome As we initially screened anti-SC5 mAb for its reactivity with a CTCL cell line, we further studied the expression of SC5 molecule in the periphcral blood lymphocytes isolated from 8 patients with Sézary syndrome.\",\n \"Two-color flow cytometry analysis showed that expression of SC5 was significantly increased in the CD4+ subset (see representative results obtained with one patient in FIG.\",\n \"6A).\",\n \"On the average, 35.5% of the CD4+ lymphocytes in SS patient blood expressed SC5 as compared to 9.4% in healthy controls (p<0.001), (FIG.\",\n \"6B).\",\n \"In the 8 SS patients studied, the expression of SC5 on CD4+ cells correlated with the percentage of circulating malignant cells detected by cytomorphology (r=0.91, p=0.0012).\",\n \"It should be noted that in these patients the percentage of total CD4+ cells (mean=88%, SD±8.8) was higher than that of Sézary cells (mean=26%, SD±12.8).\",\n \"We also studied SC5 molecule expression in peripheral blood samples from 3 MF patients without blood involvement.\",\n \"No significant difference in the levels of SC5 expression on peripheral blood CD4+ cells was observed between MF patients and healthy donors (FIG.\",\n \"6B).\",\n \"Still, an increased percentage of SC5+CD4+ was detected in the skin samples from two of these patients, that we were able to to test (26% and 19% respectively).\",\n \"In one SS patient we had previously demonstrated that the circulating malignant cells were clonal lymphocytes with a CD4+TCRVβ22+ phenotype (Poszcepczynska et al.\",\n \"2000, see supra).\",\n \"Here we demonstrated that SC5 was co-expressed on the TCRVβ22+ cells, i.e.—by the malignant cell population (FIG.\",\n \"7A).\",\n \"It should be noted that the profile of SC5-mAb binding to TCRVβ22+ cells corresponded to a weak homogeneous expression on the whole malignant clonal cell population (FIG.\",\n \"7B).\",\n \"Interestingly, the malignant TCRVβ22+ cells did not co-express common activation markers as CD69 (FIG.\",\n \"7C), CD25 or CD30 as previously described (Poszcepczynska et al.\",\n \"2000, see supra).\",\n \"Thus, the increased levels of SC5 in SS PBL were mostly due to the expression of the antigen on circulating malignant T cells.\",\n \"Inhibition of the Anti-CD3mAb-Induced CTCL Cell Line Proliferation by Anti-SC5 mAb We next investigated whether SC5 molecules were able to provide a negative signal in malignant CTCL cells.\",\n \"We used the long-term cultured Pno CTCL tumor T cell line which strongly proliferates in the presence of IL-7.As previously reported that Pno cell line expresses functional T cell receptors, since immobilized anti-CD3 mAbs or the combination of soluble anti-CD3 mAbs and PMA induced significantly their proliferation (Poszcepczynska et al.\",\n \"2000, see supra).\",\n \"We examined the effect of SC5 molecule engagement on the proliferation of Pno cells.\",\n \"FIG.\",\n \"8 shows that anti-SC5 mAb strongly inhibited the anti-CD3 mAb-induced proliferation of the CTCL cell line.\",\n \"In contrast, PMA alone (2 ng/mL) produced a low level of proliferation which was not significantly affected by anti-SC5.Notably, anti-SC5 mAb had no effect on the IL-7-dependent proliferation, since Pno cells incubated with IL-7 in the presence of anti-SC5 mAb proliferated as vigorously as in the presence of the isotype control mAb.\",\n \"Thus, we concluded that SC5 antigen is functional in CTCL malignant cells and may specifically and profoundly inhibit their proliferation triggered through the TCR/CD3 signaling pathway.\",\n \"A CTCL tumor was then successfully grafted onto transgenic mice having a substantial deficiency in functionally active NK cells and T lymphocytes (SCID mice) as described in Charley et al.\",\n \"(1990) “Establishment of a human cutaneous T cell lymphoma in CB-17 SCID mice” J.\",\n \"Invest.\",\n \"Dermatol.\",\n \"94: 381-384 (see also U.S. Pat.\",\n \"No.\",\n \"5,530,179 Cornelius P. Terhorst and Baoping Wang for obtention of SCID mice).\",\n \"Anti-SC5 mAb was intraveneously or intracutaneously administered at different times to half of the mice at various concentrations of purified mAb per gram of mouse, whereas the other half received the same amount of purified CD56 (isotype IgM) as a control.\",\n \"PBL and cutaneous cells were collected for standard cytological observations of infiltrating large T cells, and the percentage of CTCL cells was assessed for each mouse of the experiment.\",\n \"This procedure is an example of procedure that enables the is skilled person to check that anti-SC5 compounds, such as the mAb produced by the hybridoma deposited as deposit number I-2575 at the C.N.C.M., are capable of inhibiting the in vivo proliferation of human CTCL.\",\n \"Discussion A number of receptors with dynamic surface expression mediate T cell interactions with other immune cells or cytokines and, consequently modulate T cell immune responses.\",\n \"In this study, we identified a novel 96 kD functional molecule expressed by a minor subset of PBL whose surface expression is induced rapidly during in vitro T-cell activation.\",\n \"SC5 is distinct from the established molecules with similar molecular weight and/or T cell modulatory functions.\",\n \"First, SC5 was induced very early upon T cell activation, with important increase of its surface expression detected after 4 h. A similar kinetics has been observed for the very early activation antigen CD69, which is detected on the cell surface between 2 and 4 h after stimulation.\",\n \"Most established T-cell specific inducible receptors such as 4-1BB, OX-40 LIGHT or CD152 (CTLA-4) appear on T-cell surface between 10 to 24 h following stimulation.\",\n \"Further, SC5 expression is not T cell restricted.\",\n \"The antigen was detected on sub-populations of NK and B lymphocytes, monocytes and granulocytes.\",\n \"Such a wide distribution pattern is typical of certain killer cell Ig-like receptors (KIRs), as the recently described AIRM1 and IRp60, which are found on NK cells, T and B lymphocytes, myeloid and other antigen-presenting cells.\",\n \"The family of immunoglobulin—like transcript (ILT) inhibitory receptors has also a pan-leukocyte type of expression, the individual receptors being detected on different NK, T, B and myeloid sub-populations.\",\n \"Moreover, ILT4 has a molecular weight very close to SC5.However, neither the expression of AIRM1 and IRp60, nor of ILTRs depends on the activation status of cells.\",\n \"The prompt induction of SC5 molecule at the surface of activated T cells followed by its down-regulation may result from trafficking of intracellular molecules to the cell membrane and vice versa, as we detected SC5 in most non-activated T lymphocytes after cell permeabilization.\",\n \"Such a regulatory mechanism has been already reported for another T cell receptor, CD152.Further on, a heterogeneous family of lysosome associated membrane proteins (LAMP) exists, including molecules involved in endocytosis, lysosomal trafficking and secretion from intracellular granules and that may be induced on the surface of monocytes, neutrophiles, NK, B or T cells upon activation.\",\n \"Additional studies on the intracellular localization and transport of SC5 would precise its proper role in T cell activation and functions.\",\n \"Our studies rcvealed that triggering of SC5 by its specific mAb inhibited the anti-CD3 mAb-induced T cell proliferation.\",\n \"This inhibitory effect required an optimal anti-CD3 mAb stimulation, which is necessary to induce maximal SC5 surface expression in PBL (see FIG.\",\n \"5B).\",\n \"A 1:200 final dilution of anti-SC5 ascites significantly inhibits the proliferation induced by anti-CD3 mAb at 2 microgram par well and this effect of inhibition increases with lower anti-CD3 mAb concentrations −1 microgram par well; 0.5 microgram per well—A 1:400 final dilution of anti-SC5 ascites do not significantly inhibits the proliferation induced by anti-CD3 mAb at 2 microgram per well, but inhibition is significant when a lower concentration of anti-CD3 is used −1 microgram per well; 0.5 microgram per well—The person of ordinary skill in the art can proceed with such adjustments by standard interpolation from the particular results described herein, or from his own trials.\",\n \"At the same time, the inhibitory.\",\n \"threshold posed by SC5 engagement could be bypassed by excessive anti-CD3 mAb stimulation.\",\n \"It should be noted that the inhibitory functional effects obtained by ligation of SC5 molecules were observed in an APC-independent system.\",\n \"Further experiments are needed to determine whether SC5 stimulation may have different effects on the proliferation of APC or allo-stimulated T cells.\",\n \"Recently, the negative regulation of immune responses has been extensively investigated, in particular in T lymphocytes and NK cells.\",\n \"The best studied T cell specific inhibitory receptor, CD152 (CTLA-4), is a typical activation-induced antigen, functionally coupled to CD28 co-stimulatory receptor.\",\n \"Indeed, CD152 may overcome CD28 positive signals at low antigenic concentrations and prevent immune response.\",\n \"Alternatively, it may downregulate and terminate T-cell proliferation induced through CD28 by affecting IL-2R expression and IL-2 synthesis.\",\n \"Similarly to CD152, SC5 is an intracellular molecule whose surface expression is regulated by the activation status of cells.\",\n \"Both receptors mediate their effects by affecting IL-2 synthesis.\",\n \"The identification of SC5 specific ligands and signaling pathways will precise the temporal and spatial correlation between these negative T-cell regulators.\",\n \"In consideration to its wide cell distribution, SC5 functional effects are probably not restricted to T cells.\",\n \"In several aspects, SC5 is comparable to NK cell inhibitory receptors, equally expressed by T cells and other lymphocytes sub-populations.\",\n \"Both KIRs and CD94/NKG2 receptors were shown to inhibit antigen-specific and superantigen-driven activation of TCRαβ and TCRγδ T cells, including cytotoxicity, proliferation and cytokine secretion.\",\n \"The KIR-induced inhibition depends on the activation status of T cells and the potency of T cell stimulation and may be bypassed under specific in vivo conditions.\",\n \"However, SC5 is definitely distinct from KIRs, since we demonstrated that it selectively inhibited the proliferation of anti-CD3 mAb stimulated T cells without affecting their effector cytotoxic functions.\",\n \"An important characteristic of SC5 molecule is its significantly increased expression in peripheral blood lymphocytes of patients with SS in comparison to healthy subjects.\",\n \"SS is characterized by the generalization of a primary epidermotropic malignant T cell clone with mature phenotype.\",\n \"Actually, none of the established T cell functional receptors conclusively identifies peripheral blood SS tumor cells.\",\n \"MF/SS has been considered as the expansion of a T cell subset with a particular phenotype, CD4+CD7−, corresponding to a normally existing T memory subset.\",\n \"However, a recent study demonstrated that the CD4+CD7− population does not always represent the dominant T cell clone in SS patients (Dummer et al.\",\n \"1999, Arch.\",\n \"Dermatol.\",\n \"Res.\",\n \"291: 307-311).\",\n \"SS is regarded as a proliferation of specifically activated T cells although the nature of the stimulus is not known (Fargnoli et al.\",\n \"1997, Leukemia 11: 1338-1346).\",\n \"For this reason the distinct expression of activation induced antigens on SS and normal T cells is of special interest.\",\n \"Established activation antigens such as CD69, CD25, CD30 are not consistently detected on Sézary cells, as confirmed by our own investigations.\",\n \"Expression of CD25 rather correlates with the progression of the disease and the transformation of MF/SS cells into a large cell variant in a minority of the cases.\",\n \"An increased rate of expression of T cell activation molecules as HLA-DR, CD25 or CD71 in SS has been primarily associated with reactive tumor-infiltrating T cells.\",\n \"A 78 kD very late activation antigen, BE2, was reported to be specifically increased in SS peripheral blood, while not detected on normal T cells.\",\n \"In fact, BE2 was not consistently detected in all SS patients studied, a comparatively low percentage of peripheral blood cells in relation to the reported percentage of malignant cells were BE2-positive, and BE2 was equally detected in patients without clinical evidence of blood involvement.\",\n \"In contrast, SC5 was detected in 8/8 SS cases studied.\",\n \"Although the percentage of malignant cells determined by cytomorphology varied considerably from patient to patient (10-80%), the expression of SC5 on CD4+ cells correlated with this percentage.\",\n \"Finally, no elevated expression of SC5 on CD4+ cells was detected in MF patients without blood involvement.\",\n \"Further, we demonstrated that Sézary cells identified by the expression of the clonotypic TCRVβ chain co-expressed SC5 molecule, confirming that increased levels of SC5 in the peripheral blood of SS patients were due to its expression on the malignant T cell clone.\",\n \"A very important observation was that SC5 triggering on Sézary cells induced down-modulation of the CD3-mediated proliferation of tumor cells, analogous to the inhibition observed in normal T cells.\",\n \"It has been already demonstrated that malignant CTCL lymphocytes preserve certain functional properties as they may be activated through basic signaling pathways although producing aberrant profiles of cytokines.\",\n \"We now demonstrate that an inhibitory pathway is preserved in CTCL tumor cells, that may permit to counterbalance their proliferation.\",\n \"Notably, the inhibitory effect observed after anti-SC5 mAb binding on anti-CD3 mAb-stimulated CTCL Pno cells was much more important than in normal T cells.\",\n \"Anti-SC5 mAb does not seem to directly mediate cell death of neither normal nor Sézary T cells, since it does not modulate the rIL-induced proliferation.\",\n \"Moreover, T-cells that had been cultivated in the presence of anti-SC5 mAb can be re-stimulated by addition of rIL-2.At the same time, we observed a diminished IL-2 production by anti-SC5 mAb-treated T cells.\",\n \"which should account at least partially for the inhibition of their proliferation.\",\n \"Inhibitory effects of functional receptors on the proliferation of malignant cells have been already observed.\",\n \"It has been recently reported the inhibition of the proliferation of normal and leukemic myeloid cells mediated by the homologous NK cell sialoadhesin receptor AIRMI and CD33 myeloid-specific antigen (Vitale et al.\",\n \"1999, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 96: 15091-15096).\",\n \"Unlike SC5, these receptors inhibited proliferation in the presence of growvth factors and most probably by induction of apoptosis.\",\n \"Interestingly, another T cell activation antigen, CD30, expressed by a subset of normal CD45RO+ T cells, is likely to mediate the regression of primary CTCL other than MF/SS.\",\n \"This effect is related to the natural ligand of CD30 (CD30L) which is co-located within the cytoplasm of malignant cells and may induce cytolytic cell death independently of Fas/FasL system (Mori et al.\",\n \"1999, Blood 94: 3077-3083).\",\n \"In this aspect, the identification of SC5 ligand will enlighten the mechanism of its inhibitory action.\",\n \"In conclusion, we have identified an early activation antigen which is distinct, by phenotypic, biochemical and functional criteria, from the established T lymphocyte receptors.\",\n \"As it was underlined, SC5 molecule is expressed by a sub-population of the post-activation/memory peripheral blood T cell subset.\",\n \"Likewise, continuous expression of KIRs is detected on minor T cell clones, and this expression seems to depend on TCR-occupancy by specific antigens.\",\n \"It was recently proposed that such clones might be auto-reactive and maintained tolerant by KIRs signaling.\",\n \"We may speculate that SC5 surface expression is also preserved on auto-reactive T cells, suggesting that Sézary syndrome tumor cells possibly originate from such clones.\",\n \"Importantly, the expression of SC5 on CTCL malignant cells will serve to better define their origin and the nature of the signal driving mature T cells to uncontrolled proliferation.\",\n \"In addition, the inhibitory effect of anti-SC5 mAb on tumor T cells constitutes a novel therapeutic approach in patients with advanced CTCL.\",\n \"Furthermore, expression of SC5 has also been observed at the surface of CD8+ CTCL, such as CD8+ transformed MF.\",\n \"EXAMPLE 2 P140 is Expressed by CD4+ CTCL Cells, and Identification of a Novel P140 Allelic Form Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of lymphomas primarily involving the skin.\",\n \"Mycosis fungoides (MF) is characterized by skin invasion of clonally-derived malignant CD4+ T lymphocytes that phenotypically resemble mature T helper cells.\",\n \"A more aggressive form of CTCL develops when the is malignant cells become non-epidermotropic and is associated with extra-cutaneous involvement.\",\n \"Sézary syndrome (SS) is a more aggressive form of CTCL that is characterized by a clonal expansion of CD4+/CD45RO+ T cells and the appearance of these malignant T cells in the blood.\",\n \"The biology of the disease remains poorly understood, as it is difficult to identify the malignant cell, due to the lack of specific cell surface markers.\",\n \"Thus, in cutaneous lesions it is difficult to distinguish CD4+ CTCL cells from reactive infiltrating CD4+ T lymphocytes.\",\n \"Bagot et al.\",\n \"previously described a unique CD4+ T cell line derived from CTCL lesions (Bagot et al.\",\n \"1998, Blood 91: 4331-4341); this cell line and the in vivo tumor cells expressed an identical size of the complementarily-determining region 3 of TCR-Vβ transcripts.\",\n \"More recently, Poszepczynska et al.\",\n \"(Blood 2000, 96: 1056-1063) functionally characterized an IL-7-dependent CD4+CD8αα+ tumor T cell line isolated from the blood of another patient with a cutaneous erythrodermic CTCL.\",\n \"This tumor T-cell line was identical to the major circulating T cell populations, as demonstrated by the expression of TCR-Vβ22 and the identity of the TCRβ-VDJ sequences.\",\n \"Here, we show that these two different CTCL lines express the p140/KIR inhibitory receptor for HLA-A alleles.\",\n \"Importantly, this receptor is detected on freshly isolated tumor cells derived from the same patients.\",\n \"Moreover, the p140/KIR was also co-expressed by a major subset of CD4+ lymphocytes in seven other patients with SS, and by tumor skin CD4+ cells in two additional patients with advanced MF.\",\n \"The p140/KIR is detected in normal individuals on a minor NK cell-subset and on rare peripheral blood CD3+CD8+ cells.\",\n \"Moreover, T cells obtained from skin in other dermatological diseases such as inflammatory skin diseases and toxic epidermal necrolysis did not express this receptor (Le Cleach et al.\",\n \"2000, Clin.\",\n \"Exp.\",\n \"Immunol.\",\n \"119: 225-230).\",\n \"Thus, our present findings demonstrate that the p140/KIR represents a suitable marker on CD4+ cells for the identification of CTCL.\",\n \"Materials and Methods Patients After informed consent and approval by an ethic committee, we obtained skin and blood samples from eleven patients with a CTCL.\",\n \"Eight patients had a Sézary syndrome with 10 to 45% circulating Sézary cells in the blood.\",\n \"In seven cases, the phenotype of tumor cells was CD3+,CD4+,CD8−.\",\n \"In one case, the phenotype was CD3+,CD4+,CD8αα+ (patient Pno).\",\n \"Three patients had a transformed mycosis fungoides (Lez, Cou, Bic) and presented with disseminated skin tumors with a CD3+,CD4+,CD8− phenotype.\",\n \"All patients were not previously treated with chemotherapy.\",\n \"Isolation of Tumoral Lymphocytes Fresh CTCL tumor cells were obtained from tumor fragments mechanically dispersed into single-cell suspensions (Bagot et al.\",\n \"1998, Blood 91: 4331-4341).\",\n \"The mononuclear cells were then washed and frozen in human serum plus 10% dimethyl sulfoxide for later use.\",\n \"For Sézary syndrome patients, the mononuclear blood cells were isolated by the technique of Ficoll-Isopaque (Pharmacia fine Chemicals, Piscataway, N.J.).\",\n \"Long Term Culture of Tumor Cell Lines We established the long term culture of Pno cell line (TCRVβ22+,CD3+, CD4+,CD8αα+,MHC class I+,MHC class II−) in vitro from the peripheral blood of the patient as previously described (Bagot et al.\",\n \"1998, Blood 91: 4331-4341).\",\n \"We demonstrated that both the malignant clone circulating in the patient blood and the derived cultured T-cell line were identical for their cell surface phenotype and for their size and sequence of the TCRβ VDJ region (Poszepczynska et al.\",\n \"2000, cf.\",\n \"supra).\",\n \"Other sources of SS cell lines may be found at the ATCC (e.g.\",\n \"HUT 78 ATCC TIB-161).\",\n \"The Cou-L cell line (TCRVβ13+) was cultured in vitro with rIL-2 for more than three years.\",\n \"It corresponds to a subelone of the CD4+ Cou-LS CTCL line previously described (Bagot et al.\",\n \"1998, cf supra).\",\n \"The Cou-L cell line, the original TCRVβ13+, CD4+ Cou-LS CTCL, and the tumor cells freshly isolated from the skin shared the same size and sequence of the TCRβ VDJ region (Bagot et al.\",\n \"1998, cf.\",\n \"supra).\",\n \"Monoclonal Antibodies (mAbs) and Flow Cytometry Studies One- and two-color immunofluorescence analysis was performed as previously described (Bagot et al.\",\n \"1998, cf.\",\n \"supra).\",\n \"Anti-CD3 (Cell Analysis 2000 catalogue reference 6604629), anti-CD4 (Cell Analysis 2000 catalogue reference 6602138), anti-MHC Class I (Cell Analysis 2000 catalogue reference IM0107) and II (Cell Analysis 2000 catalogue reference IM0108) are obtainable from Beckman Coulter (see Cell Analysis 2000 catalogue).\",\n \"The anti-TCRVβ13+ mAb was purchased from Bioadvance (Emerainville, France, catalogue reference TCR 1738), and the anti-TCRVβ22+ mAb was obtained from Beckman-Coulter (Marseille, France, Cell Analysis 2000 catalogue reference IM 1484/IM1365).\",\n \"The anti-CD8 αβ 2ST8.5H7 mAb is obtainable from Beckman Coulter (Cell Analysis 2000 catalogue reference IM 2014.As anti-p140 mAbs, Q66 (IgM, anti-p140) and AZ158 (IgG2a, recognizing both p70/NKB1 and p140) which have been described in Pende et al.\",\n \"1996 (J. Exp.\",\n \"Med.\",\n \"184: 505-518) have been used; however any anti-140 binding compound is appropriate (e.g.\",\n \"anti-p140 antiserum, Fc-HLA-A11, Fc-HLA-A3 fuision proteins).\",\n \"Z27 (IgG1, anti-p70/NKB1), EB6 (IgG1, anti-p58.1/S50.1), GL183 (IgG1, anti-p58.2/p50.2), XA185 (IgG1, anti-CD94), Z199 and Z270 (IgG2b and IgG2a respectively, anti-NKG2A), and B9.4 (IgG2b, anti-CD8) mAbs are all obtainable from Beckman Coulter (see Cell Analysis 2000 catalogue references IM2748, IM2277, IM2278, IM1610, IM2750, IM0102).\",\n \"Goat anti-mouse IgG FITC and goat anti-mouse IgM PE are also obtainable from Beckman Coulter (see Cell Analysis Catalogue references IM08 and IM0555).\",\n \"Biochemical Characterization 20×106 cells were incubated (15′ at 20° C.) in 1 ml PBS pH8 containing 250 μg of EZ-LINK SULFO-NHS-LC-LC-BIOTIN (Pierce, Rockford, Ill.) and washed three times in Washing Buffer (10 mM TRIS pH 8, 0.14 M NaCl).\",\n \"Cells were then lysed in 1% NP-40 and immuno-precipitated with Sepharose-PA (Pharmacia Biotech Inc. Piscataway, N.J.)-coupled AZ158 (IgG2a, antip70/140) or B9.4 ([gG2b, anti-CD8) mAbs.\",\n \"Samples were analyzed by discontinuous SDS-PAGE either undigested or digested with N-glycosidase F (Boehringer Mannheim, GmbH, Germany) and transferred to Immobilon P (Millipore Corp, Bedform, Mass.).\",\n \"After staining with Neutravidin (Pierce) the Renaissance Chemiluminescence Kit (NEN, Boston, Mass.)\",\n \"was used for detection.\",\n \"NK cell clones were obtained by limiting dilution as described in Pende et al.\",\n \"1996 (J. Exp.\",\n \"Med.\",\n \"184: 505-518).\",\n \"RT-PCR Analysis Total RNA was extracted from CTCL cell lines Pno and Cou-LCD8 αα using RNA-Clean System (AGS GmbH, Heidelberg, Germany).\",\n \"cDNA synthesis was performed using oligodT priming.\",\n \"Primers used for cDNA amplification of the complete ORF (open reading frame) of KIR displaying three Ig-like domains (1395 bp) were the following: 5′CATGT(CT)GCTCA(CT)GGTCGTC (Ig3 UP; SEQ ID No.\",\n \"5) and 5′ GGTTTTGAGACAGGGCTG (Ig3 DOWN; SEQ ID No.\",\n \"6).\",\n \"Amplification was performed for 30 cycles (30 sec.\",\n \"at 94° C., 30 sec.\",\n \"at 55° C., 30 sec.\",\n \"at 72° C.), followed by a 7 min.\",\n \"incubation at 72° C., utilizing AmpliTAQ (Perkin Elmer-Applied Biosystems, Foster City, Calif.).\",\n \"PCR products were sub-cloned into pcDNA3.1/V5-His-TOPO vector (Invitrogen, Carlsbad, Calif.).\",\n \"DNA sequencing was performed using d-Rhodamine Terminator Cycle Sequencing Kit and a 377 Applied Biosystems Automatic Sequencer (Perkin Elmer-Applied Biosystems).\",\n \"Transient Transfections COS-7 cells were transfected with pcDNA3.1 TOPO-KIR3D cl.24 or with pCR3-cl.1.1 (Pende et al.\",\n \"1996, J. Exp.\",\n \"Med.\",\n \"184: 505-518) utilizing Fugene 6 (Roche, Monza Italy).\",\n \"Briefly, cells were seeded at 5×105/plate; 24 hr later they were incubated with 6 μg plasmid and 10 μl of Fugene-6 reagent in DMEM/10% FCS.\",\n \"After 48 or 72 hrs, transfected cells were used for cytofluorimetric analysis.\",\n \"Cell transfectants were stained with Q66 and AZ158 mAbs, followed by a phycoerythrin-conjugated goat antibody to mouse IgG2a or IgM and analyzed by flow cytometry using a FACSort (Becton Dickinson, San Jose, Calif.).\",\n \"Results CTCL Cell Lines are Stained by mAbs to the p140/KIR Two long-term CTCL tumor lines Pno (labeled with anti-TCR-Vβ22 mAb) (Poszepczynska et al.\",\n \"2000, cf supra) and Cou-L (labeled with anti-CD3 mAb) (Bagot et al.\",\n \"1998, cf.\",\n \"supra) were analyzed for reactivity with different anti-KIR mAbs.\",\n \"We found that both cell lines were reactive with mAbs Q66 (see FIG.\",\n \"9A) and AZ158, both recognizing the p140/KIR.\",\n \"In contrast, these cell lines did not express other inhibitory receptors specific for HLA class I molecules, including p58.1, p58.2, p70 KIRs and the CD94/NKG2A lectin-like receptor (Poszepczynska et al.\",\n \"2000, cf supra).\",\n \"Tumor T Lymph Ocytes Freshly Isolated from CTCL Patients are Stained by Anti-p140/KIR mAbs To determine whether p140/KIR was expressed by freshly isolated tumor cells, we tested the reactivity of Q66 mAb with uncultured tumor cells isolated from the blood in SS patient Pno and from tumoral skin fragments of MF patient Cou.\",\n \"We found that the majority of tumor cells were stained by this mAb (FIG.\",\n \"9B).\",\n \"In particular, we observed that most TCRVβ22+ tumor lymphocytes isolated from the blood of patient Pno were reactive with Q66 antibody, and that most of the TCRVβ13+ tumor lymphocytes isolated from the skin of patient Cou were stained by the same antibody (FIG.\",\n \"9B).\",\n \"Next, we studied the phenotype of tumor T lymphocytes from the blood of seven additional patients with a Sézary syndrome, with malignant cells representing 10 to 45% of circulating CD4+ lymphocytes, and tumor T lymphocytes isolated from skin tumors of two other patients with a MF.\",\n \"Remarkably, all patients tested exhibited a significant population co-expressing CD4 and p140/KIR (see Table 2 below).\",\n \"TABLE 2 Anti-Q66 mAb stained CD4+ lymphocytes isolated from the skin of patients with transformed mycosis fungoides and from blood of patients with Sezary syndrome.\",\n \"Percentage of positive cells with Patient anti- anti-CD4 mAb + samples CD4 mAb anti-Q66 mAb MF Lez 65 29 Bic 53 44 SS Bri 85 35 Bar 90 18 Att 95 35 Ros 98 15 Can 78 36 Pet 45 9 Riv 98 19 It should be noted that all Q66+ cells were included in the CD4+ cell population.\",\n \"Thus, the expression of p140/KIR, which in normal individuals is restricted to subsets of lymphocytes from the NK and CD8+ populations, appears to be a characteristic of CTCL tumor CD4+ T lymphocytes, both in the skin and in the blood.\",\n \"As control, skin T lymphocytes derived from another dermatological disease, toxic epidermal necrolysis, which were shown to contain small percentages of various KIR expressing T lymphocytes (Le Cleach et al.\",\n \"2000, Clin.\",\n \"Exp.\",\n \"Immunol.\",\n \"119: 225-230), failed to express p140/KIR.\",\n \"Molecular Characterization of the p140 Receptor Expressed by CTCL.\",\n \"The Pno and Cou-L cell lines were surface labeled with biotin and cell lysates were immuno-precipitated with an anti-p140 (AZ158 mAb).\",\n \"As shown in FIG.\",\n \"10, this mAb immuno-precipitated from a NK clone and from the Pno and Cou-L cell lines a molecule with a molecular mass of approximately 70 kD under reducing conditions.\",\n \"Treatment with N-glycosidase revealed a protein backbone of approximately 50 kD with a slightly higher mobility for Pno and Cou-L compared to NK clone.\",\n \"These data suggested that the p140 inhibitory receptor expressed by these CTCL tumor cell lines could be almost similar to that previously detected on normal NK cells (Pende et al.\",\n \"1996, J. Exp.\",\n \"Med.\",\n \"184: 505-518).\",\n \"Next, we determined whether the cDNA encoding the molecule recognized by Q66 and AZ158 mAbs on Pno and Cou-L CTCL cell lines corresponded to one of the already described cDNA encoding p140/KIR (SEQ ID No.\",\n \"3).\",\n \"To this end, RT-PCR was performed on RNA derived from these cell lines using a set of primers able to amplify all cDNA encoding KIR with three Ig-like domains.\",\n \"From the Pno cell line, we isolated a full-length cDNA, termed KIR3D cl.24 (SEQ ID No.\",\n \"1).\",\n \"Comparison of its nucleotide sequence with DNA sequences coding for all KIR characterized by three extra-cellular Ig-like domains revealed that KIR3D cl.24 represents a novel allelic form of p140 receptor.\",\n \"In particular, its nucleotide sequence displays five differences compared to the previously described cl.\",\n \"1.1 cDNA (Pende et al.\",\n \"1996, J. Exp.\",\n \"Med.\",\n \"184: 505-518), resulting in four amino acid substitutions in the mature protein (see FIG.\",\n \"11; SEQ ID No.\",\n \"2: clone 24 protein; SEQ ID No.\",\n \"4: clone 1.1 protein).\",\n \"Three of the four substitutions are found in the extra-cellular Ig-like domain (pos.\",\n \"20, 92 and 111 of the mature protein), whereas the other is located in the cytoplasmic region (pos.401).\",\n \"RT-PCR performed on Cou-L CTCL cell line revealed two different allelic fonns of p140/KIR one corresponding to cl.\",\n \"1.1 cDNA and the other identical to KIR3D cl.24 (isolated from Pno cell line).\",\n \"The cDNA derived from the CTCL cell lines were then transiently transfected in COS-7 cells.\",\n \"As expected, all cell transfectants were brightly stained by Q66 and AZ158 mAb, while they were unreactive with p70/KIR-specific Z27 mAb used as a negative control.\",\n \"Finally, RT-PCR was performed on RNA extracted from PBL derived from three SS patients (including patient Pno) and from skin-derived T cells of one additional MF patients.\",\n \"Also in these samples one or another allelic isoform of p140/KIR described above could be identified.\",\n \"The same results were obtained on malignant CTCL cells collected from patients.\",\n \"Discussion Skin lesions in CTCL contain a heterogeneous lymphocytic infiltrate composed of malignant T cells, which are most often CD4+, and non-neoplastic tumor infiltrating T lymphocytes.\",\n \"We previously reported CD4+ cytotoxic tumor infiltrating lymphocytes specifically directed against autologous malignant CTCL CD4+ cell line.\",\n \"However, as no tumor restricted cell surface structure, besides the clonotypic TCR expressed by tumor cells, has been identified on CTCL, it is difficult using standard methods to distinguish malignant from non-malignant reactive CD4+ lymphocytes.\",\n \"In the present study, we report for the first time that tumor cells from MF and SS patients express the p140/KIR.\",\n \"This receptor has been identified in both skin and blood tumor cells from CTCL patients as well as in two long-term culture CTCL lines.\",\n \"Two color fluorescence analysis indicated that p140/KIR expression is restricted to T cells characterized by a given TCRβ-VDJ previously identified on tumor cells.\",\n \"Thus, p140/KIR allows rapid identification of tumor cells from tumor reactive cells among the T lymphocyte CD4+ population.\",\n \"This could be particularly useful in SS patients, in which the tumor T cell clone is not always easily identified within the peripheral blood CD4+ lymphocyte population.\",\n \"Moreover, during the course of the disease or after treatment, it is crucial to assess whether an increase of the CD4+ population is due to an expansion of the tumor cell population or of reactive T lymphocytes.\",\n \"Since the anti-p140/KIR mAbs appear to recognize tumor cells in all CTCL patients analyzed, they may be considered as a suitable tool for the direct identification of CTCLs.\",\n \"In addition, the use of p140/KIR co-expression on CD4+ CTCL cells provides a unique tool to distinguish malignant cells from normal cells in every patient.\",\n \"This is in contrast to TCR determinations which is unique to an individual patient.\",\n \"Moreover, the p140/KIR represents a possible target for the development of novel specific immuno-therapy of CTCL.\",\n \"Previous studies indicated that this receptor was able to generate inhibitory signals upon recognition of HLA.A3 and HLA.A11 alleles.\",\n \"It is of note, however, that p140/KIR expression in the various patients analyzed appears apparently independent from their own HLA Class I haplotype.\",\n \"Nevertheless, the actual role of p140/KIR in the patho-physiology of CTCL is an important feature that remains to be studied by taking into account the potential role of this receptor in the tolerance to self.\",\n \"In conclusion, the present study demonstrates for the first time the expression of the p140/KIR in CD4+ CTCL cells and the isolation of a novel allelic form of this receptor in tumor cells.\",\n \"This finding is an important new issue, both for the patho-physiology and for the clinical management of CTCL patients.\",\n \"Furthermore, p140 expression has also been observed at the surface of CD8+ CTCL such as CD8+ transformed MF.\",\n \"EXAMPLE 3 Production of Anti-CTCL Drugs The person of ordinary skill in the art is enabled to produce anti-CTCL drugs pursuant to the following outlines, given for illustration purposes: use of anti-SC5 or anti-p140 mAb with a cytokine such as Interferon gamma or IL-2 for stimulating the immune system of cells in the vicinity of CTCL cells, use of these anti-SC5 and anti-p140 mAb as vectors for delivering anti-CTCL ingredients onto CTCL cells; examples of anti-CTCL ingredients comprise standard compounds used in chimiotherapy such as radioelements, toxines.\",\n \"mAb vectors for radioelements can be produced following standard techniques applied to an anti-SC5 mAb of the invention (see e.g.\",\n \"Press O W et al.\",\n \"“Phase two trial of iode131-B1 (anti-CD20) antibody therapy with autologous stem cell transplantation for relapsed B cell lymphoma”, Lancet 1995 vol.\",\n \"346 pp336-340, of which content is herein incorporated by reference).\",\n \"mAb vectors for toxine can be produced following standard techniques applied to an anti-SC5 mAb of the invention (see e.g.\",\n \"Ghetie U et al.\",\n \"“Large scale preparation of an immunoconjuguate constructed with human recombinant CD4 and deglycosylated ricin A chain”, J. Immunol.\",\n \"Methods 1990, vol.\",\n \"126 pp 135-141, of which content is herein incorporated by reference).\",\n \"combined use of an antimitotic pro-drug and of anti-SC5 mAb and/or anti-p140 mAb vectors delivering an enzyme capable of transforming an antimitotic pro-drug to the active drug form (mitotic agent in a functional form).\",\n \"Anti-SC5 mAb and anti-p140 mAb vectors delivering an enzyme capable of transforming an antimitotic pro-drug to the active drug form can be produced following standard techniques applied to an anti-SC5 mAb of the invention and to an anti-p140 mAb (see e.g.\",\n \"the ADEPT technique, for Antibody Directed Enzymre Prodrug Therapy, described in Plakey T C et al.\",\n \"“Anti-tumor effects of an antibody-carboxypeptidase G2 conjuguate in combination with phenol mustard prodrugs”, British J.\",\n \"Cancer 1995 vol.\",\n \"72 pp 1083-1088, of which content is herein incorporated by reference).\",\n \"use of anti-SC5 or anti-p140 mAb as ligand agonists (SC5 stimulation or p140 stimulation) use of anti-SC5 or anti-p140 mAb as complement recruiting agent For the above-mentioned uses, mAbs with a double anti-SC5/anti-CD4 or anti-p140/anti-CD4 specificity are preferred.\",\n \"use of anti-SC5 or anti-p140 (no CD4+ specificity needed) as activators of ADCC (Antibody Dependent Cell Cytotoxicity): polynuclear cells, macrophages, NK cells bear Fc receptors which are activated by the Fc portion of the anti-SC5 and anti-p140 mAb, these mAb therefore enable the ADCC activation in the vicinity of CTCL cells.\",\n \"Abbreviations: APC: Antigen Presenting Cell, CTCL: cutaneous T cell lymphoma, DIG: detergent-insoluble glycolipid-enriched fraction, ILT: immunoglobulin-like transcript, KIR: killer cell immunoglobulin receptor, mAb: monoclonal antibody, MF: mycosis fungoides, PBL: peripheral blood lymphocytes, PBMC: peripheral blood mononuclear cells, PHA: phytohemagglutinin, PMA: phorbo112 beta-myristate13 alpha-acetate, SS: Sézary syndrome, TCR: T cell receptor\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10450818"}}},{"rowIdx":284,"cells":{"text":{"kind":"list like","value":[["Anti-Cd28 Antibody","The invention concerns an antibody directed against the CD28 receptor and capable of blocking CD28/B7 interaction, and proteins derived from said antibody, for use in particular to block CD28-dependent activation of lymphocytes."],["1.A protein capable of binding specifically to the CD28 lymphocyte receptor and of blocking the CD28/B7 interaction, comprising at least the CDRs of the heavy chain and of the light chain of the immunoglobulin CD28.3, produced by the hybridoma CNCM I-2582.2.The protein as claimed in claim 1, selected from the group consisting of: a) the antibody CD28.3 produced by the hybridoma CNCM I-2582; b) Fv, Fab, Fab′2 or scFv fragments of the antibody CD28.3; c) chimeric or humanized antibodies obtained from the variable regions of CD28.3; d) Fv, Fab, Fab′2 or scFv fragments of an antibody b); and e) recombinant proteins comprising a fragment b) or d) and a heterologous polypeptide.","3.A nucleic acid molecule encoding a protein as claimed claim 1.4.An expression vector comprising a nucleic acid molecule as claimed in claim 3.5.The expression vector as claimed in claim 4, wherein the expression vector is the plasmid CNCM I-2762.6.A cell expressing a protein as claimed in claim 1.7.The cell as claimed in claim 6, wherein the cell is the hybridoma CNCM I-2582.8.A cell transformed with a nucleic acid molecule as claimed in claim 3, and expressing a protein capable of binding specifically to the CD28 lymphocyte receptor and of blocking the CO28/B7 interaction and comprising at least the CDRs of the heavy chain and of the light chain of the immunoglobulin CD28.3, produced by the hybridoma CNCM I-2582.9.A method for preparing a protein as claimed in claim 1, comprising culturing at least one cell as claimed in claim 6, and recovering said protein from said culture.","10.A medicinal product comprising the protein as claimed in claim 1.11.The medicinal product as claimed in claim 10, wherein in that said protein has a single CD28 receptor-binding site, and said medicinal product is an immunosuppressor which selectively blocks T cell activation via the CD28 receptor.","12.A method of treating a pathological condition selected from the group consisting of transplant rejection, graft-versus-host disease, T-lymphocyte-mediated autoimmune diseases, allergic phenomena and chronic inflammatory diseases wherein the method comprises administering to a patient in need thereof an effective amount of the medicinal product of claim 11.13.A nucleic acid molecule encoding a protein as claimed in claim 2.14.A cell expressing a protein as claimed in claim 2."],["<SOH> Construction of Expression Vectors Comprising the Sequence Encoding α1-Antitrypsin and Allowing the Introduction of a Sequence Encoding an scFv Fragment <EOH>"],["The invention relates to antibodies directed against the CD28 lymphocyte receptor and to their fragments, and to their therapeutic uses, in particular in the context of regulating T cell activation.","Abnormal activation of T cells is involved in the pathogenesis of many autoimmune diseases, and also in transplant rejection phenomena, where they cause an immune response directed against the transplanted organ to develop.","T lymphocyte activation requires an activating signal, induced by the recognition, by T receptors (TCRs) of the antigen associated with the class II major histocompatibility complex (MHC) and presented by antigen-presenting cells (APCs).","However, this activation only causes proliferation of T cells and secretion of specific immunomodulatory cytokines (such as interleukin 2, gamma interferon or interleukin 4) if other T co-stimulation systems are also activated.","One of the most important systems for regulating T lymphocyte activation is the molecular system B7/CD28/CTLA4.This system plays, for example, an essential role in the mechanisms of transplant rejection [Woodward et al., Transplantation, 66, 14-20, (1998)].","The molecules B7.1 (CD80) and B7.2 (CD86) borne by the APCs can activate the CD28 receptor and also the CTLA4 receptor of T lymphocytes.","The activation of CD28 sends the T lymphocyte a positive signal which stimulates the cell; on the other hand, the activation of CTLA4 sends a negative signal which leads to a non-response (anergy) [FALLARINO et al., J. Exp.","Med., 188, 205-210, (1998)].","Dormant T lymphocytes express a large amount of CD28 and very little CTLA4.When there is a first cognitive contact between an APC and a T lymphocyte, the CD28/B7 interaction is favored, which activates the cell.","It is only several hours after the initiation of activation that, due to the increase in membrane expression of CTLA4, the affinity of which for B7 is 5 to 10 times greater than that of CD28, the B7/CD28 interaction shifts in favor of a B7/CTLA4 interaction.","Currently, cyclosporin is mainly used to block T lymphocyte activation, in particular in the context of organ transplants.","Despite the effectiveness of this medicinal product, it does not, however, confer absolute protection.","In addition, it acts by blocking all the calcium-dependent cell activation pathways, and therefore has a biological activity which is not strictly T lyphocyte specific and leads to a considerable number of side effects.","It is therefore desirable to develop new immunosuppressants which have a defined method of action and greater specificity.","It has been postulated that selective inhibition of the agonist signal given to the T cell by CD28, leaving the antagonist system consisting of the pair CTLA4/B7 intact, via specific blocking of the CD28/B7 interaction, would make it possible to prevent T lymphocyte activation.","Such specific blocking of the CD28/B7 interaction can be obtained using an antibody directed against CD28.Anti-CD28 antibodies capable of preventing CD28 binding to B7 are known.","However, they have the drawback, when they are used in their divalent native form, of bringing about the dimerization and the activation of CD28 via their binding with this receptor.","However, monovalent fragments derived from these antibodies are capable of blocking the CD28 receptor without activating it [DAMLE et al., J. Immunol.","140, 1753-1761, (1988); NUNES et al., Int.","Immunol., 5, 311-315 (1993); PAGES et al., J. Biol.","Chem., 271, 9403, (1996)].","It has thus been reported [PERRIN et al., J. Immunol.","163, 1704-1710, (1999)] that Fab fragments derived from an anti-CD28 antibody can curb the clinical symptoms of experimental autoimmune encephalitis induced in mice by the administration of myelin or the transfer of T cells from an affected animal.","Monovalent Fab or scFv fragments derived from an anti-CD28 antibody can potentially be used to prevent T lymphocyte activation via specific blocking of the CD28/B7 interaction.","Fab fragments result from the action of papain on an immunoglobulin molecule, and each contain a light chain and the first half of a heavy chain; scfv fragments consist of the variable portions of the heavy and light chains of an antibody, connected to one another via a flexible linker [CLACKSON et al., Nature, 352, 624-628, (1991)], thus forming a single-chain protein.","These monovalent fragments frequently exhibit less affinity for the antigen than the native antibodies, which can limit their possibilities for use in diagnostic or therapeutic applications.","The inventors have succeeded in selecting, among various antibodies which recognize the CD28 antigen, an antibody capable of blocking the CD28/B7 interaction, the monovalent fragments of which exhibit sufficient affinity for the antigen so that they can be used, in vitro or in vivo, to block the CD28 receptor without activation of this receptor.","This antibody, called CD28.3, is produced by the hybridoma deposited, according to the terms of the Treaty of Budapest, on Nov. 28, 2000, with the CNCM (Collection Nationale de Cultures de Microorganismes [National Collection of Cultures and Microorganisms], 25 rue du Docteur Roux, 75724 PARIS CEDEX 15), under the number I-2582.A subject of the present invention is a protein capable of binding specifically to the CD28 lymphocyte receptor and of blocking the CD28/B7 interaction, characterized in that it comprises at least the CDRs of the heavy chain and of the light chain of the immunoglobulin CD28.3.CDRs (complementarity determining regions) are the portions of the variable regions of an immunoglobulin which are involved in antigen recognition specificity.","Proteins in accordance with the invention thus encompass in particular: a) the antibody CD28.3 produced by the hybridoma CNCM I-2582; b) the Fv, Fab, Fab′2 or scfv fragments of the antibody CD28.3; c) the chimeric or humanized antibodies obtained from the variable regions of CD28.3; d) the fragments of the antibodies b) above comprising the CDRs of the antibody CD28.3, with a monovalent Fv, Fab or scfv fragments, or divalent Fab′2 fragments; e) the recombinant proteins comprising a fragment b) or d) and a heterologous polypeptide.","They may, for example, be: divalent or plurivalent derivatives of scfv fragments, such as “diabodies” or “triabodies”, resulting from the association of 2 or 3 scFv fragments; proteins combining at least one antibody fragment comprising the CDRs of the antibody CD28.3, with at least one antibody fragment comprising the CDRs of an antibody of different specificity; by way of examples, mention will be made of bispecific immunoglobulins, conjugates of an Fv or Fab fragment containing the CDRs of CD28.3 with an Fv or Fab fragment of an antibody of different specificity, and “bispecific diabodies” resulting from the association of an scFv fragment containing the CDRs of CD28.3 with an Fv or Fab fragment of an antibody of different specificity; proteins combining at least one antibody fragment comprising the CDRs of the antibody CD28.3, with a molecule having pharmacological activity (for example a toxin) or effector properties (for example an Fc fragment); proteins combining at least one antibody fragment comprising the CDRs of the antibody CD28.3, with a molecule which makes it possible to prolong its plasma halflife when it is administered in vivo; it is, for example, possible to combine said antibody fragment with a water-soluble polypeptide of sufficient molecular mass for the molecular mass of the fusion polypeptide thus obtained to be greater than the renal filtration threshold.","In this case, a polypeptide will be chosen which, unlike Fc fragments, cannot associate as dimers, and which does not have its own effector activity liable to cause unfortunate side effects.","Polypeptides which have these properties can advantageously be obtained from water-soluble serum proteins, namely, in particular, serum albumin, haptoglobulin, ITIH2 (inter-alpha (globulin) inhibitor, H2 polypeptide), transferrin, CBG (corticosteroid binding globulin), α1-antitrypsin, ITIH4 (inter-alpha (globulin) inhibitor, H4 polypeptide), AACT (alpha-1-antichymotrypsin), TBG (thyroxine binding globulin), fibrinogen and prothrombin, in order to prepare fusion proteins with scFv fragments derived from anti-CD28 antibodies.","It is also possible to conjugate a protein in accordance with the invention with a polyol, for example polyethylene glycol, as described, for example, in U.S. Pat.","No.","4,179,337.An example of a protein in accordance with the invention is illustrated in FIG.","1, which represents an scFv fragment derived from the antibody CD28.3.The sequences of the CDRs of the antibody CD28.3 are boxed in the sequence represented in FIG.","1.The nucleotide sequence encoding this scFv fragment is represented in the attached sequence listing under the number SEQ ID No.","1, and the corresponding peptide sequence is represented under the number SEQ ID No.","2.Fv, Fab or Fab′2 fragments in accordance with the invention can be obtained by the conventional techniques of enzyme digestion, from the antibody CD28.3.A plasmid containing a polynucleotide encoding an scFv fragment of CD28.3, fused to a polynucleotide encoding amino acids 53 to 425 of α1-antitrypsin was deposited, according to the terms of the Treaty of Budapest, on Dec. 11, 2001, with the CNCM (Collection Nationale de Cultures de Microorganismes [National Collection of Cultures and Microorganisms], 25 rue du Docteur Roux, 75724 PARIS CEDEX 15), under the number I-2762.Proteins in accordance with the invention such as chimeric or recombinant antibodies, scFv fragments and their derivatives, etc., can be obtained by conventional genetic engineering techniques, such as those described by SAMBROOK et al.","[MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989)].","Polynucleotides encoding the variable regions of the anti-CD28.3 antibody can, for example, be obtained by cloning said variable regions from a cDNA library of the hybridoma CD28.3, or from the plasmid CNCM I-2762.They can also be prepared, completely or partially, by nucleic acid synthesis, based on the nucleotide sequences of said variable regions.","It is, for example, possible to synthesize polynucleotides encoding the CDRs of CD28.3, and to incorporate them into the framework regions (FRs) of another antibody, in particular of an antibody of human origin, using techniques, known in themselves, of CDR grafting, such as those described by ROUTLEDGE et al.","[“Reshaping antibodies for therapy”, in Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, 13-44, Academic Titles, Nottingham, England (1993)] or by ROGUSKA et al.","Protein Engineering, 9(10), 895-904, (1996)].","A subject of the present invention is also any nucleic acid molecule encoding a protein in accordance with the invention comprising the CDRs of the antibody CD28.3, and also any recombinant vector, in particular any expression vector, comprising said nucleic acid molecule.","A subject of the present invention is also any cell expressing a protein in accordance with the invention comprising the CDRs of the antibody CD28.3.This encompasses in particular the hybridoma CNCM I-2582, and also the host cells transformed with a nucleic acid molecule in accordance with the invention.","Nucleic acid molecules in accordance with the invention may advantageously comprise, besides a sequence encoding a protein in accordance with the invention, a sequence encoding a signal peptide allowing secretion of said protein; they may also comprise one or more sequence(s) encoding one or more marker peptide(s) for detecting, and/or facilitating the purification of, said protein.","Expression vectors in accordance with the invention comprise at least one nucleic acid sequence encoding a protein in accordance with the invention, associated with transcription- and translation-controlling elements which are active in the host cell chosen.","Vectors which can be used to construct expression vectors in accordance with the invention are known in themselves, and will be chosen in particular as a function of the host cell intended to be used.","Host cells which can be used in the context of the present invention can be prokaryotic or eukaryotic cells.","Among the eukaryotic cells which can be used, mention will in particular be made of plant cells, cells from yeast, such as Saccharomyces, insect cells, such as Drosophila or Spodoptera cells, and mammalian cells such as HeLa, CHO, 3T3, C127, BHK, COS, etc., cells.","The construction of expression vectors in accordance with the invention and the transformation of the host cells can be carried out by the conventional techniques of molecular biology.","A subject of the invention is also a method for producing a protein in accordance with the invention, characterized in that it comprises culturing at least one cell in accordance with the invention, and recovering said protein from said culture.","If the protein is secreted, it can be recovered directly from the culture medium; if not, cell lysis will be carried out beforehand.","The protein can then be purified from the culture medium or from the cell lysate, by conventional procedures, known in themselves to those skilled in the art, for example by fractionated precipitation, in particular precipitation with ammonium sulfate, electrophoresis, gel filtration, affinity chromatography, etc.","The proteins in accordance with the invention can be used, in vitro, to study the proliferative response or the differentiation of T lymphocytes responding to an antigenic, viral, allogenic or xenogenic stimulation.","They can also be used, in vitro, to induce the differentiation of T lymphocytes taken from a patient, for example the induction of tolerance with respect to an antigen or to an alloantigen, intended to be subsequently re-administered in vivo.","They may also be used to obtain medicinal products, or diagnostic reagents.","Proteins in accordance with the invention which are divalent, i.e.","which have 2 CD28 receptor-binding sites, and thus capable of inducing dimerization of this receptor, can be used in all situations where it is desired to activate this CD28 receptor, i.e.","to increase the response of a T lymphocyte with respect to an antigen.","Proteins in accordance with the invention which are monovalent, i.e.","which have a single CD28 receptor-binding site, can be used in all situations where it is desired to selectively block this receptor without activating it, in order to induce immunosuppression.","A protein in accordance with the invention, comprising a monovalent fragment derived from an anti-CD28 antibody, can in particular be used to obtain an immunosuppressant medicinal product which selectively blocks T cell activation phenomena involving the CD28 receptor, and which does not have the drawbacks of known immunosuppressants such as cyclosporin.","The T immunosuppression by selective blocking of CD28 with protein in accordance with the invention has applications in all T lymphocyte-dependent pathological conditions.","These are essentially transplant rejection, graft-versus-host disease, T lymphocyte-mediated autoimmune diseases, such as type I diabetes or multiple sclerosis, and type IV hypersensitivity, which is involved in allergic phenomena and also in the pathogenesis of chronic inflammatory diseases following infection with a pathogenic agent (in particular leprosy, tuberculosis, leishmaniasis, listeriosis, etc.).","The present invention will be understood more clearly from the further description which follows, which refers to nonlimiting examples of preparation and of use of antibodies in accordance with the invention.","EXAMPLE 1 Choice of an Antibody Producing Monovalent Fragments, Properties of Monovalent Fab Fragments Derived from CD28.3 Some of the properties of several anti-CD28 antibodies (CD28.1, CD28.2, CD28.3, CD28.4, CD28.5 and CD28.6) are described in the publication by NUNES et al.","[Int.","Immunol., 5, 311, (1993)].","These various antibodies, which are not accessible to the public, were provided by the laboratory of Daniel OLIVE (INSERM).","The antigen-binding properties of the monovalent Fab fragments of these various antibodies were compared.","5 mg of Fab fragments of each of these antibodies were prepared by digestion with papain (papain/antibody molar ratio= 1/100) for 24 hours at 37° C., followed by inactivation of the enzyme with 0.03M iodoacetamide, and dialysis against PBS to remove the iodoacetamide.","1) Binding of the Fab Fragments to CD28+ Jurkat T Cells: 100,000 CD28+ Jurkat cells in 100 μl are incubated in PBS-1% BSA-0.1% NaN3 at 4° C. for 30 minutes with increasing concentrations of anti-CD28 antibodies or of their Fab fragments.","After washing, the cells are incubated in a similar way with an FITC-conjugated anti-mouse IgG goat antibody, washed, and analyzed by cytofluorometry.","The results are given in FIG.","2: Legend of FIG.","2: X-axis: antibody or Fab fragments concentration Y-axis: mean fluorescence intensity (MFI) —⋄—: F1=Fab fragments of the antibody CD28.1 —▪—: F2=Fab fragments of the antibody CD28.2 —Δ—: F3=Fab fragments of the antibody CD28.3 —x—: F5=Fab fragments of the antibody CD28.5 —◯—: F6=Fab fragments of the antibody CD28.6 ····: W132 whole antibody CD28.1 ··-··: W2=whole antibody CD28.2 : W3=whole antibody CD28.3 ——: W5=whole antibody CD28.5 —*—: W6=whole antibody CD28.6 —▴—: Mara-1=negative control (mouse IgG1).","These results show that, among the Fab fragments, only those derived from CD28.3 are capable of significantly binding to the CD28+ Jurkat cells at concentrations of less than 10 μg/ml.","2) Effect of the Fab Fragments on the Adhesion of CD28+ Jurkat T Cells to Transfected Murine L Cells Expressing the B7-1 Molecule: 4×105 human T cells (Jurkat, CD28-positive) labeled with 51Cr are incubated for 2 hours in a microtitration plate in which 105 adherent LTK− or LB7+ cells (murine fibroblasts transfected with human B7.1 [PAGES et al., J. Biol.","Chem., 271, 9403, (1996)] have been seeded 24 hours beforehand.","These incubations are carried out in the presence of the Fab fragments derived from the antibodies CD28.1 to CD28.6, or of the antibody CD28.3, diluted to various dilutions in PBS buffer without Ca2+ or Mg2+.","The adherent cells after washing are quantified by reading the residual radioactivity in a beta counter (PACKARD TOPCOUNT).","The results are given in FIG.","3: Legend of FIG.","3: X-axis: percentage of adherent cells Y-axis: antibody concentration —♦—: F1=Fab fragments of the antibody CD28.1 —▪—: F2=Fab fragments of the antibody CD28.2 —▴—: F3=Fab fragments of the antibody CD28.3 —x—: F5=Fab fragments of the antibody CD28.5 —*—: F6=Fab fragments of the antibody CD28.6 ····: whole antibody CD28.3 ◯: no antibody.","These results show that the Fab fragments derived from CD28.3 are the most effective for inhibiting CD28/B7 interactions.","They give 90% inhibition of adhesion at a concentration of 3 μg/ml, and with an effectiveness comparable to that of the whole antibody CD28.3, whereas, at this concentration, the Fab fragments derived from the other antibodies give no more than 50% inhibition of adhesion.","3) Effect of the Fab Fragments on Proliferation in a Mixed Lymphocyte Reaction: 105 peripheral blood mononuclear cells (PBMCs) are mixed with 105 allogenic mononuclear cells irradiated at 35 Gy, in the presence of varying concentrations of the antibodies CD28.1 to CD28.6 or of the Fab fragments derived from these antibodies.","The proliferative response in these cultures is evaluated after 3 days, by incorporation of (3H) thymidine for a period of 16 hours.","The results are given in FIG.","4: Legend of FIG.","4: X-axis: antibody concentration Y-axis: proliferative response (cpm) Basal level of proliferation=6 500 cpm.","—♦—: 28.1=antibody CD28.1 —▪—: 28.2=antibody CD28.2 —▴—: 28.3=antibody CD28.3 —x—: 28.5=antibody CD28.5 ···*···: 28.6=antibody CD28.6 ——: Fab.","1=Fab fragments of the antibody CD28.1 ··+··: Fab.","2=Fab fragments of the antibody CD28.2 ——: Fab.","3=Fab fragments of the antibody CD28.3 —+—: Fab.","5=Fab fragment of the antibody CD28.5 —⋄—: Fab.","6=Fab fragment of the antibody CD28.6.These results show that the Fab fragments derived from CD28.3 or from CD28.6 are the most effective for inhibiting mononuclear cell proliferation.","The whole antibodies CD28.1 to CD28.6, tested in parallel, have no inhibitory effect or indeed stimulate the proliferation by virtue of their stimulator action on CD28.Effect of the Fab Fragments Derived from CD28.3 on Proliferation Induced by a Superantigen For this experiment, responder CD4+ T cells were mixed with irradiated isogenic PBMCs, in the presence of 50 ng/ml of toxic shock syndrome toxin-1 (TSST-1), which specifically stimulates the vβ2+ T cell, either in the absence of antibody or in the presence of anti-B7-1 (1 μg/ml), of anti-B7-2 (0.5 μg/ml), of CTLA4Ig (10 μg/ml), or of Fab fragments derived from CD28.3 (10 μg/ml).","The proliferative response in these cultures is evaluated after 1, 3, 6 and 8 days, by incorporation of (3H) thymidine for a period of 16 hours.","The results are given in FIG.","5: Legend of FIG.","5: X-axis: culturing time Y-axis: proliferation index=PI P I = cpm mixed lumphocyte reaction - cpm irradiated stimulating cells only cpm unstimulated responder cells —♦—: anti-CD28.3 Fab —X—: anti-B7-2 —▪—: CTLA-4 Ig ··*··: anti-B7-1+2 —▴—: anti-B7-1 —◯—: no antibody TSST-1 induces considerable proliferation of CD4+ T cells.","In the presence of anti-B7, of CTLA4Ig or of the Fab fragments of CD28.3, 70% inhibition of this proliferation is observed after 6 days.","Effect of the Fab Fragments Derived from CD28.3 on Cytokine Production In order to determine whether the Fab fragments derived from CD28.3 could induce an immune deviation in vitro, a mixed lymphocyte reaction (PBMCs derived from a donor A/irradiated PBMCs derived from a donor B) was carried out, in the presence of Fab fragments derived from CD28.3.105 peripheral blood mononuclear cells from a donor are mixed with 105 allogenic mononuclear cells irradiated at 35 Gy, and cultured for 5 days in the presence or absence of 10 μg/ml of Fab derived from the antibody CD28.3.The RNA of the responder cells was extracted, and the amount of cytokine mRNA was evaluated by quantitative measurement of the number of transcripts, related to the amount of HPRT, using a TaqMan (Perkin Elmer).","In the presence of Fab fragments derived from CD28.3, a decrease in the production of γIFN and of IL2, and an increase in the production of IL10 are observed.","This deviation in the immune response suggests an orientation toward a Th2-type response.","This result is unexpected insofar as it has been reported that the involvement of CTLA4 (which is supposed to intervene in the blocking of CD28 alone) leads to a Th1-type response.","In Vitro Processing of the Antibody CD28.3 and of the Fab Fragments Derived Therefrom, by Human T Cells A possible internalization of the Fab fragments of the antibody CD28.3 in human T cells was investigated, in comparison with the whole antibody CD28.3.Jurkat T cells were incubated in culture medium with 100 μg/ml of antibody CD28.3, at 37° C. or at 0° C. At various times, the cells were washed with cold PBS buffer containing 0.1% of bovine serum albumin, and NaN3, in order to block membrane motility.","The bound antibodies were revealed with a fluorescein-labeled goat anti-mouse secondary antibody.","The cells were mounted in MOVIOL and analyzed by confocal microscopy.","It is thus observed that the whole CD28.3 antibodies which bind to the Jurkat T cells are captured and disappear from the cell surface at 37° C., but not at 0° C. On the other hand, the Fab fragments remain attached at the surface of the cell.","This indicates that the attachment of the divalent antibodies CD28.3 leads to dimerization of CD28, which brings about their entry into the cell, whereas the monovalent Fab fragments, which do not induce this dimerization, remain at the surface.","EXAMPLE 2 Properties of an scFv Fragment Derived from the Antibody CD28.3 FIG.","1 represents the nucleotide sequence and the deduced polypeptide sequence of an scFv fragment derived from the antibody CD28.3.The portions of this sequence corresponding to the variable fragment of the heavy chain and of the light chain are represented in capital letters.","The sequence corresponding to the variable fragment of the light chain is also underlined.","The sequence of the linker is represented in lower case letters.","The sequences of the CDRs of the heavy chain and of the light chain are boxed in.","The nucleotide sequence encoding this scFv fragment is also represented in the attached sequence listing, under the number SEQ ID No.1.The cDNA encoding this scFv fragment was inserted into the vector pIG6 (Biochemisches Institut, University of Zurich).","This vector comprises in particular an ampicillin resistance marker and an expression cassette which comprises an inducible lac promoter under the control of which are placed: a sequence encoding an ompA signal peptide, a sequence encoding a marker peptide of sequence (1-letter code) DYKD, a sequence encoding a c-myc marker peptide, and a sequence encoding a polyhistidine-5 marker.","The cDNA encoding the scFv fragment described above was introduced between the EcoRI and EcoRV sites of pIG6, downstream of the sequence encoding the peptide DYKD and upstream of the sequence encoding the c-myc marker.","The construct obtained is called pIg6-28.3.Production in Prokaryotic Cells The vector pIg6-28.3 was used to transform E.coli JM83 cells.","The cells are cultured at 25° C., up to an OD550 of 0.5.After induction with IPTG, the scFv fragment is produced in soluble form in the periplasma.","After electrophoresis and Western blotting, it appears in the form of a band at approximately 30 kDa.","It is purified from the periplasmic extracts of the bacteria, obtained after osmotic shock in 50 mM Tris-Cl, and ultracentrifugation of the insoluble material, by chromatography on an Ni-NTA matrix and ion exchange on DEAE-Sepharose.","The binding of the scFv fragments present in the eluate of the NiNTA column, to CD28+ Jurkat cells, is comparable to that obtained with Fab fragments obtained from the antibody CD28.3 by digestion with papain.","Production in Eukaryotic Cells The vector pSec-28.3 was used to transfect Cos cells.","The cells are cultured at 37° C. for 3 days.","The scFv fragment is produced in soluble form in the supernatant.","This supernatant inhibits the mixed lymphocyte reaction: 105 peripheral blood mononuclear cells from a healthy donor are mixed with 105 peripheral blood mononuclear cells from another, healthy allogenic donor.","The proliferative response in these cultures is evaluated after 5 days by incorporation of (3H) thymidine for a period of 16 hours.","Considerable inhibition of the incorporation, dependent on the supernatant dilution used, is observed.","A control supernatant exhibits no proliferation-inhibiting activity.","EXAMPLE 3 Production of a Fusion Protein Comprising an scFv Fragment of CD28.3 The nucleotide sequence encoding the scfv fragment described in example 2 was linked to the 5′ end of a portion of the cDNA of human α1-antitrypsin (GENBANK accession number K01396) corresponding to amino acids 53 to 425, via a hinged peptide of sequence VAAPS.","The resulting sequence is represented in the attached sequence listing under the number SEQ ID No.","3, and the corresponding polypeptide under the number SEQ ID No.","4.EXAMPLE 4 Construction of Expression Vectors Comprising the Sequence Encoding α1-Antitrypsin and Allowing the Introduction of a Sequence Encoding an scFv Fragment Prokaryotic Expression Vector: The vector pIG6 was used (Biochemisches Institut, University of Zurich).","This vector comprises in particular an ampicillin resistance marker, and an expression cassette which comprises an inducible lac promoter under the control of which are placed: a sequence encoding an ompA signal peptide, a sequence encoding a marker peptide of sequence (1-letter code) DYKD, a sequence encoding a c-myc marker peptide, and a sequence encoding a polyhistidine-5 marker.","The cDNA encoding a fragment of human α1-antitrypsin corresponding to amino acids 53 to 425 was introduced between the EcoRI and EcoRV sites of pIg6, downstream of the sequence encoding the peptide DYKD and upstream of the sequence encoding the c-myc marker.","FIG.","1 illustrates diagrammatically the construct obtained, called pIg6-Haat.","Eukaryotic Expression Vector: The vector pSECTagB (Invitrogen, De Schelp, The Netherlands) was used.","This vector comprises in particular an ampicillin resistance marker, a zeocin resistance marker, and an expression cassette which comprises a CMV promoter under the control of which are placed: a sequence encoding a signal peptide of the IgG kappa light chain, a sequence encoding a c-myc marker peptide, and a sequence encoding a polyhistidine-6 marker.","The cDNA encoding a fragment of human α1-antitrypsin corresponding to amino acids 53 to 425 was introduced between the BamHI and EcoRI sites of the vector PSEC B Tag, upstream of the sequence encoding the c-myc marker.","FIG.","2 illustrates diagrammatically the construct obtained, called pSecHaat.","EXAMPLE 5 Construction of Expression Vectors Integrating the Sequence Encoding the CD28.3 scFv/α1-Antitrypsin Fusion Protein Prokaryotic Expression Vector: The cDNA encoding the CD28.3 ScFv/α1-antitrypsin fusion protein described in example 3 above was introduced between the EcoRI and XhoI sites of pIG6, downstream of the sequence encoding the peptide DYKD and upstream of the sequence encoding the c-myc marker.","FIG.","3 illustrates diagrammatically the construct obtained, called pIg6-28.3Haat.","Eukaryotic Expression Vector: The cDNA encoding the CD28.3 ScFv/α1-antitrypsin fusion protein described in example 3 above was introduced between the BamHI and XhoI sites of the vector PSEC B Tag, upstream of the sequence encoding the c-myc marker.","FIG.","4 illustrates diagrammatically the construct obtained, called pSec-28.3Haat.","This vector, harbored by E.coli DH5α, was deposited with the CNCM on Dec. 11, 2001, under the number I-2762.EXAMPLE 6 Expression and Purification of the Fusion Proteins In Prokaryotic Cells: The vector pIg6-28.3Haat was used to transform E. coli JM83 cells.","The cells are cultured at 25° C., up to an OD550 of 0.5.After induction with IPTG, the protein is produced in soluble form in the periplasm.","After electrophoresis and Western blotting, it appears in the form of a band at approximately 74 kDa.","It can be purified from the periplasmic extracts using an NI-NTA affinity chromatography matrix and/or an anti-c-myc affinity chromatography matrix.","It can also be purified using an anti-α1-antitrypsin affinity column.","In Eukaryotic Cells: The vector pSec-28.3Haat was used to transfect CHO cells by lipofection.","The cells are cultured in the presence of 200 μg/ml on zeocin in MEM medium containing 10% of fetal calf serum.","The protein is secreted into the culture medium.","After separation by electrophoresis, Western blotting, and revelation with an anti-c-myc antibody, it appears in the form of a band at approximately 80 kDa.","EXAMPLE 7 Assays for Activity of an scFv/α1-Antitrypsin Fusion Protein The anti-CD28 activity of the CD28.3 scFv/α1-antitrypsin fusion protein obtained in example 6 above was evaluated by its binding to the CD28 molecule, or to cells expressing CD28 on their membrane, and its lack of binding to cells which do not express CD28.The immunosuppressor activity of the CD28.3 scFv/α1-antitrypsin fusion protein obtained in example 6 above is evaluated by the inhibition of adhesion to B7, and the inhibition of the induced activation of the T lymphocyte.","These anti-CD28 and immunosuppresor activities were measured using the following assays: Anti-CD28 Activity Biosensor Measurement of the CD28-Binding Parameters: Recombinant human CD28 was immobilized on the biosensor (BIACORE) detector.","A CD28.3 scFv/α1-antitrypsin fusion protein obtained as described in example 6 above was brought into contact with the detector.","The binding parameters are: KA (1/M)2.86e9; KD (M): 3.49e-10.In comparison, these parameters measured for the Fab fragment of the antibody CD28.3 are: KA (1/M): 9.69e8; KD (M): 1.03e-9.The affinity for CD28, of the Fab fragment of the antibody CD28.3 and of the fusion protein, are therefore comparable.","Cytofluorometry Assay for Specific Recognition of CD28: 105 Jurkat (Cd28+) and U937 (CD28−) cells are incubated in PBS-1% BSA-0.1% NaN3, at 4° C., for 1 hour with increasing concentrations of the CD28.3 scFv/α1-antitrypsin fusion protein.","After washing, the cells are incubated with an anti-alpha-1-antitrypsin rabbit antibody and then with an FITC-conjugated goat anti-rabbit antibody, washed, and analyzed by cytofluorometry.","Binding dependent on the dose of the Jurkat cells (CD28+), and no binding to the U937 (CD28−) cells, were observed.","This shows the specificity of the fusion protein for the CD28 molecule and its lack of reactivity toward other molecules expressed by human hematopoietic cells.","Immunosuppressor Activity CD28/B7-Dependent Adhesion Assay: 4×105 human T cells (CD28-positive Jurkat cells) labeled with 51Cr are incubated for 2 hours in a microtitration plate in which 105 adherent LTK− or LB7+ cells (murine fibroblasts transfected with human B7.1 [PAGES et al., J. Biol.","Chem., 271, 9403 (1996)] had been seeded 24 hours beforehand.","These incubations are carried out in the absence or in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein, diluted to various concentrations in PBS buffer without Ca2+ or Mg2+.","The adherent cells after washing are quantified by reading the residual radioactivity using a beta counter (PACKARD TOPCOUNT).","Inhibition of the adhesion in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein is observed, which inhibition is directly dependent on the dose of fusion protein used.","Inhibition of the Activation: 5×104 T cells (human polyclonal cells depleted of CD11b cells) are stimulated with 1×104 irradiated OKT3 hybridoma cells (anti-CD3), or with allogenic CD28− B cells (depleted of CD28+ cells), in the absence or in the presence of varying amounts of the CD28.3 scFv/α1-antitrypsin fusion protein.","The proliferative response in these cultures is evaluated after 3 days when the stimulation is performed with anti-CD3s, or after 7 days when the stimulation is performed with allogenic cells, by incorporation of (3H) thymidine for a period of 16 hours.","Considerable inhibition of the incorporation is observed in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein, which inhibition is directly dependent on the dose of fusion protein used.","Inhibition of the Mixed Lymphocyte Reaction: 105 peripheral blood mononuclear cells from a healthy donor are mixed with 105 peripheral blood mononuclear cells from another, allogenic, healthy donor.","The proliferative response in these cultures is evaluated after 5 days by incorporation of (3H) thymidine for a period of 16 hours.","Considerable inhibition of the incorporation is observed in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein, which inhibition is directly dependent on the dose of fusion protein used."]],"string":"[\n [\n \"Anti-Cd28 Antibody\",\n \"The invention concerns an antibody directed against the CD28 receptor and capable of blocking CD28/B7 interaction, and proteins derived from said antibody, for use in particular to block CD28-dependent activation of lymphocytes.\"\n ],\n [\n \"1.A protein capable of binding specifically to the CD28 lymphocyte receptor and of blocking the CD28/B7 interaction, comprising at least the CDRs of the heavy chain and of the light chain of the immunoglobulin CD28.3, produced by the hybridoma CNCM I-2582.2.The protein as claimed in claim 1, selected from the group consisting of: a) the antibody CD28.3 produced by the hybridoma CNCM I-2582; b) Fv, Fab, Fab′2 or scFv fragments of the antibody CD28.3; c) chimeric or humanized antibodies obtained from the variable regions of CD28.3; d) Fv, Fab, Fab′2 or scFv fragments of an antibody b); and e) recombinant proteins comprising a fragment b) or d) and a heterologous polypeptide.\",\n \"3.A nucleic acid molecule encoding a protein as claimed claim 1.4.An expression vector comprising a nucleic acid molecule as claimed in claim 3.5.The expression vector as claimed in claim 4, wherein the expression vector is the plasmid CNCM I-2762.6.A cell expressing a protein as claimed in claim 1.7.The cell as claimed in claim 6, wherein the cell is the hybridoma CNCM I-2582.8.A cell transformed with a nucleic acid molecule as claimed in claim 3, and expressing a protein capable of binding specifically to the CD28 lymphocyte receptor and of blocking the CO28/B7 interaction and comprising at least the CDRs of the heavy chain and of the light chain of the immunoglobulin CD28.3, produced by the hybridoma CNCM I-2582.9.A method for preparing a protein as claimed in claim 1, comprising culturing at least one cell as claimed in claim 6, and recovering said protein from said culture.\",\n \"10.A medicinal product comprising the protein as claimed in claim 1.11.The medicinal product as claimed in claim 10, wherein in that said protein has a single CD28 receptor-binding site, and said medicinal product is an immunosuppressor which selectively blocks T cell activation via the CD28 receptor.\",\n \"12.A method of treating a pathological condition selected from the group consisting of transplant rejection, graft-versus-host disease, T-lymphocyte-mediated autoimmune diseases, allergic phenomena and chronic inflammatory diseases wherein the method comprises administering to a patient in need thereof an effective amount of the medicinal product of claim 11.13.A nucleic acid molecule encoding a protein as claimed in claim 2.14.A cell expressing a protein as claimed in claim 2.\"\n ],\n [\n \"<SOH> Construction of Expression Vectors Comprising the Sequence Encoding α1-Antitrypsin and Allowing the Introduction of a Sequence Encoding an scFv Fragment <EOH>\"\n ],\n [\n \"The invention relates to antibodies directed against the CD28 lymphocyte receptor and to their fragments, and to their therapeutic uses, in particular in the context of regulating T cell activation.\",\n \"Abnormal activation of T cells is involved in the pathogenesis of many autoimmune diseases, and also in transplant rejection phenomena, where they cause an immune response directed against the transplanted organ to develop.\",\n \"T lymphocyte activation requires an activating signal, induced by the recognition, by T receptors (TCRs) of the antigen associated with the class II major histocompatibility complex (MHC) and presented by antigen-presenting cells (APCs).\",\n \"However, this activation only causes proliferation of T cells and secretion of specific immunomodulatory cytokines (such as interleukin 2, gamma interferon or interleukin 4) if other T co-stimulation systems are also activated.\",\n \"One of the most important systems for regulating T lymphocyte activation is the molecular system B7/CD28/CTLA4.This system plays, for example, an essential role in the mechanisms of transplant rejection [Woodward et al., Transplantation, 66, 14-20, (1998)].\",\n \"The molecules B7.1 (CD80) and B7.2 (CD86) borne by the APCs can activate the CD28 receptor and also the CTLA4 receptor of T lymphocytes.\",\n \"The activation of CD28 sends the T lymphocyte a positive signal which stimulates the cell; on the other hand, the activation of CTLA4 sends a negative signal which leads to a non-response (anergy) [FALLARINO et al., J. Exp.\",\n \"Med., 188, 205-210, (1998)].\",\n \"Dormant T lymphocytes express a large amount of CD28 and very little CTLA4.When there is a first cognitive contact between an APC and a T lymphocyte, the CD28/B7 interaction is favored, which activates the cell.\",\n \"It is only several hours after the initiation of activation that, due to the increase in membrane expression of CTLA4, the affinity of which for B7 is 5 to 10 times greater than that of CD28, the B7/CD28 interaction shifts in favor of a B7/CTLA4 interaction.\",\n \"Currently, cyclosporin is mainly used to block T lymphocyte activation, in particular in the context of organ transplants.\",\n \"Despite the effectiveness of this medicinal product, it does not, however, confer absolute protection.\",\n \"In addition, it acts by blocking all the calcium-dependent cell activation pathways, and therefore has a biological activity which is not strictly T lyphocyte specific and leads to a considerable number of side effects.\",\n \"It is therefore desirable to develop new immunosuppressants which have a defined method of action and greater specificity.\",\n \"It has been postulated that selective inhibition of the agonist signal given to the T cell by CD28, leaving the antagonist system consisting of the pair CTLA4/B7 intact, via specific blocking of the CD28/B7 interaction, would make it possible to prevent T lymphocyte activation.\",\n \"Such specific blocking of the CD28/B7 interaction can be obtained using an antibody directed against CD28.Anti-CD28 antibodies capable of preventing CD28 binding to B7 are known.\",\n \"However, they have the drawback, when they are used in their divalent native form, of bringing about the dimerization and the activation of CD28 via their binding with this receptor.\",\n \"However, monovalent fragments derived from these antibodies are capable of blocking the CD28 receptor without activating it [DAMLE et al., J. Immunol.\",\n \"140, 1753-1761, (1988); NUNES et al., Int.\",\n \"Immunol., 5, 311-315 (1993); PAGES et al., J. Biol.\",\n \"Chem., 271, 9403, (1996)].\",\n \"It has thus been reported [PERRIN et al., J. Immunol.\",\n \"163, 1704-1710, (1999)] that Fab fragments derived from an anti-CD28 antibody can curb the clinical symptoms of experimental autoimmune encephalitis induced in mice by the administration of myelin or the transfer of T cells from an affected animal.\",\n \"Monovalent Fab or scFv fragments derived from an anti-CD28 antibody can potentially be used to prevent T lymphocyte activation via specific blocking of the CD28/B7 interaction.\",\n \"Fab fragments result from the action of papain on an immunoglobulin molecule, and each contain a light chain and the first half of a heavy chain; scfv fragments consist of the variable portions of the heavy and light chains of an antibody, connected to one another via a flexible linker [CLACKSON et al., Nature, 352, 624-628, (1991)], thus forming a single-chain protein.\",\n \"These monovalent fragments frequently exhibit less affinity for the antigen than the native antibodies, which can limit their possibilities for use in diagnostic or therapeutic applications.\",\n \"The inventors have succeeded in selecting, among various antibodies which recognize the CD28 antigen, an antibody capable of blocking the CD28/B7 interaction, the monovalent fragments of which exhibit sufficient affinity for the antigen so that they can be used, in vitro or in vivo, to block the CD28 receptor without activation of this receptor.\",\n \"This antibody, called CD28.3, is produced by the hybridoma deposited, according to the terms of the Treaty of Budapest, on Nov. 28, 2000, with the CNCM (Collection Nationale de Cultures de Microorganismes [National Collection of Cultures and Microorganisms], 25 rue du Docteur Roux, 75724 PARIS CEDEX 15), under the number I-2582.A subject of the present invention is a protein capable of binding specifically to the CD28 lymphocyte receptor and of blocking the CD28/B7 interaction, characterized in that it comprises at least the CDRs of the heavy chain and of the light chain of the immunoglobulin CD28.3.CDRs (complementarity determining regions) are the portions of the variable regions of an immunoglobulin which are involved in antigen recognition specificity.\",\n \"Proteins in accordance with the invention thus encompass in particular: a) the antibody CD28.3 produced by the hybridoma CNCM I-2582; b) the Fv, Fab, Fab′2 or scfv fragments of the antibody CD28.3; c) the chimeric or humanized antibodies obtained from the variable regions of CD28.3; d) the fragments of the antibodies b) above comprising the CDRs of the antibody CD28.3, with a monovalent Fv, Fab or scfv fragments, or divalent Fab′2 fragments; e) the recombinant proteins comprising a fragment b) or d) and a heterologous polypeptide.\",\n \"They may, for example, be: divalent or plurivalent derivatives of scfv fragments, such as “diabodies” or “triabodies”, resulting from the association of 2 or 3 scFv fragments; proteins combining at least one antibody fragment comprising the CDRs of the antibody CD28.3, with at least one antibody fragment comprising the CDRs of an antibody of different specificity; by way of examples, mention will be made of bispecific immunoglobulins, conjugates of an Fv or Fab fragment containing the CDRs of CD28.3 with an Fv or Fab fragment of an antibody of different specificity, and “bispecific diabodies” resulting from the association of an scFv fragment containing the CDRs of CD28.3 with an Fv or Fab fragment of an antibody of different specificity; proteins combining at least one antibody fragment comprising the CDRs of the antibody CD28.3, with a molecule having pharmacological activity (for example a toxin) or effector properties (for example an Fc fragment); proteins combining at least one antibody fragment comprising the CDRs of the antibody CD28.3, with a molecule which makes it possible to prolong its plasma halflife when it is administered in vivo; it is, for example, possible to combine said antibody fragment with a water-soluble polypeptide of sufficient molecular mass for the molecular mass of the fusion polypeptide thus obtained to be greater than the renal filtration threshold.\",\n \"In this case, a polypeptide will be chosen which, unlike Fc fragments, cannot associate as dimers, and which does not have its own effector activity liable to cause unfortunate side effects.\",\n \"Polypeptides which have these properties can advantageously be obtained from water-soluble serum proteins, namely, in particular, serum albumin, haptoglobulin, ITIH2 (inter-alpha (globulin) inhibitor, H2 polypeptide), transferrin, CBG (corticosteroid binding globulin), α1-antitrypsin, ITIH4 (inter-alpha (globulin) inhibitor, H4 polypeptide), AACT (alpha-1-antichymotrypsin), TBG (thyroxine binding globulin), fibrinogen and prothrombin, in order to prepare fusion proteins with scFv fragments derived from anti-CD28 antibodies.\",\n \"It is also possible to conjugate a protein in accordance with the invention with a polyol, for example polyethylene glycol, as described, for example, in U.S. Pat.\",\n \"No.\",\n \"4,179,337.An example of a protein in accordance with the invention is illustrated in FIG.\",\n \"1, which represents an scFv fragment derived from the antibody CD28.3.The sequences of the CDRs of the antibody CD28.3 are boxed in the sequence represented in FIG.\",\n \"1.The nucleotide sequence encoding this scFv fragment is represented in the attached sequence listing under the number SEQ ID No.\",\n \"1, and the corresponding peptide sequence is represented under the number SEQ ID No.\",\n \"2.Fv, Fab or Fab′2 fragments in accordance with the invention can be obtained by the conventional techniques of enzyme digestion, from the antibody CD28.3.A plasmid containing a polynucleotide encoding an scFv fragment of CD28.3, fused to a polynucleotide encoding amino acids 53 to 425 of α1-antitrypsin was deposited, according to the terms of the Treaty of Budapest, on Dec. 11, 2001, with the CNCM (Collection Nationale de Cultures de Microorganismes [National Collection of Cultures and Microorganisms], 25 rue du Docteur Roux, 75724 PARIS CEDEX 15), under the number I-2762.Proteins in accordance with the invention such as chimeric or recombinant antibodies, scFv fragments and their derivatives, etc., can be obtained by conventional genetic engineering techniques, such as those described by SAMBROOK et al.\",\n \"[MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989)].\",\n \"Polynucleotides encoding the variable regions of the anti-CD28.3 antibody can, for example, be obtained by cloning said variable regions from a cDNA library of the hybridoma CD28.3, or from the plasmid CNCM I-2762.They can also be prepared, completely or partially, by nucleic acid synthesis, based on the nucleotide sequences of said variable regions.\",\n \"It is, for example, possible to synthesize polynucleotides encoding the CDRs of CD28.3, and to incorporate them into the framework regions (FRs) of another antibody, in particular of an antibody of human origin, using techniques, known in themselves, of CDR grafting, such as those described by ROUTLEDGE et al.\",\n \"[“Reshaping antibodies for therapy”, in Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, 13-44, Academic Titles, Nottingham, England (1993)] or by ROGUSKA et al.\",\n \"Protein Engineering, 9(10), 895-904, (1996)].\",\n \"A subject of the present invention is also any nucleic acid molecule encoding a protein in accordance with the invention comprising the CDRs of the antibody CD28.3, and also any recombinant vector, in particular any expression vector, comprising said nucleic acid molecule.\",\n \"A subject of the present invention is also any cell expressing a protein in accordance with the invention comprising the CDRs of the antibody CD28.3.This encompasses in particular the hybridoma CNCM I-2582, and also the host cells transformed with a nucleic acid molecule in accordance with the invention.\",\n \"Nucleic acid molecules in accordance with the invention may advantageously comprise, besides a sequence encoding a protein in accordance with the invention, a sequence encoding a signal peptide allowing secretion of said protein; they may also comprise one or more sequence(s) encoding one or more marker peptide(s) for detecting, and/or facilitating the purification of, said protein.\",\n \"Expression vectors in accordance with the invention comprise at least one nucleic acid sequence encoding a protein in accordance with the invention, associated with transcription- and translation-controlling elements which are active in the host cell chosen.\",\n \"Vectors which can be used to construct expression vectors in accordance with the invention are known in themselves, and will be chosen in particular as a function of the host cell intended to be used.\",\n \"Host cells which can be used in the context of the present invention can be prokaryotic or eukaryotic cells.\",\n \"Among the eukaryotic cells which can be used, mention will in particular be made of plant cells, cells from yeast, such as Saccharomyces, insect cells, such as Drosophila or Spodoptera cells, and mammalian cells such as HeLa, CHO, 3T3, C127, BHK, COS, etc., cells.\",\n \"The construction of expression vectors in accordance with the invention and the transformation of the host cells can be carried out by the conventional techniques of molecular biology.\",\n \"A subject of the invention is also a method for producing a protein in accordance with the invention, characterized in that it comprises culturing at least one cell in accordance with the invention, and recovering said protein from said culture.\",\n \"If the protein is secreted, it can be recovered directly from the culture medium; if not, cell lysis will be carried out beforehand.\",\n \"The protein can then be purified from the culture medium or from the cell lysate, by conventional procedures, known in themselves to those skilled in the art, for example by fractionated precipitation, in particular precipitation with ammonium sulfate, electrophoresis, gel filtration, affinity chromatography, etc.\",\n \"The proteins in accordance with the invention can be used, in vitro, to study the proliferative response or the differentiation of T lymphocytes responding to an antigenic, viral, allogenic or xenogenic stimulation.\",\n \"They can also be used, in vitro, to induce the differentiation of T lymphocytes taken from a patient, for example the induction of tolerance with respect to an antigen or to an alloantigen, intended to be subsequently re-administered in vivo.\",\n \"They may also be used to obtain medicinal products, or diagnostic reagents.\",\n \"Proteins in accordance with the invention which are divalent, i.e.\",\n \"which have 2 CD28 receptor-binding sites, and thus capable of inducing dimerization of this receptor, can be used in all situations where it is desired to activate this CD28 receptor, i.e.\",\n \"to increase the response of a T lymphocyte with respect to an antigen.\",\n \"Proteins in accordance with the invention which are monovalent, i.e.\",\n \"which have a single CD28 receptor-binding site, can be used in all situations where it is desired to selectively block this receptor without activating it, in order to induce immunosuppression.\",\n \"A protein in accordance with the invention, comprising a monovalent fragment derived from an anti-CD28 antibody, can in particular be used to obtain an immunosuppressant medicinal product which selectively blocks T cell activation phenomena involving the CD28 receptor, and which does not have the drawbacks of known immunosuppressants such as cyclosporin.\",\n \"The T immunosuppression by selective blocking of CD28 with protein in accordance with the invention has applications in all T lymphocyte-dependent pathological conditions.\",\n \"These are essentially transplant rejection, graft-versus-host disease, T lymphocyte-mediated autoimmune diseases, such as type I diabetes or multiple sclerosis, and type IV hypersensitivity, which is involved in allergic phenomena and also in the pathogenesis of chronic inflammatory diseases following infection with a pathogenic agent (in particular leprosy, tuberculosis, leishmaniasis, listeriosis, etc.).\",\n \"The present invention will be understood more clearly from the further description which follows, which refers to nonlimiting examples of preparation and of use of antibodies in accordance with the invention.\",\n \"EXAMPLE 1 Choice of an Antibody Producing Monovalent Fragments, Properties of Monovalent Fab Fragments Derived from CD28.3 Some of the properties of several anti-CD28 antibodies (CD28.1, CD28.2, CD28.3, CD28.4, CD28.5 and CD28.6) are described in the publication by NUNES et al.\",\n \"[Int.\",\n \"Immunol., 5, 311, (1993)].\",\n \"These various antibodies, which are not accessible to the public, were provided by the laboratory of Daniel OLIVE (INSERM).\",\n \"The antigen-binding properties of the monovalent Fab fragments of these various antibodies were compared.\",\n \"5 mg of Fab fragments of each of these antibodies were prepared by digestion with papain (papain/antibody molar ratio= 1/100) for 24 hours at 37° C., followed by inactivation of the enzyme with 0.03M iodoacetamide, and dialysis against PBS to remove the iodoacetamide.\",\n \"1) Binding of the Fab Fragments to CD28+ Jurkat T Cells: 100,000 CD28+ Jurkat cells in 100 μl are incubated in PBS-1% BSA-0.1% NaN3 at 4° C. for 30 minutes with increasing concentrations of anti-CD28 antibodies or of their Fab fragments.\",\n \"After washing, the cells are incubated in a similar way with an FITC-conjugated anti-mouse IgG goat antibody, washed, and analyzed by cytofluorometry.\",\n \"The results are given in FIG.\",\n \"2: Legend of FIG.\",\n \"2: X-axis: antibody or Fab fragments concentration Y-axis: mean fluorescence intensity (MFI) —⋄—: F1=Fab fragments of the antibody CD28.1 —▪—: F2=Fab fragments of the antibody CD28.2 —Δ—: F3=Fab fragments of the antibody CD28.3 —x—: F5=Fab fragments of the antibody CD28.5 —◯—: F6=Fab fragments of the antibody CD28.6 ····: W132 whole antibody CD28.1 ··-··: W2=whole antibody CD28.2 : W3=whole antibody CD28.3 ——: W5=whole antibody CD28.5 —*—: W6=whole antibody CD28.6 —▴—: Mara-1=negative control (mouse IgG1).\",\n \"These results show that, among the Fab fragments, only those derived from CD28.3 are capable of significantly binding to the CD28+ Jurkat cells at concentrations of less than 10 μg/ml.\",\n \"2) Effect of the Fab Fragments on the Adhesion of CD28+ Jurkat T Cells to Transfected Murine L Cells Expressing the B7-1 Molecule: 4×105 human T cells (Jurkat, CD28-positive) labeled with 51Cr are incubated for 2 hours in a microtitration plate in which 105 adherent LTK− or LB7+ cells (murine fibroblasts transfected with human B7.1 [PAGES et al., J. Biol.\",\n \"Chem., 271, 9403, (1996)] have been seeded 24 hours beforehand.\",\n \"These incubations are carried out in the presence of the Fab fragments derived from the antibodies CD28.1 to CD28.6, or of the antibody CD28.3, diluted to various dilutions in PBS buffer without Ca2+ or Mg2+.\",\n \"The adherent cells after washing are quantified by reading the residual radioactivity in a beta counter (PACKARD TOPCOUNT).\",\n \"The results are given in FIG.\",\n \"3: Legend of FIG.\",\n \"3: X-axis: percentage of adherent cells Y-axis: antibody concentration —♦—: F1=Fab fragments of the antibody CD28.1 —▪—: F2=Fab fragments of the antibody CD28.2 —▴—: F3=Fab fragments of the antibody CD28.3 —x—: F5=Fab fragments of the antibody CD28.5 —*—: F6=Fab fragments of the antibody CD28.6 ····: whole antibody CD28.3 ◯: no antibody.\",\n \"These results show that the Fab fragments derived from CD28.3 are the most effective for inhibiting CD28/B7 interactions.\",\n \"They give 90% inhibition of adhesion at a concentration of 3 μg/ml, and with an effectiveness comparable to that of the whole antibody CD28.3, whereas, at this concentration, the Fab fragments derived from the other antibodies give no more than 50% inhibition of adhesion.\",\n \"3) Effect of the Fab Fragments on Proliferation in a Mixed Lymphocyte Reaction: 105 peripheral blood mononuclear cells (PBMCs) are mixed with 105 allogenic mononuclear cells irradiated at 35 Gy, in the presence of varying concentrations of the antibodies CD28.1 to CD28.6 or of the Fab fragments derived from these antibodies.\",\n \"The proliferative response in these cultures is evaluated after 3 days, by incorporation of (3H) thymidine for a period of 16 hours.\",\n \"The results are given in FIG.\",\n \"4: Legend of FIG.\",\n \"4: X-axis: antibody concentration Y-axis: proliferative response (cpm) Basal level of proliferation=6 500 cpm.\",\n \"—♦—: 28.1=antibody CD28.1 —▪—: 28.2=antibody CD28.2 —▴—: 28.3=antibody CD28.3 —x—: 28.5=antibody CD28.5 ···*···: 28.6=antibody CD28.6 ——: Fab.\",\n \"1=Fab fragments of the antibody CD28.1 ··+··: Fab.\",\n \"2=Fab fragments of the antibody CD28.2 ——: Fab.\",\n \"3=Fab fragments of the antibody CD28.3 —+—: Fab.\",\n \"5=Fab fragment of the antibody CD28.5 —⋄—: Fab.\",\n \"6=Fab fragment of the antibody CD28.6.These results show that the Fab fragments derived from CD28.3 or from CD28.6 are the most effective for inhibiting mononuclear cell proliferation.\",\n \"The whole antibodies CD28.1 to CD28.6, tested in parallel, have no inhibitory effect or indeed stimulate the proliferation by virtue of their stimulator action on CD28.Effect of the Fab Fragments Derived from CD28.3 on Proliferation Induced by a Superantigen For this experiment, responder CD4+ T cells were mixed with irradiated isogenic PBMCs, in the presence of 50 ng/ml of toxic shock syndrome toxin-1 (TSST-1), which specifically stimulates the vβ2+ T cell, either in the absence of antibody or in the presence of anti-B7-1 (1 μg/ml), of anti-B7-2 (0.5 μg/ml), of CTLA4Ig (10 μg/ml), or of Fab fragments derived from CD28.3 (10 μg/ml).\",\n \"The proliferative response in these cultures is evaluated after 1, 3, 6 and 8 days, by incorporation of (3H) thymidine for a period of 16 hours.\",\n \"The results are given in FIG.\",\n \"5: Legend of FIG.\",\n \"5: X-axis: culturing time Y-axis: proliferation index=PI P I = cpm mixed lumphocyte reaction - cpm irradiated stimulating cells only cpm unstimulated responder cells —♦—: anti-CD28.3 Fab —X—: anti-B7-2 —▪—: CTLA-4 Ig ··*··: anti-B7-1+2 —▴—: anti-B7-1 —◯—: no antibody TSST-1 induces considerable proliferation of CD4+ T cells.\",\n \"In the presence of anti-B7, of CTLA4Ig or of the Fab fragments of CD28.3, 70% inhibition of this proliferation is observed after 6 days.\",\n \"Effect of the Fab Fragments Derived from CD28.3 on Cytokine Production In order to determine whether the Fab fragments derived from CD28.3 could induce an immune deviation in vitro, a mixed lymphocyte reaction (PBMCs derived from a donor A/irradiated PBMCs derived from a donor B) was carried out, in the presence of Fab fragments derived from CD28.3.105 peripheral blood mononuclear cells from a donor are mixed with 105 allogenic mononuclear cells irradiated at 35 Gy, and cultured for 5 days in the presence or absence of 10 μg/ml of Fab derived from the antibody CD28.3.The RNA of the responder cells was extracted, and the amount of cytokine mRNA was evaluated by quantitative measurement of the number of transcripts, related to the amount of HPRT, using a TaqMan (Perkin Elmer).\",\n \"In the presence of Fab fragments derived from CD28.3, a decrease in the production of γIFN and of IL2, and an increase in the production of IL10 are observed.\",\n \"This deviation in the immune response suggests an orientation toward a Th2-type response.\",\n \"This result is unexpected insofar as it has been reported that the involvement of CTLA4 (which is supposed to intervene in the blocking of CD28 alone) leads to a Th1-type response.\",\n \"In Vitro Processing of the Antibody CD28.3 and of the Fab Fragments Derived Therefrom, by Human T Cells A possible internalization of the Fab fragments of the antibody CD28.3 in human T cells was investigated, in comparison with the whole antibody CD28.3.Jurkat T cells were incubated in culture medium with 100 μg/ml of antibody CD28.3, at 37° C. or at 0° C. At various times, the cells were washed with cold PBS buffer containing 0.1% of bovine serum albumin, and NaN3, in order to block membrane motility.\",\n \"The bound antibodies were revealed with a fluorescein-labeled goat anti-mouse secondary antibody.\",\n \"The cells were mounted in MOVIOL and analyzed by confocal microscopy.\",\n \"It is thus observed that the whole CD28.3 antibodies which bind to the Jurkat T cells are captured and disappear from the cell surface at 37° C., but not at 0° C. On the other hand, the Fab fragments remain attached at the surface of the cell.\",\n \"This indicates that the attachment of the divalent antibodies CD28.3 leads to dimerization of CD28, which brings about their entry into the cell, whereas the monovalent Fab fragments, which do not induce this dimerization, remain at the surface.\",\n \"EXAMPLE 2 Properties of an scFv Fragment Derived from the Antibody CD28.3 FIG.\",\n \"1 represents the nucleotide sequence and the deduced polypeptide sequence of an scFv fragment derived from the antibody CD28.3.The portions of this sequence corresponding to the variable fragment of the heavy chain and of the light chain are represented in capital letters.\",\n \"The sequence corresponding to the variable fragment of the light chain is also underlined.\",\n \"The sequence of the linker is represented in lower case letters.\",\n \"The sequences of the CDRs of the heavy chain and of the light chain are boxed in.\",\n \"The nucleotide sequence encoding this scFv fragment is also represented in the attached sequence listing, under the number SEQ ID No.1.The cDNA encoding this scFv fragment was inserted into the vector pIG6 (Biochemisches Institut, University of Zurich).\",\n \"This vector comprises in particular an ampicillin resistance marker and an expression cassette which comprises an inducible lac promoter under the control of which are placed: a sequence encoding an ompA signal peptide, a sequence encoding a marker peptide of sequence (1-letter code) DYKD, a sequence encoding a c-myc marker peptide, and a sequence encoding a polyhistidine-5 marker.\",\n \"The cDNA encoding the scFv fragment described above was introduced between the EcoRI and EcoRV sites of pIG6, downstream of the sequence encoding the peptide DYKD and upstream of the sequence encoding the c-myc marker.\",\n \"The construct obtained is called pIg6-28.3.Production in Prokaryotic Cells The vector pIg6-28.3 was used to transform E.coli JM83 cells.\",\n \"The cells are cultured at 25° C., up to an OD550 of 0.5.After induction with IPTG, the scFv fragment is produced in soluble form in the periplasma.\",\n \"After electrophoresis and Western blotting, it appears in the form of a band at approximately 30 kDa.\",\n \"It is purified from the periplasmic extracts of the bacteria, obtained after osmotic shock in 50 mM Tris-Cl, and ultracentrifugation of the insoluble material, by chromatography on an Ni-NTA matrix and ion exchange on DEAE-Sepharose.\",\n \"The binding of the scFv fragments present in the eluate of the NiNTA column, to CD28+ Jurkat cells, is comparable to that obtained with Fab fragments obtained from the antibody CD28.3 by digestion with papain.\",\n \"Production in Eukaryotic Cells The vector pSec-28.3 was used to transfect Cos cells.\",\n \"The cells are cultured at 37° C. for 3 days.\",\n \"The scFv fragment is produced in soluble form in the supernatant.\",\n \"This supernatant inhibits the mixed lymphocyte reaction: 105 peripheral blood mononuclear cells from a healthy donor are mixed with 105 peripheral blood mononuclear cells from another, healthy allogenic donor.\",\n \"The proliferative response in these cultures is evaluated after 5 days by incorporation of (3H) thymidine for a period of 16 hours.\",\n \"Considerable inhibition of the incorporation, dependent on the supernatant dilution used, is observed.\",\n \"A control supernatant exhibits no proliferation-inhibiting activity.\",\n \"EXAMPLE 3 Production of a Fusion Protein Comprising an scFv Fragment of CD28.3 The nucleotide sequence encoding the scfv fragment described in example 2 was linked to the 5′ end of a portion of the cDNA of human α1-antitrypsin (GENBANK accession number K01396) corresponding to amino acids 53 to 425, via a hinged peptide of sequence VAAPS.\",\n \"The resulting sequence is represented in the attached sequence listing under the number SEQ ID No.\",\n \"3, and the corresponding polypeptide under the number SEQ ID No.\",\n \"4.EXAMPLE 4 Construction of Expression Vectors Comprising the Sequence Encoding α1-Antitrypsin and Allowing the Introduction of a Sequence Encoding an scFv Fragment Prokaryotic Expression Vector: The vector pIG6 was used (Biochemisches Institut, University of Zurich).\",\n \"This vector comprises in particular an ampicillin resistance marker, and an expression cassette which comprises an inducible lac promoter under the control of which are placed: a sequence encoding an ompA signal peptide, a sequence encoding a marker peptide of sequence (1-letter code) DYKD, a sequence encoding a c-myc marker peptide, and a sequence encoding a polyhistidine-5 marker.\",\n \"The cDNA encoding a fragment of human α1-antitrypsin corresponding to amino acids 53 to 425 was introduced between the EcoRI and EcoRV sites of pIg6, downstream of the sequence encoding the peptide DYKD and upstream of the sequence encoding the c-myc marker.\",\n \"FIG.\",\n \"1 illustrates diagrammatically the construct obtained, called pIg6-Haat.\",\n \"Eukaryotic Expression Vector: The vector pSECTagB (Invitrogen, De Schelp, The Netherlands) was used.\",\n \"This vector comprises in particular an ampicillin resistance marker, a zeocin resistance marker, and an expression cassette which comprises a CMV promoter under the control of which are placed: a sequence encoding a signal peptide of the IgG kappa light chain, a sequence encoding a c-myc marker peptide, and a sequence encoding a polyhistidine-6 marker.\",\n \"The cDNA encoding a fragment of human α1-antitrypsin corresponding to amino acids 53 to 425 was introduced between the BamHI and EcoRI sites of the vector PSEC B Tag, upstream of the sequence encoding the c-myc marker.\",\n \"FIG.\",\n \"2 illustrates diagrammatically the construct obtained, called pSecHaat.\",\n \"EXAMPLE 5 Construction of Expression Vectors Integrating the Sequence Encoding the CD28.3 scFv/α1-Antitrypsin Fusion Protein Prokaryotic Expression Vector: The cDNA encoding the CD28.3 ScFv/α1-antitrypsin fusion protein described in example 3 above was introduced between the EcoRI and XhoI sites of pIG6, downstream of the sequence encoding the peptide DYKD and upstream of the sequence encoding the c-myc marker.\",\n \"FIG.\",\n \"3 illustrates diagrammatically the construct obtained, called pIg6-28.3Haat.\",\n \"Eukaryotic Expression Vector: The cDNA encoding the CD28.3 ScFv/α1-antitrypsin fusion protein described in example 3 above was introduced between the BamHI and XhoI sites of the vector PSEC B Tag, upstream of the sequence encoding the c-myc marker.\",\n \"FIG.\",\n \"4 illustrates diagrammatically the construct obtained, called pSec-28.3Haat.\",\n \"This vector, harbored by E.coli DH5α, was deposited with the CNCM on Dec. 11, 2001, under the number I-2762.EXAMPLE 6 Expression and Purification of the Fusion Proteins In Prokaryotic Cells: The vector pIg6-28.3Haat was used to transform E. coli JM83 cells.\",\n \"The cells are cultured at 25° C., up to an OD550 of 0.5.After induction with IPTG, the protein is produced in soluble form in the periplasm.\",\n \"After electrophoresis and Western blotting, it appears in the form of a band at approximately 74 kDa.\",\n \"It can be purified from the periplasmic extracts using an NI-NTA affinity chromatography matrix and/or an anti-c-myc affinity chromatography matrix.\",\n \"It can also be purified using an anti-α1-antitrypsin affinity column.\",\n \"In Eukaryotic Cells: The vector pSec-28.3Haat was used to transfect CHO cells by lipofection.\",\n \"The cells are cultured in the presence of 200 μg/ml on zeocin in MEM medium containing 10% of fetal calf serum.\",\n \"The protein is secreted into the culture medium.\",\n \"After separation by electrophoresis, Western blotting, and revelation with an anti-c-myc antibody, it appears in the form of a band at approximately 80 kDa.\",\n \"EXAMPLE 7 Assays for Activity of an scFv/α1-Antitrypsin Fusion Protein The anti-CD28 activity of the CD28.3 scFv/α1-antitrypsin fusion protein obtained in example 6 above was evaluated by its binding to the CD28 molecule, or to cells expressing CD28 on their membrane, and its lack of binding to cells which do not express CD28.The immunosuppressor activity of the CD28.3 scFv/α1-antitrypsin fusion protein obtained in example 6 above is evaluated by the inhibition of adhesion to B7, and the inhibition of the induced activation of the T lymphocyte.\",\n \"These anti-CD28 and immunosuppresor activities were measured using the following assays: Anti-CD28 Activity Biosensor Measurement of the CD28-Binding Parameters: Recombinant human CD28 was immobilized on the biosensor (BIACORE) detector.\",\n \"A CD28.3 scFv/α1-antitrypsin fusion protein obtained as described in example 6 above was brought into contact with the detector.\",\n \"The binding parameters are: KA (1/M)2.86e9; KD (M): 3.49e-10.In comparison, these parameters measured for the Fab fragment of the antibody CD28.3 are: KA (1/M): 9.69e8; KD (M): 1.03e-9.The affinity for CD28, of the Fab fragment of the antibody CD28.3 and of the fusion protein, are therefore comparable.\",\n \"Cytofluorometry Assay for Specific Recognition of CD28: 105 Jurkat (Cd28+) and U937 (CD28−) cells are incubated in PBS-1% BSA-0.1% NaN3, at 4° C., for 1 hour with increasing concentrations of the CD28.3 scFv/α1-antitrypsin fusion protein.\",\n \"After washing, the cells are incubated with an anti-alpha-1-antitrypsin rabbit antibody and then with an FITC-conjugated goat anti-rabbit antibody, washed, and analyzed by cytofluorometry.\",\n \"Binding dependent on the dose of the Jurkat cells (CD28+), and no binding to the U937 (CD28−) cells, were observed.\",\n \"This shows the specificity of the fusion protein for the CD28 molecule and its lack of reactivity toward other molecules expressed by human hematopoietic cells.\",\n \"Immunosuppressor Activity CD28/B7-Dependent Adhesion Assay: 4×105 human T cells (CD28-positive Jurkat cells) labeled with 51Cr are incubated for 2 hours in a microtitration plate in which 105 adherent LTK− or LB7+ cells (murine fibroblasts transfected with human B7.1 [PAGES et al., J. Biol.\",\n \"Chem., 271, 9403 (1996)] had been seeded 24 hours beforehand.\",\n \"These incubations are carried out in the absence or in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein, diluted to various concentrations in PBS buffer without Ca2+ or Mg2+.\",\n \"The adherent cells after washing are quantified by reading the residual radioactivity using a beta counter (PACKARD TOPCOUNT).\",\n \"Inhibition of the adhesion in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein is observed, which inhibition is directly dependent on the dose of fusion protein used.\",\n \"Inhibition of the Activation: 5×104 T cells (human polyclonal cells depleted of CD11b cells) are stimulated with 1×104 irradiated OKT3 hybridoma cells (anti-CD3), or with allogenic CD28− B cells (depleted of CD28+ cells), in the absence or in the presence of varying amounts of the CD28.3 scFv/α1-antitrypsin fusion protein.\",\n \"The proliferative response in these cultures is evaluated after 3 days when the stimulation is performed with anti-CD3s, or after 7 days when the stimulation is performed with allogenic cells, by incorporation of (3H) thymidine for a period of 16 hours.\",\n \"Considerable inhibition of the incorporation is observed in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein, which inhibition is directly dependent on the dose of fusion protein used.\",\n \"Inhibition of the Mixed Lymphocyte Reaction: 105 peripheral blood mononuclear cells from a healthy donor are mixed with 105 peripheral blood mononuclear cells from another, allogenic, healthy donor.\",\n \"The proliferative response in these cultures is evaluated after 5 days by incorporation of (3H) thymidine for a period of 16 hours.\",\n \"Considerable inhibition of the incorporation is observed in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein, which inhibition is directly dependent on the dose of fusion protein used.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10450832"}}},{"rowIdx":285,"cells":{"text":{"kind":"list like","value":[["Deflector Devices","A deflector device (22) for use with a tow line between a seismic survey vessel and a tow, in particular a seismic streamer or streamer array, in the water behind the vessel comprises a vertically oriented wing-shaped body (28) shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel.","The wing-shaped body (28) includes one or more buoyancy elements, and a rearwardly extending boom (32).","A pivotable control surface (54) extends sideways from the boom (32), and is shaped to produce in use a force having a substantial vertical component.","The angle of the control surface is remotely controllable, in order to control the depth of the deflector device."],["1.A deflector device for use with a tow line between a towing vessel and a tow in water behind the vessel, the device comprising: a principal wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel; one or more buoyancy elements disposed within and/or secured to the upper end of the principal wing-shaped body; a boom extending rearwardly from the principal wing-shaped body; a pivotable control surface extending sideways from the boom and shaped to produce in use a force having a substantial vertical component; and remotely-operable means for pivoting the control surface, thereby to control the depth of the deflector device substantially independently of the lateral position of the deflector device.","2.A deflector device as claimed in claim 1, wherein the one or more buoyancy elements have a buoyancy selected to give the complete device a small positive buoyancy.","3.A deflector device as claimed in claim 1, wherein the remotely-operable means comprises a telescopic member connected to pivot the control surface.","4.A deflector device as claimed in claim 3, wherein the telescopic member is hydraulically operated.","5.A deflector device as claimed in claim 1, further comprising an auxiliary wing-shaped body, smaller than the principal wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body.","6.A deflector device as claimed in claim 5, further comprising additional remotely-operable means for varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body.","7.A deflector device as claimed in claim 6, wherein the additional remotely-operable adjusting means comprises a further telescopic member connected to the auxiliary wing-shaped body.","8.A deflector device as claimed in claim 7, wherein the further telescopic member is hydraulically operated.","9.A deflector device as claimed in claim 5, wherein the auxiliary wing-shaped body is provided with a trailing edge flap angled away from the boom, typically at about 35°.","10.A method of performing a marine seismic survey, comprising: towing a plurality of laterally-spaced seismic streamers over an area to be surveyed, wherein the lateral position and the depth of at least one of the streamers are controlled by a deflector device comprising: a principal wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel; one or more buoyancy elements disposed within and/or secured to the upper end of the principal wing-shaped body; a boom extending rearwardly from the principal wing-shaped body; a pivotable control surface extending sideways from the boom and shaped to produce in use a force having a substantial vertical component; and remotely-operable means for pivoting the control surface, thereby to control the depth of the deflector device substantially independently of the lateral position of the deflector device.","11.The method of claim 10, further comprising selecting a buoyancy of the one or more buoyancy elements to give the complete device a small positive buoyancy.","12.The method of claim 10, wherein the remotely-operable means comprises a telescopic member connected to the control surface, and further comprising pivoting the control surface using the telescopic member.","13.The method of claim 10, wherein the deflector device includes an auxiliary wing-shaped body, smaller than the principal wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in: use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body, and further comprising varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body."],["This invention relates to deflector devices of the kind used between a towing vessel and a tow located in water, for example a seismic streamer or streamer array, or a seismic source array, in order to pull the tow out to one side of the vessel, so as to position it at a desired lateral offset from the course followed by the vessel.","A deflector device of this kind is described in detail in our U.S. Pat.","No.","5,357,892, and comprises a wing-shaped deflector body having a remotely-operable pivotal lever or “boom” which extends rearwardly from a point near the middle of the trailing edge of the wing-shaped body.","In use, the wing-shaped body is suspended beneath a float so as to be completely submerged and positioned generally vertically in the water, and is connected to the towing vessel by means of a tow line, while the tow is connected to the end of the boom remote from the wing-shaped body.","As the device is pulled through the water, the wing-shaped body produces a sideways force, or “lift”, which moves the tow laterally.","This lift can be varied by adjusting the angle of the boom from the vessel, thus permitting the lateral offset of the tow from the course of the vessel to be varied in use.","The deflector device of U.S. Pat.","No.","5,357,892 has been successfully commercialised by the Applicant as its MONOWING deflector device.","In use, rolling stability of the device is provided by the connection to the float, while stability of the device about a vertical axis is provided by the drag produced by the tow.","The MONOWING deflector devices in current use are very large, typically 7.5 m high by 2.5 m wide, and weigh several tonnes.","They are usually suspended around 2 m to 8 m below the float by means such as a fibre rope, and are also provided with a safety chain intended to prevent separation of the float and wing-shaped body in the event that the rope breaks.","In rough weather, the upper part of the wing-shaped body may rise up out of the water, allowing the rope connecting the wing-shaped body and the float to go slack.","If the wing-shaped body then drops abruptly, the rope, and possibly even the safety chain, may break, and/or their attachment points on the wing-shaped body may be badly damaged.","Additionally, the depth at which the current deflector device operates is effectively determined by the length of the rope connecting it to the float.","As a result of this, the operating depth of the deflector device cannot readily be varied while the device is deployed in the water.","And since the normal operating depth of the current deflector device is typically a few meters, in the event of the onset of bad weather during a survey, the device and all the streamers and other equipment directly or indirectly attached to it have to be recovered onto the towing vessel, and then re-deployed when the bad weather has passed, both of which operations are very time consuming.","It is an object of the present invention to alleviate the drawbacks arising from the connection of the deflector device to the float.","According to the present invention, there is provided a deflector device for use with a tow line between a towing vessel and a tow in water behind the vessel, the device comprising a wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel, one or more buoyancy elements disposed within and/or secured to the upper end of the wing-shaped body, a boom extending rearwardly from the wing-shaped body, and a remotely-operable pivotable control surface extending sideways from the boom and shaped to produce in use a force having a vertical component, whereby to control the depth of the deflector device.","It will be appreciated that since the deflector device of the invention can generate a controllable vertical force, this force, together with the buoyancy of the one or more buoyancy elements, can be selected and adjusted so that the separate surface float is no longer required, and the operating depth of the device can be remotely controlled while the device is deployed in the water.","In particular, at the onset of bad weather, the deflector device and its tow can be caused to dive to a greater depth, where the effects of the bad weather are much reduced, until the weather improves.","Advantageously, the one or more buoyancy elements has or have a buoyancy selected to give the complete device a small positive buoyancy.","In a preferred embodiment of the invention, the deflector device further comprises an auxiliary wing-shaped body, smaller than the firstmentioned (or principal) wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body.","Advantageously, this embodiment further includes remotely-operable means for varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body.","The pivotable control surface and the remotely-operable means are preferably both hydraulically operated.","Advantageously, the auxiliary wing-shaped body is provided with a trailing edge flap angled away from the boom, typically at about 35°.","The invention also includes a method of performing a marine seismic survey, the method including towing a plurality of laterally spaced seismic steamers over an area to be surveyed, wherein the lateral position and the depth of at least one of the streamers are controlled by a deflector device in accordance with any one of the preceding statements of invention.","The invention will now be described by way of example only, with reference to the accompanying drawings, of which: FIG.","1 is a somewhat schematic view of a seismic survey vessel carrying out a marine seismic survey; FIG.","2 is a somewhat schematic part-sectional view of a first embodiment of a deflector device in accordance with the present invention, for use in carrying out the survey of FIG.","1; and FIGS.","3A and 3B are respective perspective view of the deflector device of FIG.","2.The seismic survey vessel shown in FIG.","1 is indicated generally at 10, and is preferably as described in our PCT Patent Application No.","PCT/GB98/01832 (WO 99/00295).","The vessel 10 is shown towing a seismic source 15, typically a TRISOR multiple air gun source of the kind described in our U.S. Pat.","No.","4,757,482, and an array 16 of four substantially identical streamers 18.However, it will be appreciated that, in practice, many more than four streamers can be towed, for example by using the techniques described in our PCT Patent Application No.","PCT/IB98/01435 (WO 99/15913).","The streamers 18 are towed by means of their respective lead-ins 20 (ie the high strength steel- or fibre-reinforced electrical or electro-optical cables which convey electrical power, control and data signals between the vessel 10 and the streamers), and their spread is controlled by two deflector devices, indicated at 22, connected to the respective forward ends 24 of the two outermost streamers.","The deflector devices 22 act in co-operation with respective spreader lines 26 connected between the forward end 24 of each outermost streamer 18 and the forward end 24 of its adjacent streamer to maintain a substantially uniform spacing between the streamers.","One of the deflector devices 22 is shown in more detail in FIGS.","2, 3A and 3B.","The deflector device 22 is similar in general principle to the deflector device of our U.S. Pat.","No.","5,357,892, but is a much improved version of it.","In particular, the deflector device 22 has a main wing-shaped body 28 which is coupled in use to a respective outer lead-in 20 via a towing bridle 27, and which corresponds to the deflector body 2 of U.S. Pat.","No.","5,357,892.However, the main wing-shaped body 28 is of improved hydrodynamic cross-sectional shape and includes a fixed-angle trailing edge flap 29, both of which features enhance lift.","Also, the main wing-shaped body 28 is provided with vortex controlling end plates 30 (see FIGS.","3A and 3B) of the kind described in our PCT Patent Application No.","PCT/FR99/02272, to reduce drag and improve stability, and is largely made of titanium to reduce weight, while the towing bridle 27 comprises a pair of titanium chains 52 (see FIGS.","3A and 3B).","Additionally, the angle lever 10 of U.S. Pat.","No.","5,357,892 is replaced by a rearwardly extending fixed angle boom 32, which is detachably connected at one end 34 to the low pressure side 36 of the body 28 near the trailing edge flap 29, at a mounting bracket 38.The boom 32 is of sandwich construction, and is made from two similarly shaped plates 39 which are bolted together at intervals along their length and which sandwich between them the mounting bracket 38.Typically, the boom 32 is detached from the bracket 38 whenever the deflector device 22 is on the vessel 10, for ease of stowage.","The other end 40 of the boom 32 has a towing eye 42, coupled in use to the forward end 24 of a respective one of the two outermost streamers 18.An auxiliary wing-shaped body 44, which is much smaller than the body 28 in length, thickness and chord, is pivotally secured as will be explained hereinafter to the end 40 of the boom 32, with its longitudinal axis (which lies in a plane perpendicular to the plane of FIG.","2) extending parallel to the longitudinal axis of the body 28.The shape of the body 44 is designed to produce, in use, a sideways force in a direction approximately opposite to that produced by the body 28 (approximately opposite, because as will become apparent, the direction of the force varies in use).","This sideways force is increased by providing the body 44 with a fixed trailing edge flap 46, angled away from the boom 32 at an angle of about 35°.","As best seen in FIGS.","3A and 3B, the auxiliary wing-shaped body 44 is implemented in two symmetrical halves 44a and 44b, which each have vortex-reducing end plates 45 and which are disposed on opposite sides of the boom 32.These two halves 44a, 44b are rotatable in unison about a common axis perpendicular to the plane of the boom 32, so as to vary the angle of the chord of the auxiliary wing-shaped body 44 with respect to the boom.","Rotation of the auxiliary wing-shaped body 44 is effected by a telescopic actuator 48 pivotally mounted between the plates 39 of the boom 32, the actuator being pivotally connected to a lever arm or eccentric 50 secured to each of the two halves 44a, 44b of the auxiliary wing-shaped body.","The telescopic actuator 48 is hydraulically operated by a remotely controllable electro-hydraulic control pack 52 also mounted between the plates 39 of the boom 32.It will be appreciated that varying the angle of the auxiliary wing-shaped body 44 of the deflector device 22 changes the angle of the main wing-shaped body 28 with respect to the direction of tow, and so changes the lift produced by the main wing-shaped body.","This in turn changes the lateral offset produced by the deflector device 22.In accordance with the present invention, the deflector device 22 is made approximately neutrally buoyant, by including gas-filled pipe-like buoyancy elements 58 extending longitudinally within it from top to bottom, and/or by providing an integral buoyancy element at its upper end similar to but smaller than that described in our co-pending United Kingdom Patent Applications Nos.","0023775.0, 0025719.6 and 0029451.2.In practice, the deflector device 22 is preferably designed to be slightly positively buoyant, so that in the event of a malfunction, it tends to float rather than to sink.","Additionally, the deflector device 22 is provided with a pivotable control surface (or flap) 54, which is secured to the boom 32 in the region of the auxiliary wing-shaped body 44 by a generally triangular bracket 56, and which is pivotable about an axis perpendicular to both the pivot axis of the body 44 and the direction of tow (indicated by the arrow 58 in FIG.","2).","The flap 54 and the bracket 56 are both made from titanium.","The angular position of the flap 54 is controlled by a further telescopic actuator 60, which is connected to a lever arm 62 provided on the flap, and which is hydraulically operated by the electro-hydraulic control pack 52.It will be appreciated that rotation of the flap 54 about its pivot axis produces in use an upward or downward force at the end 40 of the boom 32, and thus enables the depth of the deflector device 22 to be controlled.","It will be appreciated that as a result of making the deflector device 22 approximately neutrally buoyant and capable of generating a remotely-controllable vertical force, a separate surface float is no longer required, and the operating depth of the device can be remotely controlled while the device is deployed in the water.","In particular, in the event of the onset of bad weather, the deflector device 22 and the streamers 18 attached to it can be caused to dive to a greater depth, where the effects of the bad weather are much reduced, until the bad weather passes.","Many modifications can be made to the described embodiment of the invention.","In particular, the flap 54 and the auxiliary wing-shaped body 44 can be made from a plastics material reinforced with high strength fibres, eg Kevlar fibres, and can be electrically actuated rather than hydraulically actuated, Additionally, the devices 22 and 60 can be used with tows other than streamers, for example seismic sources, and the tow need not be connected to the end 40 of the boom 32 (it could instead be connected to the lead-in 20, at a point near where the bridle 24 is connected to the lead-in).","Also, the invention can if desired be used with a deflector device in which the auxiliary wing-shaped body 44 is fixed, and the boom 32 is pivotable towards and away from the main deflector body 28, as described in our United Kingdom Patent Applications Nos.","0023755.2, 0025711.3 and 0029452.0.Indeed, the invention can even be used with a deflector device like that described in our U.S. Pat.","No.","5,357,892, ie a deflector device without the auxiliary wing-shaped body 44, by mounting a pivotable flap analogous to the flap 54 on a pivotable boom analogous to the angle lever 10 of the deflector device of the US patent.","Finally, although the invention has been described in relation to deflector devices whose lift can be varied by varying the angle of the device with respect to the direction of tow, it is also applicable in its broadest aspect to a fixed angle deflector device, eg of the kind referred to as a “door”."]],"string":"[\n [\n \"Deflector Devices\",\n \"A deflector device (22) for use with a tow line between a seismic survey vessel and a tow, in particular a seismic streamer or streamer array, in the water behind the vessel comprises a vertically oriented wing-shaped body (28) shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel.\",\n \"The wing-shaped body (28) includes one or more buoyancy elements, and a rearwardly extending boom (32).\",\n \"A pivotable control surface (54) extends sideways from the boom (32), and is shaped to produce in use a force having a substantial vertical component.\",\n \"The angle of the control surface is remotely controllable, in order to control the depth of the deflector device.\"\n ],\n [\n \"1.A deflector device for use with a tow line between a towing vessel and a tow in water behind the vessel, the device comprising: a principal wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel; one or more buoyancy elements disposed within and/or secured to the upper end of the principal wing-shaped body; a boom extending rearwardly from the principal wing-shaped body; a pivotable control surface extending sideways from the boom and shaped to produce in use a force having a substantial vertical component; and remotely-operable means for pivoting the control surface, thereby to control the depth of the deflector device substantially independently of the lateral position of the deflector device.\",\n \"2.A deflector device as claimed in claim 1, wherein the one or more buoyancy elements have a buoyancy selected to give the complete device a small positive buoyancy.\",\n \"3.A deflector device as claimed in claim 1, wherein the remotely-operable means comprises a telescopic member connected to pivot the control surface.\",\n \"4.A deflector device as claimed in claim 3, wherein the telescopic member is hydraulically operated.\",\n \"5.A deflector device as claimed in claim 1, further comprising an auxiliary wing-shaped body, smaller than the principal wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body.\",\n \"6.A deflector device as claimed in claim 5, further comprising additional remotely-operable means for varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body.\",\n \"7.A deflector device as claimed in claim 6, wherein the additional remotely-operable adjusting means comprises a further telescopic member connected to the auxiliary wing-shaped body.\",\n \"8.A deflector device as claimed in claim 7, wherein the further telescopic member is hydraulically operated.\",\n \"9.A deflector device as claimed in claim 5, wherein the auxiliary wing-shaped body is provided with a trailing edge flap angled away from the boom, typically at about 35°.\",\n \"10.A method of performing a marine seismic survey, comprising: towing a plurality of laterally-spaced seismic streamers over an area to be surveyed, wherein the lateral position and the depth of at least one of the streamers are controlled by a deflector device comprising: a principal wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel; one or more buoyancy elements disposed within and/or secured to the upper end of the principal wing-shaped body; a boom extending rearwardly from the principal wing-shaped body; a pivotable control surface extending sideways from the boom and shaped to produce in use a force having a substantial vertical component; and remotely-operable means for pivoting the control surface, thereby to control the depth of the deflector device substantially independently of the lateral position of the deflector device.\",\n \"11.The method of claim 10, further comprising selecting a buoyancy of the one or more buoyancy elements to give the complete device a small positive buoyancy.\",\n \"12.The method of claim 10, wherein the remotely-operable means comprises a telescopic member connected to the control surface, and further comprising pivoting the control surface using the telescopic member.\",\n \"13.The method of claim 10, wherein the deflector device includes an auxiliary wing-shaped body, smaller than the principal wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in: use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body, and further comprising varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body.\"\n ],\n [\n \"This invention relates to deflector devices of the kind used between a towing vessel and a tow located in water, for example a seismic streamer or streamer array, or a seismic source array, in order to pull the tow out to one side of the vessel, so as to position it at a desired lateral offset from the course followed by the vessel.\",\n \"A deflector device of this kind is described in detail in our U.S. Pat.\",\n \"No.\",\n \"5,357,892, and comprises a wing-shaped deflector body having a remotely-operable pivotal lever or “boom” which extends rearwardly from a point near the middle of the trailing edge of the wing-shaped body.\",\n \"In use, the wing-shaped body is suspended beneath a float so as to be completely submerged and positioned generally vertically in the water, and is connected to the towing vessel by means of a tow line, while the tow is connected to the end of the boom remote from the wing-shaped body.\",\n \"As the device is pulled through the water, the wing-shaped body produces a sideways force, or “lift”, which moves the tow laterally.\",\n \"This lift can be varied by adjusting the angle of the boom from the vessel, thus permitting the lateral offset of the tow from the course of the vessel to be varied in use.\",\n \"The deflector device of U.S. Pat.\",\n \"No.\",\n \"5,357,892 has been successfully commercialised by the Applicant as its MONOWING deflector device.\",\n \"In use, rolling stability of the device is provided by the connection to the float, while stability of the device about a vertical axis is provided by the drag produced by the tow.\",\n \"The MONOWING deflector devices in current use are very large, typically 7.5 m high by 2.5 m wide, and weigh several tonnes.\",\n \"They are usually suspended around 2 m to 8 m below the float by means such as a fibre rope, and are also provided with a safety chain intended to prevent separation of the float and wing-shaped body in the event that the rope breaks.\",\n \"In rough weather, the upper part of the wing-shaped body may rise up out of the water, allowing the rope connecting the wing-shaped body and the float to go slack.\",\n \"If the wing-shaped body then drops abruptly, the rope, and possibly even the safety chain, may break, and/or their attachment points on the wing-shaped body may be badly damaged.\",\n \"Additionally, the depth at which the current deflector device operates is effectively determined by the length of the rope connecting it to the float.\",\n \"As a result of this, the operating depth of the deflector device cannot readily be varied while the device is deployed in the water.\",\n \"And since the normal operating depth of the current deflector device is typically a few meters, in the event of the onset of bad weather during a survey, the device and all the streamers and other equipment directly or indirectly attached to it have to be recovered onto the towing vessel, and then re-deployed when the bad weather has passed, both of which operations are very time consuming.\",\n \"It is an object of the present invention to alleviate the drawbacks arising from the connection of the deflector device to the float.\",\n \"According to the present invention, there is provided a deflector device for use with a tow line between a towing vessel and a tow in water behind the vessel, the device comprising a wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel, one or more buoyancy elements disposed within and/or secured to the upper end of the wing-shaped body, a boom extending rearwardly from the wing-shaped body, and a remotely-operable pivotable control surface extending sideways from the boom and shaped to produce in use a force having a vertical component, whereby to control the depth of the deflector device.\",\n \"It will be appreciated that since the deflector device of the invention can generate a controllable vertical force, this force, together with the buoyancy of the one or more buoyancy elements, can be selected and adjusted so that the separate surface float is no longer required, and the operating depth of the device can be remotely controlled while the device is deployed in the water.\",\n \"In particular, at the onset of bad weather, the deflector device and its tow can be caused to dive to a greater depth, where the effects of the bad weather are much reduced, until the weather improves.\",\n \"Advantageously, the one or more buoyancy elements has or have a buoyancy selected to give the complete device a small positive buoyancy.\",\n \"In a preferred embodiment of the invention, the deflector device further comprises an auxiliary wing-shaped body, smaller than the firstmentioned (or principal) wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body.\",\n \"Advantageously, this embodiment further includes remotely-operable means for varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body.\",\n \"The pivotable control surface and the remotely-operable means are preferably both hydraulically operated.\",\n \"Advantageously, the auxiliary wing-shaped body is provided with a trailing edge flap angled away from the boom, typically at about 35°.\",\n \"The invention also includes a method of performing a marine seismic survey, the method including towing a plurality of laterally spaced seismic steamers over an area to be surveyed, wherein the lateral position and the depth of at least one of the streamers are controlled by a deflector device in accordance with any one of the preceding statements of invention.\",\n \"The invention will now be described by way of example only, with reference to the accompanying drawings, of which: FIG.\",\n \"1 is a somewhat schematic view of a seismic survey vessel carrying out a marine seismic survey; FIG.\",\n \"2 is a somewhat schematic part-sectional view of a first embodiment of a deflector device in accordance with the present invention, for use in carrying out the survey of FIG.\",\n \"1; and FIGS.\",\n \"3A and 3B are respective perspective view of the deflector device of FIG.\",\n \"2.The seismic survey vessel shown in FIG.\",\n \"1 is indicated generally at 10, and is preferably as described in our PCT Patent Application No.\",\n \"PCT/GB98/01832 (WO 99/00295).\",\n \"The vessel 10 is shown towing a seismic source 15, typically a TRISOR multiple air gun source of the kind described in our U.S. Pat.\",\n \"No.\",\n \"4,757,482, and an array 16 of four substantially identical streamers 18.However, it will be appreciated that, in practice, many more than four streamers can be towed, for example by using the techniques described in our PCT Patent Application No.\",\n \"PCT/IB98/01435 (WO 99/15913).\",\n \"The streamers 18 are towed by means of their respective lead-ins 20 (ie the high strength steel- or fibre-reinforced electrical or electro-optical cables which convey electrical power, control and data signals between the vessel 10 and the streamers), and their spread is controlled by two deflector devices, indicated at 22, connected to the respective forward ends 24 of the two outermost streamers.\",\n \"The deflector devices 22 act in co-operation with respective spreader lines 26 connected between the forward end 24 of each outermost streamer 18 and the forward end 24 of its adjacent streamer to maintain a substantially uniform spacing between the streamers.\",\n \"One of the deflector devices 22 is shown in more detail in FIGS.\",\n \"2, 3A and 3B.\",\n \"The deflector device 22 is similar in general principle to the deflector device of our U.S. Pat.\",\n \"No.\",\n \"5,357,892, but is a much improved version of it.\",\n \"In particular, the deflector device 22 has a main wing-shaped body 28 which is coupled in use to a respective outer lead-in 20 via a towing bridle 27, and which corresponds to the deflector body 2 of U.S. Pat.\",\n \"No.\",\n \"5,357,892.However, the main wing-shaped body 28 is of improved hydrodynamic cross-sectional shape and includes a fixed-angle trailing edge flap 29, both of which features enhance lift.\",\n \"Also, the main wing-shaped body 28 is provided with vortex controlling end plates 30 (see FIGS.\",\n \"3A and 3B) of the kind described in our PCT Patent Application No.\",\n \"PCT/FR99/02272, to reduce drag and improve stability, and is largely made of titanium to reduce weight, while the towing bridle 27 comprises a pair of titanium chains 52 (see FIGS.\",\n \"3A and 3B).\",\n \"Additionally, the angle lever 10 of U.S. Pat.\",\n \"No.\",\n \"5,357,892 is replaced by a rearwardly extending fixed angle boom 32, which is detachably connected at one end 34 to the low pressure side 36 of the body 28 near the trailing edge flap 29, at a mounting bracket 38.The boom 32 is of sandwich construction, and is made from two similarly shaped plates 39 which are bolted together at intervals along their length and which sandwich between them the mounting bracket 38.Typically, the boom 32 is detached from the bracket 38 whenever the deflector device 22 is on the vessel 10, for ease of stowage.\",\n \"The other end 40 of the boom 32 has a towing eye 42, coupled in use to the forward end 24 of a respective one of the two outermost streamers 18.An auxiliary wing-shaped body 44, which is much smaller than the body 28 in length, thickness and chord, is pivotally secured as will be explained hereinafter to the end 40 of the boom 32, with its longitudinal axis (which lies in a plane perpendicular to the plane of FIG.\",\n \"2) extending parallel to the longitudinal axis of the body 28.The shape of the body 44 is designed to produce, in use, a sideways force in a direction approximately opposite to that produced by the body 28 (approximately opposite, because as will become apparent, the direction of the force varies in use).\",\n \"This sideways force is increased by providing the body 44 with a fixed trailing edge flap 46, angled away from the boom 32 at an angle of about 35°.\",\n \"As best seen in FIGS.\",\n \"3A and 3B, the auxiliary wing-shaped body 44 is implemented in two symmetrical halves 44a and 44b, which each have vortex-reducing end plates 45 and which are disposed on opposite sides of the boom 32.These two halves 44a, 44b are rotatable in unison about a common axis perpendicular to the plane of the boom 32, so as to vary the angle of the chord of the auxiliary wing-shaped body 44 with respect to the boom.\",\n \"Rotation of the auxiliary wing-shaped body 44 is effected by a telescopic actuator 48 pivotally mounted between the plates 39 of the boom 32, the actuator being pivotally connected to a lever arm or eccentric 50 secured to each of the two halves 44a, 44b of the auxiliary wing-shaped body.\",\n \"The telescopic actuator 48 is hydraulically operated by a remotely controllable electro-hydraulic control pack 52 also mounted between the plates 39 of the boom 32.It will be appreciated that varying the angle of the auxiliary wing-shaped body 44 of the deflector device 22 changes the angle of the main wing-shaped body 28 with respect to the direction of tow, and so changes the lift produced by the main wing-shaped body.\",\n \"This in turn changes the lateral offset produced by the deflector device 22.In accordance with the present invention, the deflector device 22 is made approximately neutrally buoyant, by including gas-filled pipe-like buoyancy elements 58 extending longitudinally within it from top to bottom, and/or by providing an integral buoyancy element at its upper end similar to but smaller than that described in our co-pending United Kingdom Patent Applications Nos.\",\n \"0023775.0, 0025719.6 and 0029451.2.In practice, the deflector device 22 is preferably designed to be slightly positively buoyant, so that in the event of a malfunction, it tends to float rather than to sink.\",\n \"Additionally, the deflector device 22 is provided with a pivotable control surface (or flap) 54, which is secured to the boom 32 in the region of the auxiliary wing-shaped body 44 by a generally triangular bracket 56, and which is pivotable about an axis perpendicular to both the pivot axis of the body 44 and the direction of tow (indicated by the arrow 58 in FIG.\",\n \"2).\",\n \"The flap 54 and the bracket 56 are both made from titanium.\",\n \"The angular position of the flap 54 is controlled by a further telescopic actuator 60, which is connected to a lever arm 62 provided on the flap, and which is hydraulically operated by the electro-hydraulic control pack 52.It will be appreciated that rotation of the flap 54 about its pivot axis produces in use an upward or downward force at the end 40 of the boom 32, and thus enables the depth of the deflector device 22 to be controlled.\",\n \"It will be appreciated that as a result of making the deflector device 22 approximately neutrally buoyant and capable of generating a remotely-controllable vertical force, a separate surface float is no longer required, and the operating depth of the device can be remotely controlled while the device is deployed in the water.\",\n \"In particular, in the event of the onset of bad weather, the deflector device 22 and the streamers 18 attached to it can be caused to dive to a greater depth, where the effects of the bad weather are much reduced, until the bad weather passes.\",\n \"Many modifications can be made to the described embodiment of the invention.\",\n \"In particular, the flap 54 and the auxiliary wing-shaped body 44 can be made from a plastics material reinforced with high strength fibres, eg Kevlar fibres, and can be electrically actuated rather than hydraulically actuated, Additionally, the devices 22 and 60 can be used with tows other than streamers, for example seismic sources, and the tow need not be connected to the end 40 of the boom 32 (it could instead be connected to the lead-in 20, at a point near where the bridle 24 is connected to the lead-in).\",\n \"Also, the invention can if desired be used with a deflector device in which the auxiliary wing-shaped body 44 is fixed, and the boom 32 is pivotable towards and away from the main deflector body 28, as described in our United Kingdom Patent Applications Nos.\",\n \"0023755.2, 0025711.3 and 0029452.0.Indeed, the invention can even be used with a deflector device like that described in our U.S. Pat.\",\n \"No.\",\n \"5,357,892, ie a deflector device without the auxiliary wing-shaped body 44, by mounting a pivotable flap analogous to the flap 54 on a pivotable boom analogous to the angle lever 10 of the deflector device of the US patent.\",\n \"Finally, although the invention has been described in relation to deflector devices whose lift can be varied by varying the angle of the device with respect to the direction of tow, it is also applicable in its broadest aspect to a fixed angle deflector device, eg of the kind referred to as a “door”.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10450862"}}},{"rowIdx":286,"cells":{"text":{"kind":"list like","value":[["Methods for the production of multimeric proteins and related compositions","Improved methods for the production of multimeric-protein-complexes, such as redox proteins and immunoglobins, in association with oil bodies are described.","The redox protein is enzymatically active when prepared in association with the oil bodies.","Also provided are related nucleic acids, proteins, cells, plants, and compositions."],["1-266.","(canceled) 267.A method of producing an oil body associated with a recombinant multimeric-protein-complex, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide.","268.A method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex; and (d) associating said first recombinant polypeptide with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide.","269.A method of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.","270.A chimeric nucleic acid sequence encoding a multimeric-fusion-protein, said nucleic acid comprising: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","271.A recombinant multimeric-fusion-protein comprising (i) an oil-body-targeting-protein, or fragment thereof, (ii) a first recombinant polypeptide and a (iii) second recombinant polypeptide, wherein said first and second recombinant polypeptides are capable of forming a multimeric-protein-complex.","272.Isolated oil bodies comprising a multimeric-protein-complex comprising (i) an oil-body-targeting-protein and (ii) a first recombinant polypeptide, said oil bodies further comprising a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","273.Isolated oil bodies comprising (a) a first fusion protein comprising a first oil-body-targeting-protein fused to a first recombinant polypeptide; and (b) a second fusion protein comprising a second oil-body-targeting-protein fused to a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","274.A cell comprising oil bodies and (i) an oil-body-targeting-protein, (ii) a first recombinant polypeptide and (iii) a second recombinant polypeptide wherein (1) said first recombinant polypeptide is capable of associating with said oil-body-targeting-protein; and (2) said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form a multimeric-protein-complex.","275.A composition comprising isolated oil bodies, thioredoxin and thioredoxin-reductase.","276.A food product, personal care product or pharmaceutical composition comprising the composition of claim 275.277.A method of reducing allergenicity of a food comprising the steps of: providing the isolated oil bodies of claim 273; and adding the isolated oil bodies to the food, whereby allergenicity of the food is reduced.","278.A method of treating or protecting a target against oxidative stress, comprising the steps of: providing the recombinant fusion polypeptide of claim 269; and contacting the recombinant fusion polypeptide with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.","279.A method for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.","280.A method for preparing a redox protein associated with oil bodies comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide in a progeny cell comprising oil bodies; and c) isolating from said progeny cell said oil bodies comprising said redox fusion polypeptide.","281.A chimeric nucleic acid comprising: 1) a first nucleic acid sequence capable of regulating transcription in a host cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.","282.A transgenic plant comprising the chimeric nucleic acid sequence of claim 281.283.A safflower plant comprising the chimeric nucleic acid of anyone of claim 281.284.A plant seed comprising the chimeric nucleic acid of claim 281.285.A safflower seed comprising the chimeric nucleic acid of claim 281.286.An oil body preparation obtained by the method of claim 279.287.A food product comprising an oil body preparation of claim 286.288.A composition comprising an oil body preparation of claim 286 (New).","289.A personal care product comprising an oil body preparation of claim 286.290.A product capable of treating oxidative stress in a target comprising an oil body preparation of claim 286.291.A product capable of chemically reducing a target comprising an oil body preparation of claim 286.292.A detergent composition comprising the product of claim 286.293.A method of cleansing an item, comprising administering the product of claim 290 to said item under conditions that promote cleansing.","294.An emulsion formulation prepared by the method of claim 279.295.A nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one thioredoxin-related protein or an active fragment thereof.","296.The seed of the plant of claim 281.297.An extract of the seed of claim 296, wherein the extract comprises an activity of a thioredoxin-related protein.","298.An oil body from the seed of claim 296.299.Oil produced from the seed of claim 296.300.A method of making a fusion protein comprising a thioredoxin-related activity, the method comprising the steps of: providing a transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, the gene fusion encoding a fusion protein comprising a thioredoxin-related activity; obtaining seeds from the plant; and recovering the fusion protein by isolating oil bodies from the seeds.","301.Oil bodies in association with a fusion protein, obtained by the method of claim 300.302.A method of reducing allergenicity of a food comprising the steps of: providing a preparation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and adding the preparation to the food, whereby allergenicity of the food is reduced due to activity of the thioredoxin-related protein or fragment.","303.A composition comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.","304.A cosmetic formulation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in an acceptable carrier.","305.A method of treating or protecting a target against oxidative stress, comprising the steps of: providing a preparation comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and contacting the preparation with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.","306.A nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one recombinant polypeptide and an oil-body-surface-avoiding linker in frame between the first and second region polypeptides."],["<SOH> BACKGROUND <EOH>Multimeric proteins (i.e.","proteins comprising multiple polypeptide chains) are a biologically and commercially important class of proteins.","Antibodies for example are multimeric proteins which are used to treat a wide range of disease conditions.","However in view of their complexity, multimeric proteins frequently represent significant manufacturing challenges.","Redox proteins are also a commercially important class of proteins with applications in a variety of different industries including the pharmaceutical, personal care and food industry.","For example, the redox protein thioredoxin may be used in the manufacture of personal care products (Japanese Patent Applications JP9012471A2, JP103743A2, JP1129785A2), pharmaceutical compositions/products (Aota et al.","(1996) J. Cardiov.","Pharmacol.","(1996) 27: 727-732) as well as to reduce protein allergens present in food products such as milk (del Val et al.","(1999) J. Allerg.","Vlin.","Immunol.","103: 690-697) and wheat (Buchanan et al.","(1997) Proc.","Natl.","Acad.","Sci.","USA 94: 5372-5377).","However, there is a need in the art to further improve the methods for the recombinant expression of multimeric proteins, including redox proteins.","The present invention satisfies this need and provides related advantages as well."],["<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to novel and improved methods of producing a first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulin-polypeptide-chains, immunoglobulins, redox-fusion-polypeptides, and/or thioredoxin-related proteins; in association with oil bodies.","Accordingly, provided herein are methods of producing a recombinant multimeric-protein-complex, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.","The method further contemplates isolating the oil bodies associated with said recombinant multimeric-protein-complex.","The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and said second recombinant polypeptide.","Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.","The oil-body-targeting-protein can be an oleosin or caleosin.","When the oil-body-targeting-protein can be an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.","Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.","The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.","The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.","In one embodiment, the first recombinant polypeptide is capable of associating with said second recombinant polypeptide in the cell.","In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.","In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.","Also provided herein is a method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex; and (d) associating said first recombinant polypeptide with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.","This method further contemplates isolating from the progeny cell, oil bodies comprising the multimeric-protein-complex.","The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and second recombinant polypeptide.","Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.","The oil-body-targeting-protein can be an oleosin or caleosin.","When the oil-body-targeting-protein is an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.","Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.","The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.","The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.","In one embodiment, the first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex in said progeny cell.","In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.","In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 60 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.","Also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.","The second recombinant polypeptide can be associated with oil bodies through a second oil-body-targeting-protein in the second plant.","The oil bodies can be isolated from the progeny plant comprising said multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, wherein the oil-body-protein can be an oleosin or caleosin.","The first recombinant polypeptide can be fused to the oleosin or caleosin; and the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.","The first and second recombinant polypeptide can form a multimeric-protein-complex, such as a heteromultimeric-protein-complex, wherein the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The plant can be a safflower plant.","Also provided herein are chimeric nucleic acids encoding a multimeric-fusion-protein as described herein, said nucleic acid comprising: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin.","The oil-body-protein can be an oleosin or caleosin.","The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the first and second recombinant polypeptide can form an enzymatically active heteromultimeric redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence that is also a non-proteolytic linker and said sequence encoding the first recombinant polypeptide.","Also provided herein are recombinant multimeric-fusion-proteins comprising (i) an oil-body-targeting-protein, or fragment thereof, (ii) a first recombinant polypeptide and a (iii) second recombinant polypeptide, wherein said first and second recombinant polypeptides are capable of forming a multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.","The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said sequence encoding the first recombinant polypeptide.","Also provided herein are isolated oil bodies comprising a multimeric-protein-complex comprising (i) an oil-body-targeting-protein and (ii) a first recombinant polypeptide, said oil bodies further comprising a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.","The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are isolated oil bodies comprising (a) a first fusion protein comprising a first oil-body-targeting-protein fused to a first recombinant polypeptide; and (b) a second fusion protein comprising a second oil-body-targeting-protein fused to a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.","The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are cells and transgenic plants comprising oil bodies, multimeric-protein-complexes, and multimeric-fusion-proteins, set forth herein.","In one embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In embodiments, wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase, the methods described herein can be used to formulate the oil bodies for use in the preparation of a food product, personal care product or pharmaceutical composition.","These formulations can further comprise the addition of NADP or NADPH.","The food product can be a milk or wheat based food product.","The personal care product can reduce the oxidative stress to the surface area of the human body or can be used to lighten the skin.","The pharmaceutical composition can be used to treat chronic obstructive pulmonary disease (COPD), cataracts, diabetes, envenomation, bronchiopulmonary disease, malignancies, psoriasis, reperfusion injury, wound healing, sepsis, GI bleeding, intestinal bowel disease (IBD), ulcers, GERD (gastro esophageal reflux disease).","Also provided herein are compositions comprising isolated oil bodies, thioredoxin and thioredoxin-reductase, wherein said thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194, and said thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.The composition can further comprise NADP or NADPH.","In another embodiment, the composition comprises a first recombinant polypeptide that can be an immunoglobulin-polypeptide-chain and a second recombinant polypeptide.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are multimeric-fusion-proteins, wherein the fusion-protein contains two or more polypeptide chains selected from the group of proteins set forth in FIG.","5 .","Methods are also provided of reducing allergenicity of a food comprising the steps of providing the isolated oil bodies set forth herein; and adding the isolated oil bodies to the food, whereby allergenicity of the food is reduced.","The food can be selected from the group consisting of wheat flour, wheat dough, milk, cheese, yogurt and ice cream.","The various methods of treating food can further comprise providing NADH as a co-factor in the substantial absence of NADPH.","Also provided herein are methods of treating or protecting a target against oxidative stress, comprising the steps of providing the recombinant redox fusion polypeptide comprising thioredoxin and thioredoxin-reductase; and contacting the recombinant fusion polypeptide with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.","The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.","Also provided herein are methods for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.","The first redox protein can be a thioredoxin and the second redox protein can be a thioredoxin-reductase.","Also, provided herein are methods of producing an immunoglobulin, said method comprising: (a) producing in a cell comprising oil bodies, a first immunoglobulin-polypeptide-chain and a second immunoglobulin-polypeptide-chain wherein said first immunoglobulin-polypeptide-chain is capable of associating with said second immunoglobulin-polypeptide-chain to form said immunoglobulin; and (b) associating said immunoglobulin with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first immunoglobulin-polypeptide-chain.","For example, the first immunoglobulin-polypeptide-chain can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second immunoglobulin-polypeptide-chain can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are methods for preparing a redox protein or an immunoglobulin associated with oil bodies comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein, or a nucleic acid sequence encoding a immunoglobulin comprising a first immunoglobulin-polypeptide-chain linked to a second immunoglobulin-polypeptide-chain, operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide or immunoglobulin in a progeny cell comprising oil bodies; and c) isolating from said progeny cell said oil bodies comprising said redox fusion polypeptide or immunoglobulin.","In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide or immunoglobulin can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged or have a molecular weight of at least 35 kd.","Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.","In this optional embodiment, also contemplated is the introduction of an enzyme or chemical that cleaves said redox fusion polypeptide from said oil body, thereby obtaining isolated redox fusion polypeptide.","The first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.","In one embodiment, the thioredoxin and thioredoxin-reductase can be obtained from Arabidopsis .","In another embodiment, the first redox protein is at least 5 times more active when produced as a redox fusion polypeptide as compared to the production of the first redox protein without the second redox protein.","Also provided herein, for use with the various methods set forth herein is the formulation of an emulsion of the oil bodies associated with the redox fusion polypeptide for use in the preparation of a product capable of treating oxidative stress in a target, a product capable of chemically reducing a target, pharmaceutical composition, a personal care product or a food product.","Accordingly, an emulsion formulation composition is provided.","Also provided herein is a chimeric nucleic acid comprising: 1) a first nucleic acid sequence capable of regulating transcription in a host cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.","The oil-body-protein can be an oleosin or a caleosin, the first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.","In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide is a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","In one embodiment, the gene fusion optionally further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.","Also provided herein are transgenic plants, e.g., safflower plants, comprising any of the chimeric nucleic acid sequences and constructs described herein.","The chimeric nucleic acids can be contained within a plastid.","Accordingly, isolated plastids are provided having chimeric nucleic acids therein.","Also provided are plant seeds comprising the chimeric nucleic acids provided herein.","Also provided are oil body preparations obtained using any of the methods provided herein, and food products, pharmaceutical compositions, and personal care products containing the oil body preparations.","The products and/or compositions provided herein are capable of treating oxidative stress in a target, capable of chemically reducing a target.","Also provided is a detergent composition comprising an oil body preparation capable of chemically reducing a target, and related methods of cleansing an item, comprising administering such product to the item under conditions that promote cleansing.","Also provided herein are nucleic acid constructs comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one thioredoxin-related protein or an active fragment thereof.","In one embodiment, the at least one thioredoxin-related protein can be thioredoxin.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194.The thioredoxin can be obtained from Arabidopsis or wheat.","In another embodiment, the at least one thioredoxin-related protein can be thioredoxin-reductase.","The thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313 and/or derived from Arabidopsis or wheat.","The thioredoxin-reductase can be an NADPH-dependent thioredoxin-reductase.","The second region can encode a thioredoxin and thioredoxin-reductase.","In one embodiment, the thioredoxin and thioredoxin-reductase is obtained from Mycobacterium leprae .","In another embodiment, the at least one thioredoxin-related protein can be an engineered fusion protein.","The first region can precede, in a 5′ to 3′ direction, the second region.","Alternatively, the first region follows, in a 5′ to 3′ direction, the second region.","The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.","A seed-specific promoter, such as a phaseolin promoter, can be operably linked to the gene fusion.","In one embodiment, at least one thioredoxin-related protein is derived from a plant species selected from the group consisting of Arabidopsis and wheat.","In another embodiment, at least one thioredoxin-related protein can be derived from E. coli.","In one embodiment, the gene fusion further comprises a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the linker amino acid sequence is positioned between the first region and the second region.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","In addition, the gene fusion can further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the second region.","Also provided herein are transgenic plants containing a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof.","The thioredoxin-related protein can be thioredoxin.","The nucleic acid construct can be contained within a plastid.","In one embodiment, when the first thioredoxin-related protein is thioredoxin and the construct can further comprise a region encoding a thioredoxin-reductase.","The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.","The gene fusion can optionally further comprise a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the nucleic acid encoding the linker amino acid sequence is positioned between the region encoding an oil-body-protein and the region encoding a first thioredoxin-related protein.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","The gene fusion can optionally further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the region encoding a first thioredoxin-related protein.","Also provided is a transgenic plant comprising a nucleic acid construct, a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, wherein a fusion protein comprising activities of oleosin and the thioredoxin-related protein is produced in a seed of the plant.","In another embodiment, a thioredoxin-related protein having concentration of at least about 0.5% of total cellular seed protein is provided.","Also provided herein is an extract comprising an activity of a thioredoxin-related protein.","Also provided are oil bodies and/or oil obtained from various seeds.","Also provided herein are methods of making a fusion protein comprising a thioredoxin-related activity, the method comprising the steps of: a) providing a transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, the gene fusion encoding a fusion protein comprising a thioredoxin-related activity; b) obtaining seeds from the plant; and c) recovering the fusion protein by isolating oil bodies from the seeds.","In one embodiment, the oil bodies are fractionated to achieve partial purification of the fusion protein.","The oil bodies can be in association with a fusion protein.","The oil-body-protein can be cleaved from the thioredoxin-related protein after fractionation of the oil bodies.","The cleaving step can make use of a protease or chemical proteolysis.","Also provided herein are methods of reducing allergenicity of a food comprising the steps of: a) providing a preparation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) adding the preparation to the food, whereby allergenicity of the food is reduced due to activity of the thioredoxin-related protein or fragment.","The food can be wheat flour, wheat dough, milk, cheese, yogurt and ice cream.","In one embodiment, NADH is used as a co-factor in the substantial absence of NADPH.","Also provided herein are pharmaceutical compositions comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.","The oil bodies can be associated with the fusion protein.","Also provided is a cosmetic formulation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.","Also provided are methods of treating or protecting a target against oxidative stress, comprising the steps of: a) providing a preparation comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) contacting the preparation with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.","The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.","Also provided is a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one polypeptide or an active fragment thereof, and an oil-body-surface-avoiding linker in frame between the first and second region polypeptides.","Also provided are methods of expressing this construct into the encoded amino acid sequence; and oil bodies, formulations, emulsions, cells, and plants comprising the construct and encoded amino acid sequence.","These particular constructs, oil bodies, formulations, emulsions, cells, and plants can be produced according to the methods described herein.","The second region can encode any polypeptide, for example, a therapeutically, nutritionally, industrially or cosmetically useful peptide as set forth herein.","For example, the second region can encode a redox protein, an immunoglobulin, a thioredoxin-related protein or any one or more recombinant polypeptides of a multimeric-protein-complex.","Other features and advantages of the present invention will become readily apparent from the following detailed description.","It should be understood however that the detailed description and the specific examples while indicating particular embodiments of the invention are given by way of illustration only."],["RELATED APPLICATIONS Benefit of priority under 35 U.S.C.","§119(e) is claimed to U.S. provisional application Ser.","No.","60/302,885, filed Jul.","5, 2001, to van Rooijen, et al., entitled “METHODS FOR THE PRODUCTION OF REDOX PROTEINS”.","This application is also a continuation-in-part of U.S. utility application Ser.","No.","10/006,038, filed Dec. 4, 2001 to van Rooijen, et al., entitled “METHODS FOR THE PRODUCTION OF REDOX PROTEINS”; which is a continuation-in-part of U.S. utility application Ser.","No.","09/742,900, filed Dec. 19, 2000 to Heifetz, et al., entitled “METHOD OF PRODUCTION AND DELIVERY OF THIOREDOXIN”.","This application is also a continuation-in-part of U.S. utility application Ser.","No.","09/742,900.The subject matter of each of the provisional and utility applications is incorporated herein by reference in its entirety.","FIELD OF THE INVENTION The present invention relates to multimeric-protein-complexes, redox proteins, and recombinant polypeptides; and improved methods for their production.","BACKGROUND Multimeric proteins (i.e.","proteins comprising multiple polypeptide chains) are a biologically and commercially important class of proteins.","Antibodies for example are multimeric proteins which are used to treat a wide range of disease conditions.","However in view of their complexity, multimeric proteins frequently represent significant manufacturing challenges.","Redox proteins are also a commercially important class of proteins with applications in a variety of different industries including the pharmaceutical, personal care and food industry.","For example, the redox protein thioredoxin may be used in the manufacture of personal care products (Japanese Patent Applications JP9012471A2, JP103743A2, JP1129785A2), pharmaceutical compositions/products (Aota et al.","(1996) J. Cardiov.","Pharmacol.","(1996) 27: 727-732) as well as to reduce protein allergens present in food products such as milk (del Val et al.","(1999) J. Allerg.","Vlin.","Immunol.","103: 690-697) and wheat (Buchanan et al.","(1997) Proc.","Natl.","Acad.","Sci.","USA 94: 5372-5377).","However, there is a need in the art to further improve the methods for the recombinant expression of multimeric proteins, including redox proteins.","The present invention satisfies this need and provides related advantages as well.","SUMMARY OF THE INVENTION The present invention relates to novel and improved methods of producing a first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulin-polypeptide-chains, immunoglobulins, redox-fusion-polypeptides, and/or thioredoxin-related proteins; in association with oil bodies.","Accordingly, provided herein are methods of producing a recombinant multimeric-protein-complex, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.","The method further contemplates isolating the oil bodies associated with said recombinant multimeric-protein-complex.","The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and said second recombinant polypeptide.","Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.","The oil-body-targeting-protein can be an oleosin or caleosin.","When the oil-body-targeting-protein can be an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.","Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.","The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.","The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.","In one embodiment, the first recombinant polypeptide is capable of associating with said second recombinant polypeptide in the cell.","In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.","In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.","Also provided herein is a method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex; and (d) associating said first recombinant polypeptide with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.","This method further contemplates isolating from the progeny cell, oil bodies comprising the multimeric-protein-complex.","The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and second recombinant polypeptide.","Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.","The oil-body-targeting-protein can be an oleosin or caleosin.","When the oil-body-targeting-protein is an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.","Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.","The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.","The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.","In one embodiment, the first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex in said progeny cell.","In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.","In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 60 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.","Also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.","The second recombinant polypeptide can be associated with oil bodies through a second oil-body-targeting-protein in the second plant.","The oil bodies can be isolated from the progeny plant comprising said multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, wherein the oil-body-protein can be an oleosin or caleosin.","The first recombinant polypeptide can be fused to the oleosin or caleosin; and the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.","The first and second recombinant polypeptide can form a multimeric-protein-complex, such as a heteromultimeric-protein-complex, wherein the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The plant can be a safflower plant.","Also provided herein are chimeric nucleic acids encoding a multimeric-fusion-protein as described herein, said nucleic acid comprising: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin.","The oil-body-protein can be an oleosin or caleosin.","The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the first and second recombinant polypeptide can form an enzymatically active heteromultimeric redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence that is also a non-proteolytic linker and said sequence encoding the first recombinant polypeptide.","Also provided herein are recombinant multimeric-fusion-proteins comprising (i) an oil-body-targeting-protein, or fragment thereof, (ii) a first recombinant polypeptide and a (iii) second recombinant polypeptide, wherein said first and second recombinant polypeptides are capable of forming a multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.","The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said sequence encoding the first recombinant polypeptide.","Also provided herein are isolated oil bodies comprising a multimeric-protein-complex comprising (i) an oil-body-targeting-protein and (ii) a first recombinant polypeptide, said oil bodies further comprising a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.","The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are isolated oil bodies comprising (a) a first fusion protein comprising a first oil-body-targeting-protein fused to a first recombinant polypeptide; and (b) a second fusion protein comprising a second oil-body-targeting-protein fused to a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.","The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are cells and transgenic plants comprising oil bodies, multimeric-protein-complexes, and multimeric-fusion-proteins, set forth herein.","In one embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In embodiments, wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase, the methods described herein can be used to formulate the oil bodies for use in the preparation of a food product, personal care product or pharmaceutical composition.","These formulations can further comprise the addition of NADP or NADPH.","The food product can be a milk or wheat based food product.","The personal care product can reduce the oxidative stress to the surface area of the human body or can be used to lighten the skin.","The pharmaceutical composition can be used to treat chronic obstructive pulmonary disease (COPD), cataracts, diabetes, envenomation, bronchiopulmonary disease, malignancies, psoriasis, reperfusion injury, wound healing, sepsis, GI bleeding, intestinal bowel disease (IBD), ulcers, GERD (gastro esophageal reflux disease).","Also provided herein are compositions comprising isolated oil bodies, thioredoxin and thioredoxin-reductase, wherein said thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194, and said thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.The composition can further comprise NADP or NADPH.","In another embodiment, the composition comprises a first recombinant polypeptide that can be an immunoglobulin-polypeptide-chain and a second recombinant polypeptide.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are multimeric-fusion-proteins, wherein the fusion-protein contains two or more polypeptide chains selected from the group of proteins set forth in FIG.","5.Methods are also provided of reducing allergenicity of a food comprising the steps of providing the isolated oil bodies set forth herein; and adding the isolated oil bodies to the food, whereby allergenicity of the food is reduced.","The food can be selected from the group consisting of wheat flour, wheat dough, milk, cheese, yogurt and ice cream.","The various methods of treating food can further comprise providing NADH as a co-factor in the substantial absence of NADPH.","Also provided herein are methods of treating or protecting a target against oxidative stress, comprising the steps of providing the recombinant redox fusion polypeptide comprising thioredoxin and thioredoxin-reductase; and contacting the recombinant fusion polypeptide with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.","The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.","Also provided herein are methods for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.","The first redox protein can be a thioredoxin and the second redox protein can be a thioredoxin-reductase.","Also, provided herein are methods of producing an immunoglobulin, said method comprising: (a) producing in a cell comprising oil bodies, a first immunoglobulin-polypeptide-chain and a second immunoglobulin-polypeptide-chain wherein said first immunoglobulin-polypeptide-chain is capable of associating with said second immunoglobulin-polypeptide-chain to form said immunoglobulin; and (b) associating said immunoglobulin with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first immunoglobulin-polypeptide-chain.","For example, the first immunoglobulin-polypeptide-chain can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second immunoglobulin-polypeptide-chain can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are methods for preparing a redox protein or an immunoglobulin associated with oil bodies comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein, or a nucleic acid sequence encoding a immunoglobulin comprising a first immunoglobulin-polypeptide-chain linked to a second immunoglobulin-polypeptide-chain, operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide or immunoglobulin in a progeny cell comprising oil bodies; and c) isolating from said progeny cell said oil bodies comprising said redox fusion polypeptide or immunoglobulin.","In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide or immunoglobulin can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged or have a molecular weight of at least 35 kd.","Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.","In this optional embodiment, also contemplated is the introduction of an enzyme or chemical that cleaves said redox fusion polypeptide from said oil body, thereby obtaining isolated redox fusion polypeptide.","The first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.","In one embodiment, the thioredoxin and thioredoxin-reductase can be obtained from Arabidopsis.","In another embodiment, the first redox protein is at least 5 times more active when produced as a redox fusion polypeptide as compared to the production of the first redox protein without the second redox protein.","Also provided herein, for use with the various methods set forth herein is the formulation of an emulsion of the oil bodies associated with the redox fusion polypeptide for use in the preparation of a product capable of treating oxidative stress in a target, a product capable of chemically reducing a target, pharmaceutical composition, a personal care product or a food product.","Accordingly, an emulsion formulation composition is provided.","Also provided herein is a chimeric nucleic acid comprising: 1) a first nucleic acid sequence capable of regulating transcription in a host cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.","The oil-body-protein can be an oleosin or a caleosin, the first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.","In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide is a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","In one embodiment, the gene fusion optionally further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.","Also provided herein are transgenic plants, e.g., safflower plants, comprising any of the chimeric nucleic acid sequences and constructs described herein.","The chimeric nucleic acids can be contained within a plastid.","Accordingly, isolated plastids are provided having chimeric nucleic acids therein.","Also provided are plant seeds comprising the chimeric nucleic acids provided herein.","Also provided are oil body preparations obtained using any of the methods provided herein, and food products, pharmaceutical compositions, and personal care products containing the oil body preparations.","The products and/or compositions provided herein are capable of treating oxidative stress in a target, capable of chemically reducing a target.","Also provided is a detergent composition comprising an oil body preparation capable of chemically reducing a target, and related methods of cleansing an item, comprising administering such product to the item under conditions that promote cleansing.","Also provided herein are nucleic acid constructs comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one thioredoxin-related protein or an active fragment thereof.","In one embodiment, the at least one thioredoxin-related protein can be thioredoxin.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194.The thioredoxin can be obtained from Arabidopsis or wheat.","In another embodiment, the at least one thioredoxin-related protein can be thioredoxin-reductase.","The thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313 and/or derived from Arabidopsis or wheat.","The thioredoxin-reductase can be an NADPH-dependent thioredoxin-reductase.","The second region can encode a thioredoxin and thioredoxin-reductase.","In one embodiment, the thioredoxin and thioredoxin-reductase is obtained from Mycobacterium leprae.","In another embodiment, the at least one thioredoxin-related protein can be an engineered fusion protein.","The first region can precede, in a 5′ to 3′ direction, the second region.","Alternatively, the first region follows, in a 5′ to 3′ direction, the second region.","The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.","A seed-specific promoter, such as a phaseolin promoter, can be operably linked to the gene fusion.","In one embodiment, at least one thioredoxin-related protein is derived from a plant species selected from the group consisting of Arabidopsis and wheat.","In another embodiment, at least one thioredoxin-related protein can be derived from E. coli.","In one embodiment, the gene fusion further comprises a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the linker amino acid sequence is positioned between the first region and the second region.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","In addition, the gene fusion can further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the second region.","Also provided herein are transgenic plants containing a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof.","The thioredoxin-related protein can be thioredoxin.","The nucleic acid construct can be contained within a plastid.","In one embodiment, when the first thioredoxin-related protein is thioredoxin and the construct can further comprise a region encoding a thioredoxin-reductase.","The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.","The gene fusion can optionally further comprise a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the nucleic acid encoding the linker amino acid sequence is positioned between the region encoding an oil-body-protein and the region encoding a first thioredoxin-related protein.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","The gene fusion can optionally further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the region encoding a first thioredoxin-related protein.","Also provided is a transgenic plant comprising a nucleic acid construct, a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, wherein a fusion protein comprising activities of oleosin and the thioredoxin-related protein is produced in a seed of the plant.","In another embodiment, a thioredoxin-related protein having concentration of at least about 0.5% of total cellular seed protein is provided.","Also provided herein is an extract comprising an activity of a thioredoxin-related protein.","Also provided are oil bodies and/or oil obtained from various seeds.","Also provided herein are methods of making a fusion protein comprising a thioredoxin-related activity, the method comprising the steps of: a) providing a transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, the gene fusion encoding a fusion protein comprising a thioredoxin-related activity; b) obtaining seeds from the plant; and c) recovering the fusion protein by isolating oil bodies from the seeds.","In one embodiment, the oil bodies are fractionated to achieve partial purification of the fusion protein.","The oil bodies can be in association with a fusion protein.","The oil-body-protein can be cleaved from the thioredoxin-related protein after fractionation of the oil bodies.","The cleaving step can make use of a protease or chemical proteolysis.","Also provided herein are methods of reducing allergenicity of a food comprising the steps of: a) providing a preparation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) adding the preparation to the food, whereby allergenicity of the food is reduced due to activity of the thioredoxin-related protein or fragment.","The food can be wheat flour, wheat dough, milk, cheese, yogurt and ice cream.","In one embodiment, NADH is used as a co-factor in the substantial absence of NADPH.","Also provided herein are pharmaceutical compositions comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.","The oil bodies can be associated with the fusion protein.","Also provided is a cosmetic formulation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.","Also provided are methods of treating or protecting a target against oxidative stress, comprising the steps of: a) providing a preparation comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) contacting the preparation with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.","The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.","Also provided is a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one polypeptide or an active fragment thereof, and an oil-body-surface-avoiding linker in frame between the first and second region polypeptides.","Also provided are methods of expressing this construct into the encoded amino acid sequence; and oil bodies, formulations, emulsions, cells, and plants comprising the construct and encoded amino acid sequence.","These particular constructs, oil bodies, formulations, emulsions, cells, and plants can be produced according to the methods described herein.","The second region can encode any polypeptide, for example, a therapeutically, nutritionally, industrially or cosmetically useful peptide as set forth herein.","For example, the second region can encode a redox protein, an immunoglobulin, a thioredoxin-related protein or any one or more recombinant polypeptides of a multimeric-protein-complex.","Other features and advantages of the present invention will become readily apparent from the following detailed description.","It should be understood however that the detailed description and the specific examples while indicating particular embodiments of the invention are given by way of illustration only.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 shows a ClustalW Formatted Alignment comparison of the published NADPH thioredoxin-reductase nucleic acid sequence (SEQ ID NO:9) (ATTHIREDB-Jacquot et al.","J. Mol.","Biol.","(1994) 235 (4):1357-63.)","with the sequence isolated herein in Example 1 (TR; SEQ ID NO:8).","FIG.","2 shows a ClustalW Formatted Alignment comparison of the deduced amino acid sequence of the published NADPH thioredoxin-reductase sequence (SEQ ID NO:12) (ATTHIREDB Jacquot et al.","J. Mol.","Biol.","(1994) 235 (4):1357-63.)","with the sequence isolated herein in Example 1 (TR; SEQ ID NO:13).","FIG.","3 shows a clustal alignment comparing the amino acid sequence of the Arabidopsis thaliana thioredoxin-reductase-linker-thioredoxin synthetic fusion (Arab TR-link-Trxh; SEQ ID NO:37) to the Mycobacterium leprae thioredoxin-reductase-thioredoxin natural fusion (M. lep TR/Trxh; SEQ ID NO:36) natural fusion.","Overall, the proteins are approximately 50% identical at the amino acid level.","FIG.","4 is a bar graph showing the thioredoxin/thioredoxin-reductase activity measurements for the various transgenic Arabidopsis seed fractions.","Relative specific activity is expressed as a percentage of the E. coli thioredoxin and thioredoxin-reductase activities.","The numbered bars in the graph correspond to the following: 1.W.T.+oleosin-thioredoxin 2.W.T.+thioredoxin-oleosin 3.W.T.+thioredoxin 4.W.T.+oleosin-thioredoxin-reductase 5.W.T.+thioredoxin-reductase-oleosin 6.W.T.+thioredoxin-reductase 7.thioredoxin+oleosin-thioredoxin-reductase 8.thioredoxin+thioredoxin-reductase-oleosin 9.thioredoxin+thioredoxin-reductase 10.thioredoxin-reductase+oleosin-thioredoxin 11.thioredoxin-reductase+thioredoxin-oleosin 12.oleosin-M. lep TR/Trxh 13.E.","coli thioredoxin-reductase+thioredoxin FIG.","5 provides a listing of exemplary proteins for use in the heteromultimeric-fusion-proteins and heteromultimeric-protein-complexes provided herein.","DETAILED DESCRIPTION As hereinbefore mentioned, the present invention relates to novel and improved methods for the production of multimeric proteins, including a first and second recombinant polypeptide, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulin-polypeptide-chains, immunoglobulins, redox-fusion-polypeptides, and a first and second thioredoxin-related protein; and related products.","These methods permit the production of active multimeric-protein-complexes in association with oil bodies.","The oil bodies in association with the multimeric-protein-complex may be used to prepare various useful emulsions.","Accordingly, provided herein are methods of producing a recombinant multimeric-protein-complex associated with an oil body, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide in the cell to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide.","DEFINITIONS AND TERMS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.","Where permitted, all patents, applications, published applications and other publications and sequences from GenBank, SwissPro and other data bases referred to throughout in the disclosure herein are incorporated by reference in their entirety.","As used herein, the phrase “multimeric-protein-complex”, refers to two or more polypeptide chains that permanently or repeatedly interact or permanently or repeatedly coordinate to form a biologically active assembly comprising said two or more polypeptide chains.","It should be noted that the polypeptides may be independently biologically active without interaction or coordination to form the complex.","The multimeric-protein-complex may provide a biological structure, or it may be capable of facilitating a chemical or biological reaction.","For example, one of the protein regions within the multimeric-protein-complex can repeatedly activate or repeatedly inactivate the biological or metabolic activity of one or more of the other proteins contained within the multimeric-protein-complex.","In one embodiment, the first and second recombinant polypeptide contained in a multimeric-protein-complex may either associate or interact as independent non-contiguous polypeptide chains or the multimeric-protein-complex may be prepared as a fusion polypeptide (multimeric-fusion-protein) between the first and second recombinant polypeptide.","One example of a repeated (e.g., reoccurring) interaction or association between the two or more polypeptides of a multimeric-protein-complex provided herein is the interaction between two or more non-identical redox proteins to form a heteromultimeric-protein-complex.","Exemplary redox proteins for use in this regard are thioredoxin and the thioredoxin-reductase.","A further example is the interaction between two or more immunoglobulin-polypeptide-chains to form an immunoglobulin.","As used herein, the phrase “heteromultimeric-protein-complex”, refers to two or more non-identical polypeptide chains that permanently or repeatedly interact or permanently or repeatedly coordinate to form a biologically active assembly comprising said two or more polypeptide chains.","Other examples of multimeric-protein-complexes provided herein include a first and second recombinant polypeptide, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, first and second immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and a first and second thioredoxin-related protein.","The recombinant polypeptide or multimeric-protein-complex is associated with an oil body.","As used herein, the phrase “oil body” or “oil bodies” refers to any oil or fat storage organelle in any cell type.","Accordingly, the oil bodies may be obtained from any cell comprising oil bodies, including plant cells (described in for example: Huang (1992) Ann.","Rev.","Plant Mol.","Biol.","43: 177-200), animal cells (described in for example: Murphy (1990) Prog Lipid Res 29(4): 299-324), including adipocytes, hepatocytes, steroidogenic cells, mammary epithelial cells, macrophages, algae cells (described in for example: Rossler (1988) J. Physiol.","London, 24: 394-400) fungal cells, including yeast cells (described in for example Leber et al.","(1994) Yeast 10: 1421-1428) and bacterial cells (described in for example: Pieper-Furst et al.","(1994) J. Bacteriol.","176: 4328-4337).","Preferably the oil bodies used herein are oil bodies obtainable from plant cells and more preferably the oil bodies obtainable from plant seed cells.","As used herein, the phrase “is capable of associating with”, “associate” or grammatical variations thereof, refers to any interaction between two or more polypeptides, including any covalent interactions (e.g.","multimeric-fusion-proteins) as well as non-covalent interactions.","Exemplary non-covalent interactions can be between the oil-body-targeting-protein and a redox protein or immunoglobulin-polypeptide-chain, as well as between two or more different proteins contained within two or more separate oil-body-protein fusion proteins (e.g., the redox proteins in oleosin-thioredoxin and oleosin-thioredoxin-reductase).","As used herein, the term “recombinant” (also referred to as heterologous) in the context of recombinant proteins and amino acids, means “of different natural origin” or represents a non-natural state.","For example, if a host cell is transformed with a nucleotide sequence derived from another organism, particularly from another species, that nucleotide sequence and amino acid sequence encoded thereby, is recombinant (heterologous) with respect to that host cell and also with respect to descendants of the host cell which carry that gene.","Similarly, recombinant (or heterologous) refers to a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g., a different copy number, or under the control of different regulatory elements.","A transforming nucleotide sequence may include a recombinant coding sequence, or recombinant regulatory elements.","Alternatively, the transforming nucleotide sequence may be completely heterologous or may include any possible combination of heterologous and endogenous nucleic acid sequences.","In various embodiments of the present invention, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins, are produced in a cell comprising oil bodies.","As used herein the phrase “in a cell”, “in the cell”, or grammatical variations thereof, mean that the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins, may be produced in any cellular compartment of that cell, so long as that cell comprises oil bodies therein.","In embodiments of the invention in which plant cells are used, the phrase is intended to include the plant apoplast.","In various embodiments provided herein, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and thioredoxin-related proteins, associate with an oil body through an oil-body-targeting-protein.","As used herein, the phrase “oil-body-targeting-protein” refers to any protein, protein fragment or peptide capable of associating with an oil body.","Exemplary oil-body-targeting-proteins for use herein include oil-body-proteins, such as oleosin and caleosin; immunoglobulins, such as bi-specific antibodies; and the like.","In embodiments described herein in which an oil-body-protein is used, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and thioredoxin-related proteins, are preferably fused to the oil-body-protein.","The term “oil-body-protein” refers to any protein naturally present in cells and having the capability of association with oil bodies, including any oleosin or caleosin.","Accordingly, provided herein a method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide, such as a redox protein, an immunoglobulin-polypeptide-chain or an thioredoxin-related protein, fused to an oil-body-protein; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide, such as a second redox protein, a second immunoglobulin-polypeptide-chain or a second thioredoxin-related protein; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex, preferably in said progeny cell; and (d) associating said first recombinant polypeptide with an oil body through said oil-body-protein.","The term “nucleic acid” as used herein refers to a sequence of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages.","The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof, which function similarly.","The nucleic acid sequences may be ribonucleic acids (RNA) or deoxyribonucleic acids (DNA) and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil.","The sequences also may contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo-uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-amino adenine, 8-thiol-adenine, 8-thio-alkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8 amino guanine, 8 thiol guanine, 8-thioalkyl guanines, 8 hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.","Multimeric-Protein-Complexes In accordance with the methods and compositions provided herein, any two recombinant polypeptides capable of forming a multimeric-protein-complex may be used.","The nucleic acid sequences encoding the two recombinant polypeptides may be obtained from any biological source or may be prepared synthetically.","In general nucleic acid sequence encoding multimeric proteins are known to the art and readily available.","Known nucleic acid sequences encoding multimeric-protein-complexes may be used to design and construct nucleic acid sequence based probes in order to uncover and identify previously undiscovered nucleic acid sequences encoding multimeric-protein-complexes, for example, by screening cDNA or genomic libraries or using 2- or multi-hybrid systems.","Thus, additional nucleic acid sequences encoding multimeric-protein-complexes may be discovered and used as described herein.","The first and/or second recombinant polypeptides that are comprised within a multimeric-protein-complex provided herein, can themselves be in the form of heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or a first and/or second thioredoxin-related protein.","The nucleic acid sequence encoding the first and second recombinant polypeptide, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or a first and/or second thioredoxin-related protein may be obtained from separate sources or may be obtained from the same source.","In general however, such nucleic acid sequence is obtained from the same or a similar biological source.","In certain embodiments wherein the nucleic acid sequence encoding the first and second recombinant polypeptide protein are obtained from the same source, the nucleic acid sequence encoding the first recombinant polypeptide and second recombinant polypeptide may be naturally fused.","In accordance with a particular embodiment, the nucleic acid sequences encoding the first and second recombinant polypeptide are obtained from a plant source.","Oil-Body-Surface-Avoiding Linkers Polypeptide spacers or linkers of variable length and/or negative charge can be used herein to separate the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and the first and/or second thioredoxin-related proteins from the in-frame oil-body-targeting-protein, to improve activity of and/or the accessibility of the polypeptide or complex.","For example, in one embodiment set forth herein, positioned between a nucleic acid sequence encoding a sufficient portion of an oil-body-protein and a nucleic acid sequence encoding either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and the first and/or second thioredoxin-related proteins; is a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","Oil-body-surface-avoiding linkers are positioned between the oil-body targeting sequence and an in-frame recombinant polypeptide of interest, e.g., the multimeric-protein-complexes provided herein, serve to increase the distance and or decrease the interaction between the negatively charged oil body surface and the recombinant polypeptide of interest.","A negatively charged linker is repelled by the negatively charged oil body surface, in turn increasing the distance or decreasing the interaction of its attached recombinant polypeptide with the oil body surface.","As a consequence of the increased distance from the oil body surface, the recombinant polypeptide will be more accessible, e.g.","to its target(s) substrate, protein substrate, protein partner, and less affected by the charged oil body surface.","Exemplary linker sequences for use herein can be either a negatively charged linker, or a linker having a molecular weight of at least about 35 kd or more.","As used herein, a “negatively charged linker” sequence, refers to any amino acid segment, or nucleic acid encoding such, that has a pI less than or equal to the pI of an oil body.","In certain embodiments, the pI of the negatively charged linker is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, down to about 25% or more, below that of the pI of an oil body in the particular plant or cell system being used.","Exemplary negatively charged linkers can be prepared comprising any combination of the negatively charged amino acid residues.","For example, in one embodiment, a negatively charged linker comprises either a poly-glutamate or poly-aspartate sequence, or any combination of both amino acid residues.","The negatively charged linker is typically at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length.","The negatively charged linkers are preferably non-proteolytic (e.g., non-proteolytic linkers), having no site for efficient proteolysis.","When linker size rather than charge is used to minimize interaction of the recombinant polypeptide of interest with the oil body surface, then the linker is non-proteolytic and ranges in molecular weight from about 35 kd up to about 100 kd.","The upper size limit is chosen such that the expression of, the activity of, the conformation of, and/or the access to target of, the recombinant polypeptide of interest is not significantly affected by the linker.","In certain embodiments, described herein where a non-proteolytic linker amino acid sequence is employed, the gene-fusion or protein fusion (multimeric-fusion-protein) can optionally further comprise a linker nucleic or amino acid sequence encoding a sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the non-proteolytic linker sequence and sequence encoding the desired recombinant protein region, e.g., the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins set forth herein.","When a cleavable linker sequence is used herein, in a particular embodiment, it is further downstream than the non-proteolytic linker sequence from the oil-body-targeting-protein region of the fusion protein.","By virtue of cleavable linker, the recombinant fusion polypeptides provided herein, such as the multimeric-fusion-proteins and redox fusion polypeptides, can be isolated and purified by introducing an enzyme or chemical that cleaves said multimeric-fusion-protein and/or redox fusion polypeptide from said oil body, thereby obtaining and/or isolating the desired protein.","It is contemplated herein that the use of cleavable linker sequence downstream of the non-proteolytic linker/spacer sequence will improve the yield of protein recovery when isolating or purifying proteins using the methods provided herein.","The nucleic acid sequences encoding the first or second recombinant polypeptide may be altered to improve expression levels for example, by optimizing the nucleic acids sequence in accordance with the preferred codon usage for the particular cell type which is selected for expression of the first and second recombinant polypeptide, or by altering of motifs known to destabilize mRNAs (see for example: PCT Patent Application 97/02352).","Comparison of the codon usage of the first and second recombinant polypeptide with codon usage of the host will enable the identification of codons that may be changed.","For example, typically plant evolution has tended towards a preference for CG rich nucleotide sequences while bacterial evolution has resulted in bias towards AT rich nucleotide sequences.","By modifying the nucleic acid sequences to incorporate nucleic acid sequences preferred by the host cell, expression may be optimized.","Construction of synthetic genes by altering codon usage is described in for example PCT patent Application 93/07278.The first and second recombinant polypeptide can be altered using for example targeted mutagenesis, random mutagenesis (Shiraishi et al.","(1998) Arch.","Biochem.","Biophys.","358: 104-115; Galkin et al.","(1997) Protein Eng.","10: 687-690; Carugo et al.","(1997) Proteins 28: 10-28; Hurley et al.","(1996) Biochemistry 35: 5670-5678), gene shuffling, and/or by the addition of organic solvent (Holmberg et al.","(1999) Protein Eng.","12: 851-856).","Any polypeptide spacers that are used in accordance with the methods and products provided herein may be altered in similar ways.","In particular embodiments provided herein, the recombinant polypeptides or thioredoxin-related proteins capable of forming a multimeric-protein-complex are capable of forming a heteromultimeric-protein-complex.","Examples of heteromultimeric-protein-complexes that contain polypeptide chains that repeatedly interact, either to activate, inactivate, oxidize, reduce, stabilize, etc., with one another, that can be produced in association with oil bodies using the methods provided herein include those set forth in FIG.","5.Accordingly, exemplary proteins for use in the heteromultimeric-protein-complexes and nucleic acid constructs encoding such, provided herein include, among others described herein, those set forth in FIG.","5.Other polypeptide regions that can be used in the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, provided herein include, among other, those immunoglobulin regions set forth in Table 1.TABLE 1 ANTIBODY HETERODIMERS Class or molecule Subunits Fab Variable region and first constant region of heavy chain and complete light chain Fv Variable regions of heavy and light antibody chains IgA heavy chains, light chains and J (joining) chain IgG, IgD, IgE heavy and light chains IgM heavy chains, light chains and J (joining) chain Antibody chain(s) and a toxin Antibody chain(s) and a toxin Autoantigens, allergens and Autoantigens, allergens and transplant transplant antigens with an antigens with an adjuvant or tolerogen adjuvant or tolerogen Chimeras using antibody Fc Receptor subunits fused to the constant domain region of antibody heavy chains As set forth above, in one embodiment, exemplary heteromultimeric-protein-complexes and exemplary heteromultimeric-fusion-proteins provided herein comprise redox proteins, such as the thioredoxins and thioredoxin-reductases and immunoglobulins.","Oil-Body-Targeting-Proteins The nucleic acid sequence encoding the oil-body-targeting-protein that may be used in the methods and compositions provided herein may be any nucleic acid sequence encoding an oil-body-targeting-protein, protein fragment or peptide capable of association with first recombinant polypeptide, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or a first and/or second thioredoxin-related protein and the oil bodies.","The nucleic acid sequence encoding the oil body targeting peptide may be synthesized or obtained from any biological source.","For example, in one embodiment the oil-body-targeting-protein is an immunoglobulin or an immunoglobulin derived molecule, for example, a bispecific single chain antibody.","The generation of single chain antibodies and bi-specific single chain antibodies is known to the art (see, e.g., U.S. Pat.","No.","5,763,733, U.S. Pat.","No.","5,767,260 and U.S. Pat.","No.","5,260,203).","Nucleic acid sequences encoding single chain antibodies functioning as oil-body-targeting-proteins may be prepared from hybridoma cell lines expressing monoclonal antibodies raised against an oleosin as described by Alting-Mees et al (2000) IBC's Annual International Conference on Antibody Engineering, Poster #1.In order to attain specificity for the first recombinant polypeptide a nucleic acid sequence encoding a second single chain antibody prepared from a monoclonal raised against the first recombinant polypeptide may be prepared and linked to the anti-oleosin single chain antibody.","In this embodiment the oil body associates with the first recombinant polypeptide through non-covalent interactions of the oil-body-targeting-protein with the first recombinant polypeptide and the oil body.","Alternatively the first recombinant polypeptide may be prepared as a fusion protein with an oil-body-targeting-protein.","For example, a nucleic acid sequence encoding a single chain antibody raised against an oleosin may be fused to a nucleic acid sequence encoding the first recombinant polypeptide Non-immunoglobulin-based oil-body-targeting-proteins capable of association with the first recombinant polypeptide may be discovered and prepared using for example phage display techniques (Pharmacia Biotech Catalogue Number 27-9401-011 Recombinant Phage Antibody System Expression Kit).","Oil-body-targeting-proteins may also be chemically modified.","For example, oleosins may be modified by changing chemical modification of the lysine residues using chemical agents such as biotinyl-N-hydroxysuccinimide ester resulting in a process referred to as biotinylation.","Conveniently this is accomplished by in vitro biotinylation of the oil bodies.","In vivo biotinylation may be accomplished using the biotinylation domain peptide from the biotin carboxy carrier protein of E. coli acetyl-CoA carboxylase (Smith et al.","(1998) Nucl.","Acids.","Res.","26: 1414-1420).","Avidin or streptavidin may subsequently be used to accomplish association of the redox protein with the oil body.","In a particular embodiment the oil-body-targeting-protein is an oil-body-protein such as for example an oleosin or a caleosin or a sufficient portion derived thereof capable of targeting to an oil body.","Nucleic acid sequences encoding oleosins are known to the art.","These include for example the Arabidopsis oleosin (van Rooijen et al (1991) Plant Mol.","Bio.","18:1177-1179); the maize oleosin (Qu and Huang (1990) J. Biol.","Chem.","Vol.","265 4:2238-2243); rapeseed oleosin (Lee and Huang (1991) Plant Physiol.","96:1395-1397); and the carrot oleosin (Hatzopoulos et al (1990) Plant Cell Vol.","2, 457-467.).","Caleosin nucleic acid sequences are also known to the art (Naested et al (2000) Plant Mol.","Biol.","44(4):463-476; Chen et al (1999) Plant Cell Physiol.","40(10):1079-1086).","Animal cell derived oil body proteins that may be used herein include adopihilin (Brasaemle et al, (1997) J. Lipid Res., 38: 2249-2263; Heid et al.","(1998) Cell Tissue Research 294: 309-321), perilipin (Blanchette-Mackie et al.","(1995), J. Lipid Res.","36: 1211-1226; Servetnick et al.","(1995) J. Biol.","Chem.","270: 16970-16973), apolipoproteins such as apo A-I, A-II, A-IV, C-I, C-II, CIII (Segrest et al.","(1990), Proteins 8:103-117) and apoB (Chatterton et al.","(1995) J. Lipid Res.","36: 2027-2037; Davis, R A in: Vance D E, Vance J. editors.","Lipoprotein structure and secretion.","The Netherlands, Elsevier, 191: 403-426.In one embodiment, the first recombinant polypeptide is fused to an oil-body-protein.","The methodology is further described in U.S. Pat.","No.","5,650,554, which is incorporated herein by reference in its entirety.","The first recombinant polypeptide may be fused to the N-terminus as well as to the C-terminus of the oil-body-protein (as described in: Moloney and van Rooijen (1996) INFORM 7:107-113) and fragments of the oil-body-protein such as for example the central domain of an oleosin molecule, or modified versions of the oil-body-protein may be used.","In this embodiment, the second recombinant polypeptide is expressed intracellularly and then intracellularly associates with the first recombinant polypeptide to form the multimeric-protein-complex in the cell.","Oil bodies comprising the multimeric-protein-complex are then conveniently isolated from the cells.","In a further embodiment both the first and second recombinant polypeptide are separately fused to an oil-body-protein.","In this embodiment nucleic acid sequences encoding the first and second polypeptides may be prepared separately and introduced in separate cell lines or they may be introduced in the same cell lines.","Where the nucleic acid sequences are introduced in the same cell line, these nucleic acid sequence may be prepared using two separate expression vectors, or they may be prepared using a single vector comprising nucleic acid sequences encoding both the first polypeptide fused to an oil body protein and the second polypeptide fused to an oil-body-protein.","Where separate cell lines are used subsequent mating of the offspring (e.g.","mating of plants) is used to prepare a generation of cells comprising oil bodies which comprise both the first and second recombinant polypeptide fused to an oil-body-protein.","In further alternate embodiment, the first and second recombinant polypeptide are fused to form a multimeric-fusion-protein comprising the multimeric-protein-complex.","In such an embodiment, the first and second polypeptide is associated with the oil body through an oil-body-targeting-protein capable of associating with both the fusion protein and with the oil body.","In a particular embodiment, the fusion protein comprising the multimeric-protein-complex is fused to an oil-body-protein, for example, an oleosin or caleosin.","In embodiments provided herein in which the multimeric-protein-complex is an immunoglobulin (e.g., a multimeric-immunoglobulin-complex), a particularly preferred oil body targeting protein is an oleosin or caleosin associated with an immunoglobulin binding protein, such as for example protein A (U.S. Pat.","No.","5,151,350), protein L (U.S. Pat.","No.","5,965,390) and protein G (U.S. Pat.","No.","4,954,618), or active fragments of such immunoglobulin binding proteins.","New oil-body-proteins may be discovered for example by preparing oil bodies (described in further detail below) and identifying proteins in these preparations using for example SDS gel electrophoresis.","Polyclonal antibodies may be raised against these proteins and used to screen cDNA libraries in order to identify nucleic acid sequences encoding oil-body-proteins.","The methodologies are familiar to the skilled artisan (Huynh et al.","(1985) in DNA Cloning Vol.","1.a Practical Approach ed.","D M Glover, IRL Press, pp 49-78).","New oil-body-proteins may further be discovered using known nucleic acid sequences encoding oil-body-proteins (e.g.","the Arabidopsis, rapeseed, carrot and corn nucleic acid sequences) to probe for example cDNA and genomic libraries for the presence of nucleic acid sequences encoding oil-body-proteins.","In one embodiment, the first and second polypeptide are a first and second redox protein.","Accordingly, one embodiment provided herein relates to novel and improved methods for the production of redox proteins.","It has unexpectedly been found that a redox protein when prepared as a fusion protein with a second redox protein is fully enzymatically active when produced in association with an oil body.","In contrast, when the redox protein is prepared without the second redox protein it has reduced enzymatic activity.","In one embodiment, the first redox protein is at least 5 times more active when produced as a redox fusion polypeptide relative to production as a non-fusion polypeptide.","Accordingly, provided herein are methods for producing an oil body associated with a heteromultimeric redox protein complex, said method comprising: (a) producing in a cell comprising oil bodies, a first redox protein and a second redox protein wherein said first redox protein is capable of interacting with said second redox protein, preferably in the cell, to form said heteromultimeric redox protein complex; and (b) associating said heteromultimeric redox protein complex with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said heteromultimeric redox protein complex.","In a particular embodiment the first and second redox protein are prepared as a fusion protein to form a redox fusion polypeptide.","Accordingly, provided herein are methods for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.","The oil bodies in association with the redox protein may be used to prepare a variety of useful emulsions.","As used herein the phrase “redox proteins” or grammatical variations thereof, refers to any protein or active protein fragment capable of participating in electron transport.","For example, redox proteins are capable of catalyzing the transfer of an electron from an electron donor (also frequently referred to as the reducing agent) to an electron acceptor (also frequently referred to as the oxidizing agent).","In the process of electron transfer, the reducing agent (electron donor) is oxidized and the oxidizing agent (electron acceptor) is reduced.","Exemplary redox proteins for use herein include iron-sulfur proteins, cytochromes, redox active thiol proteins and redox-active flavoproteins.","To carry out their function as conduits for electrons, redox proteins, such as thioredoxin and thioredoxin-reductase for example, are known to function by interacting or associating with one another in multimeric-protein-complexes (e.g., heteromultimeric-protein-complexes).","The term “redox fusion polypeptide” as used herein refers to any fusion polypeptide comprising a first redox protein linked to a second redox protein (e.g., an in-frame translational fusion).","The redox proteins that may be used with the methods and compositions provided herein may be any redox protein.","In one embodiment the first and second redox proteins are a pair of redox proteins that would normally occur together from the same source, in nature.","In a particular embodiment, the first redox protein is a thioredoxin and the second redox protein is a thioredoxin-reductase.","The redox fusion polypeptide may be produced in any cell comprising oil bodies, including any animal cell, plant cell, algae cell, fungal cell or bacterial cell.","In certain embodiments the redox fusion polypeptide is produced in a plant cell and in particular embodiments the redox fusion polypeptide is produced in the seed cells of a seed plant.","In particular embodiments the oil-body-targeting-protein that is used is an oil-body-protein.","In embodiments of the present invention in which an oil-body-protein is used, the first and second redox protein are preferably covalently fused to the oil-body-protein.","Accordingly, provided herein are methods for the preparation of a redox protein In association with an oil body comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a first nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked in reading frame to (ii) a second nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide in a progeny cell comprising oil bodies; and c) isolating said oil bodies comprising said redox fusion polypeptide from said progeny cell.","Redox Proteins In accordance with various methods and compositions provided herein, any nucleic acid sequence encoding a redox protein may be used.","The nucleic acid sequence encoding the first and/or second redox protein may be obtained from any biological source or may be prepared synthetically.","In general, nucleic acid sequences encoding redox proteins are well known in the art and readily available.","See, for example: Cristiano et al.","(1993) Genomics 17: (2) 348-354, Doyama et al.","(1998) Plant Sci.","137: 53-62, Hoeoeg et al.","(1984) Biosci.","Rep. 4: 917-923; as well as the Swiss Protein sequences set forth in Table 5.Known nucleic acid sequences encoding redox proteins may be used to design and construct nucleic acid sequence based probes in order to uncover and identify previously undiscovered nucleic acid sequences Encoding redox proteins, for example by screening cDNA or genomic libraries.","Thus, additional nucleic acid sequences may be discovered and used in accordance with the present invention.","The nucleic acid sequence encoding the first and/or second redox protein may be obtained from separate sources or may be obtained from the same source.","In general however, the nucleic acid sequence encoding a redox-fusion polypeptide comprises nucleic acid sequences encoding a first and a second redox protein obtained from the same or a similar biological source.","In certain embodiments provided herein, wherein the nucleic acid sequence encoding the first and second redox protein is obtained from the same source, the nucleic acid sequence encoding the first redox protein and second redox protein may be naturally fused.","In accordance with a particular embodiment, the nucleic acid sequences encoding the first and second redox protein are preferably obtained from a plant source.","As set forth above, a polypeptide spacer or linker of variable length may separate the first and second redox proteins from each other and/or from the oil-body-targeting-protein; and additional redox proteins (e.g., one or more) may be fused to the first and/or second redox protein.","The nucleic acid sequences encoding the redox proteins may be altered to improve expression levels for example by optimizing the nucleic acids sequence in accordance with the preferred codon usage for the particular cell type which is selected for expression of the redox proteins, or by altering of motifs known to destabilize mRNAs (see for example: PCT Patent Application 97/02352).","Comparison of the codon usage of the redox protein with codon usage of the host will enable the identification of codons that may be changed.","For example, typically plant evolution has tended towards a preference for CG rich nucleotide sequences while bacterial evolution has resulted in bias towards AT rich nucleotide sequences.","By modifying the nucleic acid sequences to incorporate nucleic acid sequences preferred by the host cell, expression may be optimized.","Construction of synthetic genes by altering codon usage is described in for example PCT patent Application 93/07278.The redox proteins may be altered using for example, targeted mutagenesis, random mutagenesis (Shiraishi et al.","(1998) Arch.","Biochem.","Biophys.","358: 104-115; Galkin et al.","(1997) Protein Eng.","10: 687-690; Carugo et al.","(1997) Proteins 28: 10-28; Hurley et al.","(1996) Biochemistry 35: 5670-5678) (and/or by the addition of organic solvent (Holmberg et al.","(1999) Protein Eng.","12: 851-856).","The polypeptide spacer between the first and second redox protein may be altered in similar ways.","The first and second redox protein may be selected by developing a two-dimensional matrix and determining which combination of first and second redox protein is most effective in electron transport using for example, a colorometric reduction assay (Johnson et al (1984) J. of Bact.","Vol.","158 3:1061-1069, Luthman et al (1982) Biochemistry Vol 21 26:6628-2233).","Combinations of thioredoxin and thioredoxin-reductase may be tested by determining the reduction of wheat storage proteins and milk storage protein beta-lactoglobulin in vitro (Del Val et al.","(1999) J. Allerg.","Clin.","Immunol.","103: 690-697).","Using the same strategy polypeptide spacers between the first and second redox proteins may be evaluated for their efficiency.","First and second redox proteins that may be used herein include without limitation any first redox protein and second redox protein selected from the group of redox proteins consisting of cytochromes, such as cytochrome a, cytochrome b and cytochrome c; porphyrin containing proteins, for example hemoglobin; iron-sulfur proteins, such as ferredoxin; flavoproteins such as thioredoxin-reductase, NADH dehydrogenase, succinate dehydrogenase, dihydrolipoyl dehydrogenase, acyl-CoA dehydrogenase, D-amino acid oxidase, xanthine oxidase, orotate reductase and aldehyde oxidase; pyridine-linked dehydrogenases, for example, lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, and beta-hydroxy-butarate dehydrogenase; and redox active thiol containing proteins such as thioredoxin.","In particular embodiments, the redox proteins provided herein are thioredoxin and its reductant thioredoxin-reductase (which are jointly also referred to herein as “thioredoxin-related” protein(s)).","As used herein, the term “thioredoxin” refers to relatively small proteins (typically approximately 12 kDa) that belong to the family of thioltransferases which catalyze oxido-reductions via the formation or hydrolysis of disulfide bonds and are widely, if not universally, distributed throughout the animal plant and bacterial kingdom.","The reduces form of thioredoxin is an excellent catalyst for the reduction of even the most intractable disulfide bonds.","In order to reduce the oxidized thioredoxin, two cellular reductants provide the reduction equivalents: reduced ferredoxin and NADPH.","These reduction equivalents are supplied to thioredoxin via interaction or association with different thioredoxin-reductases including the NADPH thioredoxin-reductase and ferredoxin thioredoxin-reductase.","The supply of these reduction equivalents requires the formation of a heteromultimeric-protein-complex comprising thioredoxin and thioredoxin-reductase.","Ferredoxin thioredoxin-reductase is involved in the reduction of plant thioredoxins designated as Trxf and Trxm, both of which are involved in the regulation of photosynthetic processes in the chloroplast.","The NADPH/thioredoxin active in plant seeds is designated Trxh (also referred to herein as thioredoxin h-type) and is capable of the reduction of a wide range of proteins thereby functioning as an important cellular redox buffer.","Generally, only one kind of thioredoxin, which analogous to the plant Trxh type, is found in bacterial or animal cells.","The h-type thioredoxins are capable of being reduced by NADPH and NADPH-thioredoxin reductase.","Exemplary thioredoxins are further characterized as a protein having a core of 5 beta-sheets surrounded by 4 to 6 alpha helixes.","Exemplary thioredoxins are further characterized by having an active site containing the consensus amino acid sequence: XCYYCZ, wherein Y is any amino acid, such as hydrophobic or non-polar amino acids, wherein X can be any of the 20 amino acids, preferably a hydrophobic amino acid, such as a tryptophan, and Z can be any amino acid, preferably polar amino acids.","In certain embodiments, the thioredoxins for use herein comprise an active site having the amino acid sequence XCGPCZ.","When the cysteines in the active site of thioredoxin or thioredoxin-like proteins are oxidized, they form an intramolecular disulfide bond.","In the reduced state, the same active sites are capable of participating in redox reactions through the reversible oxidation of its active site dithiol, to a disulfide and catalyzes dithioldisulfide exchange reactions.","Exemplary thioredoxins are well-known in the art and can be obtained from several organisms including Arabidopsis thaliana (Riveira Madrid et al.","(1995) Proc.","Natl.","Acad.","Sci.","92: 5620-5624), wheat (Gautier et al.","(1998) Eur.","J. Biochem.","252: 314-324); Escherichia coli (Hoeoeg et al (1984) Biosci.","Rep. 4: 917-923) and thermophylic microorganisms such as Methanococcus jannaschii and Archaeoglobus fulgidus (PCT Patent Application 00/36126).","Thioredoxins have also been recombinantly expressed in several host systems including bacteria (Gautier et al.","(1998) Eur J. Biochem.","252: 314-324) and plants (PCT Patent Application WO 00/58453) Commercial preparations of E. coli sourced Thioredoxins are readily available from for example: Sigma Cat No.","T 0910 Thioredoxin (E. coli, recombinant; expressed in E. coli).","Exemplary nucleic acid sequences encoding thioredoxin polypeptides for use herein are readily available from a variety of diverse biological sources including E. coli (Hoeoeg et al.","(1984) Biosci.","Rep.: 4 917-923); Methanococcus jannaschii and Archaeoglobus fulgidus (PCT Patent Application 00/36126); Arabidopsis thaliana (Rivera-Madrid (1995) Proc.","Natl.","Acad.","Sci.","92: 5620-5624); wheat (Gautier et al (1998) Eur.","J. Biochem.","252(2): 314-324); tobacco (Marty et al.","(1991) Plant Mol.","Biol.","17: 143-148); barley (PCT Patent Application 00/58352); rice (Ishiwatari et al.","(1995) Planta 195: 456-463); soybean (Shi et al.","(1996) Plant Mol.","Biol.","32: 653-662); rapeseed (Bower et al.","Plant Cell 8: 1641-1650) and calf (Terashima et al.","(1999) DNA Seq.","10(3): 203-205); and the like.","In yet other embodiments, exemplary nucleic acids for use herein include those encoding the thioredoxin and thioredoxin-like polypeptide chains set forth as SEQ ID NOs:38, 42, 46 and 50; and those encoding the thioredoxin and thioredoxin-like polypeptide chains set forth in Table 5 as SEQ ID NOs:52-194.The respective nucleic acid sequences encoding the amino acids set forth in SEQ ID NOs:52-194 can be readily identified via the Swiss Protein identifier (accession) numbers provided in Table 5 (in parenthesis).","As used herein, the term “thioredoxin-reductase” refers to a protein that complexes with a flavin, such as FAD.","The flavin compound serves as an electron donor for the thioredoxin-reductase protein active site.","Thioredoxin reductases have a redox active, disulfide bond site capable of reducing thioredoxin.","The active site of thioredoxin-reductase contains 2 cysteines.","The type of amino acids surrounding the 2 cysteine residues forming the active site can vary as hydrophobic, non-polar or polar.","An exemplary thioredoxin-reductase is NADPH-thioredoxin-reductase (NTR), which is a cytosolic homodimeric enzyme comprising typically 300-500 amino acids.","Crystal structures of both E. coli and plant thioredoxin-reductase have been obtained (Waksman et al.","(1994) J. Mol.","Biol.","236: 800-816; Dai et al.","(1996) J. Mol.","Biol.","264:1044-1057).","NADPH-thioredoxin-reductases have been expressed in heterologous hosts, for example the Arabidopsis NADPH-thioredoxin-reductase has been expressed in E. coli (Jacquot et al.","(1994) J. Mol.","Biol.","235: 1357-1363) and wheat (PCT Patent Application 00/58453).","Exemplary nucleic acid sequences encoding thioredoxin-reductase proteins can readily be obtained from a variety of sources, such as from the sequence set forth in Table 5 and the Sequence Listing provide herein, from Arabidopsis (Riveira Madrid et al.","(1995) Proc.","Natl.","Acad.","Sci.","USA 92: 5620-5624), E. coli (Russel et al.","(1988) J. Biol.","Chem.","263: 9015-9019); barley (PCT Patent Application 00158352 and wheat (Gautier et al., (1998) Eur.","J. Biochem.","252: 314-324); and the like.","In yet other embodiments, exemplary nucleic acids for use herein include those encoding the thioredoxin-reductase polypeptide chains set forth as SEQ ID NOs:8, 9, 10, 40, 44, 48 and 50; and those encoding the thioredoxin-reductase polypeptide chains set forth in Table 5 as SEQ ID NOs:195-313.The respective nucleic acid sequences encoding the amino acids set forth in SEQ ID NOs:195-313 can be readily identified via the Swiss Protein identifier (accession) numbers provided in Table 5 (in parenthesis).","Also contemplated for use in the methods and compositions provided herein are nucleic acid and amino acid homologs that are “substantially homologous” to the thioredoxin and thioredoxin-reductase nucleic and amino acids set forth herein, which includes thioredoxin and thioredoxin-reductase polypeptides encoded by a sequence of nucleotides that hybridizes under conditions of low, moderate or high stringency to the sequence of nucleotides encoding the thioredoxin and thioredoxin-reductase nucleic and amino acids set forth herein (e.g., in the Examples, Sequence Listing and/or Table 5).","As used herein, a DNA or nucleic acid homolog refers to a nucleic acid that includes a preselected conserved nucleotide sequence, such as a sequence encoding a therapeutic polypeptide.","By the term “substantially homologous” is meant having at least 80%, preferably at least 90%, most preferably at least 95% homology therewith or a less percentage of homology or identity and conserved biological activity or function.","The terms “homology” and “identity” are often used interchangeably.","In this regard, percent homology or identity may be determined, for example, by comparing sequence information using a GAP computer program.","The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol.","Biol.","48:443 (1970), as revised by Smith and Waterman (Adv.","Appl.","Math.","2:482 (1981).","Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.","The preferred default parameters for the GAP program may include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl.","Acids Res.","14:6745 (1986), as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp.","353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.","By sequence identity, the number of conserved amino acids are determined by standard alignment algorithms programs, and are used with default gap penalties established by each supplier.","Substantially homologous nucleic acid molecules would hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid of interest.","Preferably the two molecules will hybridize under conditions of high stringency.","Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.","Whether any two nucleic acid molecules have nucleotide sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can be determined using known computer algorithms such as the “FAST A” program, using for example, the default parameters as in Pearson and Lipman, Proc.","Natl.","Acad.","Sci.","USA 85:2444 (1988).","Alternatively the BLAST function of the National Center for Biotechnology Information database may be used to determine relative sequence identity.","In general, sequences are aligned so that the highest order match is obtained.","“Identity” per se has an art-recognized meaning and can be calculated using published techniques.","(See, e.g.",": Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).","While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988)).","Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988).","Methods to determine identity and similarity are codified in computer programs.","Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)).","Therefore, as used herein, the term “identity” represents a comparison between a test and a reference polypeptide or polynucleotide.","For example, a test polypeptide may be defined as any polypeptide that is 90% or more identical to a reference polypeptide.","As used herein, the term at least “90% identical to” refers to percent identities from 90 to 99.99 relative to the reference polypeptides.","Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids are compared.","No more than 10% (i.e., 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptides.","Similar comparisons may be made between a test and reference polynucleotides.","Such differences may be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they may be clustered in one or more locations of varying length up to the maximum allowable, e.g.","10/100 amino acid difference (approximately 90% identity).","Differences are defined as nucleic acid or amino acid substitutions, or deletions.","As used herein: stringency of hybridization in determining percentage mismatch is as follows: 1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C. 2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C. 3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C. Those of skill in this art know that the washing step selects for stable hybrids and also know the ingredients of SSPE (see, e.g., Sambrook, E. F. Fritsch, T. Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), vol.","3, p. B.","13, see, also, numerous catalogs that describe commonly used laboratory solutions).","SSPE is pH 7.4 phosphate-buffered 0.18 NaCl.","Further, those of skill in the art recognize that the stability of hybrids is determined by Tm, which is a function of the sodium ion concentration and temperature (Tm=81.5° C.-16.6(log10[Na+])+0.41 (% G+C)−600/l)), so that the only parameters in the wash conditions critical to hybrid stability are sodium ion concentration in the SSPE (or SSC) and temperature.","It is understood that equivalent stringencies may be achieved using alternative buffers, salts and temperatures.","By way of example and not limitation, procedures using conditions of low stringency are as follows (see also Shilo and Weinberg, Proc.","Natl.","Acad.","Sci.","USA, 78:6789-6792 (1981)): Filters containing DNA are pretreated for 6 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA (10×SSC is 1.5 M sodium chloride, and 0.15 M sodium citrate, adjusted to a pH of 7).","In a particular embodiment, a heteromultimeric-protein-complex is produced as a fusion polypeptide between the first and second redox protein, wherein the first redox protein is thioredoxin and the second redox protein is a thioredoxin-reductase.","In one embodiment, the second recombinant polypeptide, e.g., the thioredoxin-reductase is positioned N-terminal relative to the first recombinant polypeptide, e.g., the thioredoxin.","Accordingly, any protein which is classified as thioredoxin, such as the thioredoxin component of the NADPH thioredoxin system and the thioredoxin present in the ferredoxin/thioredoxin system also known as TRx and TRm may be used in combination with any thioredoxin-reductase such as the NADPH thioredoxin-reductase and the ferredoxin-thioredoxin-reductase and any other proteins having the capability of reducing thioredoxin.","In particular embodiments the thioredoxin and thioredoxin-reductase are plant derived.","In an alternate embodiment, the naturally occurring nucleic acid sequence encoding the thioredoxin/thioredoxin-reductase protein fusion obtainable from Mycobacterium leprae (Wieles et al.","(1995) J. Biol.","Chem.","27:25604-25606) is used, as set forth in the Examples herein.","Immunoglobulins In another embodiment of the present invention, the multimeric-protein-complexes are immunoglobulins.","As used herein “immunoglobulin-polypeptide-chain” refers to a first polypeptide that is capable of associating with a second polypeptide to form an immunologically active (i.e.","capable of antigen binding multimeric-protein-complex.","The types of immunoglobulins and immunoglobulin-polypeptide-chains contemplated for use herein include the immunologically active (i.e.","antigen binding) portions of a light and heavy chain.","Exemplary immunoglobulins and immunoglobulin-polypeptide-chains for use herein include substantially intact immunoglobulins, including any IgG, IgA, IgD, IgE and IgM, as well as any portion of an immunoglobulin, including those portions well-known as Fab fragments, Fab′ fragments, F(ab′).sub2.fragments and Fv fragments.","In this embodiment, the first recombinant polypeptide may be any immunoglobulin heavy chain, including any IgG, IgA, IgD, IgE or IgM heavy chain, and the second recombinant polypeptide may be a kappa or lambda immunoglobulin light chain.","Accordingly, provided herein are methods of producing an immunoglobulin, said method comprising: (a) producing in a cell comprising oil bodies, a first immunoglobulin-polypeptide-chain and a second immunoglobulin-polypeptide-chain wherein said first immunoglobulin-polypeptide-chain is capable of associating with said second immunoglobulin-polypeptide-chain to form said immunoglobulin; and (b) associating said immunoglobulin with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first immunoglobulin-polypeptide-chain.","As set forth herein, the multimeric immunoglobulin is associated with an oil body through an oil-body-targeting-protein.","In particular embodiments, the oil-body-targeting-protein may be a fusion polypeptide comprising an oil-body-protein and an immunoglobulin binding protein, such as for example protein A, protein L, and protein G. In yet another embodiment involving immunoglobulins, the first and second recombinant polypeptides (immunoglobulins) are separately fused to an oil body protein, for example an oleosin or caleosin.","For example, a) the first recombinant polypeptide may be an immunoglobulin heavy chain, including any IgG, IgA, IgD, IgE or IgM heavy chain, and the second recombinant polypeptide may be a kappa or lambda immunoglobulin light chain; or b) the first recombinant polypeptide may be the variable and first constant domain from an immunoglobulin heavy chain and the second recombinant polypeptide may be a kappa or lambda immunoglobulin light chain; or c) the first recombinant polypeptide may be the variable domain from an immunoglobulin heavy chain and the second recombinant polypeptide may be the variable domain from a kappa or lambda immunoglobulin light chain.","In certain embodiments, the fusion polypeptides are designed or selected to allow the heteromultimeric-protein-complex formation between immunoglobulin light and heavy chain sequences on the oil bodies within the cell comprising oil bodies.","Preparation of Expression Vectors Comprising Oil-Body-Targeting-Proteins and the First and/or Second Recombinant Polypeptides, Multimeric-Protein-Complexes, Heteromultimeric-Protein-Complexes, Multimeric-Fusion-Proteins, Heteromultimeric-Fusion-Proteins, Immunoglobulins, Immunoglobulin-Polypeptide-Chains, Redox-Fusion-Polypeptides, or the First and/or Second Thioredoxin-Related Proteins.","In accordance with the present invention, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and the oil-body-targeting-protein are conveniently produced in a cell.","In order to produce the recombinant polypeptides or multimeric-protein-complexes, a nucleic acid sequence encoding either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and/or the oil-body-targeting-protein are incorporated in a recombinant expression vector.","Accordingly, provided herein are recombinant expression vectors comprising the chimeric nucleic acids provided herein suitable for expression of the oil-body-targeting-protein and the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, suitable for the selected cell.","The term “suitable for expression in the selected cell” means that the recombinant expression vector contains all nucleic acid sequences required to ensure expression in the selected cell.","Accordingly, the recombinant expression vectors further contain regulatory nucleic acid sequences selected on the basis of the cell which is used for expression and ensuring initiation and termination of transcription operatively linked to the nucleic acid sequence encoding the recombinant polypeptide or multimeric-protein-complex and/or the oil-body-targeting-protein.","Regulatory nucleic acid sequences include promoters, enhancers, silencing elements, ribosome binding sites, Shine-Dalgarno sequences, introns and other expression elements.","“Operatively linked” is intended to mean that the nucleic acid sequences comprising the regulatory regions linked to the nucleic acid sequences encoding the recombinant polypeptide or multimeric-protein-complex and/or the oil-body-targeting-protein allow expression in the cell.","A typical nucleic acid construct comprises in the 5′ to 3′ direction a promoter region capable of directing expression, a coding region comprising the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and/or an oil-body-targeting-protein and a termination region functional in the selected cell.","The selection of regulatory sequences will depend on the organism and the cell type in which the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and/or the oil-body-targeting-protein is expressed, and may influence the expression levels of the polypeptide.","Regulatory sequences are art-recognized and selected to direct expression of the oil-body-targeting-protein and the recombinant polypeptides or multimeric-protein-complexes in the cell.","Promoters that may be used in bacterial cells include the lac promoter (Blackman et al.","(1978) Cell: 13: 65-71), the trp promoter (Masuda et al.","(1996) Protein Eng: 9: 101-106) and the T7 promoters (Studier et al.","(1986) J. Mol.","Biol.","189: 113-130).","Promoters functional in plant cells that may be used herein include constitutive promoters such as the 35S CaMV promoter (Rothstein et al.","(1987) Gene: 53: 153-161) the actin promoter (McElroy et al.","(1990) Plant Cell 2: 163-171) and the ubiquitin promoter (European Patent Application 0 342 926).","Other promoters are specific to certain tissues or organs (for example, roots, leaves, flowers or seeds) or cell types (for example, leaf epidermal cells, mesophyll cells or root cortex cells) and or to certain stages of plant development.","Timing of expression may be controlled by selecting an inducible promoter, for example the PR-a promoter described in U.S. Pat.","No.","5,614,395.Selection of the promoter therefore depends on the desired location and timing of the accumulation of the desired polypeptide.","In a particular embodiment, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and the oil-body-targeting-protein are expressed in a seed cell and seed specific promoters are utilized.","Seed specific promoters that may be used herein include for example the phaseolin promoter (Sengupta-Gopalan et al.","(1985) Proc.","Natl.","Acad.","Sci.","USA: 82: 3320-3324), and the Arabidopsis 18 kDa oleosin promoter (van Rooijen et al.","(1992) Plant.","Mol.","Biol.","18: 1177-1179).","New promoters useful in various plant cell types are constantly discovered.","Numerous examples of plant promoters may be found in Ohamuro et al.","(Biochem of Pl.","(1989) 15: 1-82).","Genetic elements capable of enhancing expression of the polypeptide may be included in the expression vectors.","In plant cells these include for example, the untranslated leader sequences from viruses such as the AMV leader sequence (Jobling and Gehrke (1987) Nature: 325: 622-625) and the intron associated with the maize ubiquitin promoter (See: U.S. Pat.","No.","5,504,200).","Transcriptional terminators are generally art recognized and besides serving as a signal for transcription termination serve as a protective element serving to extend the mRNA half-life (Guarneros et al.","(1982) Proc.","Natl.","Acad.","Sci.","USA: 79: 238-242).","In nucleic acid sequences for the expression in plant cells, the transcriptional terminator typically is from about 200 nucleotide to about 1000 nucleotides in length.","Terminator sequences that may be used herein include for example, the nopaline synthase termination region (Bevan et al.","(1983) Nucl.","Acid.","Res.",": 11: 369-385), the phaseolin terminator (van der Geest et al.","(1994) Plant J.: δ: 413-423), the terminator for the octopine synthase gene of Agrobacterium tumefaciens or other similarly functioning elements.","Transcriptional terminators can be obtained as described by An (1987) Methods in Enzym.","153: 292).","The selection of the transcriptional terminator may have an effect on the rate of transcription.","Accordingly, provided herein are chimeric nucleic acid sequences encoding a first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins.","In one embodiment, said nucleic acid comprises: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide, immunoglobulin-polypeptide-chain, or redox protein; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, immunoglobulin-polypeptide-chain or redox protein, wherein said first and second recombinant polypeptides, immunoglobulin-polypeptide-chains or redox proteins are capable of forming a multimeric-protein-complex.","In another embodiment, provided herein is an expression vector comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked in reading frame to (ii) a nucleic acid sequence encoding a multimeric-fusion-protein, such as a redox fusion polypeptide or immunoglobulin, comprising a first recombinant polypeptide, such as a redox protein or immunoglobulin-polypeptide-chain, linked to a second recombinant polypeptide, such as a second redox protein or a second immunoglobulin-polypeptide-chain, operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.","The recombinant expression vector further may contain a marker gene.","Marker genes that may be used in accordance with the present invention include all genes that allow the distinction of transformed cells from non-transformed cells including all selectable and screenable marker genes.","A marker may be a resistance marker such as an antibiotic resistance marker against for example kanamycin, ampicillin, G418, bleomycin hygromycin, chloramphenicol which allows selection of a trait by chemical means or a tolerance marker against for example a chemical agent such as the normally phytotoxic sugar mannose (Negrotto et al.","(2000) Plant Cell Rep. 19: 798-803).","In plant recombinant expression vectors herbicide resistance markers may conveniently be used for example markers conferring resistance against glyphosate (U.S. Pat.","Nos.","4,940,935 and 5,188,642) or phosphinothricin (White et al.","(1990) Nucl.","Acids Res.","18: 1062; Spencer et al.","(1990) Theor.","Appl.","Genet.","79: 625-631).","Resistance markers to a herbicide when linked in close proximity to the redox protein or oil-body-targeting-protein may be used to maintain selection pressure on a population of plant cells or plants for those plants that have not lost the protein of interest.","Screenable markers that may be employed to identify transformants through visual observation include beta-glucuronidase (GUS) (see U.S. Pat.","No.","5,268,463 and U.S. Pat.","No.","5,599,670) and green fluorescent protein (GFP) (Niedz et al.","(1995) Plant Cell Rep.: 14: 403).","The recombinant expression vectors further may contain nucleic acid sequences encoding targeting signals ensuring targeting to a cell compartment or organelle.","Suitable targeting signals that may be used herein include those that are capable of targeting polypeptides to the endomembrane system.","Exemplary targeting signals that may be used herein include targeting signals capable of directing the protein to the periplasm, the cytoplasm, the golgi apparatus, the apoplast (Sijmons et al., 1990, Bio/Technology, 8:217-221) the chloroplast (Comai et al.","(1988) J. Biol.","Chem.","263: 15104-15109), the mitochondrion, the peroxisome (Unger et al.","(1989) Plant Mol.","Biol.","13: 411-418), the ER, the vacuole (Shinshi et al.","(1990) Plant Mol.","Biol.","14: 357-368 and the oil body.","By the inclusion of the appropriate targeting sequences it is possible to direct the oil-body-targeting-protein or the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins, to the desired organelle or cell compartment.","The recombinant expression vectors of the present invention may be prepared in accordance with methodologies well known to those of skill in the art of molecular biology (see for example: Sambrook et al.","(1990) Molecular Cloning, 2nd ed.","Cold Spring Harbor Press).","The preparation of these constructs may involve techniques such as restriction digestion, ligation, gel electrophoresis, DNA sequencing and PCR.","A wide variety of cloning vectors is available to perform the necessary cloning steps resulting in a recombinant expression vector ensuring expression of the polypeptide.","Especially suitable for this purpose are vectors with a replication system that is functional in Escherichia coli such as pBR322, the PUC series of vectors, the M13 mp series of vectors, pBluescript etc.","Typically these vectors contain a marker allowing the selection of transformed cells for example by conferring antibiotic resistance.","Nucleic acid sequences may be introduced in these vectors and the vectors may be introduced in E. coli grown in an appropriate medium.","Vectors may be recovered from cells upon harvesting and lysing the cells.","Recombinant expression vectors suitable for the introduction of nucleic acid sequences in plant cells include Agrobacterium and Rhizobium based vectors such as the Ti and Ri plasmids.","Agrobacterium based vectors typically carry at least one T-DNA border sequence and include vectors such pBIN 19 (Bevan (1984) Nucl Acids Res.","Vol.","12, 22:8711-8721) and other binary vector systems (for example: U.S. Pat.","No.","4,940,838).","Production of Cells Comprising a First and/or Second Recombinant Polypeptides, Multimeric-Protein-Complexes, Heteromultimeric-Protein-Complexes, Multimeric-Fusion-Proteins, Heteromultimeric-Fusion-Proteins, Immunoglobulins, Immunoglobulin-Polypeptide-Chains, Redox-Fusion-Polypeptides, and/or a First and/or Second Thioredoxin-Related Protein and Oil-Body-Targeting-Proteins In accordance with the present invention, the recombinant expression vectors are introduced into the cell that is selected and the selected cells are grown to produce the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, a first and/or second thioredoxin-related protein; and the oil-body-targeting-protein either directly or in a progeny cell.","Methodologies to introduce recombinant expression vectors into a cell also referred to herein as “transformation” are well known to the art and vary depending on the cell type that is selected.","General techniques to transfer the recombinant expression vectors into the cell include electroporation; chemically mediated techniques, for example CaCl2 mediated nucleic acid uptake; particle bombardment (biolistics); the use of naturally infective nucleic acid sequences for example virally derived nucleic acid sequences or when plant cells are used Agrobacterium or Rhizobium derived nucleic acid sequences; PEG mediated nucleic acid uptake, microinjection, and the use of silicone carbide whiskers (Kaeppler et al.","(1990) Plant Cell Rep. 9:415-418) all of which may be used herein.","Introduction of the recombinant expression vector into the cell may result in integration of its whole or partial uptake into host cell genome including the chromosomal DNA or the plastid genome.","Alternatively the recombinant expression vector may not be integrated into the genome and replicate independently of the host cell's genomic DNA.","Genomic integration of the nucleic acid sequence is typically used as it will allow for stable inheritance of the introduced nucleic acid sequences by subsequent generations of cells and the creation of cell, plant or animal lines.","Particular embodiments involve the use of plant cells.","Particular plant cells used herein include cells obtainable from Brazil nut (Betholletia excelsa); castor (Riccinus communis); coconut (Cocus nucifera); coriander (Coriandrum sativum); cotton (Gossypium spp.","); groundnut (Arachis hypogaea); jojoba (Simmondsia chinensis); linseed/flax (Linum usitatissimum); maize (Zea mays); mustard (Brassica spp.","and Sinapis alba); oil palm (Elaeis guineeis); olive (Olea europaea); rapeseed (Brassica spp.","); safflower (Carthamus tinctorius); soybean (Glycine max); squash (Cucurbita maxima); barley (Hordeum vulgare); wheat (Traeticum aestivum) and sunflower (Helianthus annuus).","Transformation methodologies for dicotelydenous plant species are well known.","Generally Agrobacterium mediated transformation is utilized because of its high efficiency as well as the general susceptibility by many, if not all dicotelydenous plant species.","Agrobacterium transformation generally involves the transfer of a binary vector (e.g.","pBIN19) comprising the DNA of interest to an appropriate Agrobacterium strain (e.g.","CIB542) by for example tri-parental mating with an E. coli strain carrying the recombinant binary vector and an E. coli strain carrying a helper plasmid capable of mobilization of the binary vector to the target Agrobacterium strain, or by DNA transformation of the Agrobacterium strain (Hofgen et al.","Nucl.","Acids.","Res.","(1988) 16: 9877.Other transformation methodologies that may be used to transform dicotelydenous plant species include biolistics (Sanford (1988) Trends in Biotechn.","6: 299-302); electroporation (Fromm et al.","(1985) Proc.","Natl.","Acad.","Sci.","USA 82: 5824-5828); PEG mediated DNA uptake (Potrykus et al.","(1985) Mol.","Gen. Genetics 199: 169-177); microinjection (Reich et al.","Bio/Techn.","(1986) 4: 1001-1004) and silicone carbide whiskers (Kaeppler et al.","(1990) Plant Cell Rep. 9: 415-418).","The exact transformation methodologies typically vary somewhat depending on the plant species that is used.","In a particular embodiment the oil bodies are obtained from safflower and the recombinant proteins are expressed in safflower.","Safflower transformation has been described by Baker and Dyer (Plant Cell Rep. (1996) 16: 106-110).","Monocotelydenous plant species may now also be transformed using a variety of methodologies including particle bombardment (Christou et al.","(1991) Biotechn.","9: 957-962; Weeks et al.","Plant Physiol.","(1993) 102: 1077-1084; Gordon-Kamm et al.","Plant Cell (1990) 2: 603-618) PEG mediated DNA uptake (EP 0 292 435; 0 392 225) or Agrobacterium-mediated transformation (Goto-Fumiyuki et al (1999) Nature-Biotech.","17 (3):282-286).","Plastid transformation is described in U.S. Pat.","Nos.","5,451,513; 5,545,817 and 5,545,818; and PCT Patent Applications 95/16783; 98/11235 and 00/39313) Basic chloroplast transformation involves the introduction of cloned plastid DNA flanking a selectable marker together with the nucleic acid sequence of interest into a suitable target tissue using for example biolistics or protoplast transformation.","Selectable markers that may be used include for example the bacterial aadA gene (Svab et al.","(1993) Proc.","Natl.","Acad.","Sci.","USA 90: 913-917).","Plastid promoters that may be used include for example the tobacco clpP gene promoter (PCT Patent Application 97/06250).","In another embodiment, the invention chimeric nucleic acid constructs provided herein are directly transformed into the plastid genome.","Plastid transformation technology is described extensively in U.S. Pat.","Nos.","5,451,513, 5,545,817, 5,545,818 and 5,576,198; in PCT application nos.","WO 95/16783 and WO 97/32977; and in McBride et.","al., Proc Natl Acad Sci USA 91: 7301-7305 (1994), the entire disclosures of all of which are hereby incorporated by reference.","In one embodiment, plastid transformation is achieved via biolistics, first carried out in the unicellular green alga Chlamydomonas reinhardtii (Boynton et al.","(1988) Science 240:1534-1537)) and then extended to Nicotiana tabacum (Svab et al.","(1990) Proc Natl Acad Sci USA 87:8526-8530), combined with selection for cis-acting antibiotic resistance loci (spectinomycin or streptomycin resistance) or complementation of non-photosynthetic mutant phenotypes.","In another embodiment, tobacco plastid transformation is carried out by particle bombardment of leaf or callus tissue, or polyethylene glycol (PEG)-mediated uptake of plasmid DNA by protoplasts, using cloned plastid DNA flanking a selectable antibiotic resistance marker.","For example, 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and allow the replacement or modification of specific regions of the 156 kb tobacco plastid genome.","In one embodiment, point mutations in the plastid 16S rDNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin can be utilized as selectable markers for transformation (Svab et al.","(1990) Proc Natl Acad Sci USA 87:8526-8530; Staub et al.","(1992) Plant Cell 4:39-45, the entire disclosures of which are hereby incorporated by reference), resulting in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves.","The presence of cloning sites between these markers allows creation of a plastid targeting vector for introduction of foreign genes (Staub et al.","(1993) EMBO J 12:601-606, the entire disclosure of which is hereby incorporated by reference).","In another embodiment, substantial increases in transformation frequency can be obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3′-adenyltransferase (Svab et al.","(1993) Proc Natl Acad Sci USA 90: 913-917, the entire disclosure of which is hereby incorporated by reference).","This marker has also been used successfully for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl Acids Res 19, 4083-4089, the entire disclosure of which is hereby incorporated by reference).","In other embodiments, plastid transformation of protoplasts from tobacco and the moss Physcomitrella can be attained using PEG-mediated DNA uptake (O'Neill et al.","(1993) Plant J 3:729-738; Koop et al.","(1996) Planta 199:193-201, the entire disclosures of which are hereby incorporated by reference).","Both particle bombardment and protoplast transformation are also contemplated for use herein.","Plastid transformation of oilseed plants has been successfully carried out in the genera Arabidopsis and Brassica (Sikdar et al.","(1998) Plant Cell Rep 18:20-24; PCT Application WO 00/39313, the entire disclosures of which are hereby incorporated by reference).","A chimeric nucleic sequence construct is inserted into a plastid expression cassette including a promoter capable of expressing the construct in plant plastids.","A particular promoter capable of expression in a plant plastid is, for example, a promoter isolated from the 5′ flanking region upstream of the coding region of a plastid gene, which may come from the same or a different species, and the native product of which is typically found in a majority of plastid types including those present in non-green tissues.","Gene expression in plastids differs from nuclear gene expression and is related to gene expression in prokaryotes (Stern et al.","(1997) Trends in Plant Sci 2:308-315, the entire disclosure of which is hereby incorporated by reference).","Plastid promoters generally contain the −35 and −10 elements typical of prokaryotic promoters, and some plastid promoters called PEP (plastid-encoded RNA polymerase) promoters are recognized by an E. coli-like RNA polymerase mostly encoded in the plastid genome, while other plastid promoters called NEP promoters are recognized by a nuclear-encoded RNA polymerase.","Both types of plastid promoters are suitable for use herein.","Examples of plastid promoters include promoters of clpP genes such as the tobacco clpP gene promoter (WO 97/06250, the entire disclosure of which is hereby incorporated by reference) and the Arabidopsis clpP gene promoter (U.S. application Ser.","No.","09/038,878, the entire disclosure of which is hereby incorporated by reference).","Another promoter capable of driving expression of a chimeric nucleic acid construct in plant plastids comes from the regulatory region of the plastid 16S ribosomal RNA operon (Harris et al., (1994) Microbiol Rev 58:700-754; Shinozaki et al.","(1986) EMBO J 5:2043-2049, the entire disclosures of both of which are hereby incorporated by reference).","Other examples of promoters capable of driving expression of a nucleic acid construct in plant plastids include a psbA promoter or am rbcL promoter.","A plastid expression cassette preferably further includes a plastid gene 3′ untranslated sequence (3′ UTR) operatively linked to a chimeric nucleic acid construct of the present invention.","The role of untranslated sequences is preferably to direct the 3′ processing of the transcribed RNA rather than termination of transcription.","An exemplary 3′ UTR is a plastid rps16 gene 3′ untranslated sequence, or the Arabidopsis plastid psbA gene 3′ untranslated sequence.","In a further embodiment, a plastid expression cassette includes a poly-G tract instead of a 3′ untranslated sequence.","A plastid expression cassette also preferably further includes a 5′ untranslated sequence (5′ UTR) functional in plant plastids, operatively linked to a chimeric nucleic acid construct provided herein.","A plastid expression cassette is contained in a plastid transformation vector, which preferably further includes flanking regions for integration into the plastid genome by homologous recombination.","The plastid transformation vector may optionally include at least one plastid origin of replication.","The present invention also encompasses a plant plastid transformed with such a plastid transformation vector, wherein the chimeric nucleic acid construct is expressible in the plant plastid.","Also encompassed herein is a plant or plant cell, including the progeny thereof, including this plant plastid.","In a particular embodiment, the plant or plant cell, including the progeny thereof, is homoplasmic for transgenic plastids.","Other promoters capable of driving expression of a chimeric nucleic acid construct in plant plastids include transactivator-regulated promoters, preferably heterologous with respect to the plant or to the subcellular organelle or component of the plant cell in which expression is effected.","In these cases, the DNA molecule encoding the transactivator is inserted into an appropriate nuclear expression cassette which is transformed into the plant nuclear DNA.","The transactivator is targeted to plastids using a plastid transit peptide.","The transactivator and the transactivator-driven DNA molecule are brought together either by crossing a selected plastid-transformed line with and a transgenic line containing a DNA molecule encoding the transactivator supplemented with a plastid-targeting sequence and operably linked to a nuclear promoter, or by directly transforming a plastid transformation vector containing the desired DNA molecule into a transgenic line containing a chimeric nucleic acid construct encoding the transactivator supplemented with a plastid-targeting sequence operably linked to a nuclear promoter.","If the nuclear promoter is an inducible promoter, in particular a chemically inducible embodiment, expression of the chimeric nucleic acid construct in the plastids of plants is activated by foliar application of a chemical inducer.","Such an inducible transactivator-mediated plastid expression system is preferably tightly regulatable, with no detectable expression prior to induction and exceptionally high expression and accumulation of protein following induction.","A particular transactivator is, for example, viral RNA polymerase.","Particular promoters of this type are promoters recognized by a single sub-unit RNA polymerase, such as the T7 gene 10 promoter, which is recognized by the bacteriophage T7 DNA-dependent RNA polymerase.","The gene encoding the T7 polymerase is preferably transformed into the nuclear genome and the T7 polymerase is targeted to the plastids using a plastid transit peptide.","Promoters suitable for nuclear expression of a gene, for example a gene encoding a viral RNA polymerase such as the T7 polymerase, are described above and elsewhere in this application.","Expression of chimeric nucleic acid constructs in plastids can be constitutive or can be inducible, and such plastid expression can be also organ- or tissue-specific.","Examples of various expression systems are extensively described in WO 98/11235, the entire disclosure of which is hereby incorporated by reference.","Thus, in one aspect, the present invention utilizes coupled expression in the nuclear genome of a chloroplast-targeted phage T7 RNA polymerase under the control of the chemically inducible PR-1a promoter, for example of the PR-1 promoter of tobacco, operably linked with a chloroplast reporter transgene regulated by T7 gene 10 promoter/terminator sequences, for example as described in as in U.S. Pat.","No.","5,614,395 the entire disclosure of which is hereby incorporated by reference.","In another embodiment, when plastid transformants homoplasmic for the maternally inherited TR or NTR genes are pollinated by lines expressing the T7 polymerase in the nucleus, F1 plants are obtained that carry both transgene constructs but do not express them until synthesis of large amounts of enzymatically active protein in the plastids is triggered by foliar application of the PR-1a inducer compound benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH).","In a particular embodiment, two or more genes, for example TR and NTR genes, are transcribed from the plastid genome from a single promoter in an operon-like polycistronic gene.","In one embodiment, the operon-like polycistronic gene includes an intervening DNA sequence between two genes in the operon-like polycistronic gene.","In a particular embodiment, the intervening DNA sequence is not present in the plastid genome to avoid homologous recombination with plastid sequences.","In another embodiment, the DNA sequence is derived from the 5′ untranslated (UTR) region of a non-eukaryotic gene, preferably from a viral 5′UTR, preferably from a 6′UTR derived from a bacterial phage, such as a T7, T3 or SP6 phage.","In one embodiment, a portion of the DNA sequence may be modified to prevent the formation of RNA secondary structures in an RNA transcript of the operon-like polycistronic gene, for example between the DNA sequence and the RBS of the downstream gene.","Such secondary structures may inhibit or repress the expression of the downstream gene, particularly the initiation of translation.","Such RNA secondary structures are predicted by determining their melting temperatures using computer models and programs such a the “mfold” program version 3 (available from Zuker and Turner, Washington University School of Medicine, St-Louis, Mo.)","and other methods known to one skilled in the art.","The presence of the intervening DNA sequence in the operon-like polycistronic gene increases the accessibility of the RBS of the downstream gene, thus resulting in higher rates of expression.","Such strategy is applicable to any two or more genes to be transcribed from the plastid genome from a single promoter in an operon-like chimeric heteromultimeric gene.","Following transformation the cells are grown, typically in a selective medium allowing the identification of transformants.","Cells may be harvested in accordance with methodologies known to the art.","In order to associate the oil bodies with the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and a first and/or second thioredoxin-related protein, the integrity of cells may be disrupted using any physical, chemical or biological methodology capable of disrupting the cells' integrity.","These methodologies are generally cell-type dependent and known to the skilled artisan.","Where plants are employed they may be regenerated into mature plants using plant tissue culture techniques generally known to the skilled artisan.","Seeds may be harvested from mature transformed plants and used to propagate the plant line.","Plants may also be crossed and in this manner, contemplated herein is the breeding of cells lines and transgenic plants that vary in genetic background.","It is also possible to cross a plant line comprising the first recombinant polypeptide with a plant line comprising the second recombinant polypeptide.","Accordingly, also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide, such as a redox protein (e.g., a thioredoxin-related protein, and the like) or an immunoglobulin-polypeptide-chain, wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide, such as a second redox protein (e.g., a thioredoxin-related protein, and the like) or a second immunoglobulin-polypeptide-chain; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.","The second recombinant polypeptide may also associate with the oil bodies.","Accordingly, also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide, such as a redox (or thioredoxin-related) protein or immunoglobulin-polypeptide-chain, wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide, such as a second redox (thioredoxin-related) protein or a second immunoglobulin-polypeptide-chain, wherein said second recombinant polypeptide is capable of associating with said oil bodies through an oil body targeting protein; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.","Isolation of Oil Bodies The oil bodies provided herein may be obtained from any cell containing oil bodies, including any animal cell; plant cell; fungal cell; for example a yeast cell, algae cell; or bacterial cell.","Any process suitable for the isolation oil bodies from cells may be used herein.","Processes for the isolation of oil bodies from plant seed cells have been described in U.S. Pat.","Nos.","(6,146,645 and 6,183,762) and the isolation of oil bodies from yeast cells has been described by Ting et al.","(1997) J. Biol.","Chem.","272: 3699-3706).","In certain embodiments, the oil bodies are obtained from a plant cell such as for example a pollen cell; a fruit cell; a spore cell; a nut cell; mesocarp cell; for example the mesocarp cells obtainable from olive (Olea europaea) or avocado (Persea americana); or a seed cell.","In particular embodiments the oil bodies are obtained from a plant seed cell.","The seeds can be obtained from a transgenic plant according to the present invention.","In particular embodiments, a seed of a transgenic plant according to the present invention contains the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or first and/or second thioredoxin-related proteins in a concentration of at least about 0.5% of total cellular seed protein.","In further embodiments, a seed of a transgenic plant provided herein contains a recombinant polypeptide or multimeric-protein-complex in a concentration of at least about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more, of total cellular seed protein.","The upper limits of the recombinant polypeptide or multimeric-protein-complex concentration can be up to about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%.","Thus, the ranges at least about 0.5% up to about 15%; at least about 1.0% up to about 10%; and at least about 5% up to about 8% are among the various ranges contemplated herein.","Among the plant seeds useful in this regard are plant seeds obtainable from the group of plant species consisting of Brazil nut (Betholletia excelsa); castor (Riccinus communis); coconut (Cocus nucifera); coriander (Coriandrum sativum); cotton (Gossypium spp.","); groundnut (Arachis hypogaea); jojoba (Simmondsia chinensis); linseed/flax (Linum usitatissimum); maize (Zea mays); mustard (Brassica spp.","and Sinapis alba); oil palm (Elaeis guineeis); olive (Olea europaea); rapeseed (Brassica spp.","); safflower (Carthamus tinctorius); soybean (Glycine max); squash (Cucurbita maxima); sunflower (Helianthus annuus); barley (Hordeum vulgare); wheat (Traeticum aestivum) and mixtures thereof.","In a particular embodiment, oil bodies are obtainable from the seeds obtainable from safflower (Carthamus tinctorius).","In order to prepare oil bodies from plant seeds, plants are grown and allowed to set seed in accordance with common agricultural practices.","Thus, the present invention also provides seeds comprising oil bodies, wherein said oil bodies further comprise invention multimeric-protein-complexes described herein.","Upon harvesting the seed and, if necessary the removal of large insoluble materials such as stones or seed hulls, by for example sieving or rinsing, any process suitable for the isolation of oil bodies from seeds may be used herein.","A typical process involves grinding of the seeds followed by an aqueous extraction process.","Seed grinding may be accomplished by any comminuting process resulting in a substantial disruption of the seed cell membrane and cell walls without compromising the structural integrity of the oil bodies present in the seed cell.","Suitable grinding processes in this regard include mechanical pressing and milling of the seed.","Wet milling processes such as described for cotton (Lawhon et al.","(1977) J.","Am.","Oil Chem.","Soc.","63: 533-534) and soybean (U.S. Pat.","No.","3,971,856; Carter et al.","(1974) J.","Am.","Oil Chem.","Soc.","51: 137-141) are particularly useful in this regard.","Suitable milling equipment capable of industrial scale seed milling include colloid mills, disc mills, pin mills, orbital mills, IKA mills and industrial scale homogenizers.","The selection of the milling equipment will depend on the seed, which is selected, as well as the throughput requirement.","Solid contaminants such as seed hulls, fibrous materials, undissolved carbohydrates, proteins and other insoluble contaminants are subsequently preferably removed from the ground seed fraction using size exclusion based methodologies such as filtering or gravitational based methods such as a centrifugation based separation process.","Centrifugation may be accomplished using for example a decantation centrifuge such as a HASCO 200 2-phase decantation centrifuge or an NX310B (Alpha Laval).","Operating conditions are selected such that a substantial portion of the insoluble contaminants and sediments and may be separated from the soluble fraction.","Following the removal of insolubles the oil body fraction may be separated from the aqueous fraction.","Gravitational based methods as well as size exclusion based technologies may be used.","Gravitational based methods that may be used include centrifugation using for example a tubular bowl centrifuge such as a Sharples AS-16 or AS-46 (Alpha Laval), a disc stack centrifuge or a hydrocyclone, or separation of the phases under natural gravitation.","Size exclusion methodologies that may be used include membrane ultra filtration and crossflow microfiltration.","Separation of solids and separation of the oil body phase from the aqueous phase may also be carried out concomitantly using gravity based separation methods or size exclusion based methods.","The oil body preparations obtained at this stage in the process are generally relatively crude and depending on the application of the oil bodies, it may be desirable to remove additional contaminants.","Any process capable of removing additional seed contaminants may be used in this regard.","Conveniently the removal of these contaminants from the oil body preparation may be accomplished by resuspending the oil body preparation in an aqueous phase and re-centrifuging the resuspended fraction, a process referred to herein as “washing the oil bodies”.","The washing conditions selected may vary depending on the desired purity of the oil body fractions.","For example where oil bodies are used in pharmaceutical compositions, generally a higher degree of purity may be desirable than when the oil bodies are used in food preparations.","The oil bodies may be washed one or more times depending on the desired purity and the ionic strength, pH and temperature may all be varied.","Analytical techniques may be used to monitor the removal of contaminants.","For example SDS gel electrophoresis may be employed to monitor the removal of seed proteins.","The entire oil body isolation process may be performed in a batch wise fashion or continuous flow.","In a particular embodiment, industrial scale continuous flow processes are utilized.","Through the application of these and similar techniques the skilled artisan is able to obtain oil bodies from any cell comprising oil bodies.","The skilled artisan will recognize that generally the process will vary somewhat depending on the cell type that is selected.","However, such variations may be made without departing from the scope and spirit of the present invention.","Association of the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, the first and/or second thioredoxin-related proteins with oil bodies.","In accordance with the present invention, the oil bodies are associated with either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, the first and/or second thioredoxin-related proteins through association with an oil-body-targeting-protein capable of association with these multimeric-protein-complexes and the oil bodies.","As used herein the phrase “associating the oil bodies with the multimeric-protein-complex” means that the oil bodies are brought in proximity of the multimeric-protein-complexes in a manner that allows the association of the oil bodies with either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins.","The association of the oil bodies with the multimeric-protein-complexes is accomplished by association of the oil-body-targeting-protein with both the oil body and with the multimeric-protein-complex.","In particular embodiments, the cells expressing the multimeric-protein-complex associate with the oil bodies that are obtainable from these same cells, which permits the convenient production and isolation of the multimeric-protein-complex, including the first and/or second recombinant polypeptides, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, in an oil body-comprising host cell system.","Accordingly, in one embodiment, the association of the oil body with the multimeric-protein-complex is accomplished intracellularly during the growth of the cell.","For example, a redox fusion polypeptide may be fused to an oil-body-protein and the chimeric protein may be expressed in oil body-containing plant seeds.","Isolation of the oil bodies from the seeds in this case results in isolation of oil bodies comprising either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins.","In another embodiment, in which the multimeric-protein-complex associates with oil bodies obtainable from the same cells in which the complex is produced, the association of the oil bodies with the multimeric-protein-complex is accomplished upon disrupting the cell's integrity.","For example, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins may be expressed in such a manner that it is targeted to the endomembrane system of the seed cells.","Oil bodies present in the same seed cells comprising an oil-body-targeting-protein capable of association with these multimeric-protein-complexes, for example an oleosin linked to a single chain antibody capable of association with a recombinant polypeptide or multimeric-protein-complex, may then associate with the recombinant polypeptide or multimeric-protein-complex upon grinding of the seed.","In accordance with this embodiment, plant seed cells comprising a light and heavy chain of an immunoglobulin targeted to the plant apoplast can be prepared.","These particular seed cells are prepared to further comprise oil bodies associated with an oil-body-targeting-protein capable of association with the immunoglobulin, such as for example, an oleosin-protein A fusion protein, and the like.","Upon grinding of the seed, the oil bodies comprising protein A associate with the immunoglobulin through binding.","In yet another embodiment, the oil bodies used to associate with the multimeric-protein-complex are obtained from a cellular source different from the cell comprising the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, such as from a separate plant line.","For example, oil bodies associated with protein A may be prepared from one plant line.","These oil bodies may then be mixed with ground seeds comprising an apoplastically expressed light and heavy chain constituting an immunoglobulin.","Alternatively, a plant line comprising oil bodies associated with protein A may be crossed with a plant line comprising an immunoglobulin.","The first recombinant polypeptide, second recombinant polypeptide and oil-body-targeting-protein may also be prepared in separate cellular compartments.","Association of the first polypeptide, second polypeptide, and oil body then may occur upon disruption of the cell's integrity.","For example, various mechanisms for targeting gene products are known to exist in plants, and the sequences controlling the functioning of these mechanisms have been characterized in some detail.","For example, the targeting of gene products to the chloroplast is controlled by a transit sequence found at the amino terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein (Comai et al.","(1988) J Biol Chem 263: 15104-15109).","Other gene products are localized to other organelles such as the mitochondrion and the peroxisome (Unger et al.","(1989) Plant Mol Biol 13:411-418).","The cDNAs encoding these products can be manipulated to target heterologous gene products to these organelles.","In addition, sequences have been characterized which cause the targeting of gene products to other cell compartments.","Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho (1990) Plant Cell 2:769-783).","Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al., (1990) Plant Mol Biol 14:357-368).","By the fusion of the appropriate targeting sequences described above to transgene sequences of interest it is possible to direct the transgene product to the desired organelle or cell compartment.","As hereinbefore mentioned, the redox protein obtained using the methods provided herein is enzymatically active while associated with the oil body.","Preferably the redox protein is at least 5 times more active when produced as a redox fusion polypeptide with a second redox protein relative to its production in association with an oil body as a non-fusion polypeptide (i.e.","without the second redox protein).","More preferably the redox protein is at least 10 times more active when produced as a redox fusion polypeptide.","The activity of the redox fusion polypeptide may be determined in accordance with methodologies generally known to the art (see for example: Johnson et al (1984) J. of Bact.","Vol.","158 3:1061-1069) and may be optimized by for example the addition of detergents, including ionic and non-ionic detergents.","Formulation of Oil Bodies In accordance with a particular embodiment, the oil bodies comprising the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, are preferably formulated into an emulsion.","The emulsion is preferably used in the preparation of a pharmaceutical composition, personal care or a food product.","In emulsified form, the oil body offers certain desirable properties, such as for example excellent compatibility with the human skin.","It particular embodiments, the oil body formulation is stabilized so that a final product may be obtained which may be stored and preserved for longer periods of time.","As used herein, the term “stabilized oil body preparation” refers to an oil body preparation that is prepared so that the formulation does not undergo undesirable physical or chemical alterations when the oil body preparation is stored.","The stabilization requirements may vary depending on the final product.","For example personal care products are preferably stable for at least one year at room temperature while additionally being able to withstand short temperature fluctuations.","Pharmaceutical formulations may in some cases be less stable as they may be stored at lower temperatures thereby preventing the occurrence of undesirable reactions.","In general, stabilization techniques that may be used herein include any and all methods for the preservation of biological material including the addition of chemical agents, temperature modulation based methodologies, radiation-based technologies and combinations thereof.","In particular embodiments small amounts of stabilizing chemical agents are mixed with the oil body formulation to achieve stabilization.","These chemical agents include inter alia preservatives, antioxidants, acids, salts, bases, viscosity modifying agents, emulsifiers, gelling agents and mixtures thereof and may all be used to stabilize the oil body preparation.","In view of the presence of the redox fusion polypeptide the stabilizing agent is generally selected to be compatible with and resulting in good enzymatic function of the redox fusion polypeptide.","Diagnostic parameters to assess the stability of the oil body preparation may be as desired and include all parameters indicative of undesirable qualitative or quantitative changes with respect to chemical or physical stability.","Typical parameters to assess the oil body preparation over time include color, odor, viscosity, texture, pH and microbial growth, and enzymatic activity.","In particular embodiments, the oil body formulation is stabilized prior to the addition of further ingredients that may be used to prepare the final product.","However, in other embodiments, it is nevertheless possible to formulate the final formulation using non-stabilized oil bodies and stabilize the final formulation.","The final preparations may be obtained using one or more additional ingredients and any formulation process suitable for the preparation of a formulation comprising oil bodies.","Ingredients and processes employed will generally vary depending on the desired use of the final product, will be art recognized and may be as desired.","Ingredients and processes that may be used herein include those described in (U.S. Pat.","Nos.","6,146,645 and 6,183,762) which are incorporated by reference herein.","In particular embodiments, the redox fusion polypeptide comprises a thioredoxin and a thioredoxin-reductase.","Accordingly, provided herein are oil bodies comprising a thioredoxin/thioredoxin-reductase fusion polypeptide.","Also provided herein is a formulation containing oil bodies comprising a thioredoxin/thioredoxin-reductase fusion capable of treating or protecting a target against oxidative stress.","The stress of the target is treated or prevented by contacting the target with the formulation.","The target may be any substance susceptible to oxidative stress, including any molecule, molecular complex, cell, tissue or organ.","In another embodiment, provided herein is a formulation containing oil bodies comprising a thioredoxin/thioredoxin-reductase fusion capable of chemically reducing a target.","Contacting the target with the formulation reduces the target.","The target may be any substance susceptible to reduction, including any molecule or molecular complex.","Particularly susceptible targets in this regard are the disulfide bonds present in proteins.","The oil bodies comprising thioredoxin/thioredoxin-reductase may be used to prepare formulations used to reduce the allergenicity of food or increase the digestibility of food.","Preferably, the method of reducing the food allergenicity is practiced by mixing the thioredoxin/thioredoxin-reductase comprising oil bodies with food or food ingredients selected from a variety of sources including for example wheat flour, wheat dough, milk, cheese, soya, yogurt and ice cream.","The thioredoxin/thioredoxin-reductase comprising oil bodies may also be used to increase the digestibility of milk as well as other disulfide containing proteins (Jiao, J. et al.","(1992) J. Agric.","Food Chem 40: 2333-2336).","Further food applications include the use of the oil thioredoxin/thioredoxin-reductase comprising oil bodies as a food additive to enhance dough strength and bread quality properties (Wong et al., (1993) J. Cereal Chem.","70: 113-114; Kobrehel et al.","(1994) Gluten Proteins: Association of Cereal Research; Detmold, Germany).","Also provided herein are pharmaceutical compositions comprising, in a pharmaceutically active carrier: oil bodies comprising a thioredoxin/thioredoxin-reductase; oil bodies comprising multimeric-protein-complexes, such as heteromultimeric-protein-complexes; isolated thioredoxin/thioredoxin-reductase fusion proteins; or isolated multimeric-protein-complexes.","These pharmaceutical compositions may be used for the treatment of reperfusion injury (Aota et al.","(1996) J. Cardiov.","Pharmacol.","(1996) 27: 727-732), cataracts (U.S. Pat.","No.","4,771,036), chronic obstructive pulmonary disease (COPD) (MacNee et al.","(1999) Am.","J. Respir.","Crit.","Care Med.","160:S58-S65), diabetes (Hotta et al.","J. Exp.","Med.","188: 1445-1451), envenomation (PCT Patent Application 99/20122; U.S. Pat.","No.","5,792,506), bronchiopulmonary disease (MacNee (2000) Chest 117:3035-3175); malignancies (PCT Patent Application 91/04320) and the alleviation of the allergenic potential of airborne, for example pollen-derived, and contact allergens (PCT Patent Application 00/44781).","Other diseases or conditions that may be treated with the pharmaceutical compositions provided herein include: psoriasis, wound healing, sepsis, GI bleeding, intestinal bowel disease (IBD), ulcers, transplantation, GERD (gastro esophageal reflux disease).","In another embodiment, the pharmaceutical compositions provided herein, particularly those comprising one or more redox proteins alone or in combination with oil bodies, can be used in the treatment of inflammatory and viral diseases by reductively inactivating phospholipase A2, one of the contributing factors in inflammatory diseases.","Additionally, the redox fusion polypeptide system has been found to function as a self-defense mechanism in response to environmental stimuli, including oxidative stress caused by UV-generated free radicals.","Consequently, redox proteins, e.g., oleosin-thioredoxin, oleosin-thioredoxin-reductase, the various redox fusion polypeptides described herein, provide beneficial effects in certain skin conditions such as psoriasis, skin cancer, dandruff, diaper rash, dermatitis, acne, sun damage, aging, inflammation, and the like.","In another embodiment, oil-body-thioredoxin-related fusion proteins, e.g., oleosin-Thioredoxin-reductase, can also be used as a venom antidote.","Many animal venoms and other toxins contain disulfide bonds, including all snake venom neurotoxins, some bacterial neurotoxins including tetanus and botulinum A, bee venom phospholipase A2, and scorpion venom.","In a further embodiment, the redox protein related pharmaceutical compositions provided herein can be used to inactivate venom toxins by reduction of disulfide bonds.","A method of treating an individual suffering from the effects of a venom or toxin can include the step of administering an effective dose of a pharmaceutical composition, in a pharmaceutically effective carrier in an amount sufficient to relieve or reverse the effects of the venom toxin on the individual.","The pharmaceutical compositions provided herein are preferably formulated for single dosage administration.","The concentrations of the compounds in the formulations are effective for delivery of an amount, upon administration, that is effective for the intended treatment.","Typically, the compositions are formulated for single dosage administration.","To formulate a composition, the weight fraction of a compound or mixture thereof is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration such that the treated condition is relieved or ameliorated.","Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.","In addition, the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.","Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers.","These may be prepared according to methods known to those skilled in the art.","For example, liposome formulations may be prepared as described in U.S. Pat.","No.","4,522,811.The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.","The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo systems, such as the assays provided herein.","The concentration of active compound in the drug composition will depend on absorption, inactivation and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.","Typically a therapeutically effective dosage is contemplated.","The amounts administered may be on the order of 0.001 to 1 mg/ml, preferably about 0.005-0.05 mg/ml, more preferably about 0.01 mg/ml, of blood volume.","Pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 1000 mg and preferably from about 10 to about 500 mg, more preferably about 25-75 mg of the essential active ingredient or a combination of essential ingredients per dosage unit form.","The precise dosage can be empirically determined.","The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time.","It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data.","It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated.","It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or use of the claimed compositions and combinations containing them.","Preferred pharmaceutically acceptable derivatives include acids, salts, esters, hydrates, solvates and prodrug forms.","The derivative is typically selected such that its pharmacokinetic properties are superior to the corresponding neutral compound.","Thus, effective concentrations or amounts of one or more of the compounds provided herein or pharmaceutically acceptable derivatives thereof are mixed with a suitable pharmaceutical carrier or vehicle for systemic, topical or local administration to form pharmaceutical compositions.","Compounds are included in an amount effective for ameliorating or treating the disorder for which treatment is contemplated.","The concentration of active compound in the composition will depend on absorption, inactivation, excretion rates of the active compound, the dosage schedule, amount administered, particular formulation as well as other factors known to those of skill in the art.","Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.","Parenteral preparations can be enclosed in ampules, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable material.","In instances in which the compounds exhibit insufficient solubility, methods for solubilizing compounds may be used.","Such methods are known to those of skill in this art, and include, but are not limited to, using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such as Tween®, or dissolution in aqueous sodium bicarbonate.","Derivatives of the compounds, such as prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions.","For ophthalmic indications, the compositions are formulated in an ophthalmically acceptable carrier.","For the ophthalmic uses herein, local administration, either by topical administration or by injection is preferred.","Time release formulations are also desirable.","Typically, the compositions are formulated for single dosage administration, so that a single dose administers an effective amount.","Upon mixing or addition of the compound with the vehicle, the resulting mixture may be a solution, suspension, emulsion or other composition.","The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.","If necessary, pharmaceutically acceptable salts or other derivatives of the compounds are prepared.","The compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.","It is understood that number and degree of side effects depends upon the condition for which the compounds are administered.","For example, certain toxic and undesirable side effects are tolerated when treating life-threatening illnesses that would not be tolerated when treating disorders of lesser consequence.","The compounds can also be mixed with other active materials, that do not impair the desired action, or with materials that supplement the desired action known to those of skill in the art.","The formulations of the compounds and agents for use herein include those suitable for oral, rectal, topical, inhalational, buccal (e.g., sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), transdermal administration or any route.","The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active compound which is being used.","The formulations are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof.","The pharmaceutically therapeutically active compounds and derivatives thereof are typically formulated and administered in unit-dosage forms or multiple-dosage forms.","Unit-dose forms as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art.","Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required pharmaceutically acceptable carrier, vehicle or diluent.","Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules.","Unit-dose forms may be administered in fractions or multiples thereof.","A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form.","Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons.","Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.","The composition can contain along with the active ingredient: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acaciagelatin, glucose, molasses, polivinylpyrrolidine, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art.","Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.","If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.","Methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975).","The composition or formulation to be administered will contain a quantity of the active compound in an amount sufficient to alleviate the symptoms of the treated subject.","Dosage forms or compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared.","For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).","The tablets may be coated by methods well-known in the art.","The pharmaceutical preparation may also be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use.","Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).","Formulations suitable for rectal administration are preferably presented as unit dose suppositories.","These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.","Formulations suitable for topical application to the skin or to the eye preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol and oil.","Carriers which may be used include vaseline, lanoline, polyethylene glycols, alcohols, and combinations of two or more thereof.","The topical formulations may further advantageously contain 0.05 to 15 percent by weight of thickeners selected from among hydroxypropyl methyl cellulose, methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, poly(alkylene glycols), poly/hydroxyalkyl, (meth)acrylates or poly(meth)acrylamides.","A topical formulation is often applied by instillation or as an ointment into the conjunctival sac.","It can also be used for irrigation or lubrication of the eye, facial sinuses, and external auditory meatus.","It may also be injected into the anterior eye chamber and other places.","The topical formulations in the liquid state may be also present in a hydrophilic three-dimensional polymer matrix in the form of a strip, contact lens, and the like from which the active components are released.","For administration by inhalation, the compounds for use herein can be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.","In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.","Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.","Formulations suitable for buccal (sublingual) administration include, for example, lozenges containing the active compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles containing the compound in an inert base such as gelatin and glycerin or sucrose and acacia.","The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.","Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.","The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.","Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water or other solvents, before use.","Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.","Such patches suitably contain the active compound as an optionally buffered aqueous solution of, for example, 0.1 to 0.2 M concentration with respect to the active compound.","Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, e.g., Pharmaceutical Research 3 (6), 318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound.","The pharmaceutical compositions may also be administered by controlled release means and/or delivery devices (see, e.g., in U.S. Pat.","Nos.","3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,847,770; 3,916,899; 4,008,719; 4,687,610; 4,769,027; 5,059,595; 5,073,543; 5,120,548; 5,354,566; 5,591,767; 5,639,476; 5,674,533 and 5,733,566).","Desirable blood levels may be maintained by a continuous infusion of the active agent as ascertained by plasma levels.","It should be noted that the attending physician would know how to and when to terminate, interrupt or adjust therapy to lower dosage due to toxicity, or bone marrow, liver or kidney dysfunctions.","Conversely, the attending physician would also know how to and when to adjust treatment to higher levels if the clinical response is not adequate (precluding toxic side effects).","The efficacy and/or toxicity of the pharmaceutical compositions provided herein, alone or in combination with other agents can also be assessed by the methods known in the art (See generally, O'Reilly, Investigational New Drugs, 15:5-13 (1997)).","The active compounds or pharmaceutically acceptable derivatives may be prepared with carriers that protect the compound against rapid elimination from the body, such as time release formulations or coatings.","Kits containing the compositions and/or the combinations with instructions for administration thereof are provided.","The kit may further include a needle or syringe, preferably packaged in sterile form, for injecting the complex, and/or a packaged alcohol pad.","Instructions are optionally included for administration of the active agent by a clinician or by the patient.","Finally, the pharmaceutical compositions provided herein containing any of the preceding agents may be packaged as articles of manufacture containing packaging material, a compound or suitable derivative thereof provided herein, which is effective for treatment of a diseases or disorders contemplated herein, within the packaging material, and a label that indicates that the compound or a suitable derivative thereof is for treating the diseases or disorders contemplated herein.","The label can optionally include the disorders for which the therapy is warranted.","Also provided herein are personal care formulations containing oil bodies comprising a thioredoxin/thioredoxin-reductase fusion polypeptide.","Personal care products comprising thioredoxin and thioredoxin-reductase are disclosed in for example Japanese Patent Applications JP9012471A2, JP103743A2, and JP1129785A2 Personal care formulations that may be prepared in accordance with the present invention include formulations capable of improving the physical appearance of skin exposed to detrimental environmental stimuli resulting in oxidative stress for example oxidative stress caused by UV-generated free-radicals.","The oil bodies comprising thioredoxin/thioredoxin-reductase may also be used to prepare hair care products as described in U.S. Pat.","Nos.","4,935,231 and 4,973,475 (incorporated herein by reference in their entirety).","The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.","Example 1 Isolation of Thioredoxin and NADPH Thioredoxin-Reductase Genes An Arabidopsis silique cDNA library CD4-12 was obtained from the Arabidopsis Biological Resource Centre (ABRC, http://aims.cps.msu.edu) Arabidopsis stock centre and used as a template for the isolation of the thioredoxin h (Trxh) and thioredoxin-reductase genes from Arabidopsis.","For the isolation of the Trxh gene the following primers were synthesized: GVR833: 5′ TACCATGGCTTCGGAAGAAGGA 3′ (SEQ ID NO:1) The sequence identical to the 5′ end of the Trxh gene as published in Rivera-Madrid et al, (1993) Plant Physiol 102: 327-328, is indicated in bold.","Underlined is an NcoI restriction site to facilitate cloning.","GVR834: 5′ GAAAGCTTAAGCCAAGTGTTTG 3′ (SEQ ID NO:2) The sequence complementary to the 3′ end of the Trxh gene as published in Rivera-Madrid et al, (1993) Plant Physiol 102: 327-328, is indicated in bold.","Underlined is an HindIII restriction site to facilitate cloning.","A Polymerase Chain Reaction (PCR) was carried out using GVR833 and GVR834 as primers and the cDNA library CD4-12 as a template.","The resulted PCR fragment was isolated, cloned into pBluescript and sequenced.","The isolated sequence encoding Trxh was identical to the published Trxh gene sequence (Rivera-Madrid et al, (1993) Plant Physiol 102: 327-328).","The pBluescript vector containing the Trxh gene is called pSBS2500.For the isolation of the thioredoxin-reductase gene the following primers were synthesized: GVR836: 5′ GGCCAGCACACTACCATGAATGGTCTCGAAACTCAC 3′ (SEQ ID NO:3).","The sequence identical to the 5′ end of the thioredoxin-reductase gene as published (Jacquot et al, J Mol Biol.","(1994) 235 (4):1357-63), is indicated in bold).","GVR837: 5′ TTAAGCTTCAATCACTCTTACCTTGCTG 3′.","(SEQ ID NO:4) A Polymerase Chain Reaction (PCR) was carried out using GVR836 and GVR837 as primers and the cDNA library CD4-12 as a template.","The resulted PCR fragment was isolated, cloned into pBluescript and sequenced.","The pBluescript vector containing the thioredoxin-reductase gene is called pSBS2502.A total of three clones were sequenced, the sequence of each of the three clones were identical to each other.","However, as depicted in FIG.","1 this sequence indicated several nucleotide differences compared to the published thioredoxin-reductase gene sequence published (Jacquot et al, J Mol Biol.","(1994) 235 (4):1357-63.).","The complete coding sequence and its deduced amino acid sequence is shown in SEQ ID NO:10.As a result of the nucleotide differences between the published sequence and the sequence isolated in Example 1, several amino acid changes are also predicted.","A comparison of the deduced amino acid sequence of the published NADPH thioredoxin-reductase sequence thioredoxin-reductase (ATTHIREDB, Jacquot et al, J Mol Biol.","(1994) 235 (4):1357-63.)","with the sequence isolated in Example 1 (TR) is shown in FIG.","3.Example 2 Construction of Plant Expression Vectors Expression vectors were constructed to allow for the seed specific over-expression of thioredoxin and NADPH thioredoxin-reductase in seeds.","Vectors were constructed to allow for over-expression in its natural subcellular location and for accumulation on oil bodies.","Construction of Plant Transformation Vector pSBS2520.The Arabidopsis thioredoxin h gene as described in example 1 was placed under the regulatory control of the phaseolin promoter and the phaseolin terminator derived from the common bean Phaseolus vulgaris (Slightom et al (1983) Proc.","Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324)).","A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the Trxh gene.","Standard molecular biology laboratory techniques (see eg: Sambrook et al.","(1990) Molecular Cloning, 2nd ed.","Cold Spring Harbor Press) were used to furnish the phaseolin promoter and terminator with Pst I and HindIII/KpnI sites respectively (see SEQ ID NO:14).","Standard molecular biology laboratory techniques were also used to place the phaseolin terminator downstream from the Trxh gene.","The PstI-phaseolin promoter-Trxh-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000 (pSBS3000 is a derivative from the Agrobacterium binary plasmid pPZP221 (Hajdukiewicz et al., 1994, Plant Molec.","Biol.","25: 989-994).","In pSBS3000, the CaMV35S promoter-gentamycin resistance gene-CAMV 35S terminator of pPZP221 was replaced with parsley ubiquitin promoter-phosphinothricin acetyl transferase gene-parsley ubiquitin termination sequence to confer resistance to the herbicide glufosinate ammonium.)","The resulting plasmid is called pSBS2520.The sequence of the phaseolin promoter-Arabidopsis Trxh-phaseolin terminator sequence is shown in SEQ ID NO:14.Construction of Plant Transformation Vector pSBS2510.The 3′ coding sequence of an Arabidopsis oleosin gene (van Rooijen et al (1992) Plant Mol.","Biol.","18: 1177-1179) was altered to contain an NcoI site.","The NcoI-HindIII fragment from vector pSBS2500 (Example 1) containing the Trxh was ligated to the coding sequence of this Arabidopsis oleosin utilizing this NcoI restriction site.","A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter (Slightom et al (1983) Proc.","Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324) containing a synthetic PstI site (see construction of pSBS2520) to the coding sequence of the Arabidopsis oleosin.","Standard molecular biology laboratory techniques (see eg: Sambrook et al.","(1990) Molecular Cloning, 2nd ed.","Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin terminator (see construction of pSBS2520) downstream of the Trxh gene.","The PstI-phaseolin promoter-oleosin-Trxh-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2510.The sequence of the phaseolin promoter-oleosin Trxh-phaseolin terminator sequence is shown in SEQ ID NO:16.Construction of Plant Transformation Vector pSBS2521.This vector contains the same genetic elements as the insert of pSBS2510 except the Trxh gene is fused to the 5′ end of the oleosin gene.","The 3′ oleosin coding sequence including its native stopcodon (van Rooijen et al (1992) Plant Mol.","Biol.","18: 1177-1179) was furnished with a HindIII cloning site.","Again a gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the Trxh gene and to fuse the Trxh gene to the oleosin sequence.","Standard molecular biology laboratory techniques (see eg: Sambrook et al.","(1990) Molecular Cloning, 2nd ed.","Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin terminator (see construction of pSBS2520) downstream of the oleosin gene.","The PstI-phaseolin promoter-Trxh oleosin-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2521.The sequence of the phaseolin promoter-Trxh oleosin-phaseolin terminator sequence is shown in SEQ ID NO:19.Construction of Plant Transformation Vector pSBS2527.The Arabidopsis NADPH thioredoxin-reductase gene as described in example 1 was placed under the regulatory control of the phaseolin promoter and the phaseolin terminator derived from the common bean Phaseolus vulgaris (Slightom et al (1983) Proc.","Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324).","A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the thioredoxin-reductase gene.","Standard molecular biology laboratory techniques (see eg: Sambrook et al.","(1990) Molecular Cloning, 2nd ed.","Cold Spring Harbor Press) were used to furnish the phaseolin promoter and terminator with PstI and HindIII/KpnI sites respectively (see SEQ ID NO:14).","Standard molecular biology laboratory techniques were also used to place the phaseolin terminator downstream from the thioredoxin-reductase gene.","The PstI-phaseolin promoter-thioredoxin-reductase-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000 The resulting plasmid is called pSBS2527.The sequence of the phaseolin promoter-Arabidopsis thioredoxin-reductase-phaseolin terminator sequence is shown in SEQ ID NO:22.Construction of Plant Transformation Vector pSBS2531.A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter (Slightom et al (1983) Proc.","Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324) to the coding sequence of the Arabidopsis oleosin.","The same gene splicing technique was used to fuse the oleosin gene to the thioredoxin-reductase coding sequence.","Standard molecular biology laboratory techniques (see eg: Sambrook et al.","(1990) Molecular Cloning, 2nd ed.","Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin downstream of the thioredoxin-reductase gene.","The PstI-phaseolin promoter-oleosin-thioredoxin-reductase-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2531.The sequence of the phaseolin promoter-oleosin thioredoxin-reductase-phaseolin terminator sequence is shown in SEQ ID NO:24.Construction of Plant Transformation Vector pSBS2529 This vector contains the same genetic elements as the insert of pSBS2531 except the thioredoxin-reductase gene is fused to the 5′ end of the oleosin gene.","The 3′ oleosin coding sequence including its native stopcodon (van Rooijen et al.","(1992) Plant Mol.","Biol, 18: 1177-1179) was furnished with a HindIII cloning site.","Again a gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the thioredoxin-reductase gene and to fuse the thioredoxin-reductase gene to the oleosin sequence.","Standard molecular biology laboratory techniques (see eg: Sambrook et al.","(1990) Molecular Cloning, 2nd ed.","Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin terminator (see construction of pSBS2520) downstream of the oleosin gene.","The PstI-phaseolin promoter-thioredoxin-reductase oleosin-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2529.The sequence of the phaseolin promoter-thioredoxin-reductase oleosin-phaseolin terminator sequence is shown in SEQ ID NO:27.Construction of Plant Transformation Vector pSBS2530.A plant transformation was constructed containing the Mycobacterium Leprae thioredoxin-reductase /thioredoxin gene (Mlep TR/Trxh).","A construct called pHIS/TR/Trxh (Wieles et al (1995) J Biol Chem 270:25604-25606) was obtained from the department of Immunohematology and Blood bank, Leiden University, The Netherlands and use as a template for PCR to generate pSBS2530.The construction of pSBS2530 was identical to the construction of pSBS2531 except that the Mlep TR/Trxh gene was used instead of the Arabidopsis thioredoxin-reductase gene.","A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter (Slightom et al (1983) Proc.","Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324) to the coding sequence of the Arabidopsis oleosin.","The same gene splicing technique was used to fuse the oleosin gene to the Mlep TR/Trxh coding sequence.","Standard molecular biology laboratory techniques (see eg: Sambrook et al.","(1990) Molecular Cloning, 2nd ed.","Cold Spring Harbor Press) were again used to clone the HindIII-KpnI fragment containing the phaseolin downstream of the Mlep TR/Trxh gene.","The PstI-phaseolin promoter-oleosin-Mlep TR/Trxh -phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2530.The sequence of the phaseolin promoter-oleosin Mlep TR/Trxh -phaseolin terminator sequence is shown in SEQ ID NO:30.Construction of Plant Transformation Vector pSBS2542.From initial activity assays (FIG.","4), it was apparent that oil bodies expressing the oleosin-M. lep TR/Trxh fusion protein contained considerable reducing activity.","It was anticipated that a similar oleosin fusion construct encoding the Arabidopsis thioredoxin-reductase and thioredoxin proteins would behave in an analogous manner.","Molecular modeling was used to aid in the design of such a construct.","Primers were designed (thioredoxin link-L: 5′-ACTGGAGATGTTGACTCGACGGATACTACGGATTGGTCGACGG CTATGGAAGAAGGACAAGTGATCGCCTGC-3′; (SEQ ID NO:5), and thioredoxin link-R: 5′-ATCCGTCGAGTCAACATCTCCAGTTTCCTCGGTGGTCTCGTTAGCCTTCGAT CCAGCAATCTCTTGTAAGAATGCTCTGC-3′; (SEQ ID NO:6) to code for a synthetic linker peptide between the thioredoxin-reductase and thioredoxin proteins.","These primers were used in conjunction with primers GVR 873 (5′-GTGGAAGCT TATGGAGATGGAG-3′; SEQ ID NO:7) and GVR834 (5′-GAAAGCTTAAGCCAAGTGTTTG-3′; SEQ ID NO:2) to amplify a region coding for a thioredoxin-reductase-linker region-thioredoxin utilizing a gene splicing by overlap extension technique (Horton et al (1989) 15:61-68).","The thioredoxin-reductase-linker-thioredoxin encoding sequence was then cloned into a pre-existing pSBS3000 vector using standard molecular biology techniques (Sambrook et al (1990) Molecular Cloning 2nd Edition Cold Spring Harbour Press).","The resulting plasmid was called pSBS2542.The sequence of the phaseolin promoter-oleosin-thioredoxin-reductase-linker-thioredoxin-phaseolin terminator region is shown in SEQ ID NO:33.An amino acid sequence comparison between this Arabidopsis thioredoxin-reductase-linker-thioredoxin and the M. leprae TR/Trxh protein is shown in FIG.","12.Plasmids pSBS2510, pSBS2520, pSBS2521, pSBS2527, pSBS2529, pSBS2530, pSBS2531 and pSBS2542 were electroporated into Agrobacterium strain EHA101.These Agrobacterium strains were used to transform Arabidopsis.","Arabidopsis transformation was done essentially as described in “Arabidopsis Protocols; Methods in molecular biology Vol 82.Edited by Martinez-Zapater J M and Salinas J. ISBN 0-89603-391-0 pg 259-266 (1998) except the putative transgenic plants were selected on agarose plates containing 80 μM L-phosphinothricine, after they were transplanted to soil and allowed to set seed.","Example 3 Polyacrylamide Gelelectrophoresis and Immunoblotting of Transgenic Seed Extracts Source of Arabidopsis Thioredoxin, Thioredoxin-Reductase and Oleosin Antibodies.","The Arabidopsis thioredoxin and thioredoxin-reductase genes were cloned in frame in bacterial expression vector pRSETB (Invitrogen) to allow for the overexpression of Arabidopsis thioredoxin and thioredoxin-reductase proteins.","These proteins were purified using standard protocols (see eg Invitrogen protocol) and used to raise antibodies in rabbits using standard biochemical techniques (See eg Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).","The Arabidopsis oleosin gene genes was cloned in frame in bacterial expression vector pRSETB (Invitrogen) to allow for the overexpression Arabidopsis oleosin protein.","This protein was purified using standard protocols (see eg Invitrogen protocol) and used to prepare mouse monoclonal antibodies using standard biochemical techniques (See eg Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).","Preparation of total Arabidopsis seed extracts for PAGE.","Arabidopsis seeds were ground in approximately 20 volumes of 2% SDS, 50 mM Tris-Cl, this extract was boiled, spun and the supernatant was prepared for polyacrylamide gelelectrophoresis (PAGE) using standard protocols.","Preparation of Arabidopsis Oil-Body-Protein Extracts.","Arabidopsis seeds were ground in approximately 20 volumes of water and spun in a microfuge.","The oil bodies were recovered and washed sequentially with approximately 20 volumes of water, a high stringency wash buffer, containing 8M urea and 100 mM sodiumcarbonate and water.","After this last wash the oil bodies are prepared for poly acrylamide gelelectrophoresis (PAGE) using standard protocols.","Analysis of Seed and Oil Body Extracts from Plants Transformed with pSBS2510 Total seed and oil body protein extracts from plants transformed with pSBS2510 were loaded onto polyacrylamide gels and either stained with coomassie brilliant blue or electroblotted onto PVDF membranes.","The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin, or a monoclonal antibody raised against the Arabidopsis 18.5 kDa oleosin and visualized using alkaline phosphatase.","Expression of the oleosin-thioredoxin results in an additional band of 31.2 kDa.","The results indicate that the thioredoxin antibodies are immunologically reactive with a band of the right predicted molecular weight (31.2 kDa), and the oleosin antibodies are also immunologically reactive with a band of the right predicted molecular weight for the fusion protein (31.2 kDa) in addition to a band corresponding to the native Arabidopsis oleosin (18.5 kDa).","This indicates that oleosin-thioredoxin is expressed in Arabidopsis seeds and is correctly targeted to oil bodies.","Analysis of Seed and Oil Body Extracts from Plants Transformed with pSBS2521 Total seed and oil body protein extracts from plants transformed with pSBS25121 were loaded onto polyacrylamide gels and either stained with Coomassie brilliant blue or electroblotted onto PVDF membranes.","The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin, or a monoclonal antibody raised against the Arabidopsis 18.5 kDa oleosin and visualized using alkaline phosphatase.","Expression of the thioredoxin-oleosin results in an additional band of 31.2 kDa.","The results indicate that the thioredoxin antibodies are immunologically reactive with a band of the right predicted molecular weight (31.2 kDa), and the oleosin antibodies are also immunologically reactive with a band of the right predicted molecular weight for the fusion protein (31.2 kDa) in addition to a band corresponding to the native Arabidopsis oleosin (18.5 kDa).","This indicates that thioredoxin-oleosin is expressed in Arabidopsis seeds and is correctly targeted to oil bodies.","Analysis of seed extracts from plants transformed with pSBS2520 Total seed extracts from plants transformed with pSBS2520 were loaded onto polyacrylamide gels and either stained with Coomassie brilliant blue or electroblotted onto PVDF membranes.","The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin and visualized using alkaline phosphatase.","The results indicated that the thioredoxin antibodies are immunologically reactive with a band of approximately the right predicted molecular weight (12 kDa).","Untransformed seeds do not show a detectable thioredoxin band.","Analysis of Seed and Oil Body Extracts from Plants Transformed with pSBS2529 Total seed and oil body protein extracts from plants transformed with pSBS2529 were loaded onto polyacrylamide gels and electroblotted onto PVDF membranes.","The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin-reductase, or a monoclonal antibody raised against the Arabidopsis 18.5 kDa oleosin and visualized using alkaline phosphatase.","Expression of the thioredoxin-reductase -oleosin results in an additional band of 53.8 kDa.","The results indicate that the thioredoxin-reductase antibodies are immunologically reactive with a band of the right predicted molecular weight for the fusion protein (53.8 kDa), the oleosin antibodies are also immunologically reactive with a band of the right predicted molecular weight (53.8 kDa) in addition to a band corresponding to the native Arabidopsis oleosin (18.5 kDa).","This indicates that thioredoxin-reductase-oleosin is expressed in Arabidopsis seeds.","Analysis of seed extracts from plants transformed with pSBS2527 Total seed extracts from plants transformed with pSBS2527 were loaded onto polyacrylamide gels and electroblotted onto PVDF membranes.","The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin-reductase and visualized using alkaline phosphatase.","The thioredoxin-reductase antibodies are immunologically reactive with a band of approximately the right predicted molecular weight for the (35.3 kDa).","Untransformed seeds do not show a detectable thioredoxin band.","Analysis of seed extracts from plants transformed with pSBS2531 A protein gel and immunoblot was prepared assaying the expression of oleosin-DMSR in Arabidopsis T2 seeds and correct targeting to Arabidopsis oil bodies.","The expected molecular weight based on the deduced amino acid sequence is calculated to be 53,817 Da.","In the oil body extract of the transgenic oleosin-thioredoxin-reductase sample an extra band of approximately 54 kDa was observed.","This band was confirmed to be oleosin-thioredoxin-reductase by immunoblotting.","From the polyacrylamide gel it was observed that the expression of the oleosin-Thioredoxin-reductase is about double compared to the expression of the major 18.5 kDa Arabidopsis oleosin.","This represents approximately 2-4% of total seed protein.","Analysis of seed extracts from plants transformed with pSBS2530 A protein gel and immunoblot was prepared assaying the expression of oleosin-M.lep TR/Trxh in Arabidopsis T2 seeds and the correct targeting to Arabidopsis oil bodies.","The expected molecular weight based on the deduced amino acid sequence is calculated to be 67,550 Da.","In the oil body extract of the transgenic oleosin-M.lep TR/Trxh sample an extra band of approximately 68 kDa was observed.","This band was confirmed to be oleosin-M.lep TR/Trxh by immunoblotting.","From the polyacrylamide gel it was observed that the expression of the oleosin-M.lep TR/Trxh is similar to the expression of the major 18.5 kDa Arabidopsis oleosin.","This represents approximately 1-2% of total seed protein.","Analysis of seed extracts from plants transformed with pSBS2542 Crude oil body extracts from pSBS2542 lines were prepared by grinding 100 μg of seed in 1 mL of 100 mM Tris buffer at pH 7.5.The samples were then centrifuged in order to isolate the oil body fraction.","The oil body fraction was then loaded on an SDS polyacrylamide gel for expression analysis.","A Coomassie stained gel revealed that the synthetic fusion accumulated to high levels in crude oil body extracts from 3 of the 4 lines tested.","It was estimated that the fusion protein represented approximately 2-5% of total seed protein.","Furthermore, western blots utilizing either anti-thioredoxin or anti-thioredoxin-reductase antibodies confirmed that the over expressed 70 kDa protein was indeed oleosin-thioredoxin-reductase-linker-thioredoxin.","Example 4 Biological Activity of Thioredoxin and Thioredoxin-Reductase Transformants Initial Reduction Assays: DTNB Assay The activity of the thioredoxin and thioredoxin reductase was determined using a calorimetric DTNB [5,5′-dithiolbis(2-nitrobenzoic acid)] assay.","The assay was performed in a 700 μL reaction volume containing 100 mM Tris-Cl pH 8.0, 5 mM EDTA, 200 μM DTNB [5,5′-dithiolbis(2-nitrobenzoic acid)] and 200 μM NADPH.","If thioredoxin-reductase and thioredoxin are added, NADPH will reduce the thioredoxin-reductase, which will then reduce thioredoxin, which will, in turn, reduce DTNB (see equations below).","NADPH2+thioredoxin-reductaseox→thioredoxin-reductasered+NADP+thioredoxin-reductasered+thioredoxinox→thioredoxinred+thioredoxin-reductaseox thioredoxinred+DTNBox→2(2-nitro-5-mercaptobenzoic acid)+thioredoxinox The formation of the yellow product was monitored by measuring the OD412 in a spectrophotometer after a set period of time (usually 0.5-2 hours).","The results of initial activity assays are shown in the bar graph in FIG.","4 and described below.","Initially, 100 μg of total seed proteins were added from each of the Arabidopsis transgenic lines, pSBS2520 (cytosolic thioredoxin) and pSBS2527 (cytosolic thioredoxin-reductase), which corresponds to approximately 1 μg of cytosolic thioredoxin and thioredoxin-reductase used in the assay.","In this case, the amount of DTNB reduced was comparable to the reduction caused by 1 μg each of E. coli thioredoxin and thioredoxin-reductase.","In these plant seed samples, background readings were very low when only one of the 2 extracts (either cytosolic thioredoxin or cytosolic thioredoxin-reductase; FIG.","4, bars 3 and 6, respectively) was added to the reaction, along with wild type oil bodies.","Analysis with oil body fractions from transgenic seeds revealed that Arabidopsis thioredoxin and thioredoxin-reductase were substantially less active when fused to oleosins on oil bodies.","Approximately 300 μg of crude, unwashed oil-body-protein was used in the assay (which corresponds to 10-30 μg of thioredoxin-oleosin (pSBS 2521; FIG.","4, bar 2), oleosin-thioredoxin (pSBS 2510, FIG.","4, bar 1), thioredoxin-reductase-oleosin (pSBS 2529, FIG.","4, bar 5), or oleosin-thioredoxin-reductase (pSBS 2531, FIG.","4, bar 4).","The oil-body-proteins were tested in conjunction with 100 μg of total seed protein containing approximately 1 μg of cytosolic thioredoxin (pSBS 2520) or thioredoxin-reductase (pSBS 2527).","In such assays, pSBS2529 (thioredoxin-reductase-oleosin) and pSBS2531 (oleosin-thioredoxin-reductase) do contain reductase activity when combined with cytosolic thioredoxin from pSBS2520 (see FIG.","4, bars 7 and 8, respectively).","Experiments estimated that the reductase activity of oleosin-thioredoxin-reductase was about 10-15% that of the cytosolic thioredoxin-reductase.","The addition of tween at a final concentration of 0.4% could enhance this activity 2 or 3 fold.","Interestingly, oleosin-thioredoxin-reductase (pSBS 2531) appears to be capable of reducing DTNB in the absence of added thioredoxin, although added thioredoxin causes significantly more DTNB reduction (see FIG.","4; compare bar 4 W.T.+oleosin-thioredoxin-reductase to bar 7 thioredoxin+oleosin-thioredoxin-reductase).","Experiments with pSBS2521 (thioredoxin-oleosin) or pSBS2510 (oleosin-thioredoxin) combined with cytosolic thioredoxin-reductase from pSBS2527 (see FIG.","4, bars 10 and 11, respectively) indicate that thioredoxin activity of these fusions is undetectable at these concentrations.","Oil bodies from the transgenic Arabidopsis line, pSBS2530 (oleosin-M.lep TR/Trxh) contain significant thioredoxin/thioredoxin-reductase activity (see FIG.","4, bar 12).","One hundred micrograms of crude oil body protein for pSBS2530 was tested (corresponding to approximately 5 μg of oleosin-M.lep TR/trxh fusion) in the assay.","Based on the assay, it was estimated that this fusion is about 25-40% as active as cytosolic Arabidopsis thioredoxin and thioredoxin-reductase (FIG.","4, bar 9) when comparing specific activity.","Insulin Reduction Assay The results from the DTNB assays were confirmed with insulin reduction assays.","This assay contained insulin at a final concentration of 1 mg/mL in 100 mM KH2PO4 pH 7.0+5 mM EDTA.","In the presence of NADPH (500 μM), thioredoxin, and thioredoxin-reductase, insulin is reduced and precipitates from the solution.","Normally, insulin reduction is followed by measuring turbidity at OD 650.Alternatively, one can measure the conversion of NADPH2 to NADP+by monitoring the decrease in absorbance at 340 nm.","Both of the assays are difficult to measure when oil bodies are present, due to interference with the spectrophotometer readings.","However, qualitative data could be obtained by centrifuging the tubes after a set period of time, and determining if an insulin pellet was present (oil bodies float to the top, while the insulin precipitate pellets out).","Alternatively, samples could be filtered after a set period of time, and the change in absorbance at 340 nm could be measured.","As mentioned previously, the results of the insulin reduction assays agreed with those of the DTNB assay, with the exception of the observation that pSBS2531 (oleosin-thioredoxin-reductase) only reduced insulin in the presence of free thioredoxin from pSBS2520.Assays on Seeds from Arabidopsis Crosses that Co-Express Oleosin-Thioredoxin and Oleosin-Thioredoxin-Reductase.","Based upon initial DTNB and insulin reduction assays, it was apparent that mixing oil bodies from oleosin<->thioredoxin and oleosin<->thioredoxin-reductase transgenic seeds resulted in very limited reducing activity (Note: the <-> indicates both configurations of oleosin fusions; ie.","oleosin<->thioredoxin would represent oleosin-thioredoxin and thioredoxin-oleosin fusions).","To determine whether having oleosin<->thioredoxin and oleosin<->thioredoxin-reductase proteins present on the same oil body would have a positive effect on the reducing activity of these proteins, crosses were set up to generate double transgenic Arabidopsis lines.","The crosses are illustrated in Table 2.TABLE 2 Confirmed double transgenic lines (PCR and Western Male Female Blot) oleo-thioredoxin X oleo-thioredoxin-reductase 4 oleo-thioredoxin X thioredoxin-reductase-oleo 1 thioredoxin-oleo X oleo-thioredoxin-reductase 0 thioredoxin-oleo X thioredoxin-reductase-oleo 4 oleo-thioredoxin- X oleo-thioredoxin 2 reductase oleo-thioredoxin- X thioredoxin-oleo 0 reductase thioredoxin- X oleo-thioredoxin 7 reductase-oleo thioredoxin- X thioredoxin-oleo 0 reductase-oleo Seeds from Arabidopsis crosses were germinated on PPT plates and the seedlings were transferred to soil after approximately 2 weeks.","PCR experiments on DNA isolated from the seedlings identified a number of plants which contain both an oleosin<->thioredoxin and an oleosin<->thioredoxin-reductase gene construct within their genome.","Seeds were harvested from these plants for expression and activity assays.","Western blots were carried out to confirm expression of both oleosin<->thioredoxin and oleosin<->thioredoxin-reductase in the lines.","DTNB and insulin reduction assays were also performed to compare activity between single transgenic parent lines and the double transgenic offspring and results are summarized in Table 3.Table 3 summarizes DTNB reducing activity of various transgenic lines.","The last 2 rows compare mixing oil bodies from single transgenic parent lines to using oil bodies from double transgenic offspring.","Relative activity for the E. coli thioredoxin and thioredoxin mixture is set at 100 percent.","TABLE 3 Relative Source Material Activity (%) E. coli trx + NTR 100 Arabidopsis “free” thioredoxin + 100 thioredoxin-reductase (pSBS2520 + pSBS2527) oleosin-M. lep TR/Trxh ~30 (pSBS2530) Oleosin<->thioredoxin-reductase + ~3 oleosin<->thioredoxin (mixing oil bodies from single-transgenic parents) Oleosin<->thioredoxin-reductase × ~50 oleosin<->thioredoxin (various double transgenic lines) Based on DTNB and insulin reduction assays, it is evident that double transgenic plants co-expressing oleosin<->thioredoxin and oleosin<->thioredoxin-reductase on the same, single oil body contained significantly more reducing activity compared to mixing oil bodies from single transgenic oleosin<->thioredoxin and oleosin<->thioredoxin-reductase lines.","It was additionally apparent that oil body extracts from co-expressing lines contained more reducing activity compared to line pSBS2530 (oleosin-M. lep TR/Trxh), which was previously identified as the line containing the highest reducing activity from oil bodies.","These results suggest that the creation of double transgenic lines (either through crossing or by co-transforming 2 expression constructs into plants) may represent one means by which we could solve our initial problem of not being able to generate reducing activity by mixing oil bodies from oleosin<->thioredoxin and oleosin<->thioredoxin-reductase single transgenic lines.","Assays on Seeds from Arabidopsis pSBS2542 Transgenic Lines that Express Oleosin-Thioredoxin-Reductase-Linker-Thioredoxin.","Oil body extracts from four pSBS2542 lines were tested for reducing activity in DTNB and insulin reduction assays, using standard protocols described previously.","Again, oil body extracts containing the oleosin-thioredoxin-reductase-linker-thioredoxin protein possessed significant reducing activity.","Based on such assays, it was revealed that the oleosin-thioredoxin-reductase-linker-thioredoxin synthetic fusion protein was more active than the oleosin-M. lep TR/Trxh fusion.","Furthermore, oil bodies containing the oleosin-thioredoxin-reductase-linker-thioredoxin protein appeared to have more reducing activity compared to oil bodies from double transgenic lines that co-expressed oleosin<->thioredoxin and oleosin<->thioredoxin-reductase.","The results comparing reducing activity for the various thioredoxin-reductase/thioredoxin constructs is summarized in Table 4.Table 4 summarizes DTNB reducing activity of various transgenic lines.","The pSBS2542 line expressing oleosin-thioredoxin-reductase-linker-thioredoxin contains significant reducing activity, comparable to the “free” forms of Arabidopsis thioredoxin and thioredoxin-reductase and the equivalent E. coli proteins.","Relative activity for the E. coli thioredoxin and thioredoxin mixture is set at 100 percent.","TABLE 4 Relative Source Material Activity (%) E. coli trx + NTR 100 Arabidopsis “free” thioredoxin + thioredoxin- 100 reductase (pSBS2520 + pSBS2527) oleosin-M. lep TR/Trxh ~30 (pSBS2530) Oleosin<->thioredoxin-reductase + ~3 oleosin<->thioredoxin (mixing oil bodies from single-transgenic parents) Oleosin<->thioredoxin-reductase × ~50 oleosin<->thioredoxin (various double transgenic lines) Oleosin-thioredoxin-reductase-linker-thioredoxin ~75-100 (pSBS2542) Reduction Assays Comparing the Utilization of NADH Vs. NADPH as a Cofactor (Electron Donor) for the Thioredoxin-Reductase/Thioredoxin System.","DTNB and insulin reduction assays were conducted as described previously, except that NADH was substituted for NADPH as an electron donor in the system utilizing E. coli thioredoxin-reductase and thioredoxin.","Thus, a comparison was conducted of the utilization of NADH versus NADPH as a cofactor for the E. coli thioredoxin-reductase/thioredoxin system.","For the DTNB assay, the reaction mixture consisted of 400 μM DTNB, 10 μg/mL E. coli thioredoxin, and 10 μg/mL E. coli thioredoxin-reductase in 100 mM Tris-Cl buffer pH 8.0.Either NADH or NADPH was then added to the DTNB reaction as follows: Reaction A.","200 μM NADPH (Sigma) Reaction B.","800 μM NADH (Sigma) Reaction C. 800 μM NADH (Roche) Reaction D. (−) cofactor Reaction E. 800 μM NADH (no TR or Trxh).","For the insulin reduction assay, the reaction mixture consisted of 1 mg/mL bovine pancreatic insulin, 20 μg/mL E. coli thioredoxin, and 20 μg/mL E. coli thioredoxin-reductase in 100 mM potassium phosphate buffer at pH 7.0.Either NADH or NADPH was then added to the reaction as follows: Reaction A.","800 μM NADPH (Sigma) Reaction B.","800 μM NADH (Sigma) Reaction C. 800 μM NADH (Roche) Reaction D. (−) cofactor Reaction E. 2 mM NADH (no TR or Trxh).","The results indicate that NADH, purchased from either Sigma or Roche, could act as an electron donor in both the DTNB and insulin reduction assays.","However, the rate of reduction was lower than the rate observed with NADPH as a cofactor.","It was estimated that the rate of insulin reduction utilizing NADH as an electron donor was approximately 25-50% when compared to the maximum rate using NADPH.","Furthermore, it was estimated that the rate of DTNB reduction utilizing NADH as an electron donor was approximately 5-10% of the maximum rate using NADPH.","Similar results were observed using the oleosin-thioredoxin-reductase-linker thioredoxin fusion protein on Arabidopsis oil bodies instead of the E. coli thioredoxin-reductase and thioredoxin.","Example 5 Production of Multimeric Immunoglobulin Protein in Plant Seed Cells and Capture on Oil Bodies Using Protein A-Oleosin Fusion Proteins 1—Production of Multimeric Immunoglobulin Protein in Plant Seed Cells For expression of multimeric-protein-complexes containing multimeric-immunoglobulin-complexes, the cDNA sequences encoding individual light and heavy chains can be isolated from; 1) cell lines expressing a particular antibody, such as clonal B cell lines, or a hybridoma cell line, or 2) may be a recombinant antibody, assembled by combining select light and heavy chain variable domains and available light and heavy chain constant domain sequences, respectively.","Variable domains with specific binding properties may be isolated from screening populations of such sequences, usually in the form of a single-chain Fv phage display library.","Starting from known nucleic acid sequences and a source of light and heavy chains, the mature polypeptide coding sequences of each chain is isolated with a secretion signal sequence.","The signal sequence can be the native antibody sequence or derived from a known secreted plant sequence (e.g.","a PR sequence from Arabidopsis or tobacco).","The addition of a plant secretion signal sequence to both light and heavy chain mature coding sequences is carried out by standard molecular biology techniques.","PCR fusion is used routinely to make such modifications.","Secretion signal sequences are included to target the light and heavy immunoglobulin polypeptides for secretion from the cell and further assembly of the two chains into a multimeric-immunoglobulin-complex.","For expression in transgenic plant seeds, an expression cassette is assembled comprising: 1) a regulatory promoter sequence to provide expression in plant seeds, 2) the secretion signal—light chain sequence, and 3) a regulatory sequence to terminate transcription.","A second expression cassette is assembled comprising: 1) a regulatory promoter sequence to provide expression in plant seeds, 2) the secretion signal—heavy chain sequence, and 3) a regulatory sequence to terminate transcription.","Each of the antibody chain expression cassettes is cloned individually into an Agrobacterium plant transformation vector or is combined into a single transformation vector with both expression cassettes.","In both cases, the expression cassettes are cloned into plant transformation vectors, between the left and right delineating border sequences, and adjacent to a plant selectable marker cassette.","Each plant transformation vector is transformed into Agrobacterium.","The resulting Agrobacterium strains are used to infect plant tissues.","Transgenic plant material is regenerated and viable transgenic plants are selected.","When individual transformation vectors are used, the transgenic plant lines that are produced, expressing either light or heavy chain sequences, are crossed to generate a single plant line expressing both chains in the same plant cell.","When a single transformation vector, containing both light and heavy expression cassettes, is used, the initial transgenic plant line produces both light and heavy chain sequences in the same plant cell.","2—Production of Transgenic Oil Bodies which Display Protein a for the Capture of Immunoglobulins To capture and display immunoglobulin protein on oil bodies, oil bodies are engineered to display an immunoglobulin binding protein.","In this example, the well-known antibody-binding domains from Protein A are used.","Based on the known sequence for Protein A from Staphylococcus aureus, PCR primers are designed to isolate the five consecutive Ig-binding domains from the bacterial Protein A sequence.","Primers are designed to allow cloning of the Protein A sequence as either an N-terminal or C-terminal fusion to an oleosin sequence for targeting to oil bodies.","The sequence that encodes an in-frame translational fusion between Protein A and oleosin is cloned into a plant expression cassette for seed-specific expression.","The final cassette consists of a regulatory promoter sequence that provides expression in seeds, the Protein A-oleosin fusion sequence, and a regulatory sequence to terminate transcription.","The Protein A-oleosin expression cassette is cloned into a plant transformation vector compatible with Agrobacterium-mediated plant transformation.","The transformation vector comprises left and right border sequences flanking the Protein A-oleosin expression cassette and an adjacent plant selectable marker cassette.","The Agrobacterium strain containing this vector is used to infect plant tissues and subsequent regeneration and selection from transgenic plant material to create transgenic plants.","3—Capture and Display of Multimeric-Immunoglobulins on Oil Bodies Displaying Protein A Having produced light and heavy chain multimeric immunoglobulin complexes in one transgenic plant line and the display of Protein A on oil bodies through the oil body targeting of a Protein A-oleosin fusion protein in a second plant line, at least two embodiments can be used to capture the immunoglobulin complex on the Protein A oil bodies.","In the first embodiment, transgenic seed from both the immunoglobulin and the Protein A-oleosin expression lines is combined in an optimum ratio and then ground together such that the disrupted material from both seed lines would be combined in the same extract.","The combined seed extracts are mixed and/or incubated under conditions that allow maximum recovery of the immunoglobulin by Protein A.","The oil body fraction is separated using standard phase separation techniques (e.g.","centrifugation).","The recovered oil body fraction contains both native oil bodies, from the immunoglobulin expression line, and transgenic Protein A oil bodies from the Protein A-oleosin expression line.","In a second embodiment, the plant lines expressing the immunoglobulin complex and the Protein A-oleosin fusion are crossed and individual plant lines expressing both components are identified and propagated.","In this approach, the immunoglobulin complex and the Protein A-oleosin fusion are produced in different cellular compartments of the same plant seed cell.","Seed from the double transgenic line is ground to disrupt the cellular material and mix the contents of all cellular compartments, including combining the immunoglobulin in the extracellular compartment and the Protein A-oleosin on the oil body in the cytosolic compartment.","The material is mixed and/or incubated under conditions to allow maximum recovery of the immunoglobulin by Protein A, and the oil body fraction is separated by phase separation techniques.","The recovered oil body fraction contains the displayed Protein A and the capture immunoglobulin complex.","Example 6 Production of Assembled Multimeric-Immunoglobulin-Complexes as Fusions with Oil Body Targeting Domains Individual polypeptides are produced as a fusion protein with oil body targeting sequences (e.g.","oleosin) for display on oil bodies.","It has been found that the individual subunits of naturally associating heterodimeric proteins can be co-produced as individual oleosin fusions and still associate as an active heterodimer on the surface of the oil body.","In this example, the heterodimer is the light and heavy chain subunits, or derived portions thereof, of an immunoglobulin complex.","Production of an Immunoglobulin Fab Complex on Oil Bodies.","The mature light chain sequence, lacking the secretion signal sequence, is attached as an in-frame N-terminal fusion to an oleosin sequence.","This fusion sequence is assembled into a seed-specific expression cassette consisting of a seed-specific promoter sequence, the light chain-oleosin fusion sequence, and a transcriptional terminator sequence.","The expression cassette is inserted between the left and right border markers, adjacent to a plant selectable marker cassette, of a transformation vector.","The transformation vector, in Agrobacterium, is used to infect plants and generate transgenic plants.","An equivalent construct for the heavy chain subunit, comprising the variable and constant heavy chain domains, is also attached as an in-frame fusion to oleosin and assembled into an expression cassette for seed-specific expression.","The expression cassette can be a part of a separate transformation vector for the generation of a separate transgenic line, or the heavy chain expression cassette can be combined together with the light chain cassette into a single transformation vector.","If light and heavy chain expression cassettes are transformed into plants on separate transformation vectors, the individual plant lines are crossed to create a single line expressing both heterodimer subunit-oleosin fusions in the same plant cell.","Seed from the double transgenic line, or a single transgenic line generated from the dual expression vector, is extracted to isolate oil bodies.","The seed material is ground to release the cellular contents and oil bodies are isolated by phase separation.","The targeting of both light and heavy chain sequence to oil bodies, as oleosin fusions, allows the association of the immunoglobulin complex on the surface of the oil body.","Similar configurations, using the entire heavy chain sequence in combination with the entire light chain sequence, or using the variable domains from both the light and heavy chain sequences, are constructed to assemble different types of heteromultimeric-immunoglobulin-complexes (e.g., heterodimers) on the surface of oil bodies.","The present invention should therefore not be seen as limited to the particular embodiments described herein, but rather, it should be understood that the present invention has wide applicability with respect to protein expression generally.","Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.","SUMMARY OF SEQUENCES SEQ ID NOs:1-4 set forth primers which were synthesized for the isolation of the thioredoxin h (Trxh) and thioredoxin reductase genes from Arabidopsis, as described in Example 1.SEQ ID NOs:5-7 set forth primers which were designed to code for a specific linker peptide between thioredoxin reductase and thioredoxin proteins, as described in Example 2.SEQ ID NOs:8, 10 and 11 set forth the nucleotide sequence and the deduced amino acid sequence of the NADPH thioredoxin reductase sequence isolated herein as described in Example 1.SEQ ID NOs:9 and 11, respectively, set forth the nucleotide sequence of the published NADPH thioredoxin reductase sequence (ATTHIREDB) and the deduced amino acid sequence.","SEQ ID NO:12 sets forth the deduced amino acid sequence of the published NADPH thioredoxin reductase sequence.","SEQ ID NO:13 sets forth the deduced amino acid sequence of the NADPH reductase sequence isolated in this report.","SEQ ID NOs:14 and 15 set forth the nucleotide sequence of the phaseolin promoter-Arabidopsis Trxh-phaseolin terminator sequence described in Example 2, and the deduced amino acid sequence.","The Trxh coding sequence and its deduced amino acid sequence is indicated.","The phaseolin promoter corresponds to nucleotide 6-1554, and the phaseolin terminator corresponds to nucleotide sequence 1905-3124.The promoter was furnished with a PstI site (nt 1-6) and the terminator was furnished with a HindIII site (nt 1898-1903) and a KpnI site (nt 3124-3129) to facilitate cloning.","SEQ ID NOs:16, 17 and 18 set forth the nucleotide sequence of the phaseolin promoter-oleosin Trxh-phaseolin terminator sequence described in Example 2, and the deduced amino acid sequences.","The oleosin-Trxh coding sequence and the deduced amino acid sequences are indicated in SEQ ID NO:16.As in SEQ ID NO:14, the phaseolin promoter corresponds to nucleotide 6-1554.The sequence encoding oleosin corresponds to nt 1555-2313, the intron in this sequence (nt 1908-2147) is indicated in italics.","The Trxh coding sequence corresponds to nt 2314-2658.The phaseolin terminator corresponds to nucleotide sequence 2664-3884.SEQ ID NO:19, 20 and 21 set forth the nucleotide sequence of the phaseolin promoter-Trxh oleosin-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.","The Trxh oleosin-coding sequence and its deduced amino acid sequences are indicated in SEQ ID NO:19.As in SEQ ID NOs:14 and 16, the phaseolin promoter corresponds to nucleotide 6-1554.The Trxh coding sequence corresponds to nt 1555-1896.The sequence encoding oleosin corresponds to nt 1897-2658, the intron in this sequence (nt 2250-2489) is indicated in italics.","The phaseolin terminator corresponds to nucleotide sequence 2664-3884.SEQ ID NO:22 and 23 set forth the nucleotide sequence of the phaseolin promoter-thioredoxin-reductase-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequence.","The thioredoxin-reductase coding sequence and its deduced amino acid sequence is indicated in SEQ ID NO:22.The phaseolin promoter corresponds to nucleotide 6-1554.The thioredoxin-reductase coding sequence corresponds to nt 1555-2556 and the deduced amino acid is set forth in SEQ ID NO:23.The phaseolin terminator corresponds to nucleotide sequence 2563-3782.SEQ ID NOs:24, 25 and 26 show the nucleotide sequence of the phaseolin promoter-oleosin thioredoxin-reductase-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.","The oleosin-thioredoxin-reductase coding sequence and its deduced amino acid sequence is indicated.","The phaseolin promoter corresponds to nucleotide 6-1554.The sequence encoding oleosin corresponds to nt 1555-2313, the intron in this sequence (nt 1980-2147) is indicated in italics.","The thioredoxin-reductase coding sequence corresponds to nt 2314-3315.The phaseolin terminator corresponds to nucleotide sequence 3321-4540.SEQ ID NOs:27, 28 and 29 show the nucleotide sequence of the phaseolin promoter-thioredoxin-reductase oleosin-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.","The thioredoxin-reductase coding sequence and its deduced amino acid sequence is indicated.","The phaseolin promoter corresponds to nucleotide 6-1554.The thioredoxin-reductase coding sequence corresponds to nt 1555-2553.The sequence encoding oleosin corresponds to nt 2554-3315, the intron in this sequence (nt 2751-3146) is indicated in italics.","The phaseolin terminator corresponds to nucleotide sequence 3321-4540.SEQ ID NO:30, 31 and 32 show the sequence of the phaseolin promoter-oleosin-Mlep thioredoxin-reductase/thioredoxin-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.","The oleosin-Mlep thioredoxin-reductase/thioredoxin coding sequence and its deduced amino acid sequence is indicated.","The phaseolin promoter corresponds to nucleotide 6-1554.The sequence encoding oleosin corresponds to nt 1555-2313, the intron in this sequence (nt) is indicated in italics.","The Mlep thioredoxin-reductase/thioredoxin coding sequence corresponds to nt 2314-3690.The phaseolin terminator corresponds to nucleotide sequence 3698-4917.SEQ ID NOs:33, 34 and 35 set forth the nucleotide sequence of the phaseolin promoter-oleosin-thioredoxin-reductase-linker-thioredoxin-phaseolin terminator region of pSBS2542, and the deduced amino acid sequences.","The deduced amino acid sequence of oleosin-thioredoxin-reductase-linker-thioredoxin is also shown in SEQ ID NO:33.Amino acids representing oleosin are set forth at positions 1-173, those amino acids representing thioredoxin-reductase are set forth at positions 174-501, those amino acids representing the linker or spacer peptide are set forth at positions 501-524, and those representing thioredoxin are set forth at positions 525-636.SEQ ID NOs:38 and 39 set forth the nucleotide sequence of Arabidopsis Thaliana Thioredoxin h (Trx h 1) and the encoded protein, respectively.","SEQ ID NOs:40 and 41 set forth the nucleotide sequence of Arabidopsis Thaliana Thioredoxin Reductase (NTR1) and the encoded protein, respectively.","SEQ ID NOs:42 and 43 set forth the nucleotide sequence of E. Coli Thioredoxin (TrxA) and the encoded protein, respectively.","SEQ ID NOs:44 and 45, set forth the nucleotide sequence of E. Coli Thioredoxin Reductase and the encoded protein, respectively.","SEQ ID NOs:46 and 47 set forth the nucleotide sequence of Human Thioredoxin and the encoded protein, respectively.","SEQ ID NOs:48 and 49, set forth the nucleotide sequence of Human Thioredoxin Reductase and the encoded protein, respectively.","SEQ ID NOs:50 and 51, respectively, set forth the nucleotide sequence of Mycobacterium leprae Thioredoxin-Thioredoxin Reductase and the encoded protein, respectively.","SEQ ID NOs:52-313 are described in Table 5.TABLE 5 EXAMPLES OF REDOX PROTEINS SWISS PROTEIN IDENTIFIER SEQ.","ID NO.","(in parenthesis) PLANT THIOREDOXINS Thioredoxin f-type 52 (Q9XFH8) Thioredoxin F-type 1, chloroplast precursor (TRX- F1).","- Arabidopsis thaliana (Mouse-ear cress) 53 (Q9XFH9) Thioredoxin F-type 2, chloroplast precursor (TRX- F2).","{GENE: AT5G16400 OR MQK4.13} - Arabidopsis thaliana (Mouse-ear cress) 54 (O48897) Thioredoxin F-type, chloroplast precursor (TRX-F).","{GENE: TRXF} - Brassica napus (Rape) 55 (O81332) Thioredoxin F-type, chloroplast precursor (TRX-F).","- Mesembryanthemum crystallinum (Common ice plant) 56 (P29450) Thioredoxin F-type, chloroplast precursor (TRX-F).","- Pisum sativum (Garden pea) 57 (P09856) Thioredoxin F-type, chloroplast precursor (TRX-F).","- Spinacia oleracea (Spinach) Thioredoxin m-type 58 (P06544) Thioredoxin 1 (TRX-1) (Thioredoxin M).","{GENE: TRXA} - Anabaena sp.","(strain PCC 7119) 59 (O48737) Thioredoxin M-type 1, chloroplast precursor (TRX- M1).","{GENE: AT1G03680 OR F21B7_7 OR F21B7.28} - Arabidopsis thaliana (Mouse-ear cress) 60 (Q9SEU8) Thioredoxin M-type 2, chloroplast precursor (TRX- M2).","{GENE: AT4G03520 OR F9H3.15 OR T5L23.1} - Arabidopsis thaliana (Mouse-ear cress) 61 (Q9SEU7) Thioredoxin M-type 3, chloroplast precursor (TRX- M3).","{GENE: AT2G15570 OR F9O13.12} - Arabidopsis thaliana (Mouse-ear cress) 62 (Q9SEU6) Thioredoxin M-type 4, chloroplast precursor (TRX- M4).","- Arabidopsis thaliana (Mouse-ear cress) 63 (Q9XGS0) Thioredoxin M-type, chloroplast precursor (TRX-M).","- Brassica napus (Rape) 64 (P23400) Thioredoxin M-type, chloroplast precursor (TRX-M) (Thioredoxin CH2).","{GENE: TRXM} - Chlamydomonas reinhardtii 65 (Q41864) Thioredoxin M-type, chloroplast precursor (TRX-M).","{GENE: TRM1} - Zea mays (Maize) 66 (Q9ZP20) Thioredoxin M-type, chloroplast precursor (TRX-M).","- Oryza sativa (Rice) 67 (P48384) Thioredoxin M-type, chloroplast precursor (TRX-M).","- Pisum sativum (Garden pea) 68 (P07591) Thioredoxin M-type, chloroplast precursor (TRX-M).","- Spinacia oleracea (Spinach) 69 (Q9ZP21) Thioredoxin M-type, chloroplast precursor (TRX-M).","- Triticum aestivum (Wheat) 70 (P12243) Thioredoxin 1 (TRX-1) (Thioredoxin M).","{GENE: TRXA OR TRXM} - Synechococcus sp.","(strain PCC 7942) (Anacystis nidulans R2) 71 (P37395) Thioredoxin.","{GENE: TRXA OR TRX} - Cyanidium caldarium [Chloroplast] 72 (O22022) Thioredoxin.","{GENE: TRXA OR TRXM} - Cyanidioschyzon merolae [Chloroplast] 73 (P50338) Thioredoxin.","{GENE: TRXA} - Griffithsia pacifica [Chloroplast] 74 (P50254) Thioredoxin.","{GENE: TRXA} - Porphyra yezoensis [Chloroplast] 75 (P51225) Thioredoxin.","{GENE: TRXA} - Porphyra purpurea [Chloroplast] Thioredoxin h-type 76 (P29448) Thioredoxin H-type 1 (TRX-H-1).","{GENE: TRX1 OR AT3G51030 OR F24M12.70} - Arabidopsis thaliana (Mouse- ear cress) 77 (P20857) Thioredoxin 2 (TRX-2).","{GENE: TRXB} - Anabaena sp.","(strain PCC 7120) 78 (Q42388) Thioredoxin H-type 1 (TRX-H-1) (Pollen coat protein).","{GENE: THL-1 OR BOPC17} - Brassica napus (Rape), Brassica oleracea (Cauliflower) 79 (P29449) Thioredoxin H-type 1 (TRX-H1).","- Nicotiana tabacum (Common tobacco) 80 (Q38879) Thioredoxin H-type 2 (TRX-H-2).","{GENE: TRX2 OR AT5G39950 OR MYH19.14} - Arabidopsis thaliana (Mouse-ear cress) 81 (Q39362) Thioredoxin H-type 2 (TRX-H-2).","{GENE: THL-2} - Brassica napus (Rape) 82 (Q07090) Thioredoxin H-type 2 (TRX-H2).","- Nicotiana tabacum (Common tobacco) 83 (Q42403) Thioredoxin H-type 3 (TRX-H-3).","{GENE: TRX3 OR AT5G42980 OR MBD2.18} - Arabidopsis thaliana (Mouse-ear cress) 84 (Q39239) Thioredoxin H-type 4 (TRX-H-4).","{GENE: TRX4} - Arabidopsis thaliana (Mouse-ear cress) 85 (Q39241) Thioredoxin H-type 5 (TRX-H-5).","{GENE: TRX5} - Arabidopsis thaliana (Mouse-ear cress) 86 (O64432) Thioredoxin H-type (TRX-H).","{GENE: PEC-2} - Brassica rapa (Turnip) 87 (P80028) Thioredoxin H-type (TRX-H) (Thioredoxin CH1).","{GENE: TRXH} - Chlamydomonas reinhardtii 88 (Q96419) Thioredoxin H-type (TRX-H).","- Fagopyrum esculentum (Common buckwheat) 89 (Q42443) Thioredoxin H-type (TRX-H) (Phloem sap 13 kDa protein-1).","- Oryza sativa (Rice) 90 (O65049) Thioredoxin H-type (TRX-H).","{GENE: SB09} - Picea mariana (Black spruce) 91 (Q43636) Thioredoxin H-type (TRX-H).","- Ricinus communis (Castor bean) 92 (O64394) Thioredoxin H-type (TRX-H) (TrxTa).","- Triticum aestivum (Wheat) 93 (P29429) Thioredoxin.","- Emericella nidulans (Aspergillus nidulans) VIRUSES, BACTERIA AND FUNGI THIOREDOXINS 94 (P80579) Thioredoxin (TRX).","{GENE: TRXA} - Alicyclobacillus acidocaldarius (Bacillus acidocaldarius) 95 (O28137) Thioredoxin.","{GENE: AF2145} - Archaeoglobus fulgidus 96 (P14949) Thioredoxin (TRX).","{GENE: TRXA OR TRX} - Bacillus subtilis 97 (P00276) Thioredoxin.","{GENE: NRDC} - Bacteriophage T4 98 (O51088) Thioredoxin (TRX).","{GENE: TRXA OR BB0061} - Borrelia burgdorferi (Lyme disease spirochete) 99 (P57653) Thioredoxin (TRX).","{GENE: TRXA OR BU597} - Buchnera aphidicola (subsp.","Acyrthosiphon pisum) (Acyrthosiphon pisum symbiotic bacterium) 100 (O51890) Thioredoxin (TRX).","{GENE: TRXA} - Buchnera aphidicola (subsp.","Schizaphis graminum) 101 (P10472) Thioredoxin (TRX).","{GENE: TRXA} - Chlorobium limicola f.sp.","thiosulfatophilum 102 (Q9PJK3) Thioredoxin (TRX).","{GENE: TRXA OR TC0826} - Chlamydia muridarum 103 (Q9Z7P5) Thioredoxin (TRX).","{GENE: TRXA OR CPN0659 OR CP0088} - Chlamydia pneumoniae (Chlamydophila pneumoniae) 104 (P52227) Thioredoxin (TRX).","{GENE: TRXA} - Chlamydia psittaci (Chlamydophila psittaci) 105 (O84544) Thioredoxin (TRX).","{GENE: TRXA OR CT539} - Chlamydia trachomatis 106 (P00275) Thioredoxin C-1.- Corynebacterium nephridii 107 (P07887) Thioredoxin C-2.- Corynebacterium nephridii 108 (P52228) Thioredoxin C-3.- Corynebacterium nephridii 109 (P09857) Thioredoxin (TRX).","{GENE: TRXA} - Chromatium vinosum 110 (P21609) Thioredoxin (TRX).","{GENE: TRXA} - Clostridium litorale (Bacterium W6) 111 (P81108) Thioredoxin (TRX) (Fragment).","{GENE: TRXA} - Clostridium sporogenes 112 (P81109) Thioredoxin (TRX) (Fragment).","{GENE: TRXA} - Clostridium sticklandii 113 (Q9UW02) Thioredoxin (Allergen Cop c 2).","- Coprinus comatus (Shaggy mane) 114 (P29445) Thioredoxin 1.","{GENE: TRXA OR TRX1} - Dictyostelium discoideum (Slime mold) 115 (P29446) Thioredoxin 2 (Fragment).","{GENE: TRXB OR TRX2} - Dictyostelium discoideum (Slime mold) 116 (P29447) Thioredoxin 3.","{GENE: TRXC OR TRX3} - Dictyostelium discoideum (Slime mold) 117 (P00274) Thioredoxin 1 (TRX1) (TRX).","{GENE: TRXA OR TSNC OR FIPA OR B3781} - Escherichia coli, Salmonella typhimurium 118 (P52232) Thioredoxin-like protein SLR0233.","{GENE: SLR0233} - Synechocystis sp.","(strain PCC 6803) 119 (P33636) Thioredoxin 2 (Trx2).","{GENE: TRXC OR B2582 OR Z3867 OR ECS3448} - Escherichia coli, Escherichia coli O157:H7 120 (P21610) Thioredoxin (TRX).","{GENE: TRXA} - Eubacterium acidaminophilum 121 (P43785) Thioredoxin (TRX).","{GENE: TRXA OR TRXM OR HI0084} - Haemophilus influenzae 122 (P43787) Thioredoxin-like protein HI1115.","{GENE: HI1115} - Haemophilus influenzae 123 (P56430) Thioredoxin (TRX).","{GENE: TRXA OR HP0824 OR JHP0763} - Helicobacter pylori (Campylobacter pylori), Helicobacter pylori J99 (Campylobacter pylori J99) 124 (Q9S386) Thioredoxin (EC 1.6.4.5) {GENE: TRXA} - Listeria monocytogenes 125 (Q57755) Thioredoxin.","{GENE: TRX OR MJ0307} - Methanococcus jannaschii 126 (P47370) Thioredoxin (TRX).","{GENE: TRXA OR TRX OR MG124} - Mycoplasma genitalium 127 (P46843) Bifunctional thioredoxin-reductase/thioredoxin [Includes: Thioredoxin-reductase (EC 1.6.4.5) (TRXR); Thioredoxin].","{GENE: TRXB/A OR TRX OR ML2703} - Mycobacterium leprae 128 (P75512) Thioredoxin (TRX).","{GENE: TRXA OR TRX OR MPN263 OR MP570} - Mycoplasma pneumoniae 129 (O30974) Thioredoxin (TRX).","{GENE: TRXA} - Mycobacterium smegmatis 130 (P52229) Thioredoxin (TRX) (MPT46).","{GENE: TRXA OR TRX OR TRXC OR RV3914 OR MT4033 OR MTV028.05} - Mycobacterium tuberculosis 131 (P42115) Thioredoxin.","{GENE: TRX} - Neurospora crassa 132 (P34723) Thioredoxin.","{GENE: TRXA} - Penicillium chrysogenum 133 (Q9X2T1) Thioredoxin (TRX).","{GENE: TRXA OR TRX OR PA5240} - Pseudomonas aeruginosa 134 (P10473) Thioredoxin (TRX).","{GENE: TRXA} - Rhodospirillum rubrum 135 (P08058) Thioredoxin (TRX).","{GENE: TRXA} - Rhodobacter sphaeroides (Rhodopseudomonas sphaeroides) 136 (Q9ZEE0) Thioredoxin (TRX).","{GENE: TRXA OR RP002} - Rickettsia prowazekii 137 (P33791) Thioredoxin (TRX) (Fragment).","{GENE: TRXA} - Streptomyces aureofaciens 138 (P52230) Thioredoxin (TRX).","{GENE: TRXA OR SCH24.11C} - Streptomyces coelicolor 139 (Q05739) Thioredoxin (TRX).","{GENE: TRXA} - Streptomyces clavuligerus 140 (P52231) Thioredoxin (TRX).","{GENE: TRXA OR SLR0623} - Synechocystis sp.","(strain PCC 6803) 141 (P73263) Thioredoxin-like protein SLR1139.","{GENE: SLR1139} - Synechocystis sp.","(strain PCC 6803) 142 (P52233) Thioredoxin (TRX).","{GENE: TRXA} - Thiobacillus ferrooxidans 143 (P96132) Thioredoxin (TRX) (Fragment).","{GENE: TRXA} - Thiocapsa roseopersicina 144 (P81110) Thioredoxin (TRX) (Fragment).","{GENE: TRXA} - Tissierella creatinophila 145 (O83889) Thioredoxin (TRX).","{GENE: TRXA OR TP0919} - Treponema pallidum ANIMAL THIOREDOXIN 146 (O97680) Thioredoxin.","{GENE: TXN} - Bos taurus (Bovine) 147 (Q95108) Thioredoxin, mitochondrial precursor (MT-TRX).","{GENE: TXN2} - Bos taurus (Bovine) 148 (Q09433) Thioredoxin.","{GENE: B0228.5} - Caenorhabditis elegans 149 (P99505) Thioredoxin (Fragment).","{GENE: TXN} - Canis familiaris (Dog 150 (P08629) Thioredoxin.","{GENE: TXN} - Gallus gallus (Chicken) 151 (P47938) Thioredoxin (Deadhead protein).","{GENE: DHD OR CG4193} - Drosophila melanogaster (Fruit fly) 152 (P10599) Thioredoxin (ATL-derived factor) (ADF) (Surface associated sulphydryl protein) (SASP).","{GENE: TXN OR TRDX OR TRX} - Homo sapiens (Human) 153 (Q99757) Thioredoxin, mitochondrial precursor (MT-TRX).","{GENE: TXN2} - Homo sapiens (Human) 154 (P29451) Thioredoxin.","{GENE: TXN} - Macaca mulatta (Rhesus macaque) 155 (P10639) Thioredoxin (ATL-derived factor) (ADF).","{GENE: TXN} - Mus musculus (Mouse) 156 (P97493) Thioredoxin, mitochondrial precursor (MT-TRX).","{GENE: TXN2} - Mus musculus (Mouse) 157 (P82460) Thioredoxin (Fragment).","{GENE: TXN} - Sus scrofa (Pig) 158 (P08628) Thioredoxin.","{GENE: TXN} - Oryctolagus cuniculus (Rabbit) 159 (P11232) Thioredoxin.","{GENE: TXN} - Rattus norvegicus (Rat) 160 (P97615) Thioredoxin, mitochondrial precursor (MT-TRX).","{GENE: TXN2 OR TRX2} - Rattus norvegicus (Rat) 161 (P50413) Thioredoxin.","{GENE: TXN} - Ovis aries (Sheep) PLANTS THIOREDOXIN-LIKE PROTEINS 162 (O23166) THIOL-DISULFIDE INTERCHANGE LIKE PROTEIN (THIOREDOXIN-LIKE PROTEIN) {GENE: C7A10.160 OR AT4G37200 OR HCF164} - Arabidopsis thaliana (Mouse-ear cress) 163 (Q9C9Y6) Thioredoxin-like protein {GENE: F17O14.18} - Arabidopsis thaliana (Mouse-ear cress) 164 (Q9FYD5) Thioredoxin-like protein {GENE: F21E1_180} - Arabidopsis thaliana (Mouse-ear cress) 165 (Q38878) THIOREDOXIN-LIKE PROTEIN {GENE: TRX6 OR T7D17.3} - Arabidopsis thaliana (Mouse-ear cress) 166 (Q9LVI2) Thioredoxin-like protein - Arabidopsis thaliana (Mouse-ear cress) 167 (Q9SCN9) Thioredoxin-like protein {GENE: T4D2.150} - Arabidopsis thaliana (Mouse-ear cress) 168 (Q9SRD7) Thioredoxin-like protein, 49720-48645 {GENE: F28O16.13} - Arabidopsis thaliana (Mouse-ear cress) 169 (Q9SU84) THIOREDOXIN-LIKE PROTEIN {GENE: T16L4.180 OR AT4G29670} - Arabidopsis thaliana (Mouse-ear cress) 170 (Q9SWG6) Thioredoxin-like protein {GENE: TRX} - Hordeum bulbosum 171 (Q9SWG4) Thioredoxin-like protein {GENE: TRX} - Lolium perenne (Perennial ryegrass) 172 (Q9AS75) Thioredoxin-like protein {GENE: P0028E10.17} - Oryza sativa (Rice) 173 (O04002) CDSP32 protein (Chloroplast Drought-induced Stress Protein of 32 kDa) - Solanum tuberosum (Potato) 174 (Q9SWG5) Thioredoxin-like protein {GENE: TRX} - Secale cereale (Rye) 175 (Q9SP36) Thioredoxin-like protein (Fragment) {GENE: TRX} - Secale cereale (Rye) 176 (Q9U515) Thioredoxin-like protein - Manduca sexta (Tobacco hawkmoth) (Tobacco hornworm) VIRUSES, BACTERIA AND FUNGI THIOREDOXIN-LIKE PROTEINS 177 (P43221) Thiol: disulfide interchange protein tlpA (Cytochrome c biogenesis protein tIpA).","{GENE: TLPA} - Bradyrhizobium japonicum 178 (P43787) Thioredoxin-like protein HI1115.","{GENE: HI1115} - Haemophilus influenzae 179 (Q9GUP7) Thioredoxin-like protein {GENE: TRXLP} - Leishmania major 180 (Q9UVH0) Thioredoxin-like protein - Mortierella alpina 181 (P95355) Thioredoxin-like protein - Neisseria gonorrhoeae 182 (Q98G37) Thioredoxin-like protein {GENE: MLL3505} - Rhizobium loti (Mesorhizobium loti) 183 (P36893) Thiol: disulfide interchange protein helX precursor (Cytochrome c biogenesis protein helX).","{GENE: HELX} - Rhodobacter capsulatus (Rhodopseudomonas capsulata) 184 (P52232) Thioredoxin-like protein SLR0233.","{GENE: SLR0233} - Synechocystis sp.","(strain PCC 6803) 185 (P73263) Thioredoxin-like protein SLR1139.","{GENE: SLR1139} - Synechocystis sp.","(strain PCC 6803) 186 (Q9USR1) Thioredoxin-like protein {GENE: SPBC577.08C} - Schizosaccharomyces pombe (Fission yeast) 187 (Q9R788) Thioredoxin {GENE: TPTRX} - Treponema pallidum ANIMALS THIOREDOXIN-LIKE PROTEINS 188 (Q9UAV4) F46E10.9 PROTEIN (THIOREDOXIN-LIKE PROTEIN DPY-11) {GENE: F46E10.9 OR DPY-11} - Caenorhabditis elegans 189 (Q9N2K6) Thioredoxin-like protein (Y54E10A.3 protein) (Thioredoxin-like protein TXL) {GENE: TXL OR Y54E10A.3} - Caenorhabditis elegans 190 (Q9VRP3) THIOREDOXIN-LIKE PROTEIN TXL (CG5495 PROTEIN) {GENE: TXL OR CG5495} - Drosophila melanogaster (Fruit fly) 191 (O43396) Thioredoxin-like protein (32 kDa thioredoxin-related protein).","{GENE: TXNL OR TRP32 OR TXL} - Homo sapiens (Human) 192 (O76003) Thioredoxin-like protein - Homo sapiens (Human) 193 (Q9S753) THIOREDOXIN-LIKE PROTEIN {GENE: TRX} - Phalaris coerulescens 194 (O77404) TRYPAREDOXIN - Trypanosoma brucei brucei PLANT THIOREDOXIN-REDUCTASES 195 (Q39243) Thioredoxin-reductase 1 (EC 1.6.4.5) (NADPH- dependent thioredoxin-reductase 1) (NTR 1).","{GENE: NTR1 OR AT4G35460 OR F15J1.30} - Arabidopsis thaliana (Mouse-ear cress) 196 (Q39242) Thioredoxin-reductase 2 (EC 1.6.4.5) (NADPH- dependent thioredoxin-reductase 2) (NTR 2).","{GENE: NTR2 OR AT2G17420 OR F5J6.18} - Arabidopsis thaliana (Mouse-ear cress) VIRUSES, BACTERIA AND FUNGI THIOREDOXIN-REDUCTASES 197 (O66790) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR AQ_500} - Aquifex aeolicus 198 (P80880) Thioredoxin-reductase (EC 1.6.4.5) (TRXR) (General stress protein 35) (GSP35).","{GENE: TRXB} - Bacillus subtilis 199 (P94284) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR BB0515} - Borrelia burgdorferi (Lyme disease spirochete) 200 (P57399) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR BU314} - Buchnera aphidicola (subsp.","Acyrthosiphon pisum) (Acyrthosiphon pisum symbiotic bacterium) 201 (P81433) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB} - Buchnera aphidicola (subsp.","Schizaphis graminum) 202 (Q9PKT7) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR TC0375} - Chlamydia muridarum 203 (Q9Z8M4) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR CPN0314 OR CP0444} - Chiamydia pneumoniae (Chlamydophila pneumoniae) 204 (O84101) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR CT099} - Chlamydia trachomatis 205 (P52213) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB} - Clostridium litorale (Bacterium W6) 206 (P39916) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB} - Coxiella burnetii 207 (P09625) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR B0888 OR Z1232 OR ECSO973} - Escherichia coli, Escherichia coli O157: H7 208 (P50971) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB} - Eubacterium acidaminophilum 209 (P43788) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR HI1158} - Haemophilus influenzae 210 (Q9ZL18) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR JHP0764} - Helicobacter pylori J99 (Campylobacter pylori J99) 211 (P56431) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR HP0825} - Helicobacter pylori (Campylobacter pylori) 212 (O32823) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR LMO2478} - Listeria monocytogenes 213 (P47348) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR MG102} - Mycoplasma genitalium 214 (P46843) Bifunctional thioredoxin-reductase/thioredoxin [Includes: Thioredoxin-reductase (EC 1.6.4.5) (TRXR); Thioredoxin].","{GENE: TRXB/A OR TRX OR ML2703} - Mycobacterium leprae 215 (P75531) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR MPN240 OR MP591} - Mycoplasma pneumoniae 216 (O30973) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB} - Mycobacterium smegmatis 217 (P52214) Thioredoxin-reductase (EC 1.6.4.5) (TRXR) (TR).","{GENE: TRXB OR RV3913 OR MT4032 OR MTV028.04} - Mycobacterium tuberculosis 218 (P51978) Thioredoxin-reductase (EC 1.6.4.5).","{GENE: CYS-9} - Neurospora crassa 219 (P43496) Thioredoxin-reductase (EC 1.6.4.5).","{GENE: TRXB} - Penicillium chrysogenum 220 (Q9ZD97) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR RP445} - Rickettsia prowazekii 221 (Q92375) Thioredoxin-reductase (EC 1.6.4.5).","{GENE: SPBC3F6.03} - Schizosaccharomyces pombe (Fission yeast) 222 (Q05741) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB} - Streptomyces clavuligerus 223 (P52215) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR SCH24.12} - Streptomyces coelicolor 224 (O83790) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR TP0814} - Treponema pallidum 225 (P80892) Thioredoxin-reductase (EC 1.6.4.5) (TRXR) (Fragment).","{GENE: TRXB} - Vibrio fischeri 226 (P29509) Thioredoxin-reductase 1 (EC 1.6.4.5).","{GENE: TRR1 OR YDR353W OR D9476.5} - Saccharomyces cerevisiae (Baker's yeast) 227 (P38816) Thioredoxin-reductase 2, mitochondrial precursor (EC 1.6.4.5).","{GENE: TRR2 OR YHR106W} - Saccharomyces cerevisiae (Baker's yeast) ANIMAL THIOREDOXIN-REDUCTASES 228 (O62768) Thioredoxin-reductase (EC 1.6.4.5).","{GENE: TXNRD1} - Bos taurus (Bovine) 229 (Q17745) Thioredoxin-reductase (EC 1.6.4.5).","{GENE: C06G3.7} - Caenorhabditis elegans 230 (Q16881) Thioredoxin-reductase (EC 1.6.4.5).","{GENE: TXNRD1} - Homo sapiens (Human) 231 (Q25861) Thioredoxin-reductase (EC 1.6.4.5) (TrxR).","{GENE: TR OR GR} - Plasmodium falciparum (isolate FCH-5) Other thioredoxin-reductases PLANTS THIOREDOXIN-REDUCTASES 232 (O22229) Thioredoxin-reductase {GENE: AT2G41680} - Arabidopsis thaliana (Mouse-ear cress) 233 (Q39951) NADPH thioredoxin-reductase (Fragment} - Helianthus annuus (Common sunflower) VIRUSES, BACTERIA AND FUNGI THIOREDOXIN-REDUCTASES 234 (O28718) THioredoxin-reductase (TRXB) {GENE: AF1554} - Archaeoglobus fulgidus 235 (Q9K703) Thioredoxin-reductase (NADPH) (EC 1.6.4.5) {GENE: TRXB OR BH3571} - Bacillus halodurans 236 (Q9K7F3) Thioredoxin-reductase {GENE: BH3408} - Bacillus halodurans 237 (Q9KCZ0) Thioredoxin-reductase {GENE: BH1429} - Bacillus halodurans 238 (Q9KCZ1) Thioredoxin-reductase {GENE: BH1428} - Bacillus halodurans 239 (Q9PIY1) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB OR CJ0146} - Campylobacter jejuni 240 (Q9A4G3) Thioredoxin-reductase {GENE: CC2871} - Caulobacter crescentus 241 (Q97EM8) Thioredoxin-reductase {GENE: CAC3082} - Clostridium acetobutylicum 242 (Q97IU2) Thioredoxin-reductase {GENE: CAC1548} - Clostridium acetobutylicum 243 (Q9EV96) Thioredoxin-reductase {GENE: TRXB} - Clostridium sticklandii 244 (Q9RSY7) THioredoxin-reductase {GENE: DR1982} - Deinococcus radiodurans 245 (O30739) Thioredoxin-reductase (Fragment} - Enterococcus faecalis (Streptococcus faecalis) 246 (O54535) Thioredoxin-reductase {GENE: TRXB OR TRXB1_2 OR VNG6452G OR TRXB1_1 OR VNG6074G} - Halobacterium sp.","(strain NRC-1) [Plasmid pNRC100, and Plasmid pNRC200] 247 (P82854) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB2} - Halobacterium sp.","(strain NRC-1) 248 (Q9HN08) Thioredoxin-reductase {GENE: TXRB3 OR VNG2301G} - Halobacterium sp.","(strain NRC-1) 249 (O25779) THioredoxin-reductase (TRXB) {GENE: HP1164} - Helicobacter pylori (Campylobacter pylori) 250 (O86255) Thioredoxin-reductase {GENE: TRXB} - Klebsiella oxytoca 251 (Q9AEV9) Thioredoxin-reductase (Fragment) {GENE: TRXB} - Lactococcus lactis (subsp.","lactis) (Streptococcus lactis) 252 (Q9CF34) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB2} - Lactococcus lactis (subsp.","lactis) (Streptococcus lactis) 253 (Q9CH02) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB1} - Lactococcus lactis (subsp.","lactis) (Streptococcus lactis) 254 (Q9ZFC8) Thioredoxin-reductase (Fragment) {GENE: TRXB} - Lactococcus lactis 255 (O32822) Hypothetical 39.7 kDa protein (Fragment) - Listeria monocytogenes 256 (O26804) THioredoxin-reductase {GENE: MTH708} - Methanothermobacter thermautotrophicus 257 (P94397) Homologue of thioredoxin-reductase of Mycoplama genitalium {GENE: YCGT} - Bacillus subtilis 258 (Q98PK9) THioredoxin-reductase (EC 1.6.4.5) {GENE: MYPU_7130} - Mycoplasma pulmonis 259 (Q9JU23) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB OR NMA1538} - Neisseria meningitidis (serogroup A) 260 (Q9JZ28) Thioredoxin-reductase {GENE: NMB1324} - Neisseria meningitidis (serogroup B) 261 (Q9I0M2) Thioredoxin-reductase 1 {GENE: TRXB1 OR PA2616} - Pseudomonas aeruginosa 262 (Q9I592) Thioredoxin-reductase 2 {GENE: TRXB2 OR PA0849} - Pseudomonas aeruginosa 263 (Q9V0Q8) THioredoxin-reductase (TRXB) {GENE: TRXB OR PAB0500} - Pyrococcus abyssi 264 (Q9ZD33) THioredoxin-reductase (TRXB2) {GENE: RP514} - Rickettsia prowazekii 265 (O54079) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB} - Staphylococcus aureus 266 (Q9RIS2) Thioredoxin-reductase {GENE: TRXB OR TRXB2} - Streptomyces coelicolor 267 (Q9K4L6) Thioredoxin-reductase {GENE: SC5F8.08C} - Streptomyces coelicolor 268 (Q97PY2) Thioredoxin-reductase {GENE: SP1458} - Streptococcus pneumoniae 269 (Q9A0B5) Thioredoxin-reductase {GENE: SPY0850} - Streptococcus pyogenes 270 (Q97V69) Thioredoxin-reductase (trxB-2) (EC 1.6.4.5) {GENE: TRXB-2} - Sulfolobus solfataricus 271 (Q97W27) Thioredoxin-reductase (trxB-3) (EC 1.6.4.5) {GENE: TRXB-3} - Sulfolobus solfataricus 272 (Q97WJ5) Thioredoxin-reductase (trxB-1) (EC 1.6.4.5) {GENE: TRXB-1} - Sulfolobus solfataricus 273 (Q98I59) Thioredoxin-reductase {GENE: MLL2552} - Rhizobium loti (Mesorhizobium loti) 274 (Q98M06) Thioredoxin-reductase {GENE: MLL0792} - Rhizobium loti (Mesorhizobium loti) 275 (Q9UR80) 35 kDa THioredoxin-reductase HOMOLOG (FRAGMENT) {GENE: TRR1 AND YDR353W} - Saccharomyces cerevisiae (Baker's yeast) 276 (Q9ZEH4) THIOREDOXIN {GENE: TRXA OR SA0992} - Staphylococcus aureus, Staphylococcus aureus subsp.","aureus N315 277 (Q9S1H1) Thioredoxin-reductase (Fragment) {GENE: TRXB} - Staphylococcus xylosus 278 (Q9HJI4) Thioredoxin-reductase {GENE: TA0984} - Thermoplasma acidophilum 279 (Q9WZX3) THioredoxin-reductase {GENE: TM0869} - Thermotoga maritima 280 (Q979K8) Thioredoxin-reductase {GENE: TVG1183005} - Thermoplasma volcanium 281 (Q9PR71) Thioredoxin-reductase {GENE: TRXB OR UU074} - Ureaplasma parvum (Ureaplasma urealyticum biotype 1) 282 (Q9KSS4) Thioredoxin-reductase {GENE: VC1182} - Vibrio cholerae 283 (Q9PDD1) Thioredoxin-reductase {GENE: XF1448} - Xylella fastidiosa 284 (Q9X5F7) Thioredoxin-reductase {GENE: TRXB1} - Zymomonas mobilis ANIMAL THIOREDOXIN-REDUCTASES 285 (Q9GKW9) Thioredoxin-reductase 3 (Fragment) {GENE: TRXR3} - Bos taurus (Bovine) 286 (Q9N2I8) Thioredoxin-reductase (EC 1.6.4.5) - Bos taurus (Bovine) 287 (Q9N2K1) Thioredoxin-reductase homolog - Caenorhabditis elegans 288 (Q9NJH3) Thioredoxin-reductase - Caenorhabditis elegans 289 (Q9VNT5) CG11401 PROTEIN (THioredoxin-reductase 2) {GENE: TRXR-2 OR CG11401} - Drosophila melanogaster (Fruit fly) 290 (O95840) Thioredoxin-reductase - Homo sapiens (Human) 291 (Q9UES8) Thioredoxin-reductase GRIM-12 - Homo sapiens (Human) 292 (Q9UH79) Thioredoxin-reductase {GENE: TR} - Homo sapiens (Human) 293 (Q9UQU8) Thioredoxin-reductase - Homo sapiens (Human) 294 (Q9NNW6) Thioredoxin-reductase TR2 (Fragment) - Homo sapiens (Human) 295 (Q9NNW7) Thioredoxin-reductase TR3 - Homo sapiens (Human) 296 (Q9P101) Thioredoxin-reductase 3 (Fragment) {GENE: TRXR3} - Homo sapiens (Human) 297 (Q9P2Y0) Thioredoxin-reductase II beta (EC 1.6.4.5) - Homo sapiens (Human) 298 (Q9H2Z5) Mitochondrial thioredoxin-reductase {GENE: TRXR2A} - Homo sapiens (Human) 299 (Q99475) KM-102-DERIVED REDUCTASE-LIKE FACTOR (THioredoxin-reductase) - Homo sapiens (Human) 300 (Q99P49) Thioredoxin-reductase 1 {GENE: TXNRD1} - Mus musculus (Mouse) 301 (Q9CSV5) Thioredoxin-reductase 1 (Fragment) {GENE: TXNRD1} - Mus musculus (Mouse) 302 (Q9CZE5) Thioredoxin-reductase 1 {GENE: TXNRD1} - Mus musculus (Mouse) 303 (Q9JHA7) Thioredoxin-reductase TR3 {GENE: TXNRD2 OR TR3} - Mus musculus (Mouse) 304 (Q9JLT4) Thioredoxin-reductase {GENE: TXNRD2 OR TRXR2} - Mus musculus (Mouse) 305 (Q9JMH5) Thioredoxin-reductase 2 {GENE: TXNRD2 OR TXNRD2} - Mus musculus (Mouse) 306 (Q9JMH6) Thioredoxin-reductase 1 {GENE: TXNRD1 OR TXNRD1} - Mus musculus (Mouse) 307 (O89049) Thioredoxin-reductase - Rattus norvegicus (Rat) 308 (Q9JKZ3) Thioredoxin-reductase 1 (Fragment) - Rattus norvegicus (Rat) 309 (Q9JKZ4) Thioredoxin-reductase 1 - Rattus norvegicus (Rat) 310 (Q9JLE6) Thioredoxin-reductase (Fragment) - Rattus norvegicus (Rat) 311 (Q9R1I3) NADPH-dependent thioredoxin-reductase {GENE: TRR1} - Rattus norvegicus (Rat) 312 (Q9Z0J5) Thioredoxin-reductase precursor {GENE: TRXR2} - Rattus norvegicus (Rat) 313 (Q9MYY8) Redox enzyme thioredoxin-reductase - Sus scrofa (Pig)"]],"string":"[\n [\n \"Methods for the production of multimeric proteins and related compositions\",\n \"Improved methods for the production of multimeric-protein-complexes, such as redox proteins and immunoglobins, in association with oil bodies are described.\",\n \"The redox protein is enzymatically active when prepared in association with the oil bodies.\",\n \"Also provided are related nucleic acids, proteins, cells, plants, and compositions.\"\n ],\n [\n \"1-266.\",\n \"(canceled) 267.A method of producing an oil body associated with a recombinant multimeric-protein-complex, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide.\",\n \"268.A method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex; and (d) associating said first recombinant polypeptide with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide.\",\n \"269.A method of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.\",\n \"270.A chimeric nucleic acid sequence encoding a multimeric-fusion-protein, said nucleic acid comprising: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"271.A recombinant multimeric-fusion-protein comprising (i) an oil-body-targeting-protein, or fragment thereof, (ii) a first recombinant polypeptide and a (iii) second recombinant polypeptide, wherein said first and second recombinant polypeptides are capable of forming a multimeric-protein-complex.\",\n \"272.Isolated oil bodies comprising a multimeric-protein-complex comprising (i) an oil-body-targeting-protein and (ii) a first recombinant polypeptide, said oil bodies further comprising a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"273.Isolated oil bodies comprising (a) a first fusion protein comprising a first oil-body-targeting-protein fused to a first recombinant polypeptide; and (b) a second fusion protein comprising a second oil-body-targeting-protein fused to a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"274.A cell comprising oil bodies and (i) an oil-body-targeting-protein, (ii) a first recombinant polypeptide and (iii) a second recombinant polypeptide wherein (1) said first recombinant polypeptide is capable of associating with said oil-body-targeting-protein; and (2) said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form a multimeric-protein-complex.\",\n \"275.A composition comprising isolated oil bodies, thioredoxin and thioredoxin-reductase.\",\n \"276.A food product, personal care product or pharmaceutical composition comprising the composition of claim 275.277.A method of reducing allergenicity of a food comprising the steps of: providing the isolated oil bodies of claim 273; and adding the isolated oil bodies to the food, whereby allergenicity of the food is reduced.\",\n \"278.A method of treating or protecting a target against oxidative stress, comprising the steps of: providing the recombinant fusion polypeptide of claim 269; and contacting the recombinant fusion polypeptide with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.\",\n \"279.A method for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.\",\n \"280.A method for preparing a redox protein associated with oil bodies comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide in a progeny cell comprising oil bodies; and c) isolating from said progeny cell said oil bodies comprising said redox fusion polypeptide.\",\n \"281.A chimeric nucleic acid comprising: 1) a first nucleic acid sequence capable of regulating transcription in a host cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.\",\n \"282.A transgenic plant comprising the chimeric nucleic acid sequence of claim 281.283.A safflower plant comprising the chimeric nucleic acid of anyone of claim 281.284.A plant seed comprising the chimeric nucleic acid of claim 281.285.A safflower seed comprising the chimeric nucleic acid of claim 281.286.An oil body preparation obtained by the method of claim 279.287.A food product comprising an oil body preparation of claim 286.288.A composition comprising an oil body preparation of claim 286 (New).\",\n \"289.A personal care product comprising an oil body preparation of claim 286.290.A product capable of treating oxidative stress in a target comprising an oil body preparation of claim 286.291.A product capable of chemically reducing a target comprising an oil body preparation of claim 286.292.A detergent composition comprising the product of claim 286.293.A method of cleansing an item, comprising administering the product of claim 290 to said item under conditions that promote cleansing.\",\n \"294.An emulsion formulation prepared by the method of claim 279.295.A nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one thioredoxin-related protein or an active fragment thereof.\",\n \"296.The seed of the plant of claim 281.297.An extract of the seed of claim 296, wherein the extract comprises an activity of a thioredoxin-related protein.\",\n \"298.An oil body from the seed of claim 296.299.Oil produced from the seed of claim 296.300.A method of making a fusion protein comprising a thioredoxin-related activity, the method comprising the steps of: providing a transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, the gene fusion encoding a fusion protein comprising a thioredoxin-related activity; obtaining seeds from the plant; and recovering the fusion protein by isolating oil bodies from the seeds.\",\n \"301.Oil bodies in association with a fusion protein, obtained by the method of claim 300.302.A method of reducing allergenicity of a food comprising the steps of: providing a preparation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and adding the preparation to the food, whereby allergenicity of the food is reduced due to activity of the thioredoxin-related protein or fragment.\",\n \"303.A composition comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.\",\n \"304.A cosmetic formulation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in an acceptable carrier.\",\n \"305.A method of treating or protecting a target against oxidative stress, comprising the steps of: providing a preparation comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and contacting the preparation with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.\",\n \"306.A nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one recombinant polypeptide and an oil-body-surface-avoiding linker in frame between the first and second region polypeptides.\"\n ],\n [\n \"<SOH> BACKGROUND <EOH>Multimeric proteins (i.e.\",\n \"proteins comprising multiple polypeptide chains) are a biologically and commercially important class of proteins.\",\n \"Antibodies for example are multimeric proteins which are used to treat a wide range of disease conditions.\",\n \"However in view of their complexity, multimeric proteins frequently represent significant manufacturing challenges.\",\n \"Redox proteins are also a commercially important class of proteins with applications in a variety of different industries including the pharmaceutical, personal care and food industry.\",\n \"For example, the redox protein thioredoxin may be used in the manufacture of personal care products (Japanese Patent Applications JP9012471A2, JP103743A2, JP1129785A2), pharmaceutical compositions/products (Aota et al.\",\n \"(1996) J. Cardiov.\",\n \"Pharmacol.\",\n \"(1996) 27: 727-732) as well as to reduce protein allergens present in food products such as milk (del Val et al.\",\n \"(1999) J. Allerg.\",\n \"Vlin.\",\n \"Immunol.\",\n \"103: 690-697) and wheat (Buchanan et al.\",\n \"(1997) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 94: 5372-5377).\",\n \"However, there is a need in the art to further improve the methods for the recombinant expression of multimeric proteins, including redox proteins.\",\n \"The present invention satisfies this need and provides related advantages as well.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to novel and improved methods of producing a first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulin-polypeptide-chains, immunoglobulins, redox-fusion-polypeptides, and/or thioredoxin-related proteins; in association with oil bodies.\",\n \"Accordingly, provided herein are methods of producing a recombinant multimeric-protein-complex, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.\",\n \"The method further contemplates isolating the oil bodies associated with said recombinant multimeric-protein-complex.\",\n \"The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and said second recombinant polypeptide.\",\n \"Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.\",\n \"The oil-body-targeting-protein can be an oleosin or caleosin.\",\n \"When the oil-body-targeting-protein can be an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.\",\n \"Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.\",\n \"The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.\",\n \"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.\",\n \"In one embodiment, the first recombinant polypeptide is capable of associating with said second recombinant polypeptide in the cell.\",\n \"In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.\",\n \"In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.\",\n \"Also provided herein is a method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex; and (d) associating said first recombinant polypeptide with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.\",\n \"This method further contemplates isolating from the progeny cell, oil bodies comprising the multimeric-protein-complex.\",\n \"The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and second recombinant polypeptide.\",\n \"Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.\",\n \"The oil-body-targeting-protein can be an oleosin or caleosin.\",\n \"When the oil-body-targeting-protein is an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.\",\n \"Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.\",\n \"The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.\",\n \"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.\",\n \"In one embodiment, the first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex in said progeny cell.\",\n \"In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.\",\n \"In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 60 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.\",\n \"Also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.\",\n \"The second recombinant polypeptide can be associated with oil bodies through a second oil-body-targeting-protein in the second plant.\",\n \"The oil bodies can be isolated from the progeny plant comprising said multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, wherein the oil-body-protein can be an oleosin or caleosin.\",\n \"The first recombinant polypeptide can be fused to the oleosin or caleosin; and the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.\",\n \"The first and second recombinant polypeptide can form a multimeric-protein-complex, such as a heteromultimeric-protein-complex, wherein the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The plant can be a safflower plant.\",\n \"Also provided herein are chimeric nucleic acids encoding a multimeric-fusion-protein as described herein, said nucleic acid comprising: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin.\",\n \"The oil-body-protein can be an oleosin or caleosin.\",\n \"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the first and second recombinant polypeptide can form an enzymatically active heteromultimeric redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence that is also a non-proteolytic linker and said sequence encoding the first recombinant polypeptide.\",\n \"Also provided herein are recombinant multimeric-fusion-proteins comprising (i) an oil-body-targeting-protein, or fragment thereof, (ii) a first recombinant polypeptide and a (iii) second recombinant polypeptide, wherein said first and second recombinant polypeptides are capable of forming a multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.\",\n \"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said sequence encoding the first recombinant polypeptide.\",\n \"Also provided herein are isolated oil bodies comprising a multimeric-protein-complex comprising (i) an oil-body-targeting-protein and (ii) a first recombinant polypeptide, said oil bodies further comprising a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.\",\n \"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are isolated oil bodies comprising (a) a first fusion protein comprising a first oil-body-targeting-protein fused to a first recombinant polypeptide; and (b) a second fusion protein comprising a second oil-body-targeting-protein fused to a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.\",\n \"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are cells and transgenic plants comprising oil bodies, multimeric-protein-complexes, and multimeric-fusion-proteins, set forth herein.\",\n \"In one embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In embodiments, wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase, the methods described herein can be used to formulate the oil bodies for use in the preparation of a food product, personal care product or pharmaceutical composition.\",\n \"These formulations can further comprise the addition of NADP or NADPH.\",\n \"The food product can be a milk or wheat based food product.\",\n \"The personal care product can reduce the oxidative stress to the surface area of the human body or can be used to lighten the skin.\",\n \"The pharmaceutical composition can be used to treat chronic obstructive pulmonary disease (COPD), cataracts, diabetes, envenomation, bronchiopulmonary disease, malignancies, psoriasis, reperfusion injury, wound healing, sepsis, GI bleeding, intestinal bowel disease (IBD), ulcers, GERD (gastro esophageal reflux disease).\",\n \"Also provided herein are compositions comprising isolated oil bodies, thioredoxin and thioredoxin-reductase, wherein said thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194, and said thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.The composition can further comprise NADP or NADPH.\",\n \"In another embodiment, the composition comprises a first recombinant polypeptide that can be an immunoglobulin-polypeptide-chain and a second recombinant polypeptide.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are multimeric-fusion-proteins, wherein the fusion-protein contains two or more polypeptide chains selected from the group of proteins set forth in FIG.\",\n \"5 .\",\n \"Methods are also provided of reducing allergenicity of a food comprising the steps of providing the isolated oil bodies set forth herein; and adding the isolated oil bodies to the food, whereby allergenicity of the food is reduced.\",\n \"The food can be selected from the group consisting of wheat flour, wheat dough, milk, cheese, yogurt and ice cream.\",\n \"The various methods of treating food can further comprise providing NADH as a co-factor in the substantial absence of NADPH.\",\n \"Also provided herein are methods of treating or protecting a target against oxidative stress, comprising the steps of providing the recombinant redox fusion polypeptide comprising thioredoxin and thioredoxin-reductase; and contacting the recombinant fusion polypeptide with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.\",\n \"The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.\",\n \"Also provided herein are methods for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.\",\n \"The first redox protein can be a thioredoxin and the second redox protein can be a thioredoxin-reductase.\",\n \"Also, provided herein are methods of producing an immunoglobulin, said method comprising: (a) producing in a cell comprising oil bodies, a first immunoglobulin-polypeptide-chain and a second immunoglobulin-polypeptide-chain wherein said first immunoglobulin-polypeptide-chain is capable of associating with said second immunoglobulin-polypeptide-chain to form said immunoglobulin; and (b) associating said immunoglobulin with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first immunoglobulin-polypeptide-chain.\",\n \"For example, the first immunoglobulin-polypeptide-chain can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second immunoglobulin-polypeptide-chain can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are methods for preparing a redox protein or an immunoglobulin associated with oil bodies comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein, or a nucleic acid sequence encoding a immunoglobulin comprising a first immunoglobulin-polypeptide-chain linked to a second immunoglobulin-polypeptide-chain, operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide or immunoglobulin in a progeny cell comprising oil bodies; and c) isolating from said progeny cell said oil bodies comprising said redox fusion polypeptide or immunoglobulin.\",\n \"In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide or immunoglobulin can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged or have a molecular weight of at least 35 kd.\",\n \"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.\",\n \"In this optional embodiment, also contemplated is the introduction of an enzyme or chemical that cleaves said redox fusion polypeptide from said oil body, thereby obtaining isolated redox fusion polypeptide.\",\n \"The first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.\",\n \"In one embodiment, the thioredoxin and thioredoxin-reductase can be obtained from Arabidopsis .\",\n \"In another embodiment, the first redox protein is at least 5 times more active when produced as a redox fusion polypeptide as compared to the production of the first redox protein without the second redox protein.\",\n \"Also provided herein, for use with the various methods set forth herein is the formulation of an emulsion of the oil bodies associated with the redox fusion polypeptide for use in the preparation of a product capable of treating oxidative stress in a target, a product capable of chemically reducing a target, pharmaceutical composition, a personal care product or a food product.\",\n \"Accordingly, an emulsion formulation composition is provided.\",\n \"Also provided herein is a chimeric nucleic acid comprising: 1) a first nucleic acid sequence capable of regulating transcription in a host cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.\",\n \"The oil-body-protein can be an oleosin or a caleosin, the first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.\",\n \"In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide is a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"In one embodiment, the gene fusion optionally further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.\",\n \"Also provided herein are transgenic plants, e.g., safflower plants, comprising any of the chimeric nucleic acid sequences and constructs described herein.\",\n \"The chimeric nucleic acids can be contained within a plastid.\",\n \"Accordingly, isolated plastids are provided having chimeric nucleic acids therein.\",\n \"Also provided are plant seeds comprising the chimeric nucleic acids provided herein.\",\n \"Also provided are oil body preparations obtained using any of the methods provided herein, and food products, pharmaceutical compositions, and personal care products containing the oil body preparations.\",\n \"The products and/or compositions provided herein are capable of treating oxidative stress in a target, capable of chemically reducing a target.\",\n \"Also provided is a detergent composition comprising an oil body preparation capable of chemically reducing a target, and related methods of cleansing an item, comprising administering such product to the item under conditions that promote cleansing.\",\n \"Also provided herein are nucleic acid constructs comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one thioredoxin-related protein or an active fragment thereof.\",\n \"In one embodiment, the at least one thioredoxin-related protein can be thioredoxin.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194.The thioredoxin can be obtained from Arabidopsis or wheat.\",\n \"In another embodiment, the at least one thioredoxin-related protein can be thioredoxin-reductase.\",\n \"The thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313 and/or derived from Arabidopsis or wheat.\",\n \"The thioredoxin-reductase can be an NADPH-dependent thioredoxin-reductase.\",\n \"The second region can encode a thioredoxin and thioredoxin-reductase.\",\n \"In one embodiment, the thioredoxin and thioredoxin-reductase is obtained from Mycobacterium leprae .\",\n \"In another embodiment, the at least one thioredoxin-related protein can be an engineered fusion protein.\",\n \"The first region can precede, in a 5′ to 3′ direction, the second region.\",\n \"Alternatively, the first region follows, in a 5′ to 3′ direction, the second region.\",\n \"The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.\",\n \"A seed-specific promoter, such as a phaseolin promoter, can be operably linked to the gene fusion.\",\n \"In one embodiment, at least one thioredoxin-related protein is derived from a plant species selected from the group consisting of Arabidopsis and wheat.\",\n \"In another embodiment, at least one thioredoxin-related protein can be derived from E. coli.\",\n \"In one embodiment, the gene fusion further comprises a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the linker amino acid sequence is positioned between the first region and the second region.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"In addition, the gene fusion can further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the second region.\",\n \"Also provided herein are transgenic plants containing a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof.\",\n \"The thioredoxin-related protein can be thioredoxin.\",\n \"The nucleic acid construct can be contained within a plastid.\",\n \"In one embodiment, when the first thioredoxin-related protein is thioredoxin and the construct can further comprise a region encoding a thioredoxin-reductase.\",\n \"The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.\",\n \"The gene fusion can optionally further comprise a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the nucleic acid encoding the linker amino acid sequence is positioned between the region encoding an oil-body-protein and the region encoding a first thioredoxin-related protein.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"The gene fusion can optionally further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the region encoding a first thioredoxin-related protein.\",\n \"Also provided is a transgenic plant comprising a nucleic acid construct, a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, wherein a fusion protein comprising activities of oleosin and the thioredoxin-related protein is produced in a seed of the plant.\",\n \"In another embodiment, a thioredoxin-related protein having concentration of at least about 0.5% of total cellular seed protein is provided.\",\n \"Also provided herein is an extract comprising an activity of a thioredoxin-related protein.\",\n \"Also provided are oil bodies and/or oil obtained from various seeds.\",\n \"Also provided herein are methods of making a fusion protein comprising a thioredoxin-related activity, the method comprising the steps of: a) providing a transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, the gene fusion encoding a fusion protein comprising a thioredoxin-related activity; b) obtaining seeds from the plant; and c) recovering the fusion protein by isolating oil bodies from the seeds.\",\n \"In one embodiment, the oil bodies are fractionated to achieve partial purification of the fusion protein.\",\n \"The oil bodies can be in association with a fusion protein.\",\n \"The oil-body-protein can be cleaved from the thioredoxin-related protein after fractionation of the oil bodies.\",\n \"The cleaving step can make use of a protease or chemical proteolysis.\",\n \"Also provided herein are methods of reducing allergenicity of a food comprising the steps of: a) providing a preparation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) adding the preparation to the food, whereby allergenicity of the food is reduced due to activity of the thioredoxin-related protein or fragment.\",\n \"The food can be wheat flour, wheat dough, milk, cheese, yogurt and ice cream.\",\n \"In one embodiment, NADH is used as a co-factor in the substantial absence of NADPH.\",\n \"Also provided herein are pharmaceutical compositions comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.\",\n \"The oil bodies can be associated with the fusion protein.\",\n \"Also provided is a cosmetic formulation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.\",\n \"Also provided are methods of treating or protecting a target against oxidative stress, comprising the steps of: a) providing a preparation comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) contacting the preparation with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.\",\n \"The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.\",\n \"Also provided is a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one polypeptide or an active fragment thereof, and an oil-body-surface-avoiding linker in frame between the first and second region polypeptides.\",\n \"Also provided are methods of expressing this construct into the encoded amino acid sequence; and oil bodies, formulations, emulsions, cells, and plants comprising the construct and encoded amino acid sequence.\",\n \"These particular constructs, oil bodies, formulations, emulsions, cells, and plants can be produced according to the methods described herein.\",\n \"The second region can encode any polypeptide, for example, a therapeutically, nutritionally, industrially or cosmetically useful peptide as set forth herein.\",\n \"For example, the second region can encode a redox protein, an immunoglobulin, a thioredoxin-related protein or any one or more recombinant polypeptides of a multimeric-protein-complex.\",\n \"Other features and advantages of the present invention will become readily apparent from the following detailed description.\",\n \"It should be understood however that the detailed description and the specific examples while indicating particular embodiments of the invention are given by way of illustration only.\"\n ],\n [\n \"RELATED APPLICATIONS Benefit of priority under 35 U.S.C.\",\n \"§119(e) is claimed to U.S. provisional application Ser.\",\n \"No.\",\n \"60/302,885, filed Jul.\",\n \"5, 2001, to van Rooijen, et al., entitled “METHODS FOR THE PRODUCTION OF REDOX PROTEINS”.\",\n \"This application is also a continuation-in-part of U.S. utility application Ser.\",\n \"No.\",\n \"10/006,038, filed Dec. 4, 2001 to van Rooijen, et al., entitled “METHODS FOR THE PRODUCTION OF REDOX PROTEINS”; which is a continuation-in-part of U.S. utility application Ser.\",\n \"No.\",\n \"09/742,900, filed Dec. 19, 2000 to Heifetz, et al., entitled “METHOD OF PRODUCTION AND DELIVERY OF THIOREDOXIN”.\",\n \"This application is also a continuation-in-part of U.S. utility application Ser.\",\n \"No.\",\n \"09/742,900.The subject matter of each of the provisional and utility applications is incorporated herein by reference in its entirety.\",\n \"FIELD OF THE INVENTION The present invention relates to multimeric-protein-complexes, redox proteins, and recombinant polypeptides; and improved methods for their production.\",\n \"BACKGROUND Multimeric proteins (i.e.\",\n \"proteins comprising multiple polypeptide chains) are a biologically and commercially important class of proteins.\",\n \"Antibodies for example are multimeric proteins which are used to treat a wide range of disease conditions.\",\n \"However in view of their complexity, multimeric proteins frequently represent significant manufacturing challenges.\",\n \"Redox proteins are also a commercially important class of proteins with applications in a variety of different industries including the pharmaceutical, personal care and food industry.\",\n \"For example, the redox protein thioredoxin may be used in the manufacture of personal care products (Japanese Patent Applications JP9012471A2, JP103743A2, JP1129785A2), pharmaceutical compositions/products (Aota et al.\",\n \"(1996) J. Cardiov.\",\n \"Pharmacol.\",\n \"(1996) 27: 727-732) as well as to reduce protein allergens present in food products such as milk (del Val et al.\",\n \"(1999) J. Allerg.\",\n \"Vlin.\",\n \"Immunol.\",\n \"103: 690-697) and wheat (Buchanan et al.\",\n \"(1997) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 94: 5372-5377).\",\n \"However, there is a need in the art to further improve the methods for the recombinant expression of multimeric proteins, including redox proteins.\",\n \"The present invention satisfies this need and provides related advantages as well.\",\n \"SUMMARY OF THE INVENTION The present invention relates to novel and improved methods of producing a first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulin-polypeptide-chains, immunoglobulins, redox-fusion-polypeptides, and/or thioredoxin-related proteins; in association with oil bodies.\",\n \"Accordingly, provided herein are methods of producing a recombinant multimeric-protein-complex, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.\",\n \"The method further contemplates isolating the oil bodies associated with said recombinant multimeric-protein-complex.\",\n \"The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and said second recombinant polypeptide.\",\n \"Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.\",\n \"The oil-body-targeting-protein can be an oleosin or caleosin.\",\n \"When the oil-body-targeting-protein can be an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.\",\n \"Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.\",\n \"The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.\",\n \"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.\",\n \"In one embodiment, the first recombinant polypeptide is capable of associating with said second recombinant polypeptide in the cell.\",\n \"In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.\",\n \"In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.\",\n \"Also provided herein is a method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex; and (d) associating said first recombinant polypeptide with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.\",\n \"This method further contemplates isolating from the progeny cell, oil bodies comprising the multimeric-protein-complex.\",\n \"The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and second recombinant polypeptide.\",\n \"Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.\",\n \"The oil-body-targeting-protein can be an oleosin or caleosin.\",\n \"When the oil-body-targeting-protein is an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.\",\n \"Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.\",\n \"The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.\",\n \"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.\",\n \"In one embodiment, the first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex in said progeny cell.\",\n \"In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.\",\n \"In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 60 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.\",\n \"Also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.\",\n \"The second recombinant polypeptide can be associated with oil bodies through a second oil-body-targeting-protein in the second plant.\",\n \"The oil bodies can be isolated from the progeny plant comprising said multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, wherein the oil-body-protein can be an oleosin or caleosin.\",\n \"The first recombinant polypeptide can be fused to the oleosin or caleosin; and the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.\",\n \"The first and second recombinant polypeptide can form a multimeric-protein-complex, such as a heteromultimeric-protein-complex, wherein the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The plant can be a safflower plant.\",\n \"Also provided herein are chimeric nucleic acids encoding a multimeric-fusion-protein as described herein, said nucleic acid comprising: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin.\",\n \"The oil-body-protein can be an oleosin or caleosin.\",\n \"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the first and second recombinant polypeptide can form an enzymatically active heteromultimeric redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence that is also a non-proteolytic linker and said sequence encoding the first recombinant polypeptide.\",\n \"Also provided herein are recombinant multimeric-fusion-proteins comprising (i) an oil-body-targeting-protein, or fragment thereof, (ii) a first recombinant polypeptide and a (iii) second recombinant polypeptide, wherein said first and second recombinant polypeptides are capable of forming a multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.\",\n \"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said sequence encoding the first recombinant polypeptide.\",\n \"Also provided herein are isolated oil bodies comprising a multimeric-protein-complex comprising (i) an oil-body-targeting-protein and (ii) a first recombinant polypeptide, said oil bodies further comprising a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.\",\n \"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are isolated oil bodies comprising (a) a first fusion protein comprising a first oil-body-targeting-protein fused to a first recombinant polypeptide; and (b) a second fusion protein comprising a second oil-body-targeting-protein fused to a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.\",\n \"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are cells and transgenic plants comprising oil bodies, multimeric-protein-complexes, and multimeric-fusion-proteins, set forth herein.\",\n \"In one embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In embodiments, wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase, the methods described herein can be used to formulate the oil bodies for use in the preparation of a food product, personal care product or pharmaceutical composition.\",\n \"These formulations can further comprise the addition of NADP or NADPH.\",\n \"The food product can be a milk or wheat based food product.\",\n \"The personal care product can reduce the oxidative stress to the surface area of the human body or can be used to lighten the skin.\",\n \"The pharmaceutical composition can be used to treat chronic obstructive pulmonary disease (COPD), cataracts, diabetes, envenomation, bronchiopulmonary disease, malignancies, psoriasis, reperfusion injury, wound healing, sepsis, GI bleeding, intestinal bowel disease (IBD), ulcers, GERD (gastro esophageal reflux disease).\",\n \"Also provided herein are compositions comprising isolated oil bodies, thioredoxin and thioredoxin-reductase, wherein said thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194, and said thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.The composition can further comprise NADP or NADPH.\",\n \"In another embodiment, the composition comprises a first recombinant polypeptide that can be an immunoglobulin-polypeptide-chain and a second recombinant polypeptide.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are multimeric-fusion-proteins, wherein the fusion-protein contains two or more polypeptide chains selected from the group of proteins set forth in FIG.\",\n \"5.Methods are also provided of reducing allergenicity of a food comprising the steps of providing the isolated oil bodies set forth herein; and adding the isolated oil bodies to the food, whereby allergenicity of the food is reduced.\",\n \"The food can be selected from the group consisting of wheat flour, wheat dough, milk, cheese, yogurt and ice cream.\",\n \"The various methods of treating food can further comprise providing NADH as a co-factor in the substantial absence of NADPH.\",\n \"Also provided herein are methods of treating or protecting a target against oxidative stress, comprising the steps of providing the recombinant redox fusion polypeptide comprising thioredoxin and thioredoxin-reductase; and contacting the recombinant fusion polypeptide with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.\",\n \"The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.\",\n \"Also provided herein are methods for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.\",\n \"The first redox protein can be a thioredoxin and the second redox protein can be a thioredoxin-reductase.\",\n \"Also, provided herein are methods of producing an immunoglobulin, said method comprising: (a) producing in a cell comprising oil bodies, a first immunoglobulin-polypeptide-chain and a second immunoglobulin-polypeptide-chain wherein said first immunoglobulin-polypeptide-chain is capable of associating with said second immunoglobulin-polypeptide-chain to form said immunoglobulin; and (b) associating said immunoglobulin with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first immunoglobulin-polypeptide-chain.\",\n \"For example, the first immunoglobulin-polypeptide-chain can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second immunoglobulin-polypeptide-chain can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are methods for preparing a redox protein or an immunoglobulin associated with oil bodies comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein, or a nucleic acid sequence encoding a immunoglobulin comprising a first immunoglobulin-polypeptide-chain linked to a second immunoglobulin-polypeptide-chain, operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide or immunoglobulin in a progeny cell comprising oil bodies; and c) isolating from said progeny cell said oil bodies comprising said redox fusion polypeptide or immunoglobulin.\",\n \"In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide or immunoglobulin can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged or have a molecular weight of at least 35 kd.\",\n \"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.\",\n \"In this optional embodiment, also contemplated is the introduction of an enzyme or chemical that cleaves said redox fusion polypeptide from said oil body, thereby obtaining isolated redox fusion polypeptide.\",\n \"The first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.\",\n \"In one embodiment, the thioredoxin and thioredoxin-reductase can be obtained from Arabidopsis.\",\n \"In another embodiment, the first redox protein is at least 5 times more active when produced as a redox fusion polypeptide as compared to the production of the first redox protein without the second redox protein.\",\n \"Also provided herein, for use with the various methods set forth herein is the formulation of an emulsion of the oil bodies associated with the redox fusion polypeptide for use in the preparation of a product capable of treating oxidative stress in a target, a product capable of chemically reducing a target, pharmaceutical composition, a personal care product or a food product.\",\n \"Accordingly, an emulsion formulation composition is provided.\",\n \"Also provided herein is a chimeric nucleic acid comprising: 1) a first nucleic acid sequence capable of regulating transcription in a host cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.\",\n \"The oil-body-protein can be an oleosin or a caleosin, the first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.\",\n \"In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide is a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"In one embodiment, the gene fusion optionally further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.\",\n \"Also provided herein are transgenic plants, e.g., safflower plants, comprising any of the chimeric nucleic acid sequences and constructs described herein.\",\n \"The chimeric nucleic acids can be contained within a plastid.\",\n \"Accordingly, isolated plastids are provided having chimeric nucleic acids therein.\",\n \"Also provided are plant seeds comprising the chimeric nucleic acids provided herein.\",\n \"Also provided are oil body preparations obtained using any of the methods provided herein, and food products, pharmaceutical compositions, and personal care products containing the oil body preparations.\",\n \"The products and/or compositions provided herein are capable of treating oxidative stress in a target, capable of chemically reducing a target.\",\n \"Also provided is a detergent composition comprising an oil body preparation capable of chemically reducing a target, and related methods of cleansing an item, comprising administering such product to the item under conditions that promote cleansing.\",\n \"Also provided herein are nucleic acid constructs comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one thioredoxin-related protein or an active fragment thereof.\",\n \"In one embodiment, the at least one thioredoxin-related protein can be thioredoxin.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194.The thioredoxin can be obtained from Arabidopsis or wheat.\",\n \"In another embodiment, the at least one thioredoxin-related protein can be thioredoxin-reductase.\",\n \"The thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313 and/or derived from Arabidopsis or wheat.\",\n \"The thioredoxin-reductase can be an NADPH-dependent thioredoxin-reductase.\",\n \"The second region can encode a thioredoxin and thioredoxin-reductase.\",\n \"In one embodiment, the thioredoxin and thioredoxin-reductase is obtained from Mycobacterium leprae.\",\n \"In another embodiment, the at least one thioredoxin-related protein can be an engineered fusion protein.\",\n \"The first region can precede, in a 5′ to 3′ direction, the second region.\",\n \"Alternatively, the first region follows, in a 5′ to 3′ direction, the second region.\",\n \"The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.\",\n \"A seed-specific promoter, such as a phaseolin promoter, can be operably linked to the gene fusion.\",\n \"In one embodiment, at least one thioredoxin-related protein is derived from a plant species selected from the group consisting of Arabidopsis and wheat.\",\n \"In another embodiment, at least one thioredoxin-related protein can be derived from E. coli.\",\n \"In one embodiment, the gene fusion further comprises a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the linker amino acid sequence is positioned between the first region and the second region.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"In addition, the gene fusion can further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the second region.\",\n \"Also provided herein are transgenic plants containing a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof.\",\n \"The thioredoxin-related protein can be thioredoxin.\",\n \"The nucleic acid construct can be contained within a plastid.\",\n \"In one embodiment, when the first thioredoxin-related protein is thioredoxin and the construct can further comprise a region encoding a thioredoxin-reductase.\",\n \"The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.\",\n \"The gene fusion can optionally further comprise a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the nucleic acid encoding the linker amino acid sequence is positioned between the region encoding an oil-body-protein and the region encoding a first thioredoxin-related protein.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"The gene fusion can optionally further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the region encoding a first thioredoxin-related protein.\",\n \"Also provided is a transgenic plant comprising a nucleic acid construct, a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, wherein a fusion protein comprising activities of oleosin and the thioredoxin-related protein is produced in a seed of the plant.\",\n \"In another embodiment, a thioredoxin-related protein having concentration of at least about 0.5% of total cellular seed protein is provided.\",\n \"Also provided herein is an extract comprising an activity of a thioredoxin-related protein.\",\n \"Also provided are oil bodies and/or oil obtained from various seeds.\",\n \"Also provided herein are methods of making a fusion protein comprising a thioredoxin-related activity, the method comprising the steps of: a) providing a transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, the gene fusion encoding a fusion protein comprising a thioredoxin-related activity; b) obtaining seeds from the plant; and c) recovering the fusion protein by isolating oil bodies from the seeds.\",\n \"In one embodiment, the oil bodies are fractionated to achieve partial purification of the fusion protein.\",\n \"The oil bodies can be in association with a fusion protein.\",\n \"The oil-body-protein can be cleaved from the thioredoxin-related protein after fractionation of the oil bodies.\",\n \"The cleaving step can make use of a protease or chemical proteolysis.\",\n \"Also provided herein are methods of reducing allergenicity of a food comprising the steps of: a) providing a preparation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) adding the preparation to the food, whereby allergenicity of the food is reduced due to activity of the thioredoxin-related protein or fragment.\",\n \"The food can be wheat flour, wheat dough, milk, cheese, yogurt and ice cream.\",\n \"In one embodiment, NADH is used as a co-factor in the substantial absence of NADPH.\",\n \"Also provided herein are pharmaceutical compositions comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.\",\n \"The oil bodies can be associated with the fusion protein.\",\n \"Also provided is a cosmetic formulation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.\",\n \"Also provided are methods of treating or protecting a target against oxidative stress, comprising the steps of: a) providing a preparation comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) contacting the preparation with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.\",\n \"The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.\",\n \"Also provided is a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one polypeptide or an active fragment thereof, and an oil-body-surface-avoiding linker in frame between the first and second region polypeptides.\",\n \"Also provided are methods of expressing this construct into the encoded amino acid sequence; and oil bodies, formulations, emulsions, cells, and plants comprising the construct and encoded amino acid sequence.\",\n \"These particular constructs, oil bodies, formulations, emulsions, cells, and plants can be produced according to the methods described herein.\",\n \"The second region can encode any polypeptide, for example, a therapeutically, nutritionally, industrially or cosmetically useful peptide as set forth herein.\",\n \"For example, the second region can encode a redox protein, an immunoglobulin, a thioredoxin-related protein or any one or more recombinant polypeptides of a multimeric-protein-complex.\",\n \"Other features and advantages of the present invention will become readily apparent from the following detailed description.\",\n \"It should be understood however that the detailed description and the specific examples while indicating particular embodiments of the invention are given by way of illustration only.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 shows a ClustalW Formatted Alignment comparison of the published NADPH thioredoxin-reductase nucleic acid sequence (SEQ ID NO:9) (ATTHIREDB-Jacquot et al.\",\n \"J. Mol.\",\n \"Biol.\",\n \"(1994) 235 (4):1357-63.)\",\n \"with the sequence isolated herein in Example 1 (TR; SEQ ID NO:8).\",\n \"FIG.\",\n \"2 shows a ClustalW Formatted Alignment comparison of the deduced amino acid sequence of the published NADPH thioredoxin-reductase sequence (SEQ ID NO:12) (ATTHIREDB Jacquot et al.\",\n \"J. Mol.\",\n \"Biol.\",\n \"(1994) 235 (4):1357-63.)\",\n \"with the sequence isolated herein in Example 1 (TR; SEQ ID NO:13).\",\n \"FIG.\",\n \"3 shows a clustal alignment comparing the amino acid sequence of the Arabidopsis thaliana thioredoxin-reductase-linker-thioredoxin synthetic fusion (Arab TR-link-Trxh; SEQ ID NO:37) to the Mycobacterium leprae thioredoxin-reductase-thioredoxin natural fusion (M. lep TR/Trxh; SEQ ID NO:36) natural fusion.\",\n \"Overall, the proteins are approximately 50% identical at the amino acid level.\",\n \"FIG.\",\n \"4 is a bar graph showing the thioredoxin/thioredoxin-reductase activity measurements for the various transgenic Arabidopsis seed fractions.\",\n \"Relative specific activity is expressed as a percentage of the E. coli thioredoxin and thioredoxin-reductase activities.\",\n \"The numbered bars in the graph correspond to the following: 1.W.T.+oleosin-thioredoxin 2.W.T.+thioredoxin-oleosin 3.W.T.+thioredoxin 4.W.T.+oleosin-thioredoxin-reductase 5.W.T.+thioredoxin-reductase-oleosin 6.W.T.+thioredoxin-reductase 7.thioredoxin+oleosin-thioredoxin-reductase 8.thioredoxin+thioredoxin-reductase-oleosin 9.thioredoxin+thioredoxin-reductase 10.thioredoxin-reductase+oleosin-thioredoxin 11.thioredoxin-reductase+thioredoxin-oleosin 12.oleosin-M. lep TR/Trxh 13.E.\",\n \"coli thioredoxin-reductase+thioredoxin FIG.\",\n \"5 provides a listing of exemplary proteins for use in the heteromultimeric-fusion-proteins and heteromultimeric-protein-complexes provided herein.\",\n \"DETAILED DESCRIPTION As hereinbefore mentioned, the present invention relates to novel and improved methods for the production of multimeric proteins, including a first and second recombinant polypeptide, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulin-polypeptide-chains, immunoglobulins, redox-fusion-polypeptides, and a first and second thioredoxin-related protein; and related products.\",\n \"These methods permit the production of active multimeric-protein-complexes in association with oil bodies.\",\n \"The oil bodies in association with the multimeric-protein-complex may be used to prepare various useful emulsions.\",\n \"Accordingly, provided herein are methods of producing a recombinant multimeric-protein-complex associated with an oil body, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide in the cell to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide.\",\n \"DEFINITIONS AND TERMS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.\",\n \"Where permitted, all patents, applications, published applications and other publications and sequences from GenBank, SwissPro and other data bases referred to throughout in the disclosure herein are incorporated by reference in their entirety.\",\n \"As used herein, the phrase “multimeric-protein-complex”, refers to two or more polypeptide chains that permanently or repeatedly interact or permanently or repeatedly coordinate to form a biologically active assembly comprising said two or more polypeptide chains.\",\n \"It should be noted that the polypeptides may be independently biologically active without interaction or coordination to form the complex.\",\n \"The multimeric-protein-complex may provide a biological structure, or it may be capable of facilitating a chemical or biological reaction.\",\n \"For example, one of the protein regions within the multimeric-protein-complex can repeatedly activate or repeatedly inactivate the biological or metabolic activity of one or more of the other proteins contained within the multimeric-protein-complex.\",\n \"In one embodiment, the first and second recombinant polypeptide contained in a multimeric-protein-complex may either associate or interact as independent non-contiguous polypeptide chains or the multimeric-protein-complex may be prepared as a fusion polypeptide (multimeric-fusion-protein) between the first and second recombinant polypeptide.\",\n \"One example of a repeated (e.g., reoccurring) interaction or association between the two or more polypeptides of a multimeric-protein-complex provided herein is the interaction between two or more non-identical redox proteins to form a heteromultimeric-protein-complex.\",\n \"Exemplary redox proteins for use in this regard are thioredoxin and the thioredoxin-reductase.\",\n \"A further example is the interaction between two or more immunoglobulin-polypeptide-chains to form an immunoglobulin.\",\n \"As used herein, the phrase “heteromultimeric-protein-complex”, refers to two or more non-identical polypeptide chains that permanently or repeatedly interact or permanently or repeatedly coordinate to form a biologically active assembly comprising said two or more polypeptide chains.\",\n \"Other examples of multimeric-protein-complexes provided herein include a first and second recombinant polypeptide, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, first and second immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and a first and second thioredoxin-related protein.\",\n \"The recombinant polypeptide or multimeric-protein-complex is associated with an oil body.\",\n \"As used herein, the phrase “oil body” or “oil bodies” refers to any oil or fat storage organelle in any cell type.\",\n \"Accordingly, the oil bodies may be obtained from any cell comprising oil bodies, including plant cells (described in for example: Huang (1992) Ann.\",\n \"Rev.\",\n \"Plant Mol.\",\n \"Biol.\",\n \"43: 177-200), animal cells (described in for example: Murphy (1990) Prog Lipid Res 29(4): 299-324), including adipocytes, hepatocytes, steroidogenic cells, mammary epithelial cells, macrophages, algae cells (described in for example: Rossler (1988) J. Physiol.\",\n \"London, 24: 394-400) fungal cells, including yeast cells (described in for example Leber et al.\",\n \"(1994) Yeast 10: 1421-1428) and bacterial cells (described in for example: Pieper-Furst et al.\",\n \"(1994) J. Bacteriol.\",\n \"176: 4328-4337).\",\n \"Preferably the oil bodies used herein are oil bodies obtainable from plant cells and more preferably the oil bodies obtainable from plant seed cells.\",\n \"As used herein, the phrase “is capable of associating with”, “associate” or grammatical variations thereof, refers to any interaction between two or more polypeptides, including any covalent interactions (e.g.\",\n \"multimeric-fusion-proteins) as well as non-covalent interactions.\",\n \"Exemplary non-covalent interactions can be between the oil-body-targeting-protein and a redox protein or immunoglobulin-polypeptide-chain, as well as between two or more different proteins contained within two or more separate oil-body-protein fusion proteins (e.g., the redox proteins in oleosin-thioredoxin and oleosin-thioredoxin-reductase).\",\n \"As used herein, the term “recombinant” (also referred to as heterologous) in the context of recombinant proteins and amino acids, means “of different natural origin” or represents a non-natural state.\",\n \"For example, if a host cell is transformed with a nucleotide sequence derived from another organism, particularly from another species, that nucleotide sequence and amino acid sequence encoded thereby, is recombinant (heterologous) with respect to that host cell and also with respect to descendants of the host cell which carry that gene.\",\n \"Similarly, recombinant (or heterologous) refers to a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g., a different copy number, or under the control of different regulatory elements.\",\n \"A transforming nucleotide sequence may include a recombinant coding sequence, or recombinant regulatory elements.\",\n \"Alternatively, the transforming nucleotide sequence may be completely heterologous or may include any possible combination of heterologous and endogenous nucleic acid sequences.\",\n \"In various embodiments of the present invention, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins, are produced in a cell comprising oil bodies.\",\n \"As used herein the phrase “in a cell”, “in the cell”, or grammatical variations thereof, mean that the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins, may be produced in any cellular compartment of that cell, so long as that cell comprises oil bodies therein.\",\n \"In embodiments of the invention in which plant cells are used, the phrase is intended to include the plant apoplast.\",\n \"In various embodiments provided herein, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and thioredoxin-related proteins, associate with an oil body through an oil-body-targeting-protein.\",\n \"As used herein, the phrase “oil-body-targeting-protein” refers to any protein, protein fragment or peptide capable of associating with an oil body.\",\n \"Exemplary oil-body-targeting-proteins for use herein include oil-body-proteins, such as oleosin and caleosin; immunoglobulins, such as bi-specific antibodies; and the like.\",\n \"In embodiments described herein in which an oil-body-protein is used, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and thioredoxin-related proteins, are preferably fused to the oil-body-protein.\",\n \"The term “oil-body-protein” refers to any protein naturally present in cells and having the capability of association with oil bodies, including any oleosin or caleosin.\",\n \"Accordingly, provided herein a method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide, such as a redox protein, an immunoglobulin-polypeptide-chain or an thioredoxin-related protein, fused to an oil-body-protein; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide, such as a second redox protein, a second immunoglobulin-polypeptide-chain or a second thioredoxin-related protein; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex, preferably in said progeny cell; and (d) associating said first recombinant polypeptide with an oil body through said oil-body-protein.\",\n \"The term “nucleic acid” as used herein refers to a sequence of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages.\",\n \"The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof, which function similarly.\",\n \"The nucleic acid sequences may be ribonucleic acids (RNA) or deoxyribonucleic acids (DNA) and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil.\",\n \"The sequences also may contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo-uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-amino adenine, 8-thiol-adenine, 8-thio-alkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8 amino guanine, 8 thiol guanine, 8-thioalkyl guanines, 8 hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.\",\n \"Multimeric-Protein-Complexes In accordance with the methods and compositions provided herein, any two recombinant polypeptides capable of forming a multimeric-protein-complex may be used.\",\n \"The nucleic acid sequences encoding the two recombinant polypeptides may be obtained from any biological source or may be prepared synthetically.\",\n \"In general nucleic acid sequence encoding multimeric proteins are known to the art and readily available.\",\n \"Known nucleic acid sequences encoding multimeric-protein-complexes may be used to design and construct nucleic acid sequence based probes in order to uncover and identify previously undiscovered nucleic acid sequences encoding multimeric-protein-complexes, for example, by screening cDNA or genomic libraries or using 2- or multi-hybrid systems.\",\n \"Thus, additional nucleic acid sequences encoding multimeric-protein-complexes may be discovered and used as described herein.\",\n \"The first and/or second recombinant polypeptides that are comprised within a multimeric-protein-complex provided herein, can themselves be in the form of heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or a first and/or second thioredoxin-related protein.\",\n \"The nucleic acid sequence encoding the first and second recombinant polypeptide, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or a first and/or second thioredoxin-related protein may be obtained from separate sources or may be obtained from the same source.\",\n \"In general however, such nucleic acid sequence is obtained from the same or a similar biological source.\",\n \"In certain embodiments wherein the nucleic acid sequence encoding the first and second recombinant polypeptide protein are obtained from the same source, the nucleic acid sequence encoding the first recombinant polypeptide and second recombinant polypeptide may be naturally fused.\",\n \"In accordance with a particular embodiment, the nucleic acid sequences encoding the first and second recombinant polypeptide are obtained from a plant source.\",\n \"Oil-Body-Surface-Avoiding Linkers Polypeptide spacers or linkers of variable length and/or negative charge can be used herein to separate the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and the first and/or second thioredoxin-related proteins from the in-frame oil-body-targeting-protein, to improve activity of and/or the accessibility of the polypeptide or complex.\",\n \"For example, in one embodiment set forth herein, positioned between a nucleic acid sequence encoding a sufficient portion of an oil-body-protein and a nucleic acid sequence encoding either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and the first and/or second thioredoxin-related proteins; is a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"Oil-body-surface-avoiding linkers are positioned between the oil-body targeting sequence and an in-frame recombinant polypeptide of interest, e.g., the multimeric-protein-complexes provided herein, serve to increase the distance and or decrease the interaction between the negatively charged oil body surface and the recombinant polypeptide of interest.\",\n \"A negatively charged linker is repelled by the negatively charged oil body surface, in turn increasing the distance or decreasing the interaction of its attached recombinant polypeptide with the oil body surface.\",\n \"As a consequence of the increased distance from the oil body surface, the recombinant polypeptide will be more accessible, e.g.\",\n \"to its target(s) substrate, protein substrate, protein partner, and less affected by the charged oil body surface.\",\n \"Exemplary linker sequences for use herein can be either a negatively charged linker, or a linker having a molecular weight of at least about 35 kd or more.\",\n \"As used herein, a “negatively charged linker” sequence, refers to any amino acid segment, or nucleic acid encoding such, that has a pI less than or equal to the pI of an oil body.\",\n \"In certain embodiments, the pI of the negatively charged linker is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, down to about 25% or more, below that of the pI of an oil body in the particular plant or cell system being used.\",\n \"Exemplary negatively charged linkers can be prepared comprising any combination of the negatively charged amino acid residues.\",\n \"For example, in one embodiment, a negatively charged linker comprises either a poly-glutamate or poly-aspartate sequence, or any combination of both amino acid residues.\",\n \"The negatively charged linker is typically at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length.\",\n \"The negatively charged linkers are preferably non-proteolytic (e.g., non-proteolytic linkers), having no site for efficient proteolysis.\",\n \"When linker size rather than charge is used to minimize interaction of the recombinant polypeptide of interest with the oil body surface, then the linker is non-proteolytic and ranges in molecular weight from about 35 kd up to about 100 kd.\",\n \"The upper size limit is chosen such that the expression of, the activity of, the conformation of, and/or the access to target of, the recombinant polypeptide of interest is not significantly affected by the linker.\",\n \"In certain embodiments, described herein where a non-proteolytic linker amino acid sequence is employed, the gene-fusion or protein fusion (multimeric-fusion-protein) can optionally further comprise a linker nucleic or amino acid sequence encoding a sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the non-proteolytic linker sequence and sequence encoding the desired recombinant protein region, e.g., the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins set forth herein.\",\n \"When a cleavable linker sequence is used herein, in a particular embodiment, it is further downstream than the non-proteolytic linker sequence from the oil-body-targeting-protein region of the fusion protein.\",\n \"By virtue of cleavable linker, the recombinant fusion polypeptides provided herein, such as the multimeric-fusion-proteins and redox fusion polypeptides, can be isolated and purified by introducing an enzyme or chemical that cleaves said multimeric-fusion-protein and/or redox fusion polypeptide from said oil body, thereby obtaining and/or isolating the desired protein.\",\n \"It is contemplated herein that the use of cleavable linker sequence downstream of the non-proteolytic linker/spacer sequence will improve the yield of protein recovery when isolating or purifying proteins using the methods provided herein.\",\n \"The nucleic acid sequences encoding the first or second recombinant polypeptide may be altered to improve expression levels for example, by optimizing the nucleic acids sequence in accordance with the preferred codon usage for the particular cell type which is selected for expression of the first and second recombinant polypeptide, or by altering of motifs known to destabilize mRNAs (see for example: PCT Patent Application 97/02352).\",\n \"Comparison of the codon usage of the first and second recombinant polypeptide with codon usage of the host will enable the identification of codons that may be changed.\",\n \"For example, typically plant evolution has tended towards a preference for CG rich nucleotide sequences while bacterial evolution has resulted in bias towards AT rich nucleotide sequences.\",\n \"By modifying the nucleic acid sequences to incorporate nucleic acid sequences preferred by the host cell, expression may be optimized.\",\n \"Construction of synthetic genes by altering codon usage is described in for example PCT patent Application 93/07278.The first and second recombinant polypeptide can be altered using for example targeted mutagenesis, random mutagenesis (Shiraishi et al.\",\n \"(1998) Arch.\",\n \"Biochem.\",\n \"Biophys.\",\n \"358: 104-115; Galkin et al.\",\n \"(1997) Protein Eng.\",\n \"10: 687-690; Carugo et al.\",\n \"(1997) Proteins 28: 10-28; Hurley et al.\",\n \"(1996) Biochemistry 35: 5670-5678), gene shuffling, and/or by the addition of organic solvent (Holmberg et al.\",\n \"(1999) Protein Eng.\",\n \"12: 851-856).\",\n \"Any polypeptide spacers that are used in accordance with the methods and products provided herein may be altered in similar ways.\",\n \"In particular embodiments provided herein, the recombinant polypeptides or thioredoxin-related proteins capable of forming a multimeric-protein-complex are capable of forming a heteromultimeric-protein-complex.\",\n \"Examples of heteromultimeric-protein-complexes that contain polypeptide chains that repeatedly interact, either to activate, inactivate, oxidize, reduce, stabilize, etc., with one another, that can be produced in association with oil bodies using the methods provided herein include those set forth in FIG.\",\n \"5.Accordingly, exemplary proteins for use in the heteromultimeric-protein-complexes and nucleic acid constructs encoding such, provided herein include, among others described herein, those set forth in FIG.\",\n \"5.Other polypeptide regions that can be used in the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, provided herein include, among other, those immunoglobulin regions set forth in Table 1.TABLE 1 ANTIBODY HETERODIMERS Class or molecule Subunits Fab Variable region and first constant region of heavy chain and complete light chain Fv Variable regions of heavy and light antibody chains IgA heavy chains, light chains and J (joining) chain IgG, IgD, IgE heavy and light chains IgM heavy chains, light chains and J (joining) chain Antibody chain(s) and a toxin Antibody chain(s) and a toxin Autoantigens, allergens and Autoantigens, allergens and transplant transplant antigens with an antigens with an adjuvant or tolerogen adjuvant or tolerogen Chimeras using antibody Fc Receptor subunits fused to the constant domain region of antibody heavy chains As set forth above, in one embodiment, exemplary heteromultimeric-protein-complexes and exemplary heteromultimeric-fusion-proteins provided herein comprise redox proteins, such as the thioredoxins and thioredoxin-reductases and immunoglobulins.\",\n \"Oil-Body-Targeting-Proteins The nucleic acid sequence encoding the oil-body-targeting-protein that may be used in the methods and compositions provided herein may be any nucleic acid sequence encoding an oil-body-targeting-protein, protein fragment or peptide capable of association with first recombinant polypeptide, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or a first and/or second thioredoxin-related protein and the oil bodies.\",\n \"The nucleic acid sequence encoding the oil body targeting peptide may be synthesized or obtained from any biological source.\",\n \"For example, in one embodiment the oil-body-targeting-protein is an immunoglobulin or an immunoglobulin derived molecule, for example, a bispecific single chain antibody.\",\n \"The generation of single chain antibodies and bi-specific single chain antibodies is known to the art (see, e.g., U.S. Pat.\",\n \"No.\",\n \"5,763,733, U.S. Pat.\",\n \"No.\",\n \"5,767,260 and U.S. Pat.\",\n \"No.\",\n \"5,260,203).\",\n \"Nucleic acid sequences encoding single chain antibodies functioning as oil-body-targeting-proteins may be prepared from hybridoma cell lines expressing monoclonal antibodies raised against an oleosin as described by Alting-Mees et al (2000) IBC's Annual International Conference on Antibody Engineering, Poster #1.In order to attain specificity for the first recombinant polypeptide a nucleic acid sequence encoding a second single chain antibody prepared from a monoclonal raised against the first recombinant polypeptide may be prepared and linked to the anti-oleosin single chain antibody.\",\n \"In this embodiment the oil body associates with the first recombinant polypeptide through non-covalent interactions of the oil-body-targeting-protein with the first recombinant polypeptide and the oil body.\",\n \"Alternatively the first recombinant polypeptide may be prepared as a fusion protein with an oil-body-targeting-protein.\",\n \"For example, a nucleic acid sequence encoding a single chain antibody raised against an oleosin may be fused to a nucleic acid sequence encoding the first recombinant polypeptide Non-immunoglobulin-based oil-body-targeting-proteins capable of association with the first recombinant polypeptide may be discovered and prepared using for example phage display techniques (Pharmacia Biotech Catalogue Number 27-9401-011 Recombinant Phage Antibody System Expression Kit).\",\n \"Oil-body-targeting-proteins may also be chemically modified.\",\n \"For example, oleosins may be modified by changing chemical modification of the lysine residues using chemical agents such as biotinyl-N-hydroxysuccinimide ester resulting in a process referred to as biotinylation.\",\n \"Conveniently this is accomplished by in vitro biotinylation of the oil bodies.\",\n \"In vivo biotinylation may be accomplished using the biotinylation domain peptide from the biotin carboxy carrier protein of E. coli acetyl-CoA carboxylase (Smith et al.\",\n \"(1998) Nucl.\",\n \"Acids.\",\n \"Res.\",\n \"26: 1414-1420).\",\n \"Avidin or streptavidin may subsequently be used to accomplish association of the redox protein with the oil body.\",\n \"In a particular embodiment the oil-body-targeting-protein is an oil-body-protein such as for example an oleosin or a caleosin or a sufficient portion derived thereof capable of targeting to an oil body.\",\n \"Nucleic acid sequences encoding oleosins are known to the art.\",\n \"These include for example the Arabidopsis oleosin (van Rooijen et al (1991) Plant Mol.\",\n \"Bio.\",\n \"18:1177-1179); the maize oleosin (Qu and Huang (1990) J. Biol.\",\n \"Chem.\",\n \"Vol.\",\n \"265 4:2238-2243); rapeseed oleosin (Lee and Huang (1991) Plant Physiol.\",\n \"96:1395-1397); and the carrot oleosin (Hatzopoulos et al (1990) Plant Cell Vol.\",\n \"2, 457-467.).\",\n \"Caleosin nucleic acid sequences are also known to the art (Naested et al (2000) Plant Mol.\",\n \"Biol.\",\n \"44(4):463-476; Chen et al (1999) Plant Cell Physiol.\",\n \"40(10):1079-1086).\",\n \"Animal cell derived oil body proteins that may be used herein include adopihilin (Brasaemle et al, (1997) J. Lipid Res., 38: 2249-2263; Heid et al.\",\n \"(1998) Cell Tissue Research 294: 309-321), perilipin (Blanchette-Mackie et al.\",\n \"(1995), J. Lipid Res.\",\n \"36: 1211-1226; Servetnick et al.\",\n \"(1995) J. Biol.\",\n \"Chem.\",\n \"270: 16970-16973), apolipoproteins such as apo A-I, A-II, A-IV, C-I, C-II, CIII (Segrest et al.\",\n \"(1990), Proteins 8:103-117) and apoB (Chatterton et al.\",\n \"(1995) J. Lipid Res.\",\n \"36: 2027-2037; Davis, R A in: Vance D E, Vance J. editors.\",\n \"Lipoprotein structure and secretion.\",\n \"The Netherlands, Elsevier, 191: 403-426.In one embodiment, the first recombinant polypeptide is fused to an oil-body-protein.\",\n \"The methodology is further described in U.S. Pat.\",\n \"No.\",\n \"5,650,554, which is incorporated herein by reference in its entirety.\",\n \"The first recombinant polypeptide may be fused to the N-terminus as well as to the C-terminus of the oil-body-protein (as described in: Moloney and van Rooijen (1996) INFORM 7:107-113) and fragments of the oil-body-protein such as for example the central domain of an oleosin molecule, or modified versions of the oil-body-protein may be used.\",\n \"In this embodiment, the second recombinant polypeptide is expressed intracellularly and then intracellularly associates with the first recombinant polypeptide to form the multimeric-protein-complex in the cell.\",\n \"Oil bodies comprising the multimeric-protein-complex are then conveniently isolated from the cells.\",\n \"In a further embodiment both the first and second recombinant polypeptide are separately fused to an oil-body-protein.\",\n \"In this embodiment nucleic acid sequences encoding the first and second polypeptides may be prepared separately and introduced in separate cell lines or they may be introduced in the same cell lines.\",\n \"Where the nucleic acid sequences are introduced in the same cell line, these nucleic acid sequence may be prepared using two separate expression vectors, or they may be prepared using a single vector comprising nucleic acid sequences encoding both the first polypeptide fused to an oil body protein and the second polypeptide fused to an oil-body-protein.\",\n \"Where separate cell lines are used subsequent mating of the offspring (e.g.\",\n \"mating of plants) is used to prepare a generation of cells comprising oil bodies which comprise both the first and second recombinant polypeptide fused to an oil-body-protein.\",\n \"In further alternate embodiment, the first and second recombinant polypeptide are fused to form a multimeric-fusion-protein comprising the multimeric-protein-complex.\",\n \"In such an embodiment, the first and second polypeptide is associated with the oil body through an oil-body-targeting-protein capable of associating with both the fusion protein and with the oil body.\",\n \"In a particular embodiment, the fusion protein comprising the multimeric-protein-complex is fused to an oil-body-protein, for example, an oleosin or caleosin.\",\n \"In embodiments provided herein in which the multimeric-protein-complex is an immunoglobulin (e.g., a multimeric-immunoglobulin-complex), a particularly preferred oil body targeting protein is an oleosin or caleosin associated with an immunoglobulin binding protein, such as for example protein A (U.S. Pat.\",\n \"No.\",\n \"5,151,350), protein L (U.S. Pat.\",\n \"No.\",\n \"5,965,390) and protein G (U.S. Pat.\",\n \"No.\",\n \"4,954,618), or active fragments of such immunoglobulin binding proteins.\",\n \"New oil-body-proteins may be discovered for example by preparing oil bodies (described in further detail below) and identifying proteins in these preparations using for example SDS gel electrophoresis.\",\n \"Polyclonal antibodies may be raised against these proteins and used to screen cDNA libraries in order to identify nucleic acid sequences encoding oil-body-proteins.\",\n \"The methodologies are familiar to the skilled artisan (Huynh et al.\",\n \"(1985) in DNA Cloning Vol.\",\n \"1.a Practical Approach ed.\",\n \"D M Glover, IRL Press, pp 49-78).\",\n \"New oil-body-proteins may further be discovered using known nucleic acid sequences encoding oil-body-proteins (e.g.\",\n \"the Arabidopsis, rapeseed, carrot and corn nucleic acid sequences) to probe for example cDNA and genomic libraries for the presence of nucleic acid sequences encoding oil-body-proteins.\",\n \"In one embodiment, the first and second polypeptide are a first and second redox protein.\",\n \"Accordingly, one embodiment provided herein relates to novel and improved methods for the production of redox proteins.\",\n \"It has unexpectedly been found that a redox protein when prepared as a fusion protein with a second redox protein is fully enzymatically active when produced in association with an oil body.\",\n \"In contrast, when the redox protein is prepared without the second redox protein it has reduced enzymatic activity.\",\n \"In one embodiment, the first redox protein is at least 5 times more active when produced as a redox fusion polypeptide relative to production as a non-fusion polypeptide.\",\n \"Accordingly, provided herein are methods for producing an oil body associated with a heteromultimeric redox protein complex, said method comprising: (a) producing in a cell comprising oil bodies, a first redox protein and a second redox protein wherein said first redox protein is capable of interacting with said second redox protein, preferably in the cell, to form said heteromultimeric redox protein complex; and (b) associating said heteromultimeric redox protein complex with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said heteromultimeric redox protein complex.\",\n \"In a particular embodiment the first and second redox protein are prepared as a fusion protein to form a redox fusion polypeptide.\",\n \"Accordingly, provided herein are methods for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.\",\n \"The oil bodies in association with the redox protein may be used to prepare a variety of useful emulsions.\",\n \"As used herein the phrase “redox proteins” or grammatical variations thereof, refers to any protein or active protein fragment capable of participating in electron transport.\",\n \"For example, redox proteins are capable of catalyzing the transfer of an electron from an electron donor (also frequently referred to as the reducing agent) to an electron acceptor (also frequently referred to as the oxidizing agent).\",\n \"In the process of electron transfer, the reducing agent (electron donor) is oxidized and the oxidizing agent (electron acceptor) is reduced.\",\n \"Exemplary redox proteins for use herein include iron-sulfur proteins, cytochromes, redox active thiol proteins and redox-active flavoproteins.\",\n \"To carry out their function as conduits for electrons, redox proteins, such as thioredoxin and thioredoxin-reductase for example, are known to function by interacting or associating with one another in multimeric-protein-complexes (e.g., heteromultimeric-protein-complexes).\",\n \"The term “redox fusion polypeptide” as used herein refers to any fusion polypeptide comprising a first redox protein linked to a second redox protein (e.g., an in-frame translational fusion).\",\n \"The redox proteins that may be used with the methods and compositions provided herein may be any redox protein.\",\n \"In one embodiment the first and second redox proteins are a pair of redox proteins that would normally occur together from the same source, in nature.\",\n \"In a particular embodiment, the first redox protein is a thioredoxin and the second redox protein is a thioredoxin-reductase.\",\n \"The redox fusion polypeptide may be produced in any cell comprising oil bodies, including any animal cell, plant cell, algae cell, fungal cell or bacterial cell.\",\n \"In certain embodiments the redox fusion polypeptide is produced in a plant cell and in particular embodiments the redox fusion polypeptide is produced in the seed cells of a seed plant.\",\n \"In particular embodiments the oil-body-targeting-protein that is used is an oil-body-protein.\",\n \"In embodiments of the present invention in which an oil-body-protein is used, the first and second redox protein are preferably covalently fused to the oil-body-protein.\",\n \"Accordingly, provided herein are methods for the preparation of a redox protein In association with an oil body comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a first nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked in reading frame to (ii) a second nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide in a progeny cell comprising oil bodies; and c) isolating said oil bodies comprising said redox fusion polypeptide from said progeny cell.\",\n \"Redox Proteins In accordance with various methods and compositions provided herein, any nucleic acid sequence encoding a redox protein may be used.\",\n \"The nucleic acid sequence encoding the first and/or second redox protein may be obtained from any biological source or may be prepared synthetically.\",\n \"In general, nucleic acid sequences encoding redox proteins are well known in the art and readily available.\",\n \"See, for example: Cristiano et al.\",\n \"(1993) Genomics 17: (2) 348-354, Doyama et al.\",\n \"(1998) Plant Sci.\",\n \"137: 53-62, Hoeoeg et al.\",\n \"(1984) Biosci.\",\n \"Rep. 4: 917-923; as well as the Swiss Protein sequences set forth in Table 5.Known nucleic acid sequences encoding redox proteins may be used to design and construct nucleic acid sequence based probes in order to uncover and identify previously undiscovered nucleic acid sequences Encoding redox proteins, for example by screening cDNA or genomic libraries.\",\n \"Thus, additional nucleic acid sequences may be discovered and used in accordance with the present invention.\",\n \"The nucleic acid sequence encoding the first and/or second redox protein may be obtained from separate sources or may be obtained from the same source.\",\n \"In general however, the nucleic acid sequence encoding a redox-fusion polypeptide comprises nucleic acid sequences encoding a first and a second redox protein obtained from the same or a similar biological source.\",\n \"In certain embodiments provided herein, wherein the nucleic acid sequence encoding the first and second redox protein is obtained from the same source, the nucleic acid sequence encoding the first redox protein and second redox protein may be naturally fused.\",\n \"In accordance with a particular embodiment, the nucleic acid sequences encoding the first and second redox protein are preferably obtained from a plant source.\",\n \"As set forth above, a polypeptide spacer or linker of variable length may separate the first and second redox proteins from each other and/or from the oil-body-targeting-protein; and additional redox proteins (e.g., one or more) may be fused to the first and/or second redox protein.\",\n \"The nucleic acid sequences encoding the redox proteins may be altered to improve expression levels for example by optimizing the nucleic acids sequence in accordance with the preferred codon usage for the particular cell type which is selected for expression of the redox proteins, or by altering of motifs known to destabilize mRNAs (see for example: PCT Patent Application 97/02352).\",\n \"Comparison of the codon usage of the redox protein with codon usage of the host will enable the identification of codons that may be changed.\",\n \"For example, typically plant evolution has tended towards a preference for CG rich nucleotide sequences while bacterial evolution has resulted in bias towards AT rich nucleotide sequences.\",\n \"By modifying the nucleic acid sequences to incorporate nucleic acid sequences preferred by the host cell, expression may be optimized.\",\n \"Construction of synthetic genes by altering codon usage is described in for example PCT patent Application 93/07278.The redox proteins may be altered using for example, targeted mutagenesis, random mutagenesis (Shiraishi et al.\",\n \"(1998) Arch.\",\n \"Biochem.\",\n \"Biophys.\",\n \"358: 104-115; Galkin et al.\",\n \"(1997) Protein Eng.\",\n \"10: 687-690; Carugo et al.\",\n \"(1997) Proteins 28: 10-28; Hurley et al.\",\n \"(1996) Biochemistry 35: 5670-5678) (and/or by the addition of organic solvent (Holmberg et al.\",\n \"(1999) Protein Eng.\",\n \"12: 851-856).\",\n \"The polypeptide spacer between the first and second redox protein may be altered in similar ways.\",\n \"The first and second redox protein may be selected by developing a two-dimensional matrix and determining which combination of first and second redox protein is most effective in electron transport using for example, a colorometric reduction assay (Johnson et al (1984) J. of Bact.\",\n \"Vol.\",\n \"158 3:1061-1069, Luthman et al (1982) Biochemistry Vol 21 26:6628-2233).\",\n \"Combinations of thioredoxin and thioredoxin-reductase may be tested by determining the reduction of wheat storage proteins and milk storage protein beta-lactoglobulin in vitro (Del Val et al.\",\n \"(1999) J. Allerg.\",\n \"Clin.\",\n \"Immunol.\",\n \"103: 690-697).\",\n \"Using the same strategy polypeptide spacers between the first and second redox proteins may be evaluated for their efficiency.\",\n \"First and second redox proteins that may be used herein include without limitation any first redox protein and second redox protein selected from the group of redox proteins consisting of cytochromes, such as cytochrome a, cytochrome b and cytochrome c; porphyrin containing proteins, for example hemoglobin; iron-sulfur proteins, such as ferredoxin; flavoproteins such as thioredoxin-reductase, NADH dehydrogenase, succinate dehydrogenase, dihydrolipoyl dehydrogenase, acyl-CoA dehydrogenase, D-amino acid oxidase, xanthine oxidase, orotate reductase and aldehyde oxidase; pyridine-linked dehydrogenases, for example, lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, and beta-hydroxy-butarate dehydrogenase; and redox active thiol containing proteins such as thioredoxin.\",\n \"In particular embodiments, the redox proteins provided herein are thioredoxin and its reductant thioredoxin-reductase (which are jointly also referred to herein as “thioredoxin-related” protein(s)).\",\n \"As used herein, the term “thioredoxin” refers to relatively small proteins (typically approximately 12 kDa) that belong to the family of thioltransferases which catalyze oxido-reductions via the formation or hydrolysis of disulfide bonds and are widely, if not universally, distributed throughout the animal plant and bacterial kingdom.\",\n \"The reduces form of thioredoxin is an excellent catalyst for the reduction of even the most intractable disulfide bonds.\",\n \"In order to reduce the oxidized thioredoxin, two cellular reductants provide the reduction equivalents: reduced ferredoxin and NADPH.\",\n \"These reduction equivalents are supplied to thioredoxin via interaction or association with different thioredoxin-reductases including the NADPH thioredoxin-reductase and ferredoxin thioredoxin-reductase.\",\n \"The supply of these reduction equivalents requires the formation of a heteromultimeric-protein-complex comprising thioredoxin and thioredoxin-reductase.\",\n \"Ferredoxin thioredoxin-reductase is involved in the reduction of plant thioredoxins designated as Trxf and Trxm, both of which are involved in the regulation of photosynthetic processes in the chloroplast.\",\n \"The NADPH/thioredoxin active in plant seeds is designated Trxh (also referred to herein as thioredoxin h-type) and is capable of the reduction of a wide range of proteins thereby functioning as an important cellular redox buffer.\",\n \"Generally, only one kind of thioredoxin, which analogous to the plant Trxh type, is found in bacterial or animal cells.\",\n \"The h-type thioredoxins are capable of being reduced by NADPH and NADPH-thioredoxin reductase.\",\n \"Exemplary thioredoxins are further characterized as a protein having a core of 5 beta-sheets surrounded by 4 to 6 alpha helixes.\",\n \"Exemplary thioredoxins are further characterized by having an active site containing the consensus amino acid sequence: XCYYCZ, wherein Y is any amino acid, such as hydrophobic or non-polar amino acids, wherein X can be any of the 20 amino acids, preferably a hydrophobic amino acid, such as a tryptophan, and Z can be any amino acid, preferably polar amino acids.\",\n \"In certain embodiments, the thioredoxins for use herein comprise an active site having the amino acid sequence XCGPCZ.\",\n \"When the cysteines in the active site of thioredoxin or thioredoxin-like proteins are oxidized, they form an intramolecular disulfide bond.\",\n \"In the reduced state, the same active sites are capable of participating in redox reactions through the reversible oxidation of its active site dithiol, to a disulfide and catalyzes dithioldisulfide exchange reactions.\",\n \"Exemplary thioredoxins are well-known in the art and can be obtained from several organisms including Arabidopsis thaliana (Riveira Madrid et al.\",\n \"(1995) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"92: 5620-5624), wheat (Gautier et al.\",\n \"(1998) Eur.\",\n \"J. Biochem.\",\n \"252: 314-324); Escherichia coli (Hoeoeg et al (1984) Biosci.\",\n \"Rep. 4: 917-923) and thermophylic microorganisms such as Methanococcus jannaschii and Archaeoglobus fulgidus (PCT Patent Application 00/36126).\",\n \"Thioredoxins have also been recombinantly expressed in several host systems including bacteria (Gautier et al.\",\n \"(1998) Eur J. Biochem.\",\n \"252: 314-324) and plants (PCT Patent Application WO 00/58453) Commercial preparations of E. coli sourced Thioredoxins are readily available from for example: Sigma Cat No.\",\n \"T 0910 Thioredoxin (E. coli, recombinant; expressed in E. coli).\",\n \"Exemplary nucleic acid sequences encoding thioredoxin polypeptides for use herein are readily available from a variety of diverse biological sources including E. coli (Hoeoeg et al.\",\n \"(1984) Biosci.\",\n \"Rep.: 4 917-923); Methanococcus jannaschii and Archaeoglobus fulgidus (PCT Patent Application 00/36126); Arabidopsis thaliana (Rivera-Madrid (1995) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"92: 5620-5624); wheat (Gautier et al (1998) Eur.\",\n \"J. Biochem.\",\n \"252(2): 314-324); tobacco (Marty et al.\",\n \"(1991) Plant Mol.\",\n \"Biol.\",\n \"17: 143-148); barley (PCT Patent Application 00/58352); rice (Ishiwatari et al.\",\n \"(1995) Planta 195: 456-463); soybean (Shi et al.\",\n \"(1996) Plant Mol.\",\n \"Biol.\",\n \"32: 653-662); rapeseed (Bower et al.\",\n \"Plant Cell 8: 1641-1650) and calf (Terashima et al.\",\n \"(1999) DNA Seq.\",\n \"10(3): 203-205); and the like.\",\n \"In yet other embodiments, exemplary nucleic acids for use herein include those encoding the thioredoxin and thioredoxin-like polypeptide chains set forth as SEQ ID NOs:38, 42, 46 and 50; and those encoding the thioredoxin and thioredoxin-like polypeptide chains set forth in Table 5 as SEQ ID NOs:52-194.The respective nucleic acid sequences encoding the amino acids set forth in SEQ ID NOs:52-194 can be readily identified via the Swiss Protein identifier (accession) numbers provided in Table 5 (in parenthesis).\",\n \"As used herein, the term “thioredoxin-reductase” refers to a protein that complexes with a flavin, such as FAD.\",\n \"The flavin compound serves as an electron donor for the thioredoxin-reductase protein active site.\",\n \"Thioredoxin reductases have a redox active, disulfide bond site capable of reducing thioredoxin.\",\n \"The active site of thioredoxin-reductase contains 2 cysteines.\",\n \"The type of amino acids surrounding the 2 cysteine residues forming the active site can vary as hydrophobic, non-polar or polar.\",\n \"An exemplary thioredoxin-reductase is NADPH-thioredoxin-reductase (NTR), which is a cytosolic homodimeric enzyme comprising typically 300-500 amino acids.\",\n \"Crystal structures of both E. coli and plant thioredoxin-reductase have been obtained (Waksman et al.\",\n \"(1994) J. Mol.\",\n \"Biol.\",\n \"236: 800-816; Dai et al.\",\n \"(1996) J. Mol.\",\n \"Biol.\",\n \"264:1044-1057).\",\n \"NADPH-thioredoxin-reductases have been expressed in heterologous hosts, for example the Arabidopsis NADPH-thioredoxin-reductase has been expressed in E. coli (Jacquot et al.\",\n \"(1994) J. Mol.\",\n \"Biol.\",\n \"235: 1357-1363) and wheat (PCT Patent Application 00/58453).\",\n \"Exemplary nucleic acid sequences encoding thioredoxin-reductase proteins can readily be obtained from a variety of sources, such as from the sequence set forth in Table 5 and the Sequence Listing provide herein, from Arabidopsis (Riveira Madrid et al.\",\n \"(1995) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 92: 5620-5624), E. coli (Russel et al.\",\n \"(1988) J. Biol.\",\n \"Chem.\",\n \"263: 9015-9019); barley (PCT Patent Application 00158352 and wheat (Gautier et al., (1998) Eur.\",\n \"J. Biochem.\",\n \"252: 314-324); and the like.\",\n \"In yet other embodiments, exemplary nucleic acids for use herein include those encoding the thioredoxin-reductase polypeptide chains set forth as SEQ ID NOs:8, 9, 10, 40, 44, 48 and 50; and those encoding the thioredoxin-reductase polypeptide chains set forth in Table 5 as SEQ ID NOs:195-313.The respective nucleic acid sequences encoding the amino acids set forth in SEQ ID NOs:195-313 can be readily identified via the Swiss Protein identifier (accession) numbers provided in Table 5 (in parenthesis).\",\n \"Also contemplated for use in the methods and compositions provided herein are nucleic acid and amino acid homologs that are “substantially homologous” to the thioredoxin and thioredoxin-reductase nucleic and amino acids set forth herein, which includes thioredoxin and thioredoxin-reductase polypeptides encoded by a sequence of nucleotides that hybridizes under conditions of low, moderate or high stringency to the sequence of nucleotides encoding the thioredoxin and thioredoxin-reductase nucleic and amino acids set forth herein (e.g., in the Examples, Sequence Listing and/or Table 5).\",\n \"As used herein, a DNA or nucleic acid homolog refers to a nucleic acid that includes a preselected conserved nucleotide sequence, such as a sequence encoding a therapeutic polypeptide.\",\n \"By the term “substantially homologous” is meant having at least 80%, preferably at least 90%, most preferably at least 95% homology therewith or a less percentage of homology or identity and conserved biological activity or function.\",\n \"The terms “homology” and “identity” are often used interchangeably.\",\n \"In this regard, percent homology or identity may be determined, for example, by comparing sequence information using a GAP computer program.\",\n \"The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol.\",\n \"Biol.\",\n \"48:443 (1970), as revised by Smith and Waterman (Adv.\",\n \"Appl.\",\n \"Math.\",\n \"2:482 (1981).\",\n \"Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.\",\n \"The preferred default parameters for the GAP program may include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl.\",\n \"Acids Res.\",\n \"14:6745 (1986), as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp.\",\n \"353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.\",\n \"By sequence identity, the number of conserved amino acids are determined by standard alignment algorithms programs, and are used with default gap penalties established by each supplier.\",\n \"Substantially homologous nucleic acid molecules would hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid of interest.\",\n \"Preferably the two molecules will hybridize under conditions of high stringency.\",\n \"Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.\",\n \"Whether any two nucleic acid molecules have nucleotide sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can be determined using known computer algorithms such as the “FAST A” program, using for example, the default parameters as in Pearson and Lipman, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 85:2444 (1988).\",\n \"Alternatively the BLAST function of the National Center for Biotechnology Information database may be used to determine relative sequence identity.\",\n \"In general, sequences are aligned so that the highest order match is obtained.\",\n \"“Identity” per se has an art-recognized meaning and can be calculated using published techniques.\",\n \"(See, e.g.\",\n \": Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).\",\n \"While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988)).\",\n \"Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988).\",\n \"Methods to determine identity and similarity are codified in computer programs.\",\n \"Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)).\",\n \"Therefore, as used herein, the term “identity” represents a comparison between a test and a reference polypeptide or polynucleotide.\",\n \"For example, a test polypeptide may be defined as any polypeptide that is 90% or more identical to a reference polypeptide.\",\n \"As used herein, the term at least “90% identical to” refers to percent identities from 90 to 99.99 relative to the reference polypeptides.\",\n \"Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids are compared.\",\n \"No more than 10% (i.e., 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptides.\",\n \"Similar comparisons may be made between a test and reference polynucleotides.\",\n \"Such differences may be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they may be clustered in one or more locations of varying length up to the maximum allowable, e.g.\",\n \"10/100 amino acid difference (approximately 90% identity).\",\n \"Differences are defined as nucleic acid or amino acid substitutions, or deletions.\",\n \"As used herein: stringency of hybridization in determining percentage mismatch is as follows: 1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C. 2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C. 3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C. Those of skill in this art know that the washing step selects for stable hybrids and also know the ingredients of SSPE (see, e.g., Sambrook, E. F. Fritsch, T. Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), vol.\",\n \"3, p. B.\",\n \"13, see, also, numerous catalogs that describe commonly used laboratory solutions).\",\n \"SSPE is pH 7.4 phosphate-buffered 0.18 NaCl.\",\n \"Further, those of skill in the art recognize that the stability of hybrids is determined by Tm, which is a function of the sodium ion concentration and temperature (Tm=81.5° C.-16.6(log10[Na+])+0.41 (% G+C)−600/l)), so that the only parameters in the wash conditions critical to hybrid stability are sodium ion concentration in the SSPE (or SSC) and temperature.\",\n \"It is understood that equivalent stringencies may be achieved using alternative buffers, salts and temperatures.\",\n \"By way of example and not limitation, procedures using conditions of low stringency are as follows (see also Shilo and Weinberg, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 78:6789-6792 (1981)): Filters containing DNA are pretreated for 6 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA (10×SSC is 1.5 M sodium chloride, and 0.15 M sodium citrate, adjusted to a pH of 7).\",\n \"In a particular embodiment, a heteromultimeric-protein-complex is produced as a fusion polypeptide between the first and second redox protein, wherein the first redox protein is thioredoxin and the second redox protein is a thioredoxin-reductase.\",\n \"In one embodiment, the second recombinant polypeptide, e.g., the thioredoxin-reductase is positioned N-terminal relative to the first recombinant polypeptide, e.g., the thioredoxin.\",\n \"Accordingly, any protein which is classified as thioredoxin, such as the thioredoxin component of the NADPH thioredoxin system and the thioredoxin present in the ferredoxin/thioredoxin system also known as TRx and TRm may be used in combination with any thioredoxin-reductase such as the NADPH thioredoxin-reductase and the ferredoxin-thioredoxin-reductase and any other proteins having the capability of reducing thioredoxin.\",\n \"In particular embodiments the thioredoxin and thioredoxin-reductase are plant derived.\",\n \"In an alternate embodiment, the naturally occurring nucleic acid sequence encoding the thioredoxin/thioredoxin-reductase protein fusion obtainable from Mycobacterium leprae (Wieles et al.\",\n \"(1995) J. Biol.\",\n \"Chem.\",\n \"27:25604-25606) is used, as set forth in the Examples herein.\",\n \"Immunoglobulins In another embodiment of the present invention, the multimeric-protein-complexes are immunoglobulins.\",\n \"As used herein “immunoglobulin-polypeptide-chain” refers to a first polypeptide that is capable of associating with a second polypeptide to form an immunologically active (i.e.\",\n \"capable of antigen binding multimeric-protein-complex.\",\n \"The types of immunoglobulins and immunoglobulin-polypeptide-chains contemplated for use herein include the immunologically active (i.e.\",\n \"antigen binding) portions of a light and heavy chain.\",\n \"Exemplary immunoglobulins and immunoglobulin-polypeptide-chains for use herein include substantially intact immunoglobulins, including any IgG, IgA, IgD, IgE and IgM, as well as any portion of an immunoglobulin, including those portions well-known as Fab fragments, Fab′ fragments, F(ab′).sub2.fragments and Fv fragments.\",\n \"In this embodiment, the first recombinant polypeptide may be any immunoglobulin heavy chain, including any IgG, IgA, IgD, IgE or IgM heavy chain, and the second recombinant polypeptide may be a kappa or lambda immunoglobulin light chain.\",\n \"Accordingly, provided herein are methods of producing an immunoglobulin, said method comprising: (a) producing in a cell comprising oil bodies, a first immunoglobulin-polypeptide-chain and a second immunoglobulin-polypeptide-chain wherein said first immunoglobulin-polypeptide-chain is capable of associating with said second immunoglobulin-polypeptide-chain to form said immunoglobulin; and (b) associating said immunoglobulin with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first immunoglobulin-polypeptide-chain.\",\n \"As set forth herein, the multimeric immunoglobulin is associated with an oil body through an oil-body-targeting-protein.\",\n \"In particular embodiments, the oil-body-targeting-protein may be a fusion polypeptide comprising an oil-body-protein and an immunoglobulin binding protein, such as for example protein A, protein L, and protein G. In yet another embodiment involving immunoglobulins, the first and second recombinant polypeptides (immunoglobulins) are separately fused to an oil body protein, for example an oleosin or caleosin.\",\n \"For example, a) the first recombinant polypeptide may be an immunoglobulin heavy chain, including any IgG, IgA, IgD, IgE or IgM heavy chain, and the second recombinant polypeptide may be a kappa or lambda immunoglobulin light chain; or b) the first recombinant polypeptide may be the variable and first constant domain from an immunoglobulin heavy chain and the second recombinant polypeptide may be a kappa or lambda immunoglobulin light chain; or c) the first recombinant polypeptide may be the variable domain from an immunoglobulin heavy chain and the second recombinant polypeptide may be the variable domain from a kappa or lambda immunoglobulin light chain.\",\n \"In certain embodiments, the fusion polypeptides are designed or selected to allow the heteromultimeric-protein-complex formation between immunoglobulin light and heavy chain sequences on the oil bodies within the cell comprising oil bodies.\",\n \"Preparation of Expression Vectors Comprising Oil-Body-Targeting-Proteins and the First and/or Second Recombinant Polypeptides, Multimeric-Protein-Complexes, Heteromultimeric-Protein-Complexes, Multimeric-Fusion-Proteins, Heteromultimeric-Fusion-Proteins, Immunoglobulins, Immunoglobulin-Polypeptide-Chains, Redox-Fusion-Polypeptides, or the First and/or Second Thioredoxin-Related Proteins.\",\n \"In accordance with the present invention, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and the oil-body-targeting-protein are conveniently produced in a cell.\",\n \"In order to produce the recombinant polypeptides or multimeric-protein-complexes, a nucleic acid sequence encoding either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and/or the oil-body-targeting-protein are incorporated in a recombinant expression vector.\",\n \"Accordingly, provided herein are recombinant expression vectors comprising the chimeric nucleic acids provided herein suitable for expression of the oil-body-targeting-protein and the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, suitable for the selected cell.\",\n \"The term “suitable for expression in the selected cell” means that the recombinant expression vector contains all nucleic acid sequences required to ensure expression in the selected cell.\",\n \"Accordingly, the recombinant expression vectors further contain regulatory nucleic acid sequences selected on the basis of the cell which is used for expression and ensuring initiation and termination of transcription operatively linked to the nucleic acid sequence encoding the recombinant polypeptide or multimeric-protein-complex and/or the oil-body-targeting-protein.\",\n \"Regulatory nucleic acid sequences include promoters, enhancers, silencing elements, ribosome binding sites, Shine-Dalgarno sequences, introns and other expression elements.\",\n \"“Operatively linked” is intended to mean that the nucleic acid sequences comprising the regulatory regions linked to the nucleic acid sequences encoding the recombinant polypeptide or multimeric-protein-complex and/or the oil-body-targeting-protein allow expression in the cell.\",\n \"A typical nucleic acid construct comprises in the 5′ to 3′ direction a promoter region capable of directing expression, a coding region comprising the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and/or an oil-body-targeting-protein and a termination region functional in the selected cell.\",\n \"The selection of regulatory sequences will depend on the organism and the cell type in which the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and/or the oil-body-targeting-protein is expressed, and may influence the expression levels of the polypeptide.\",\n \"Regulatory sequences are art-recognized and selected to direct expression of the oil-body-targeting-protein and the recombinant polypeptides or multimeric-protein-complexes in the cell.\",\n \"Promoters that may be used in bacterial cells include the lac promoter (Blackman et al.\",\n \"(1978) Cell: 13: 65-71), the trp promoter (Masuda et al.\",\n \"(1996) Protein Eng: 9: 101-106) and the T7 promoters (Studier et al.\",\n \"(1986) J. Mol.\",\n \"Biol.\",\n \"189: 113-130).\",\n \"Promoters functional in plant cells that may be used herein include constitutive promoters such as the 35S CaMV promoter (Rothstein et al.\",\n \"(1987) Gene: 53: 153-161) the actin promoter (McElroy et al.\",\n \"(1990) Plant Cell 2: 163-171) and the ubiquitin promoter (European Patent Application 0 342 926).\",\n \"Other promoters are specific to certain tissues or organs (for example, roots, leaves, flowers or seeds) or cell types (for example, leaf epidermal cells, mesophyll cells or root cortex cells) and or to certain stages of plant development.\",\n \"Timing of expression may be controlled by selecting an inducible promoter, for example the PR-a promoter described in U.S. Pat.\",\n \"No.\",\n \"5,614,395.Selection of the promoter therefore depends on the desired location and timing of the accumulation of the desired polypeptide.\",\n \"In a particular embodiment, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and the oil-body-targeting-protein are expressed in a seed cell and seed specific promoters are utilized.\",\n \"Seed specific promoters that may be used herein include for example the phaseolin promoter (Sengupta-Gopalan et al.\",\n \"(1985) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA: 82: 3320-3324), and the Arabidopsis 18 kDa oleosin promoter (van Rooijen et al.\",\n \"(1992) Plant.\",\n \"Mol.\",\n \"Biol.\",\n \"18: 1177-1179).\",\n \"New promoters useful in various plant cell types are constantly discovered.\",\n \"Numerous examples of plant promoters may be found in Ohamuro et al.\",\n \"(Biochem of Pl.\",\n \"(1989) 15: 1-82).\",\n \"Genetic elements capable of enhancing expression of the polypeptide may be included in the expression vectors.\",\n \"In plant cells these include for example, the untranslated leader sequences from viruses such as the AMV leader sequence (Jobling and Gehrke (1987) Nature: 325: 622-625) and the intron associated with the maize ubiquitin promoter (See: U.S. Pat.\",\n \"No.\",\n \"5,504,200).\",\n \"Transcriptional terminators are generally art recognized and besides serving as a signal for transcription termination serve as a protective element serving to extend the mRNA half-life (Guarneros et al.\",\n \"(1982) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA: 79: 238-242).\",\n \"In nucleic acid sequences for the expression in plant cells, the transcriptional terminator typically is from about 200 nucleotide to about 1000 nucleotides in length.\",\n \"Terminator sequences that may be used herein include for example, the nopaline synthase termination region (Bevan et al.\",\n \"(1983) Nucl.\",\n \"Acid.\",\n \"Res.\",\n \": 11: 369-385), the phaseolin terminator (van der Geest et al.\",\n \"(1994) Plant J.: δ: 413-423), the terminator for the octopine synthase gene of Agrobacterium tumefaciens or other similarly functioning elements.\",\n \"Transcriptional terminators can be obtained as described by An (1987) Methods in Enzym.\",\n \"153: 292).\",\n \"The selection of the transcriptional terminator may have an effect on the rate of transcription.\",\n \"Accordingly, provided herein are chimeric nucleic acid sequences encoding a first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins.\",\n \"In one embodiment, said nucleic acid comprises: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide, immunoglobulin-polypeptide-chain, or redox protein; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, immunoglobulin-polypeptide-chain or redox protein, wherein said first and second recombinant polypeptides, immunoglobulin-polypeptide-chains or redox proteins are capable of forming a multimeric-protein-complex.\",\n \"In another embodiment, provided herein is an expression vector comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked in reading frame to (ii) a nucleic acid sequence encoding a multimeric-fusion-protein, such as a redox fusion polypeptide or immunoglobulin, comprising a first recombinant polypeptide, such as a redox protein or immunoglobulin-polypeptide-chain, linked to a second recombinant polypeptide, such as a second redox protein or a second immunoglobulin-polypeptide-chain, operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.\",\n \"The recombinant expression vector further may contain a marker gene.\",\n \"Marker genes that may be used in accordance with the present invention include all genes that allow the distinction of transformed cells from non-transformed cells including all selectable and screenable marker genes.\",\n \"A marker may be a resistance marker such as an antibiotic resistance marker against for example kanamycin, ampicillin, G418, bleomycin hygromycin, chloramphenicol which allows selection of a trait by chemical means or a tolerance marker against for example a chemical agent such as the normally phytotoxic sugar mannose (Negrotto et al.\",\n \"(2000) Plant Cell Rep. 19: 798-803).\",\n \"In plant recombinant expression vectors herbicide resistance markers may conveniently be used for example markers conferring resistance against glyphosate (U.S. Pat.\",\n \"Nos.\",\n \"4,940,935 and 5,188,642) or phosphinothricin (White et al.\",\n \"(1990) Nucl.\",\n \"Acids Res.\",\n \"18: 1062; Spencer et al.\",\n \"(1990) Theor.\",\n \"Appl.\",\n \"Genet.\",\n \"79: 625-631).\",\n \"Resistance markers to a herbicide when linked in close proximity to the redox protein or oil-body-targeting-protein may be used to maintain selection pressure on a population of plant cells or plants for those plants that have not lost the protein of interest.\",\n \"Screenable markers that may be employed to identify transformants through visual observation include beta-glucuronidase (GUS) (see U.S. Pat.\",\n \"No.\",\n \"5,268,463 and U.S. Pat.\",\n \"No.\",\n \"5,599,670) and green fluorescent protein (GFP) (Niedz et al.\",\n \"(1995) Plant Cell Rep.: 14: 403).\",\n \"The recombinant expression vectors further may contain nucleic acid sequences encoding targeting signals ensuring targeting to a cell compartment or organelle.\",\n \"Suitable targeting signals that may be used herein include those that are capable of targeting polypeptides to the endomembrane system.\",\n \"Exemplary targeting signals that may be used herein include targeting signals capable of directing the protein to the periplasm, the cytoplasm, the golgi apparatus, the apoplast (Sijmons et al., 1990, Bio/Technology, 8:217-221) the chloroplast (Comai et al.\",\n \"(1988) J. Biol.\",\n \"Chem.\",\n \"263: 15104-15109), the mitochondrion, the peroxisome (Unger et al.\",\n \"(1989) Plant Mol.\",\n \"Biol.\",\n \"13: 411-418), the ER, the vacuole (Shinshi et al.\",\n \"(1990) Plant Mol.\",\n \"Biol.\",\n \"14: 357-368 and the oil body.\",\n \"By the inclusion of the appropriate targeting sequences it is possible to direct the oil-body-targeting-protein or the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins, to the desired organelle or cell compartment.\",\n \"The recombinant expression vectors of the present invention may be prepared in accordance with methodologies well known to those of skill in the art of molecular biology (see for example: Sambrook et al.\",\n \"(1990) Molecular Cloning, 2nd ed.\",\n \"Cold Spring Harbor Press).\",\n \"The preparation of these constructs may involve techniques such as restriction digestion, ligation, gel electrophoresis, DNA sequencing and PCR.\",\n \"A wide variety of cloning vectors is available to perform the necessary cloning steps resulting in a recombinant expression vector ensuring expression of the polypeptide.\",\n \"Especially suitable for this purpose are vectors with a replication system that is functional in Escherichia coli such as pBR322, the PUC series of vectors, the M13 mp series of vectors, pBluescript etc.\",\n \"Typically these vectors contain a marker allowing the selection of transformed cells for example by conferring antibiotic resistance.\",\n \"Nucleic acid sequences may be introduced in these vectors and the vectors may be introduced in E. coli grown in an appropriate medium.\",\n \"Vectors may be recovered from cells upon harvesting and lysing the cells.\",\n \"Recombinant expression vectors suitable for the introduction of nucleic acid sequences in plant cells include Agrobacterium and Rhizobium based vectors such as the Ti and Ri plasmids.\",\n \"Agrobacterium based vectors typically carry at least one T-DNA border sequence and include vectors such pBIN 19 (Bevan (1984) Nucl Acids Res.\",\n \"Vol.\",\n \"12, 22:8711-8721) and other binary vector systems (for example: U.S. Pat.\",\n \"No.\",\n \"4,940,838).\",\n \"Production of Cells Comprising a First and/or Second Recombinant Polypeptides, Multimeric-Protein-Complexes, Heteromultimeric-Protein-Complexes, Multimeric-Fusion-Proteins, Heteromultimeric-Fusion-Proteins, Immunoglobulins, Immunoglobulin-Polypeptide-Chains, Redox-Fusion-Polypeptides, and/or a First and/or Second Thioredoxin-Related Protein and Oil-Body-Targeting-Proteins In accordance with the present invention, the recombinant expression vectors are introduced into the cell that is selected and the selected cells are grown to produce the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, a first and/or second thioredoxin-related protein; and the oil-body-targeting-protein either directly or in a progeny cell.\",\n \"Methodologies to introduce recombinant expression vectors into a cell also referred to herein as “transformation” are well known to the art and vary depending on the cell type that is selected.\",\n \"General techniques to transfer the recombinant expression vectors into the cell include electroporation; chemically mediated techniques, for example CaCl2 mediated nucleic acid uptake; particle bombardment (biolistics); the use of naturally infective nucleic acid sequences for example virally derived nucleic acid sequences or when plant cells are used Agrobacterium or Rhizobium derived nucleic acid sequences; PEG mediated nucleic acid uptake, microinjection, and the use of silicone carbide whiskers (Kaeppler et al.\",\n \"(1990) Plant Cell Rep. 9:415-418) all of which may be used herein.\",\n \"Introduction of the recombinant expression vector into the cell may result in integration of its whole or partial uptake into host cell genome including the chromosomal DNA or the plastid genome.\",\n \"Alternatively the recombinant expression vector may not be integrated into the genome and replicate independently of the host cell's genomic DNA.\",\n \"Genomic integration of the nucleic acid sequence is typically used as it will allow for stable inheritance of the introduced nucleic acid sequences by subsequent generations of cells and the creation of cell, plant or animal lines.\",\n \"Particular embodiments involve the use of plant cells.\",\n \"Particular plant cells used herein include cells obtainable from Brazil nut (Betholletia excelsa); castor (Riccinus communis); coconut (Cocus nucifera); coriander (Coriandrum sativum); cotton (Gossypium spp.\",\n \"); groundnut (Arachis hypogaea); jojoba (Simmondsia chinensis); linseed/flax (Linum usitatissimum); maize (Zea mays); mustard (Brassica spp.\",\n \"and Sinapis alba); oil palm (Elaeis guineeis); olive (Olea europaea); rapeseed (Brassica spp.\",\n \"); safflower (Carthamus tinctorius); soybean (Glycine max); squash (Cucurbita maxima); barley (Hordeum vulgare); wheat (Traeticum aestivum) and sunflower (Helianthus annuus).\",\n \"Transformation methodologies for dicotelydenous plant species are well known.\",\n \"Generally Agrobacterium mediated transformation is utilized because of its high efficiency as well as the general susceptibility by many, if not all dicotelydenous plant species.\",\n \"Agrobacterium transformation generally involves the transfer of a binary vector (e.g.\",\n \"pBIN19) comprising the DNA of interest to an appropriate Agrobacterium strain (e.g.\",\n \"CIB542) by for example tri-parental mating with an E. coli strain carrying the recombinant binary vector and an E. coli strain carrying a helper plasmid capable of mobilization of the binary vector to the target Agrobacterium strain, or by DNA transformation of the Agrobacterium strain (Hofgen et al.\",\n \"Nucl.\",\n \"Acids.\",\n \"Res.\",\n \"(1988) 16: 9877.Other transformation methodologies that may be used to transform dicotelydenous plant species include biolistics (Sanford (1988) Trends in Biotechn.\",\n \"6: 299-302); electroporation (Fromm et al.\",\n \"(1985) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 82: 5824-5828); PEG mediated DNA uptake (Potrykus et al.\",\n \"(1985) Mol.\",\n \"Gen. Genetics 199: 169-177); microinjection (Reich et al.\",\n \"Bio/Techn.\",\n \"(1986) 4: 1001-1004) and silicone carbide whiskers (Kaeppler et al.\",\n \"(1990) Plant Cell Rep. 9: 415-418).\",\n \"The exact transformation methodologies typically vary somewhat depending on the plant species that is used.\",\n \"In a particular embodiment the oil bodies are obtained from safflower and the recombinant proteins are expressed in safflower.\",\n \"Safflower transformation has been described by Baker and Dyer (Plant Cell Rep. (1996) 16: 106-110).\",\n \"Monocotelydenous plant species may now also be transformed using a variety of methodologies including particle bombardment (Christou et al.\",\n \"(1991) Biotechn.\",\n \"9: 957-962; Weeks et al.\",\n \"Plant Physiol.\",\n \"(1993) 102: 1077-1084; Gordon-Kamm et al.\",\n \"Plant Cell (1990) 2: 603-618) PEG mediated DNA uptake (EP 0 292 435; 0 392 225) or Agrobacterium-mediated transformation (Goto-Fumiyuki et al (1999) Nature-Biotech.\",\n \"17 (3):282-286).\",\n \"Plastid transformation is described in U.S. Pat.\",\n \"Nos.\",\n \"5,451,513; 5,545,817 and 5,545,818; and PCT Patent Applications 95/16783; 98/11235 and 00/39313) Basic chloroplast transformation involves the introduction of cloned plastid DNA flanking a selectable marker together with the nucleic acid sequence of interest into a suitable target tissue using for example biolistics or protoplast transformation.\",\n \"Selectable markers that may be used include for example the bacterial aadA gene (Svab et al.\",\n \"(1993) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 90: 913-917).\",\n \"Plastid promoters that may be used include for example the tobacco clpP gene promoter (PCT Patent Application 97/06250).\",\n \"In another embodiment, the invention chimeric nucleic acid constructs provided herein are directly transformed into the plastid genome.\",\n \"Plastid transformation technology is described extensively in U.S. Pat.\",\n \"Nos.\",\n \"5,451,513, 5,545,817, 5,545,818 and 5,576,198; in PCT application nos.\",\n \"WO 95/16783 and WO 97/32977; and in McBride et.\",\n \"al., Proc Natl Acad Sci USA 91: 7301-7305 (1994), the entire disclosures of all of which are hereby incorporated by reference.\",\n \"In one embodiment, plastid transformation is achieved via biolistics, first carried out in the unicellular green alga Chlamydomonas reinhardtii (Boynton et al.\",\n \"(1988) Science 240:1534-1537)) and then extended to Nicotiana tabacum (Svab et al.\",\n \"(1990) Proc Natl Acad Sci USA 87:8526-8530), combined with selection for cis-acting antibiotic resistance loci (spectinomycin or streptomycin resistance) or complementation of non-photosynthetic mutant phenotypes.\",\n \"In another embodiment, tobacco plastid transformation is carried out by particle bombardment of leaf or callus tissue, or polyethylene glycol (PEG)-mediated uptake of plasmid DNA by protoplasts, using cloned plastid DNA flanking a selectable antibiotic resistance marker.\",\n \"For example, 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and allow the replacement or modification of specific regions of the 156 kb tobacco plastid genome.\",\n \"In one embodiment, point mutations in the plastid 16S rDNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin can be utilized as selectable markers for transformation (Svab et al.\",\n \"(1990) Proc Natl Acad Sci USA 87:8526-8530; Staub et al.\",\n \"(1992) Plant Cell 4:39-45, the entire disclosures of which are hereby incorporated by reference), resulting in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves.\",\n \"The presence of cloning sites between these markers allows creation of a plastid targeting vector for introduction of foreign genes (Staub et al.\",\n \"(1993) EMBO J 12:601-606, the entire disclosure of which is hereby incorporated by reference).\",\n \"In another embodiment, substantial increases in transformation frequency can be obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3′-adenyltransferase (Svab et al.\",\n \"(1993) Proc Natl Acad Sci USA 90: 913-917, the entire disclosure of which is hereby incorporated by reference).\",\n \"This marker has also been used successfully for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl Acids Res 19, 4083-4089, the entire disclosure of which is hereby incorporated by reference).\",\n \"In other embodiments, plastid transformation of protoplasts from tobacco and the moss Physcomitrella can be attained using PEG-mediated DNA uptake (O'Neill et al.\",\n \"(1993) Plant J 3:729-738; Koop et al.\",\n \"(1996) Planta 199:193-201, the entire disclosures of which are hereby incorporated by reference).\",\n \"Both particle bombardment and protoplast transformation are also contemplated for use herein.\",\n \"Plastid transformation of oilseed plants has been successfully carried out in the genera Arabidopsis and Brassica (Sikdar et al.\",\n \"(1998) Plant Cell Rep 18:20-24; PCT Application WO 00/39313, the entire disclosures of which are hereby incorporated by reference).\",\n \"A chimeric nucleic sequence construct is inserted into a plastid expression cassette including a promoter capable of expressing the construct in plant plastids.\",\n \"A particular promoter capable of expression in a plant plastid is, for example, a promoter isolated from the 5′ flanking region upstream of the coding region of a plastid gene, which may come from the same or a different species, and the native product of which is typically found in a majority of plastid types including those present in non-green tissues.\",\n \"Gene expression in plastids differs from nuclear gene expression and is related to gene expression in prokaryotes (Stern et al.\",\n \"(1997) Trends in Plant Sci 2:308-315, the entire disclosure of which is hereby incorporated by reference).\",\n \"Plastid promoters generally contain the −35 and −10 elements typical of prokaryotic promoters, and some plastid promoters called PEP (plastid-encoded RNA polymerase) promoters are recognized by an E. coli-like RNA polymerase mostly encoded in the plastid genome, while other plastid promoters called NEP promoters are recognized by a nuclear-encoded RNA polymerase.\",\n \"Both types of plastid promoters are suitable for use herein.\",\n \"Examples of plastid promoters include promoters of clpP genes such as the tobacco clpP gene promoter (WO 97/06250, the entire disclosure of which is hereby incorporated by reference) and the Arabidopsis clpP gene promoter (U.S. application Ser.\",\n \"No.\",\n \"09/038,878, the entire disclosure of which is hereby incorporated by reference).\",\n \"Another promoter capable of driving expression of a chimeric nucleic acid construct in plant plastids comes from the regulatory region of the plastid 16S ribosomal RNA operon (Harris et al., (1994) Microbiol Rev 58:700-754; Shinozaki et al.\",\n \"(1986) EMBO J 5:2043-2049, the entire disclosures of both of which are hereby incorporated by reference).\",\n \"Other examples of promoters capable of driving expression of a nucleic acid construct in plant plastids include a psbA promoter or am rbcL promoter.\",\n \"A plastid expression cassette preferably further includes a plastid gene 3′ untranslated sequence (3′ UTR) operatively linked to a chimeric nucleic acid construct of the present invention.\",\n \"The role of untranslated sequences is preferably to direct the 3′ processing of the transcribed RNA rather than termination of transcription.\",\n \"An exemplary 3′ UTR is a plastid rps16 gene 3′ untranslated sequence, or the Arabidopsis plastid psbA gene 3′ untranslated sequence.\",\n \"In a further embodiment, a plastid expression cassette includes a poly-G tract instead of a 3′ untranslated sequence.\",\n \"A plastid expression cassette also preferably further includes a 5′ untranslated sequence (5′ UTR) functional in plant plastids, operatively linked to a chimeric nucleic acid construct provided herein.\",\n \"A plastid expression cassette is contained in a plastid transformation vector, which preferably further includes flanking regions for integration into the plastid genome by homologous recombination.\",\n \"The plastid transformation vector may optionally include at least one plastid origin of replication.\",\n \"The present invention also encompasses a plant plastid transformed with such a plastid transformation vector, wherein the chimeric nucleic acid construct is expressible in the plant plastid.\",\n \"Also encompassed herein is a plant or plant cell, including the progeny thereof, including this plant plastid.\",\n \"In a particular embodiment, the plant or plant cell, including the progeny thereof, is homoplasmic for transgenic plastids.\",\n \"Other promoters capable of driving expression of a chimeric nucleic acid construct in plant plastids include transactivator-regulated promoters, preferably heterologous with respect to the plant or to the subcellular organelle or component of the plant cell in which expression is effected.\",\n \"In these cases, the DNA molecule encoding the transactivator is inserted into an appropriate nuclear expression cassette which is transformed into the plant nuclear DNA.\",\n \"The transactivator is targeted to plastids using a plastid transit peptide.\",\n \"The transactivator and the transactivator-driven DNA molecule are brought together either by crossing a selected plastid-transformed line with and a transgenic line containing a DNA molecule encoding the transactivator supplemented with a plastid-targeting sequence and operably linked to a nuclear promoter, or by directly transforming a plastid transformation vector containing the desired DNA molecule into a transgenic line containing a chimeric nucleic acid construct encoding the transactivator supplemented with a plastid-targeting sequence operably linked to a nuclear promoter.\",\n \"If the nuclear promoter is an inducible promoter, in particular a chemically inducible embodiment, expression of the chimeric nucleic acid construct in the plastids of plants is activated by foliar application of a chemical inducer.\",\n \"Such an inducible transactivator-mediated plastid expression system is preferably tightly regulatable, with no detectable expression prior to induction and exceptionally high expression and accumulation of protein following induction.\",\n \"A particular transactivator is, for example, viral RNA polymerase.\",\n \"Particular promoters of this type are promoters recognized by a single sub-unit RNA polymerase, such as the T7 gene 10 promoter, which is recognized by the bacteriophage T7 DNA-dependent RNA polymerase.\",\n \"The gene encoding the T7 polymerase is preferably transformed into the nuclear genome and the T7 polymerase is targeted to the plastids using a plastid transit peptide.\",\n \"Promoters suitable for nuclear expression of a gene, for example a gene encoding a viral RNA polymerase such as the T7 polymerase, are described above and elsewhere in this application.\",\n \"Expression of chimeric nucleic acid constructs in plastids can be constitutive or can be inducible, and such plastid expression can be also organ- or tissue-specific.\",\n \"Examples of various expression systems are extensively described in WO 98/11235, the entire disclosure of which is hereby incorporated by reference.\",\n \"Thus, in one aspect, the present invention utilizes coupled expression in the nuclear genome of a chloroplast-targeted phage T7 RNA polymerase under the control of the chemically inducible PR-1a promoter, for example of the PR-1 promoter of tobacco, operably linked with a chloroplast reporter transgene regulated by T7 gene 10 promoter/terminator sequences, for example as described in as in U.S. Pat.\",\n \"No.\",\n \"5,614,395 the entire disclosure of which is hereby incorporated by reference.\",\n \"In another embodiment, when plastid transformants homoplasmic for the maternally inherited TR or NTR genes are pollinated by lines expressing the T7 polymerase in the nucleus, F1 plants are obtained that carry both transgene constructs but do not express them until synthesis of large amounts of enzymatically active protein in the plastids is triggered by foliar application of the PR-1a inducer compound benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH).\",\n \"In a particular embodiment, two or more genes, for example TR and NTR genes, are transcribed from the plastid genome from a single promoter in an operon-like polycistronic gene.\",\n \"In one embodiment, the operon-like polycistronic gene includes an intervening DNA sequence between two genes in the operon-like polycistronic gene.\",\n \"In a particular embodiment, the intervening DNA sequence is not present in the plastid genome to avoid homologous recombination with plastid sequences.\",\n \"In another embodiment, the DNA sequence is derived from the 5′ untranslated (UTR) region of a non-eukaryotic gene, preferably from a viral 5′UTR, preferably from a 6′UTR derived from a bacterial phage, such as a T7, T3 or SP6 phage.\",\n \"In one embodiment, a portion of the DNA sequence may be modified to prevent the formation of RNA secondary structures in an RNA transcript of the operon-like polycistronic gene, for example between the DNA sequence and the RBS of the downstream gene.\",\n \"Such secondary structures may inhibit or repress the expression of the downstream gene, particularly the initiation of translation.\",\n \"Such RNA secondary structures are predicted by determining their melting temperatures using computer models and programs such a the “mfold” program version 3 (available from Zuker and Turner, Washington University School of Medicine, St-Louis, Mo.)\",\n \"and other methods known to one skilled in the art.\",\n \"The presence of the intervening DNA sequence in the operon-like polycistronic gene increases the accessibility of the RBS of the downstream gene, thus resulting in higher rates of expression.\",\n \"Such strategy is applicable to any two or more genes to be transcribed from the plastid genome from a single promoter in an operon-like chimeric heteromultimeric gene.\",\n \"Following transformation the cells are grown, typically in a selective medium allowing the identification of transformants.\",\n \"Cells may be harvested in accordance with methodologies known to the art.\",\n \"In order to associate the oil bodies with the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and a first and/or second thioredoxin-related protein, the integrity of cells may be disrupted using any physical, chemical or biological methodology capable of disrupting the cells' integrity.\",\n \"These methodologies are generally cell-type dependent and known to the skilled artisan.\",\n \"Where plants are employed they may be regenerated into mature plants using plant tissue culture techniques generally known to the skilled artisan.\",\n \"Seeds may be harvested from mature transformed plants and used to propagate the plant line.\",\n \"Plants may also be crossed and in this manner, contemplated herein is the breeding of cells lines and transgenic plants that vary in genetic background.\",\n \"It is also possible to cross a plant line comprising the first recombinant polypeptide with a plant line comprising the second recombinant polypeptide.\",\n \"Accordingly, also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide, such as a redox protein (e.g., a thioredoxin-related protein, and the like) or an immunoglobulin-polypeptide-chain, wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide, such as a second redox protein (e.g., a thioredoxin-related protein, and the like) or a second immunoglobulin-polypeptide-chain; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.\",\n \"The second recombinant polypeptide may also associate with the oil bodies.\",\n \"Accordingly, also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide, such as a redox (or thioredoxin-related) protein or immunoglobulin-polypeptide-chain, wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide, such as a second redox (thioredoxin-related) protein or a second immunoglobulin-polypeptide-chain, wherein said second recombinant polypeptide is capable of associating with said oil bodies through an oil body targeting protein; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.\",\n \"Isolation of Oil Bodies The oil bodies provided herein may be obtained from any cell containing oil bodies, including any animal cell; plant cell; fungal cell; for example a yeast cell, algae cell; or bacterial cell.\",\n \"Any process suitable for the isolation oil bodies from cells may be used herein.\",\n \"Processes for the isolation of oil bodies from plant seed cells have been described in U.S. Pat.\",\n \"Nos.\",\n \"(6,146,645 and 6,183,762) and the isolation of oil bodies from yeast cells has been described by Ting et al.\",\n \"(1997) J. Biol.\",\n \"Chem.\",\n \"272: 3699-3706).\",\n \"In certain embodiments, the oil bodies are obtained from a plant cell such as for example a pollen cell; a fruit cell; a spore cell; a nut cell; mesocarp cell; for example the mesocarp cells obtainable from olive (Olea europaea) or avocado (Persea americana); or a seed cell.\",\n \"In particular embodiments the oil bodies are obtained from a plant seed cell.\",\n \"The seeds can be obtained from a transgenic plant according to the present invention.\",\n \"In particular embodiments, a seed of a transgenic plant according to the present invention contains the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or first and/or second thioredoxin-related proteins in a concentration of at least about 0.5% of total cellular seed protein.\",\n \"In further embodiments, a seed of a transgenic plant provided herein contains a recombinant polypeptide or multimeric-protein-complex in a concentration of at least about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more, of total cellular seed protein.\",\n \"The upper limits of the recombinant polypeptide or multimeric-protein-complex concentration can be up to about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%.\",\n \"Thus, the ranges at least about 0.5% up to about 15%; at least about 1.0% up to about 10%; and at least about 5% up to about 8% are among the various ranges contemplated herein.\",\n \"Among the plant seeds useful in this regard are plant seeds obtainable from the group of plant species consisting of Brazil nut (Betholletia excelsa); castor (Riccinus communis); coconut (Cocus nucifera); coriander (Coriandrum sativum); cotton (Gossypium spp.\",\n \"); groundnut (Arachis hypogaea); jojoba (Simmondsia chinensis); linseed/flax (Linum usitatissimum); maize (Zea mays); mustard (Brassica spp.\",\n \"and Sinapis alba); oil palm (Elaeis guineeis); olive (Olea europaea); rapeseed (Brassica spp.\",\n \"); safflower (Carthamus tinctorius); soybean (Glycine max); squash (Cucurbita maxima); sunflower (Helianthus annuus); barley (Hordeum vulgare); wheat (Traeticum aestivum) and mixtures thereof.\",\n \"In a particular embodiment, oil bodies are obtainable from the seeds obtainable from safflower (Carthamus tinctorius).\",\n \"In order to prepare oil bodies from plant seeds, plants are grown and allowed to set seed in accordance with common agricultural practices.\",\n \"Thus, the present invention also provides seeds comprising oil bodies, wherein said oil bodies further comprise invention multimeric-protein-complexes described herein.\",\n \"Upon harvesting the seed and, if necessary the removal of large insoluble materials such as stones or seed hulls, by for example sieving or rinsing, any process suitable for the isolation of oil bodies from seeds may be used herein.\",\n \"A typical process involves grinding of the seeds followed by an aqueous extraction process.\",\n \"Seed grinding may be accomplished by any comminuting process resulting in a substantial disruption of the seed cell membrane and cell walls without compromising the structural integrity of the oil bodies present in the seed cell.\",\n \"Suitable grinding processes in this regard include mechanical pressing and milling of the seed.\",\n \"Wet milling processes such as described for cotton (Lawhon et al.\",\n \"(1977) J.\",\n \"Am.\",\n \"Oil Chem.\",\n \"Soc.\",\n \"63: 533-534) and soybean (U.S. Pat.\",\n \"No.\",\n \"3,971,856; Carter et al.\",\n \"(1974) J.\",\n \"Am.\",\n \"Oil Chem.\",\n \"Soc.\",\n \"51: 137-141) are particularly useful in this regard.\",\n \"Suitable milling equipment capable of industrial scale seed milling include colloid mills, disc mills, pin mills, orbital mills, IKA mills and industrial scale homogenizers.\",\n \"The selection of the milling equipment will depend on the seed, which is selected, as well as the throughput requirement.\",\n \"Solid contaminants such as seed hulls, fibrous materials, undissolved carbohydrates, proteins and other insoluble contaminants are subsequently preferably removed from the ground seed fraction using size exclusion based methodologies such as filtering or gravitational based methods such as a centrifugation based separation process.\",\n \"Centrifugation may be accomplished using for example a decantation centrifuge such as a HASCO 200 2-phase decantation centrifuge or an NX310B (Alpha Laval).\",\n \"Operating conditions are selected such that a substantial portion of the insoluble contaminants and sediments and may be separated from the soluble fraction.\",\n \"Following the removal of insolubles the oil body fraction may be separated from the aqueous fraction.\",\n \"Gravitational based methods as well as size exclusion based technologies may be used.\",\n \"Gravitational based methods that may be used include centrifugation using for example a tubular bowl centrifuge such as a Sharples AS-16 or AS-46 (Alpha Laval), a disc stack centrifuge or a hydrocyclone, or separation of the phases under natural gravitation.\",\n \"Size exclusion methodologies that may be used include membrane ultra filtration and crossflow microfiltration.\",\n \"Separation of solids and separation of the oil body phase from the aqueous phase may also be carried out concomitantly using gravity based separation methods or size exclusion based methods.\",\n \"The oil body preparations obtained at this stage in the process are generally relatively crude and depending on the application of the oil bodies, it may be desirable to remove additional contaminants.\",\n \"Any process capable of removing additional seed contaminants may be used in this regard.\",\n \"Conveniently the removal of these contaminants from the oil body preparation may be accomplished by resuspending the oil body preparation in an aqueous phase and re-centrifuging the resuspended fraction, a process referred to herein as “washing the oil bodies”.\",\n \"The washing conditions selected may vary depending on the desired purity of the oil body fractions.\",\n \"For example where oil bodies are used in pharmaceutical compositions, generally a higher degree of purity may be desirable than when the oil bodies are used in food preparations.\",\n \"The oil bodies may be washed one or more times depending on the desired purity and the ionic strength, pH and temperature may all be varied.\",\n \"Analytical techniques may be used to monitor the removal of contaminants.\",\n \"For example SDS gel electrophoresis may be employed to monitor the removal of seed proteins.\",\n \"The entire oil body isolation process may be performed in a batch wise fashion or continuous flow.\",\n \"In a particular embodiment, industrial scale continuous flow processes are utilized.\",\n \"Through the application of these and similar techniques the skilled artisan is able to obtain oil bodies from any cell comprising oil bodies.\",\n \"The skilled artisan will recognize that generally the process will vary somewhat depending on the cell type that is selected.\",\n \"However, such variations may be made without departing from the scope and spirit of the present invention.\",\n \"Association of the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, the first and/or second thioredoxin-related proteins with oil bodies.\",\n \"In accordance with the present invention, the oil bodies are associated with either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, the first and/or second thioredoxin-related proteins through association with an oil-body-targeting-protein capable of association with these multimeric-protein-complexes and the oil bodies.\",\n \"As used herein the phrase “associating the oil bodies with the multimeric-protein-complex” means that the oil bodies are brought in proximity of the multimeric-protein-complexes in a manner that allows the association of the oil bodies with either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins.\",\n \"The association of the oil bodies with the multimeric-protein-complexes is accomplished by association of the oil-body-targeting-protein with both the oil body and with the multimeric-protein-complex.\",\n \"In particular embodiments, the cells expressing the multimeric-protein-complex associate with the oil bodies that are obtainable from these same cells, which permits the convenient production and isolation of the multimeric-protein-complex, including the first and/or second recombinant polypeptides, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, in an oil body-comprising host cell system.\",\n \"Accordingly, in one embodiment, the association of the oil body with the multimeric-protein-complex is accomplished intracellularly during the growth of the cell.\",\n \"For example, a redox fusion polypeptide may be fused to an oil-body-protein and the chimeric protein may be expressed in oil body-containing plant seeds.\",\n \"Isolation of the oil bodies from the seeds in this case results in isolation of oil bodies comprising either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins.\",\n \"In another embodiment, in which the multimeric-protein-complex associates with oil bodies obtainable from the same cells in which the complex is produced, the association of the oil bodies with the multimeric-protein-complex is accomplished upon disrupting the cell's integrity.\",\n \"For example, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins may be expressed in such a manner that it is targeted to the endomembrane system of the seed cells.\",\n \"Oil bodies present in the same seed cells comprising an oil-body-targeting-protein capable of association with these multimeric-protein-complexes, for example an oleosin linked to a single chain antibody capable of association with a recombinant polypeptide or multimeric-protein-complex, may then associate with the recombinant polypeptide or multimeric-protein-complex upon grinding of the seed.\",\n \"In accordance with this embodiment, plant seed cells comprising a light and heavy chain of an immunoglobulin targeted to the plant apoplast can be prepared.\",\n \"These particular seed cells are prepared to further comprise oil bodies associated with an oil-body-targeting-protein capable of association with the immunoglobulin, such as for example, an oleosin-protein A fusion protein, and the like.\",\n \"Upon grinding of the seed, the oil bodies comprising protein A associate with the immunoglobulin through binding.\",\n \"In yet another embodiment, the oil bodies used to associate with the multimeric-protein-complex are obtained from a cellular source different from the cell comprising the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, such as from a separate plant line.\",\n \"For example, oil bodies associated with protein A may be prepared from one plant line.\",\n \"These oil bodies may then be mixed with ground seeds comprising an apoplastically expressed light and heavy chain constituting an immunoglobulin.\",\n \"Alternatively, a plant line comprising oil bodies associated with protein A may be crossed with a plant line comprising an immunoglobulin.\",\n \"The first recombinant polypeptide, second recombinant polypeptide and oil-body-targeting-protein may also be prepared in separate cellular compartments.\",\n \"Association of the first polypeptide, second polypeptide, and oil body then may occur upon disruption of the cell's integrity.\",\n \"For example, various mechanisms for targeting gene products are known to exist in plants, and the sequences controlling the functioning of these mechanisms have been characterized in some detail.\",\n \"For example, the targeting of gene products to the chloroplast is controlled by a transit sequence found at the amino terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein (Comai et al.\",\n \"(1988) J Biol Chem 263: 15104-15109).\",\n \"Other gene products are localized to other organelles such as the mitochondrion and the peroxisome (Unger et al.\",\n \"(1989) Plant Mol Biol 13:411-418).\",\n \"The cDNAs encoding these products can be manipulated to target heterologous gene products to these organelles.\",\n \"In addition, sequences have been characterized which cause the targeting of gene products to other cell compartments.\",\n \"Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho (1990) Plant Cell 2:769-783).\",\n \"Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al., (1990) Plant Mol Biol 14:357-368).\",\n \"By the fusion of the appropriate targeting sequences described above to transgene sequences of interest it is possible to direct the transgene product to the desired organelle or cell compartment.\",\n \"As hereinbefore mentioned, the redox protein obtained using the methods provided herein is enzymatically active while associated with the oil body.\",\n \"Preferably the redox protein is at least 5 times more active when produced as a redox fusion polypeptide with a second redox protein relative to its production in association with an oil body as a non-fusion polypeptide (i.e.\",\n \"without the second redox protein).\",\n \"More preferably the redox protein is at least 10 times more active when produced as a redox fusion polypeptide.\",\n \"The activity of the redox fusion polypeptide may be determined in accordance with methodologies generally known to the art (see for example: Johnson et al (1984) J. of Bact.\",\n \"Vol.\",\n \"158 3:1061-1069) and may be optimized by for example the addition of detergents, including ionic and non-ionic detergents.\",\n \"Formulation of Oil Bodies In accordance with a particular embodiment, the oil bodies comprising the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, are preferably formulated into an emulsion.\",\n \"The emulsion is preferably used in the preparation of a pharmaceutical composition, personal care or a food product.\",\n \"In emulsified form, the oil body offers certain desirable properties, such as for example excellent compatibility with the human skin.\",\n \"It particular embodiments, the oil body formulation is stabilized so that a final product may be obtained which may be stored and preserved for longer periods of time.\",\n \"As used herein, the term “stabilized oil body preparation” refers to an oil body preparation that is prepared so that the formulation does not undergo undesirable physical or chemical alterations when the oil body preparation is stored.\",\n \"The stabilization requirements may vary depending on the final product.\",\n \"For example personal care products are preferably stable for at least one year at room temperature while additionally being able to withstand short temperature fluctuations.\",\n \"Pharmaceutical formulations may in some cases be less stable as they may be stored at lower temperatures thereby preventing the occurrence of undesirable reactions.\",\n \"In general, stabilization techniques that may be used herein include any and all methods for the preservation of biological material including the addition of chemical agents, temperature modulation based methodologies, radiation-based technologies and combinations thereof.\",\n \"In particular embodiments small amounts of stabilizing chemical agents are mixed with the oil body formulation to achieve stabilization.\",\n \"These chemical agents include inter alia preservatives, antioxidants, acids, salts, bases, viscosity modifying agents, emulsifiers, gelling agents and mixtures thereof and may all be used to stabilize the oil body preparation.\",\n \"In view of the presence of the redox fusion polypeptide the stabilizing agent is generally selected to be compatible with and resulting in good enzymatic function of the redox fusion polypeptide.\",\n \"Diagnostic parameters to assess the stability of the oil body preparation may be as desired and include all parameters indicative of undesirable qualitative or quantitative changes with respect to chemical or physical stability.\",\n \"Typical parameters to assess the oil body preparation over time include color, odor, viscosity, texture, pH and microbial growth, and enzymatic activity.\",\n \"In particular embodiments, the oil body formulation is stabilized prior to the addition of further ingredients that may be used to prepare the final product.\",\n \"However, in other embodiments, it is nevertheless possible to formulate the final formulation using non-stabilized oil bodies and stabilize the final formulation.\",\n \"The final preparations may be obtained using one or more additional ingredients and any formulation process suitable for the preparation of a formulation comprising oil bodies.\",\n \"Ingredients and processes employed will generally vary depending on the desired use of the final product, will be art recognized and may be as desired.\",\n \"Ingredients and processes that may be used herein include those described in (U.S. Pat.\",\n \"Nos.\",\n \"6,146,645 and 6,183,762) which are incorporated by reference herein.\",\n \"In particular embodiments, the redox fusion polypeptide comprises a thioredoxin and a thioredoxin-reductase.\",\n \"Accordingly, provided herein are oil bodies comprising a thioredoxin/thioredoxin-reductase fusion polypeptide.\",\n \"Also provided herein is a formulation containing oil bodies comprising a thioredoxin/thioredoxin-reductase fusion capable of treating or protecting a target against oxidative stress.\",\n \"The stress of the target is treated or prevented by contacting the target with the formulation.\",\n \"The target may be any substance susceptible to oxidative stress, including any molecule, molecular complex, cell, tissue or organ.\",\n \"In another embodiment, provided herein is a formulation containing oil bodies comprising a thioredoxin/thioredoxin-reductase fusion capable of chemically reducing a target.\",\n \"Contacting the target with the formulation reduces the target.\",\n \"The target may be any substance susceptible to reduction, including any molecule or molecular complex.\",\n \"Particularly susceptible targets in this regard are the disulfide bonds present in proteins.\",\n \"The oil bodies comprising thioredoxin/thioredoxin-reductase may be used to prepare formulations used to reduce the allergenicity of food or increase the digestibility of food.\",\n \"Preferably, the method of reducing the food allergenicity is practiced by mixing the thioredoxin/thioredoxin-reductase comprising oil bodies with food or food ingredients selected from a variety of sources including for example wheat flour, wheat dough, milk, cheese, soya, yogurt and ice cream.\",\n \"The thioredoxin/thioredoxin-reductase comprising oil bodies may also be used to increase the digestibility of milk as well as other disulfide containing proteins (Jiao, J. et al.\",\n \"(1992) J. Agric.\",\n \"Food Chem 40: 2333-2336).\",\n \"Further food applications include the use of the oil thioredoxin/thioredoxin-reductase comprising oil bodies as a food additive to enhance dough strength and bread quality properties (Wong et al., (1993) J. Cereal Chem.\",\n \"70: 113-114; Kobrehel et al.\",\n \"(1994) Gluten Proteins: Association of Cereal Research; Detmold, Germany).\",\n \"Also provided herein are pharmaceutical compositions comprising, in a pharmaceutically active carrier: oil bodies comprising a thioredoxin/thioredoxin-reductase; oil bodies comprising multimeric-protein-complexes, such as heteromultimeric-protein-complexes; isolated thioredoxin/thioredoxin-reductase fusion proteins; or isolated multimeric-protein-complexes.\",\n \"These pharmaceutical compositions may be used for the treatment of reperfusion injury (Aota et al.\",\n \"(1996) J. Cardiov.\",\n \"Pharmacol.\",\n \"(1996) 27: 727-732), cataracts (U.S. Pat.\",\n \"No.\",\n \"4,771,036), chronic obstructive pulmonary disease (COPD) (MacNee et al.\",\n \"(1999) Am.\",\n \"J. Respir.\",\n \"Crit.\",\n \"Care Med.\",\n \"160:S58-S65), diabetes (Hotta et al.\",\n \"J. Exp.\",\n \"Med.\",\n \"188: 1445-1451), envenomation (PCT Patent Application 99/20122; U.S. Pat.\",\n \"No.\",\n \"5,792,506), bronchiopulmonary disease (MacNee (2000) Chest 117:3035-3175); malignancies (PCT Patent Application 91/04320) and the alleviation of the allergenic potential of airborne, for example pollen-derived, and contact allergens (PCT Patent Application 00/44781).\",\n \"Other diseases or conditions that may be treated with the pharmaceutical compositions provided herein include: psoriasis, wound healing, sepsis, GI bleeding, intestinal bowel disease (IBD), ulcers, transplantation, GERD (gastro esophageal reflux disease).\",\n \"In another embodiment, the pharmaceutical compositions provided herein, particularly those comprising one or more redox proteins alone or in combination with oil bodies, can be used in the treatment of inflammatory and viral diseases by reductively inactivating phospholipase A2, one of the contributing factors in inflammatory diseases.\",\n \"Additionally, the redox fusion polypeptide system has been found to function as a self-defense mechanism in response to environmental stimuli, including oxidative stress caused by UV-generated free radicals.\",\n \"Consequently, redox proteins, e.g., oleosin-thioredoxin, oleosin-thioredoxin-reductase, the various redox fusion polypeptides described herein, provide beneficial effects in certain skin conditions such as psoriasis, skin cancer, dandruff, diaper rash, dermatitis, acne, sun damage, aging, inflammation, and the like.\",\n \"In another embodiment, oil-body-thioredoxin-related fusion proteins, e.g., oleosin-Thioredoxin-reductase, can also be used as a venom antidote.\",\n \"Many animal venoms and other toxins contain disulfide bonds, including all snake venom neurotoxins, some bacterial neurotoxins including tetanus and botulinum A, bee venom phospholipase A2, and scorpion venom.\",\n \"In a further embodiment, the redox protein related pharmaceutical compositions provided herein can be used to inactivate venom toxins by reduction of disulfide bonds.\",\n \"A method of treating an individual suffering from the effects of a venom or toxin can include the step of administering an effective dose of a pharmaceutical composition, in a pharmaceutically effective carrier in an amount sufficient to relieve or reverse the effects of the venom toxin on the individual.\",\n \"The pharmaceutical compositions provided herein are preferably formulated for single dosage administration.\",\n \"The concentrations of the compounds in the formulations are effective for delivery of an amount, upon administration, that is effective for the intended treatment.\",\n \"Typically, the compositions are formulated for single dosage administration.\",\n \"To formulate a composition, the weight fraction of a compound or mixture thereof is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration such that the treated condition is relieved or ameliorated.\",\n \"Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.\",\n \"In addition, the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.\",\n \"Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers.\",\n \"These may be prepared according to methods known to those skilled in the art.\",\n \"For example, liposome formulations may be prepared as described in U.S. Pat.\",\n \"No.\",\n \"4,522,811.The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.\",\n \"The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo systems, such as the assays provided herein.\",\n \"The concentration of active compound in the drug composition will depend on absorption, inactivation and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.\",\n \"Typically a therapeutically effective dosage is contemplated.\",\n \"The amounts administered may be on the order of 0.001 to 1 mg/ml, preferably about 0.005-0.05 mg/ml, more preferably about 0.01 mg/ml, of blood volume.\",\n \"Pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 1000 mg and preferably from about 10 to about 500 mg, more preferably about 25-75 mg of the essential active ingredient or a combination of essential ingredients per dosage unit form.\",\n \"The precise dosage can be empirically determined.\",\n \"The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time.\",\n \"It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data.\",\n \"It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated.\",\n \"It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or use of the claimed compositions and combinations containing them.\",\n \"Preferred pharmaceutically acceptable derivatives include acids, salts, esters, hydrates, solvates and prodrug forms.\",\n \"The derivative is typically selected such that its pharmacokinetic properties are superior to the corresponding neutral compound.\",\n \"Thus, effective concentrations or amounts of one or more of the compounds provided herein or pharmaceutically acceptable derivatives thereof are mixed with a suitable pharmaceutical carrier or vehicle for systemic, topical or local administration to form pharmaceutical compositions.\",\n \"Compounds are included in an amount effective for ameliorating or treating the disorder for which treatment is contemplated.\",\n \"The concentration of active compound in the composition will depend on absorption, inactivation, excretion rates of the active compound, the dosage schedule, amount administered, particular formulation as well as other factors known to those of skill in the art.\",\n \"Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.\",\n \"Parenteral preparations can be enclosed in ampules, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable material.\",\n \"In instances in which the compounds exhibit insufficient solubility, methods for solubilizing compounds may be used.\",\n \"Such methods are known to those of skill in this art, and include, but are not limited to, using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such as Tween®, or dissolution in aqueous sodium bicarbonate.\",\n \"Derivatives of the compounds, such as prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions.\",\n \"For ophthalmic indications, the compositions are formulated in an ophthalmically acceptable carrier.\",\n \"For the ophthalmic uses herein, local administration, either by topical administration or by injection is preferred.\",\n \"Time release formulations are also desirable.\",\n \"Typically, the compositions are formulated for single dosage administration, so that a single dose administers an effective amount.\",\n \"Upon mixing or addition of the compound with the vehicle, the resulting mixture may be a solution, suspension, emulsion or other composition.\",\n \"The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.\",\n \"If necessary, pharmaceutically acceptable salts or other derivatives of the compounds are prepared.\",\n \"The compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.\",\n \"It is understood that number and degree of side effects depends upon the condition for which the compounds are administered.\",\n \"For example, certain toxic and undesirable side effects are tolerated when treating life-threatening illnesses that would not be tolerated when treating disorders of lesser consequence.\",\n \"The compounds can also be mixed with other active materials, that do not impair the desired action, or with materials that supplement the desired action known to those of skill in the art.\",\n \"The formulations of the compounds and agents for use herein include those suitable for oral, rectal, topical, inhalational, buccal (e.g., sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), transdermal administration or any route.\",\n \"The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active compound which is being used.\",\n \"The formulations are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof.\",\n \"The pharmaceutically therapeutically active compounds and derivatives thereof are typically formulated and administered in unit-dosage forms or multiple-dosage forms.\",\n \"Unit-dose forms as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art.\",\n \"Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required pharmaceutically acceptable carrier, vehicle or diluent.\",\n \"Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules.\",\n \"Unit-dose forms may be administered in fractions or multiples thereof.\",\n \"A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form.\",\n \"Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons.\",\n \"Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.\",\n \"The composition can contain along with the active ingredient: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acaciagelatin, glucose, molasses, polivinylpyrrolidine, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art.\",\n \"Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.\",\n \"If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.\",\n \"Methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975).\",\n \"The composition or formulation to be administered will contain a quantity of the active compound in an amount sufficient to alleviate the symptoms of the treated subject.\",\n \"Dosage forms or compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared.\",\n \"For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).\",\n \"The tablets may be coated by methods well-known in the art.\",\n \"The pharmaceutical preparation may also be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use.\",\n \"Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).\",\n \"Formulations suitable for rectal administration are preferably presented as unit dose suppositories.\",\n \"These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.\",\n \"Formulations suitable for topical application to the skin or to the eye preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol and oil.\",\n \"Carriers which may be used include vaseline, lanoline, polyethylene glycols, alcohols, and combinations of two or more thereof.\",\n \"The topical formulations may further advantageously contain 0.05 to 15 percent by weight of thickeners selected from among hydroxypropyl methyl cellulose, methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, poly(alkylene glycols), poly/hydroxyalkyl, (meth)acrylates or poly(meth)acrylamides.\",\n \"A topical formulation is often applied by instillation or as an ointment into the conjunctival sac.\",\n \"It can also be used for irrigation or lubrication of the eye, facial sinuses, and external auditory meatus.\",\n \"It may also be injected into the anterior eye chamber and other places.\",\n \"The topical formulations in the liquid state may be also present in a hydrophilic three-dimensional polymer matrix in the form of a strip, contact lens, and the like from which the active components are released.\",\n \"For administration by inhalation, the compounds for use herein can be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.\",\n \"In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.\",\n \"Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.\",\n \"Formulations suitable for buccal (sublingual) administration include, for example, lozenges containing the active compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles containing the compound in an inert base such as gelatin and glycerin or sucrose and acacia.\",\n \"The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.\",\n \"Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.\",\n \"The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.\",\n \"Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water or other solvents, before use.\",\n \"Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.\",\n \"Such patches suitably contain the active compound as an optionally buffered aqueous solution of, for example, 0.1 to 0.2 M concentration with respect to the active compound.\",\n \"Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, e.g., Pharmaceutical Research 3 (6), 318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound.\",\n \"The pharmaceutical compositions may also be administered by controlled release means and/or delivery devices (see, e.g., in U.S. Pat.\",\n \"Nos.\",\n \"3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,847,770; 3,916,899; 4,008,719; 4,687,610; 4,769,027; 5,059,595; 5,073,543; 5,120,548; 5,354,566; 5,591,767; 5,639,476; 5,674,533 and 5,733,566).\",\n \"Desirable blood levels may be maintained by a continuous infusion of the active agent as ascertained by plasma levels.\",\n \"It should be noted that the attending physician would know how to and when to terminate, interrupt or adjust therapy to lower dosage due to toxicity, or bone marrow, liver or kidney dysfunctions.\",\n \"Conversely, the attending physician would also know how to and when to adjust treatment to higher levels if the clinical response is not adequate (precluding toxic side effects).\",\n \"The efficacy and/or toxicity of the pharmaceutical compositions provided herein, alone or in combination with other agents can also be assessed by the methods known in the art (See generally, O'Reilly, Investigational New Drugs, 15:5-13 (1997)).\",\n \"The active compounds or pharmaceutically acceptable derivatives may be prepared with carriers that protect the compound against rapid elimination from the body, such as time release formulations or coatings.\",\n \"Kits containing the compositions and/or the combinations with instructions for administration thereof are provided.\",\n \"The kit may further include a needle or syringe, preferably packaged in sterile form, for injecting the complex, and/or a packaged alcohol pad.\",\n \"Instructions are optionally included for administration of the active agent by a clinician or by the patient.\",\n \"Finally, the pharmaceutical compositions provided herein containing any of the preceding agents may be packaged as articles of manufacture containing packaging material, a compound or suitable derivative thereof provided herein, which is effective for treatment of a diseases or disorders contemplated herein, within the packaging material, and a label that indicates that the compound or a suitable derivative thereof is for treating the diseases or disorders contemplated herein.\",\n \"The label can optionally include the disorders for which the therapy is warranted.\",\n \"Also provided herein are personal care formulations containing oil bodies comprising a thioredoxin/thioredoxin-reductase fusion polypeptide.\",\n \"Personal care products comprising thioredoxin and thioredoxin-reductase are disclosed in for example Japanese Patent Applications JP9012471A2, JP103743A2, and JP1129785A2 Personal care formulations that may be prepared in accordance with the present invention include formulations capable of improving the physical appearance of skin exposed to detrimental environmental stimuli resulting in oxidative stress for example oxidative stress caused by UV-generated free-radicals.\",\n \"The oil bodies comprising thioredoxin/thioredoxin-reductase may also be used to prepare hair care products as described in U.S. Pat.\",\n \"Nos.\",\n \"4,935,231 and 4,973,475 (incorporated herein by reference in their entirety).\",\n \"The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.\",\n \"Example 1 Isolation of Thioredoxin and NADPH Thioredoxin-Reductase Genes An Arabidopsis silique cDNA library CD4-12 was obtained from the Arabidopsis Biological Resource Centre (ABRC, http://aims.cps.msu.edu) Arabidopsis stock centre and used as a template for the isolation of the thioredoxin h (Trxh) and thioredoxin-reductase genes from Arabidopsis.\",\n \"For the isolation of the Trxh gene the following primers were synthesized: GVR833: 5′ TACCATGGCTTCGGAAGAAGGA 3′ (SEQ ID NO:1) The sequence identical to the 5′ end of the Trxh gene as published in Rivera-Madrid et al, (1993) Plant Physiol 102: 327-328, is indicated in bold.\",\n \"Underlined is an NcoI restriction site to facilitate cloning.\",\n \"GVR834: 5′ GAAAGCTTAAGCCAAGTGTTTG 3′ (SEQ ID NO:2) The sequence complementary to the 3′ end of the Trxh gene as published in Rivera-Madrid et al, (1993) Plant Physiol 102: 327-328, is indicated in bold.\",\n \"Underlined is an HindIII restriction site to facilitate cloning.\",\n \"A Polymerase Chain Reaction (PCR) was carried out using GVR833 and GVR834 as primers and the cDNA library CD4-12 as a template.\",\n \"The resulted PCR fragment was isolated, cloned into pBluescript and sequenced.\",\n \"The isolated sequence encoding Trxh was identical to the published Trxh gene sequence (Rivera-Madrid et al, (1993) Plant Physiol 102: 327-328).\",\n \"The pBluescript vector containing the Trxh gene is called pSBS2500.For the isolation of the thioredoxin-reductase gene the following primers were synthesized: GVR836: 5′ GGCCAGCACACTACCATGAATGGTCTCGAAACTCAC 3′ (SEQ ID NO:3).\",\n \"The sequence identical to the 5′ end of the thioredoxin-reductase gene as published (Jacquot et al, J Mol Biol.\",\n \"(1994) 235 (4):1357-63), is indicated in bold).\",\n \"GVR837: 5′ TTAAGCTTCAATCACTCTTACCTTGCTG 3′.\",\n \"(SEQ ID NO:4) A Polymerase Chain Reaction (PCR) was carried out using GVR836 and GVR837 as primers and the cDNA library CD4-12 as a template.\",\n \"The resulted PCR fragment was isolated, cloned into pBluescript and sequenced.\",\n \"The pBluescript vector containing the thioredoxin-reductase gene is called pSBS2502.A total of three clones were sequenced, the sequence of each of the three clones were identical to each other.\",\n \"However, as depicted in FIG.\",\n \"1 this sequence indicated several nucleotide differences compared to the published thioredoxin-reductase gene sequence published (Jacquot et al, J Mol Biol.\",\n \"(1994) 235 (4):1357-63.).\",\n \"The complete coding sequence and its deduced amino acid sequence is shown in SEQ ID NO:10.As a result of the nucleotide differences between the published sequence and the sequence isolated in Example 1, several amino acid changes are also predicted.\",\n \"A comparison of the deduced amino acid sequence of the published NADPH thioredoxin-reductase sequence thioredoxin-reductase (ATTHIREDB, Jacquot et al, J Mol Biol.\",\n \"(1994) 235 (4):1357-63.)\",\n \"with the sequence isolated in Example 1 (TR) is shown in FIG.\",\n \"3.Example 2 Construction of Plant Expression Vectors Expression vectors were constructed to allow for the seed specific over-expression of thioredoxin and NADPH thioredoxin-reductase in seeds.\",\n \"Vectors were constructed to allow for over-expression in its natural subcellular location and for accumulation on oil bodies.\",\n \"Construction of Plant Transformation Vector pSBS2520.The Arabidopsis thioredoxin h gene as described in example 1 was placed under the regulatory control of the phaseolin promoter and the phaseolin terminator derived from the common bean Phaseolus vulgaris (Slightom et al (1983) Proc.\",\n \"Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324)).\",\n \"A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the Trxh gene.\",\n \"Standard molecular biology laboratory techniques (see eg: Sambrook et al.\",\n \"(1990) Molecular Cloning, 2nd ed.\",\n \"Cold Spring Harbor Press) were used to furnish the phaseolin promoter and terminator with Pst I and HindIII/KpnI sites respectively (see SEQ ID NO:14).\",\n \"Standard molecular biology laboratory techniques were also used to place the phaseolin terminator downstream from the Trxh gene.\",\n \"The PstI-phaseolin promoter-Trxh-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000 (pSBS3000 is a derivative from the Agrobacterium binary plasmid pPZP221 (Hajdukiewicz et al., 1994, Plant Molec.\",\n \"Biol.\",\n \"25: 989-994).\",\n \"In pSBS3000, the CaMV35S promoter-gentamycin resistance gene-CAMV 35S terminator of pPZP221 was replaced with parsley ubiquitin promoter-phosphinothricin acetyl transferase gene-parsley ubiquitin termination sequence to confer resistance to the herbicide glufosinate ammonium.)\",\n \"The resulting plasmid is called pSBS2520.The sequence of the phaseolin promoter-Arabidopsis Trxh-phaseolin terminator sequence is shown in SEQ ID NO:14.Construction of Plant Transformation Vector pSBS2510.The 3′ coding sequence of an Arabidopsis oleosin gene (van Rooijen et al (1992) Plant Mol.\",\n \"Biol.\",\n \"18: 1177-1179) was altered to contain an NcoI site.\",\n \"The NcoI-HindIII fragment from vector pSBS2500 (Example 1) containing the Trxh was ligated to the coding sequence of this Arabidopsis oleosin utilizing this NcoI restriction site.\",\n \"A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter (Slightom et al (1983) Proc.\",\n \"Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324) containing a synthetic PstI site (see construction of pSBS2520) to the coding sequence of the Arabidopsis oleosin.\",\n \"Standard molecular biology laboratory techniques (see eg: Sambrook et al.\",\n \"(1990) Molecular Cloning, 2nd ed.\",\n \"Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin terminator (see construction of pSBS2520) downstream of the Trxh gene.\",\n \"The PstI-phaseolin promoter-oleosin-Trxh-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2510.The sequence of the phaseolin promoter-oleosin Trxh-phaseolin terminator sequence is shown in SEQ ID NO:16.Construction of Plant Transformation Vector pSBS2521.This vector contains the same genetic elements as the insert of pSBS2510 except the Trxh gene is fused to the 5′ end of the oleosin gene.\",\n \"The 3′ oleosin coding sequence including its native stopcodon (van Rooijen et al (1992) Plant Mol.\",\n \"Biol.\",\n \"18: 1177-1179) was furnished with a HindIII cloning site.\",\n \"Again a gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the Trxh gene and to fuse the Trxh gene to the oleosin sequence.\",\n \"Standard molecular biology laboratory techniques (see eg: Sambrook et al.\",\n \"(1990) Molecular Cloning, 2nd ed.\",\n \"Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin terminator (see construction of pSBS2520) downstream of the oleosin gene.\",\n \"The PstI-phaseolin promoter-Trxh oleosin-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2521.The sequence of the phaseolin promoter-Trxh oleosin-phaseolin terminator sequence is shown in SEQ ID NO:19.Construction of Plant Transformation Vector pSBS2527.The Arabidopsis NADPH thioredoxin-reductase gene as described in example 1 was placed under the regulatory control of the phaseolin promoter and the phaseolin terminator derived from the common bean Phaseolus vulgaris (Slightom et al (1983) Proc.\",\n \"Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324).\",\n \"A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the thioredoxin-reductase gene.\",\n \"Standard molecular biology laboratory techniques (see eg: Sambrook et al.\",\n \"(1990) Molecular Cloning, 2nd ed.\",\n \"Cold Spring Harbor Press) were used to furnish the phaseolin promoter and terminator with PstI and HindIII/KpnI sites respectively (see SEQ ID NO:14).\",\n \"Standard molecular biology laboratory techniques were also used to place the phaseolin terminator downstream from the thioredoxin-reductase gene.\",\n \"The PstI-phaseolin promoter-thioredoxin-reductase-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000 The resulting plasmid is called pSBS2527.The sequence of the phaseolin promoter-Arabidopsis thioredoxin-reductase-phaseolin terminator sequence is shown in SEQ ID NO:22.Construction of Plant Transformation Vector pSBS2531.A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter (Slightom et al (1983) Proc.\",\n \"Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324) to the coding sequence of the Arabidopsis oleosin.\",\n \"The same gene splicing technique was used to fuse the oleosin gene to the thioredoxin-reductase coding sequence.\",\n \"Standard molecular biology laboratory techniques (see eg: Sambrook et al.\",\n \"(1990) Molecular Cloning, 2nd ed.\",\n \"Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin downstream of the thioredoxin-reductase gene.\",\n \"The PstI-phaseolin promoter-oleosin-thioredoxin-reductase-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2531.The sequence of the phaseolin promoter-oleosin thioredoxin-reductase-phaseolin terminator sequence is shown in SEQ ID NO:24.Construction of Plant Transformation Vector pSBS2529 This vector contains the same genetic elements as the insert of pSBS2531 except the thioredoxin-reductase gene is fused to the 5′ end of the oleosin gene.\",\n \"The 3′ oleosin coding sequence including its native stopcodon (van Rooijen et al.\",\n \"(1992) Plant Mol.\",\n \"Biol, 18: 1177-1179) was furnished with a HindIII cloning site.\",\n \"Again a gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the thioredoxin-reductase gene and to fuse the thioredoxin-reductase gene to the oleosin sequence.\",\n \"Standard molecular biology laboratory techniques (see eg: Sambrook et al.\",\n \"(1990) Molecular Cloning, 2nd ed.\",\n \"Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin terminator (see construction of pSBS2520) downstream of the oleosin gene.\",\n \"The PstI-phaseolin promoter-thioredoxin-reductase oleosin-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2529.The sequence of the phaseolin promoter-thioredoxin-reductase oleosin-phaseolin terminator sequence is shown in SEQ ID NO:27.Construction of Plant Transformation Vector pSBS2530.A plant transformation was constructed containing the Mycobacterium Leprae thioredoxin-reductase /thioredoxin gene (Mlep TR/Trxh).\",\n \"A construct called pHIS/TR/Trxh (Wieles et al (1995) J Biol Chem 270:25604-25606) was obtained from the department of Immunohematology and Blood bank, Leiden University, The Netherlands and use as a template for PCR to generate pSBS2530.The construction of pSBS2530 was identical to the construction of pSBS2531 except that the Mlep TR/Trxh gene was used instead of the Arabidopsis thioredoxin-reductase gene.\",\n \"A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter (Slightom et al (1983) Proc.\",\n \"Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324) to the coding sequence of the Arabidopsis oleosin.\",\n \"The same gene splicing technique was used to fuse the oleosin gene to the Mlep TR/Trxh coding sequence.\",\n \"Standard molecular biology laboratory techniques (see eg: Sambrook et al.\",\n \"(1990) Molecular Cloning, 2nd ed.\",\n \"Cold Spring Harbor Press) were again used to clone the HindIII-KpnI fragment containing the phaseolin downstream of the Mlep TR/Trxh gene.\",\n \"The PstI-phaseolin promoter-oleosin-Mlep TR/Trxh -phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2530.The sequence of the phaseolin promoter-oleosin Mlep TR/Trxh -phaseolin terminator sequence is shown in SEQ ID NO:30.Construction of Plant Transformation Vector pSBS2542.From initial activity assays (FIG.\",\n \"4), it was apparent that oil bodies expressing the oleosin-M. lep TR/Trxh fusion protein contained considerable reducing activity.\",\n \"It was anticipated that a similar oleosin fusion construct encoding the Arabidopsis thioredoxin-reductase and thioredoxin proteins would behave in an analogous manner.\",\n \"Molecular modeling was used to aid in the design of such a construct.\",\n \"Primers were designed (thioredoxin link-L: 5′-ACTGGAGATGTTGACTCGACGGATACTACGGATTGGTCGACGG CTATGGAAGAAGGACAAGTGATCGCCTGC-3′; (SEQ ID NO:5), and thioredoxin link-R: 5′-ATCCGTCGAGTCAACATCTCCAGTTTCCTCGGTGGTCTCGTTAGCCTTCGAT CCAGCAATCTCTTGTAAGAATGCTCTGC-3′; (SEQ ID NO:6) to code for a synthetic linker peptide between the thioredoxin-reductase and thioredoxin proteins.\",\n \"These primers were used in conjunction with primers GVR 873 (5′-GTGGAAGCT TATGGAGATGGAG-3′; SEQ ID NO:7) and GVR834 (5′-GAAAGCTTAAGCCAAGTGTTTG-3′; SEQ ID NO:2) to amplify a region coding for a thioredoxin-reductase-linker region-thioredoxin utilizing a gene splicing by overlap extension technique (Horton et al (1989) 15:61-68).\",\n \"The thioredoxin-reductase-linker-thioredoxin encoding sequence was then cloned into a pre-existing pSBS3000 vector using standard molecular biology techniques (Sambrook et al (1990) Molecular Cloning 2nd Edition Cold Spring Harbour Press).\",\n \"The resulting plasmid was called pSBS2542.The sequence of the phaseolin promoter-oleosin-thioredoxin-reductase-linker-thioredoxin-phaseolin terminator region is shown in SEQ ID NO:33.An amino acid sequence comparison between this Arabidopsis thioredoxin-reductase-linker-thioredoxin and the M. leprae TR/Trxh protein is shown in FIG.\",\n \"12.Plasmids pSBS2510, pSBS2520, pSBS2521, pSBS2527, pSBS2529, pSBS2530, pSBS2531 and pSBS2542 were electroporated into Agrobacterium strain EHA101.These Agrobacterium strains were used to transform Arabidopsis.\",\n \"Arabidopsis transformation was done essentially as described in “Arabidopsis Protocols; Methods in molecular biology Vol 82.Edited by Martinez-Zapater J M and Salinas J. ISBN 0-89603-391-0 pg 259-266 (1998) except the putative transgenic plants were selected on agarose plates containing 80 μM L-phosphinothricine, after they were transplanted to soil and allowed to set seed.\",\n \"Example 3 Polyacrylamide Gelelectrophoresis and Immunoblotting of Transgenic Seed Extracts Source of Arabidopsis Thioredoxin, Thioredoxin-Reductase and Oleosin Antibodies.\",\n \"The Arabidopsis thioredoxin and thioredoxin-reductase genes were cloned in frame in bacterial expression vector pRSETB (Invitrogen) to allow for the overexpression of Arabidopsis thioredoxin and thioredoxin-reductase proteins.\",\n \"These proteins were purified using standard protocols (see eg Invitrogen protocol) and used to raise antibodies in rabbits using standard biochemical techniques (See eg Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).\",\n \"The Arabidopsis oleosin gene genes was cloned in frame in bacterial expression vector pRSETB (Invitrogen) to allow for the overexpression Arabidopsis oleosin protein.\",\n \"This protein was purified using standard protocols (see eg Invitrogen protocol) and used to prepare mouse monoclonal antibodies using standard biochemical techniques (See eg Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).\",\n \"Preparation of total Arabidopsis seed extracts for PAGE.\",\n \"Arabidopsis seeds were ground in approximately 20 volumes of 2% SDS, 50 mM Tris-Cl, this extract was boiled, spun and the supernatant was prepared for polyacrylamide gelelectrophoresis (PAGE) using standard protocols.\",\n \"Preparation of Arabidopsis Oil-Body-Protein Extracts.\",\n \"Arabidopsis seeds were ground in approximately 20 volumes of water and spun in a microfuge.\",\n \"The oil bodies were recovered and washed sequentially with approximately 20 volumes of water, a high stringency wash buffer, containing 8M urea and 100 mM sodiumcarbonate and water.\",\n \"After this last wash the oil bodies are prepared for poly acrylamide gelelectrophoresis (PAGE) using standard protocols.\",\n \"Analysis of Seed and Oil Body Extracts from Plants Transformed with pSBS2510 Total seed and oil body protein extracts from plants transformed with pSBS2510 were loaded onto polyacrylamide gels and either stained with coomassie brilliant blue or electroblotted onto PVDF membranes.\",\n \"The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin, or a monoclonal antibody raised against the Arabidopsis 18.5 kDa oleosin and visualized using alkaline phosphatase.\",\n \"Expression of the oleosin-thioredoxin results in an additional band of 31.2 kDa.\",\n \"The results indicate that the thioredoxin antibodies are immunologically reactive with a band of the right predicted molecular weight (31.2 kDa), and the oleosin antibodies are also immunologically reactive with a band of the right predicted molecular weight for the fusion protein (31.2 kDa) in addition to a band corresponding to the native Arabidopsis oleosin (18.5 kDa).\",\n \"This indicates that oleosin-thioredoxin is expressed in Arabidopsis seeds and is correctly targeted to oil bodies.\",\n \"Analysis of Seed and Oil Body Extracts from Plants Transformed with pSBS2521 Total seed and oil body protein extracts from plants transformed with pSBS25121 were loaded onto polyacrylamide gels and either stained with Coomassie brilliant blue or electroblotted onto PVDF membranes.\",\n \"The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin, or a monoclonal antibody raised against the Arabidopsis 18.5 kDa oleosin and visualized using alkaline phosphatase.\",\n \"Expression of the thioredoxin-oleosin results in an additional band of 31.2 kDa.\",\n \"The results indicate that the thioredoxin antibodies are immunologically reactive with a band of the right predicted molecular weight (31.2 kDa), and the oleosin antibodies are also immunologically reactive with a band of the right predicted molecular weight for the fusion protein (31.2 kDa) in addition to a band corresponding to the native Arabidopsis oleosin (18.5 kDa).\",\n \"This indicates that thioredoxin-oleosin is expressed in Arabidopsis seeds and is correctly targeted to oil bodies.\",\n \"Analysis of seed extracts from plants transformed with pSBS2520 Total seed extracts from plants transformed with pSBS2520 were loaded onto polyacrylamide gels and either stained with Coomassie brilliant blue or electroblotted onto PVDF membranes.\",\n \"The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin and visualized using alkaline phosphatase.\",\n \"The results indicated that the thioredoxin antibodies are immunologically reactive with a band of approximately the right predicted molecular weight (12 kDa).\",\n \"Untransformed seeds do not show a detectable thioredoxin band.\",\n \"Analysis of Seed and Oil Body Extracts from Plants Transformed with pSBS2529 Total seed and oil body protein extracts from plants transformed with pSBS2529 were loaded onto polyacrylamide gels and electroblotted onto PVDF membranes.\",\n \"The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin-reductase, or a monoclonal antibody raised against the Arabidopsis 18.5 kDa oleosin and visualized using alkaline phosphatase.\",\n \"Expression of the thioredoxin-reductase -oleosin results in an additional band of 53.8 kDa.\",\n \"The results indicate that the thioredoxin-reductase antibodies are immunologically reactive with a band of the right predicted molecular weight for the fusion protein (53.8 kDa), the oleosin antibodies are also immunologically reactive with a band of the right predicted molecular weight (53.8 kDa) in addition to a band corresponding to the native Arabidopsis oleosin (18.5 kDa).\",\n \"This indicates that thioredoxin-reductase-oleosin is expressed in Arabidopsis seeds.\",\n \"Analysis of seed extracts from plants transformed with pSBS2527 Total seed extracts from plants transformed with pSBS2527 were loaded onto polyacrylamide gels and electroblotted onto PVDF membranes.\",\n \"The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin-reductase and visualized using alkaline phosphatase.\",\n \"The thioredoxin-reductase antibodies are immunologically reactive with a band of approximately the right predicted molecular weight for the (35.3 kDa).\",\n \"Untransformed seeds do not show a detectable thioredoxin band.\",\n \"Analysis of seed extracts from plants transformed with pSBS2531 A protein gel and immunoblot was prepared assaying the expression of oleosin-DMSR in Arabidopsis T2 seeds and correct targeting to Arabidopsis oil bodies.\",\n \"The expected molecular weight based on the deduced amino acid sequence is calculated to be 53,817 Da.\",\n \"In the oil body extract of the transgenic oleosin-thioredoxin-reductase sample an extra band of approximately 54 kDa was observed.\",\n \"This band was confirmed to be oleosin-thioredoxin-reductase by immunoblotting.\",\n \"From the polyacrylamide gel it was observed that the expression of the oleosin-Thioredoxin-reductase is about double compared to the expression of the major 18.5 kDa Arabidopsis oleosin.\",\n \"This represents approximately 2-4% of total seed protein.\",\n \"Analysis of seed extracts from plants transformed with pSBS2530 A protein gel and immunoblot was prepared assaying the expression of oleosin-M.lep TR/Trxh in Arabidopsis T2 seeds and the correct targeting to Arabidopsis oil bodies.\",\n \"The expected molecular weight based on the deduced amino acid sequence is calculated to be 67,550 Da.\",\n \"In the oil body extract of the transgenic oleosin-M.lep TR/Trxh sample an extra band of approximately 68 kDa was observed.\",\n \"This band was confirmed to be oleosin-M.lep TR/Trxh by immunoblotting.\",\n \"From the polyacrylamide gel it was observed that the expression of the oleosin-M.lep TR/Trxh is similar to the expression of the major 18.5 kDa Arabidopsis oleosin.\",\n \"This represents approximately 1-2% of total seed protein.\",\n \"Analysis of seed extracts from plants transformed with pSBS2542 Crude oil body extracts from pSBS2542 lines were prepared by grinding 100 μg of seed in 1 mL of 100 mM Tris buffer at pH 7.5.The samples were then centrifuged in order to isolate the oil body fraction.\",\n \"The oil body fraction was then loaded on an SDS polyacrylamide gel for expression analysis.\",\n \"A Coomassie stained gel revealed that the synthetic fusion accumulated to high levels in crude oil body extracts from 3 of the 4 lines tested.\",\n \"It was estimated that the fusion protein represented approximately 2-5% of total seed protein.\",\n \"Furthermore, western blots utilizing either anti-thioredoxin or anti-thioredoxin-reductase antibodies confirmed that the over expressed 70 kDa protein was indeed oleosin-thioredoxin-reductase-linker-thioredoxin.\",\n \"Example 4 Biological Activity of Thioredoxin and Thioredoxin-Reductase Transformants Initial Reduction Assays: DTNB Assay The activity of the thioredoxin and thioredoxin reductase was determined using a calorimetric DTNB [5,5′-dithiolbis(2-nitrobenzoic acid)] assay.\",\n \"The assay was performed in a 700 μL reaction volume containing 100 mM Tris-Cl pH 8.0, 5 mM EDTA, 200 μM DTNB [5,5′-dithiolbis(2-nitrobenzoic acid)] and 200 μM NADPH.\",\n \"If thioredoxin-reductase and thioredoxin are added, NADPH will reduce the thioredoxin-reductase, which will then reduce thioredoxin, which will, in turn, reduce DTNB (see equations below).\",\n \"NADPH2+thioredoxin-reductaseox→thioredoxin-reductasered+NADP+thioredoxin-reductasered+thioredoxinox→thioredoxinred+thioredoxin-reductaseox thioredoxinred+DTNBox→2(2-nitro-5-mercaptobenzoic acid)+thioredoxinox The formation of the yellow product was monitored by measuring the OD412 in a spectrophotometer after a set period of time (usually 0.5-2 hours).\",\n \"The results of initial activity assays are shown in the bar graph in FIG.\",\n \"4 and described below.\",\n \"Initially, 100 μg of total seed proteins were added from each of the Arabidopsis transgenic lines, pSBS2520 (cytosolic thioredoxin) and pSBS2527 (cytosolic thioredoxin-reductase), which corresponds to approximately 1 μg of cytosolic thioredoxin and thioredoxin-reductase used in the assay.\",\n \"In this case, the amount of DTNB reduced was comparable to the reduction caused by 1 μg each of E. coli thioredoxin and thioredoxin-reductase.\",\n \"In these plant seed samples, background readings were very low when only one of the 2 extracts (either cytosolic thioredoxin or cytosolic thioredoxin-reductase; FIG.\",\n \"4, bars 3 and 6, respectively) was added to the reaction, along with wild type oil bodies.\",\n \"Analysis with oil body fractions from transgenic seeds revealed that Arabidopsis thioredoxin and thioredoxin-reductase were substantially less active when fused to oleosins on oil bodies.\",\n \"Approximately 300 μg of crude, unwashed oil-body-protein was used in the assay (which corresponds to 10-30 μg of thioredoxin-oleosin (pSBS 2521; FIG.\",\n \"4, bar 2), oleosin-thioredoxin (pSBS 2510, FIG.\",\n \"4, bar 1), thioredoxin-reductase-oleosin (pSBS 2529, FIG.\",\n \"4, bar 5), or oleosin-thioredoxin-reductase (pSBS 2531, FIG.\",\n \"4, bar 4).\",\n \"The oil-body-proteins were tested in conjunction with 100 μg of total seed protein containing approximately 1 μg of cytosolic thioredoxin (pSBS 2520) or thioredoxin-reductase (pSBS 2527).\",\n \"In such assays, pSBS2529 (thioredoxin-reductase-oleosin) and pSBS2531 (oleosin-thioredoxin-reductase) do contain reductase activity when combined with cytosolic thioredoxin from pSBS2520 (see FIG.\",\n \"4, bars 7 and 8, respectively).\",\n \"Experiments estimated that the reductase activity of oleosin-thioredoxin-reductase was about 10-15% that of the cytosolic thioredoxin-reductase.\",\n \"The addition of tween at a final concentration of 0.4% could enhance this activity 2 or 3 fold.\",\n \"Interestingly, oleosin-thioredoxin-reductase (pSBS 2531) appears to be capable of reducing DTNB in the absence of added thioredoxin, although added thioredoxin causes significantly more DTNB reduction (see FIG.\",\n \"4; compare bar 4 W.T.+oleosin-thioredoxin-reductase to bar 7 thioredoxin+oleosin-thioredoxin-reductase).\",\n \"Experiments with pSBS2521 (thioredoxin-oleosin) or pSBS2510 (oleosin-thioredoxin) combined with cytosolic thioredoxin-reductase from pSBS2527 (see FIG.\",\n \"4, bars 10 and 11, respectively) indicate that thioredoxin activity of these fusions is undetectable at these concentrations.\",\n \"Oil bodies from the transgenic Arabidopsis line, pSBS2530 (oleosin-M.lep TR/Trxh) contain significant thioredoxin/thioredoxin-reductase activity (see FIG.\",\n \"4, bar 12).\",\n \"One hundred micrograms of crude oil body protein for pSBS2530 was tested (corresponding to approximately 5 μg of oleosin-M.lep TR/trxh fusion) in the assay.\",\n \"Based on the assay, it was estimated that this fusion is about 25-40% as active as cytosolic Arabidopsis thioredoxin and thioredoxin-reductase (FIG.\",\n \"4, bar 9) when comparing specific activity.\",\n \"Insulin Reduction Assay The results from the DTNB assays were confirmed with insulin reduction assays.\",\n \"This assay contained insulin at a final concentration of 1 mg/mL in 100 mM KH2PO4 pH 7.0+5 mM EDTA.\",\n \"In the presence of NADPH (500 μM), thioredoxin, and thioredoxin-reductase, insulin is reduced and precipitates from the solution.\",\n \"Normally, insulin reduction is followed by measuring turbidity at OD 650.Alternatively, one can measure the conversion of NADPH2 to NADP+by monitoring the decrease in absorbance at 340 nm.\",\n \"Both of the assays are difficult to measure when oil bodies are present, due to interference with the spectrophotometer readings.\",\n \"However, qualitative data could be obtained by centrifuging the tubes after a set period of time, and determining if an insulin pellet was present (oil bodies float to the top, while the insulin precipitate pellets out).\",\n \"Alternatively, samples could be filtered after a set period of time, and the change in absorbance at 340 nm could be measured.\",\n \"As mentioned previously, the results of the insulin reduction assays agreed with those of the DTNB assay, with the exception of the observation that pSBS2531 (oleosin-thioredoxin-reductase) only reduced insulin in the presence of free thioredoxin from pSBS2520.Assays on Seeds from Arabidopsis Crosses that Co-Express Oleosin-Thioredoxin and Oleosin-Thioredoxin-Reductase.\",\n \"Based upon initial DTNB and insulin reduction assays, it was apparent that mixing oil bodies from oleosin<->thioredoxin and oleosin<->thioredoxin-reductase transgenic seeds resulted in very limited reducing activity (Note: the <-> indicates both configurations of oleosin fusions; ie.\",\n \"oleosin<->thioredoxin would represent oleosin-thioredoxin and thioredoxin-oleosin fusions).\",\n \"To determine whether having oleosin<->thioredoxin and oleosin<->thioredoxin-reductase proteins present on the same oil body would have a positive effect on the reducing activity of these proteins, crosses were set up to generate double transgenic Arabidopsis lines.\",\n \"The crosses are illustrated in Table 2.TABLE 2 Confirmed double transgenic lines (PCR and Western Male Female Blot) oleo-thioredoxin X oleo-thioredoxin-reductase 4 oleo-thioredoxin X thioredoxin-reductase-oleo 1 thioredoxin-oleo X oleo-thioredoxin-reductase 0 thioredoxin-oleo X thioredoxin-reductase-oleo 4 oleo-thioredoxin- X oleo-thioredoxin 2 reductase oleo-thioredoxin- X thioredoxin-oleo 0 reductase thioredoxin- X oleo-thioredoxin 7 reductase-oleo thioredoxin- X thioredoxin-oleo 0 reductase-oleo Seeds from Arabidopsis crosses were germinated on PPT plates and the seedlings were transferred to soil after approximately 2 weeks.\",\n \"PCR experiments on DNA isolated from the seedlings identified a number of plants which contain both an oleosin<->thioredoxin and an oleosin<->thioredoxin-reductase gene construct within their genome.\",\n \"Seeds were harvested from these plants for expression and activity assays.\",\n \"Western blots were carried out to confirm expression of both oleosin<->thioredoxin and oleosin<->thioredoxin-reductase in the lines.\",\n \"DTNB and insulin reduction assays were also performed to compare activity between single transgenic parent lines and the double transgenic offspring and results are summarized in Table 3.Table 3 summarizes DTNB reducing activity of various transgenic lines.\",\n \"The last 2 rows compare mixing oil bodies from single transgenic parent lines to using oil bodies from double transgenic offspring.\",\n \"Relative activity for the E. coli thioredoxin and thioredoxin mixture is set at 100 percent.\",\n \"TABLE 3 Relative Source Material Activity (%) E. coli trx + NTR 100 Arabidopsis “free” thioredoxin + 100 thioredoxin-reductase (pSBS2520 + pSBS2527) oleosin-M. lep TR/Trxh ~30 (pSBS2530) Oleosin<->thioredoxin-reductase + ~3 oleosin<->thioredoxin (mixing oil bodies from single-transgenic parents) Oleosin<->thioredoxin-reductase × ~50 oleosin<->thioredoxin (various double transgenic lines) Based on DTNB and insulin reduction assays, it is evident that double transgenic plants co-expressing oleosin<->thioredoxin and oleosin<->thioredoxin-reductase on the same, single oil body contained significantly more reducing activity compared to mixing oil bodies from single transgenic oleosin<->thioredoxin and oleosin<->thioredoxin-reductase lines.\",\n \"It was additionally apparent that oil body extracts from co-expressing lines contained more reducing activity compared to line pSBS2530 (oleosin-M. lep TR/Trxh), which was previously identified as the line containing the highest reducing activity from oil bodies.\",\n \"These results suggest that the creation of double transgenic lines (either through crossing or by co-transforming 2 expression constructs into plants) may represent one means by which we could solve our initial problem of not being able to generate reducing activity by mixing oil bodies from oleosin<->thioredoxin and oleosin<->thioredoxin-reductase single transgenic lines.\",\n \"Assays on Seeds from Arabidopsis pSBS2542 Transgenic Lines that Express Oleosin-Thioredoxin-Reductase-Linker-Thioredoxin.\",\n \"Oil body extracts from four pSBS2542 lines were tested for reducing activity in DTNB and insulin reduction assays, using standard protocols described previously.\",\n \"Again, oil body extracts containing the oleosin-thioredoxin-reductase-linker-thioredoxin protein possessed significant reducing activity.\",\n \"Based on such assays, it was revealed that the oleosin-thioredoxin-reductase-linker-thioredoxin synthetic fusion protein was more active than the oleosin-M. lep TR/Trxh fusion.\",\n \"Furthermore, oil bodies containing the oleosin-thioredoxin-reductase-linker-thioredoxin protein appeared to have more reducing activity compared to oil bodies from double transgenic lines that co-expressed oleosin<->thioredoxin and oleosin<->thioredoxin-reductase.\",\n \"The results comparing reducing activity for the various thioredoxin-reductase/thioredoxin constructs is summarized in Table 4.Table 4 summarizes DTNB reducing activity of various transgenic lines.\",\n \"The pSBS2542 line expressing oleosin-thioredoxin-reductase-linker-thioredoxin contains significant reducing activity, comparable to the “free” forms of Arabidopsis thioredoxin and thioredoxin-reductase and the equivalent E. coli proteins.\",\n \"Relative activity for the E. coli thioredoxin and thioredoxin mixture is set at 100 percent.\",\n \"TABLE 4 Relative Source Material Activity (%) E. coli trx + NTR 100 Arabidopsis “free” thioredoxin + thioredoxin- 100 reductase (pSBS2520 + pSBS2527) oleosin-M. lep TR/Trxh ~30 (pSBS2530) Oleosin<->thioredoxin-reductase + ~3 oleosin<->thioredoxin (mixing oil bodies from single-transgenic parents) Oleosin<->thioredoxin-reductase × ~50 oleosin<->thioredoxin (various double transgenic lines) Oleosin-thioredoxin-reductase-linker-thioredoxin ~75-100 (pSBS2542) Reduction Assays Comparing the Utilization of NADH Vs. NADPH as a Cofactor (Electron Donor) for the Thioredoxin-Reductase/Thioredoxin System.\",\n \"DTNB and insulin reduction assays were conducted as described previously, except that NADH was substituted for NADPH as an electron donor in the system utilizing E. coli thioredoxin-reductase and thioredoxin.\",\n \"Thus, a comparison was conducted of the utilization of NADH versus NADPH as a cofactor for the E. coli thioredoxin-reductase/thioredoxin system.\",\n \"For the DTNB assay, the reaction mixture consisted of 400 μM DTNB, 10 μg/mL E. coli thioredoxin, and 10 μg/mL E. coli thioredoxin-reductase in 100 mM Tris-Cl buffer pH 8.0.Either NADH or NADPH was then added to the DTNB reaction as follows: Reaction A.\",\n \"200 μM NADPH (Sigma) Reaction B.\",\n \"800 μM NADH (Sigma) Reaction C. 800 μM NADH (Roche) Reaction D. (−) cofactor Reaction E. 800 μM NADH (no TR or Trxh).\",\n \"For the insulin reduction assay, the reaction mixture consisted of 1 mg/mL bovine pancreatic insulin, 20 μg/mL E. coli thioredoxin, and 20 μg/mL E. coli thioredoxin-reductase in 100 mM potassium phosphate buffer at pH 7.0.Either NADH or NADPH was then added to the reaction as follows: Reaction A.\",\n \"800 μM NADPH (Sigma) Reaction B.\",\n \"800 μM NADH (Sigma) Reaction C. 800 μM NADH (Roche) Reaction D. (−) cofactor Reaction E. 2 mM NADH (no TR or Trxh).\",\n \"The results indicate that NADH, purchased from either Sigma or Roche, could act as an electron donor in both the DTNB and insulin reduction assays.\",\n \"However, the rate of reduction was lower than the rate observed with NADPH as a cofactor.\",\n \"It was estimated that the rate of insulin reduction utilizing NADH as an electron donor was approximately 25-50% when compared to the maximum rate using NADPH.\",\n \"Furthermore, it was estimated that the rate of DTNB reduction utilizing NADH as an electron donor was approximately 5-10% of the maximum rate using NADPH.\",\n \"Similar results were observed using the oleosin-thioredoxin-reductase-linker thioredoxin fusion protein on Arabidopsis oil bodies instead of the E. coli thioredoxin-reductase and thioredoxin.\",\n \"Example 5 Production of Multimeric Immunoglobulin Protein in Plant Seed Cells and Capture on Oil Bodies Using Protein A-Oleosin Fusion Proteins 1—Production of Multimeric Immunoglobulin Protein in Plant Seed Cells For expression of multimeric-protein-complexes containing multimeric-immunoglobulin-complexes, the cDNA sequences encoding individual light and heavy chains can be isolated from; 1) cell lines expressing a particular antibody, such as clonal B cell lines, or a hybridoma cell line, or 2) may be a recombinant antibody, assembled by combining select light and heavy chain variable domains and available light and heavy chain constant domain sequences, respectively.\",\n \"Variable domains with specific binding properties may be isolated from screening populations of such sequences, usually in the form of a single-chain Fv phage display library.\",\n \"Starting from known nucleic acid sequences and a source of light and heavy chains, the mature polypeptide coding sequences of each chain is isolated with a secretion signal sequence.\",\n \"The signal sequence can be the native antibody sequence or derived from a known secreted plant sequence (e.g.\",\n \"a PR sequence from Arabidopsis or tobacco).\",\n \"The addition of a plant secretion signal sequence to both light and heavy chain mature coding sequences is carried out by standard molecular biology techniques.\",\n \"PCR fusion is used routinely to make such modifications.\",\n \"Secretion signal sequences are included to target the light and heavy immunoglobulin polypeptides for secretion from the cell and further assembly of the two chains into a multimeric-immunoglobulin-complex.\",\n \"For expression in transgenic plant seeds, an expression cassette is assembled comprising: 1) a regulatory promoter sequence to provide expression in plant seeds, 2) the secretion signal—light chain sequence, and 3) a regulatory sequence to terminate transcription.\",\n \"A second expression cassette is assembled comprising: 1) a regulatory promoter sequence to provide expression in plant seeds, 2) the secretion signal—heavy chain sequence, and 3) a regulatory sequence to terminate transcription.\",\n \"Each of the antibody chain expression cassettes is cloned individually into an Agrobacterium plant transformation vector or is combined into a single transformation vector with both expression cassettes.\",\n \"In both cases, the expression cassettes are cloned into plant transformation vectors, between the left and right delineating border sequences, and adjacent to a plant selectable marker cassette.\",\n \"Each plant transformation vector is transformed into Agrobacterium.\",\n \"The resulting Agrobacterium strains are used to infect plant tissues.\",\n \"Transgenic plant material is regenerated and viable transgenic plants are selected.\",\n \"When individual transformation vectors are used, the transgenic plant lines that are produced, expressing either light or heavy chain sequences, are crossed to generate a single plant line expressing both chains in the same plant cell.\",\n \"When a single transformation vector, containing both light and heavy expression cassettes, is used, the initial transgenic plant line produces both light and heavy chain sequences in the same plant cell.\",\n \"2—Production of Transgenic Oil Bodies which Display Protein a for the Capture of Immunoglobulins To capture and display immunoglobulin protein on oil bodies, oil bodies are engineered to display an immunoglobulin binding protein.\",\n \"In this example, the well-known antibody-binding domains from Protein A are used.\",\n \"Based on the known sequence for Protein A from Staphylococcus aureus, PCR primers are designed to isolate the five consecutive Ig-binding domains from the bacterial Protein A sequence.\",\n \"Primers are designed to allow cloning of the Protein A sequence as either an N-terminal or C-terminal fusion to an oleosin sequence for targeting to oil bodies.\",\n \"The sequence that encodes an in-frame translational fusion between Protein A and oleosin is cloned into a plant expression cassette for seed-specific expression.\",\n \"The final cassette consists of a regulatory promoter sequence that provides expression in seeds, the Protein A-oleosin fusion sequence, and a regulatory sequence to terminate transcription.\",\n \"The Protein A-oleosin expression cassette is cloned into a plant transformation vector compatible with Agrobacterium-mediated plant transformation.\",\n \"The transformation vector comprises left and right border sequences flanking the Protein A-oleosin expression cassette and an adjacent plant selectable marker cassette.\",\n \"The Agrobacterium strain containing this vector is used to infect plant tissues and subsequent regeneration and selection from transgenic plant material to create transgenic plants.\",\n \"3—Capture and Display of Multimeric-Immunoglobulins on Oil Bodies Displaying Protein A Having produced light and heavy chain multimeric immunoglobulin complexes in one transgenic plant line and the display of Protein A on oil bodies through the oil body targeting of a Protein A-oleosin fusion protein in a second plant line, at least two embodiments can be used to capture the immunoglobulin complex on the Protein A oil bodies.\",\n \"In the first embodiment, transgenic seed from both the immunoglobulin and the Protein A-oleosin expression lines is combined in an optimum ratio and then ground together such that the disrupted material from both seed lines would be combined in the same extract.\",\n \"The combined seed extracts are mixed and/or incubated under conditions that allow maximum recovery of the immunoglobulin by Protein A.\",\n \"The oil body fraction is separated using standard phase separation techniques (e.g.\",\n \"centrifugation).\",\n \"The recovered oil body fraction contains both native oil bodies, from the immunoglobulin expression line, and transgenic Protein A oil bodies from the Protein A-oleosin expression line.\",\n \"In a second embodiment, the plant lines expressing the immunoglobulin complex and the Protein A-oleosin fusion are crossed and individual plant lines expressing both components are identified and propagated.\",\n \"In this approach, the immunoglobulin complex and the Protein A-oleosin fusion are produced in different cellular compartments of the same plant seed cell.\",\n \"Seed from the double transgenic line is ground to disrupt the cellular material and mix the contents of all cellular compartments, including combining the immunoglobulin in the extracellular compartment and the Protein A-oleosin on the oil body in the cytosolic compartment.\",\n \"The material is mixed and/or incubated under conditions to allow maximum recovery of the immunoglobulin by Protein A, and the oil body fraction is separated by phase separation techniques.\",\n \"The recovered oil body fraction contains the displayed Protein A and the capture immunoglobulin complex.\",\n \"Example 6 Production of Assembled Multimeric-Immunoglobulin-Complexes as Fusions with Oil Body Targeting Domains Individual polypeptides are produced as a fusion protein with oil body targeting sequences (e.g.\",\n \"oleosin) for display on oil bodies.\",\n \"It has been found that the individual subunits of naturally associating heterodimeric proteins can be co-produced as individual oleosin fusions and still associate as an active heterodimer on the surface of the oil body.\",\n \"In this example, the heterodimer is the light and heavy chain subunits, or derived portions thereof, of an immunoglobulin complex.\",\n \"Production of an Immunoglobulin Fab Complex on Oil Bodies.\",\n \"The mature light chain sequence, lacking the secretion signal sequence, is attached as an in-frame N-terminal fusion to an oleosin sequence.\",\n \"This fusion sequence is assembled into a seed-specific expression cassette consisting of a seed-specific promoter sequence, the light chain-oleosin fusion sequence, and a transcriptional terminator sequence.\",\n \"The expression cassette is inserted between the left and right border markers, adjacent to a plant selectable marker cassette, of a transformation vector.\",\n \"The transformation vector, in Agrobacterium, is used to infect plants and generate transgenic plants.\",\n \"An equivalent construct for the heavy chain subunit, comprising the variable and constant heavy chain domains, is also attached as an in-frame fusion to oleosin and assembled into an expression cassette for seed-specific expression.\",\n \"The expression cassette can be a part of a separate transformation vector for the generation of a separate transgenic line, or the heavy chain expression cassette can be combined together with the light chain cassette into a single transformation vector.\",\n \"If light and heavy chain expression cassettes are transformed into plants on separate transformation vectors, the individual plant lines are crossed to create a single line expressing both heterodimer subunit-oleosin fusions in the same plant cell.\",\n \"Seed from the double transgenic line, or a single transgenic line generated from the dual expression vector, is extracted to isolate oil bodies.\",\n \"The seed material is ground to release the cellular contents and oil bodies are isolated by phase separation.\",\n \"The targeting of both light and heavy chain sequence to oil bodies, as oleosin fusions, allows the association of the immunoglobulin complex on the surface of the oil body.\",\n \"Similar configurations, using the entire heavy chain sequence in combination with the entire light chain sequence, or using the variable domains from both the light and heavy chain sequences, are constructed to assemble different types of heteromultimeric-immunoglobulin-complexes (e.g., heterodimers) on the surface of oil bodies.\",\n \"The present invention should therefore not be seen as limited to the particular embodiments described herein, but rather, it should be understood that the present invention has wide applicability with respect to protein expression generally.\",\n \"Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.\",\n \"SUMMARY OF SEQUENCES SEQ ID NOs:1-4 set forth primers which were synthesized for the isolation of the thioredoxin h (Trxh) and thioredoxin reductase genes from Arabidopsis, as described in Example 1.SEQ ID NOs:5-7 set forth primers which were designed to code for a specific linker peptide between thioredoxin reductase and thioredoxin proteins, as described in Example 2.SEQ ID NOs:8, 10 and 11 set forth the nucleotide sequence and the deduced amino acid sequence of the NADPH thioredoxin reductase sequence isolated herein as described in Example 1.SEQ ID NOs:9 and 11, respectively, set forth the nucleotide sequence of the published NADPH thioredoxin reductase sequence (ATTHIREDB) and the deduced amino acid sequence.\",\n \"SEQ ID NO:12 sets forth the deduced amino acid sequence of the published NADPH thioredoxin reductase sequence.\",\n \"SEQ ID NO:13 sets forth the deduced amino acid sequence of the NADPH reductase sequence isolated in this report.\",\n \"SEQ ID NOs:14 and 15 set forth the nucleotide sequence of the phaseolin promoter-Arabidopsis Trxh-phaseolin terminator sequence described in Example 2, and the deduced amino acid sequence.\",\n \"The Trxh coding sequence and its deduced amino acid sequence is indicated.\",\n \"The phaseolin promoter corresponds to nucleotide 6-1554, and the phaseolin terminator corresponds to nucleotide sequence 1905-3124.The promoter was furnished with a PstI site (nt 1-6) and the terminator was furnished with a HindIII site (nt 1898-1903) and a KpnI site (nt 3124-3129) to facilitate cloning.\",\n \"SEQ ID NOs:16, 17 and 18 set forth the nucleotide sequence of the phaseolin promoter-oleosin Trxh-phaseolin terminator sequence described in Example 2, and the deduced amino acid sequences.\",\n \"The oleosin-Trxh coding sequence and the deduced amino acid sequences are indicated in SEQ ID NO:16.As in SEQ ID NO:14, the phaseolin promoter corresponds to nucleotide 6-1554.The sequence encoding oleosin corresponds to nt 1555-2313, the intron in this sequence (nt 1908-2147) is indicated in italics.\",\n \"The Trxh coding sequence corresponds to nt 2314-2658.The phaseolin terminator corresponds to nucleotide sequence 2664-3884.SEQ ID NO:19, 20 and 21 set forth the nucleotide sequence of the phaseolin promoter-Trxh oleosin-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.\",\n \"The Trxh oleosin-coding sequence and its deduced amino acid sequences are indicated in SEQ ID NO:19.As in SEQ ID NOs:14 and 16, the phaseolin promoter corresponds to nucleotide 6-1554.The Trxh coding sequence corresponds to nt 1555-1896.The sequence encoding oleosin corresponds to nt 1897-2658, the intron in this sequence (nt 2250-2489) is indicated in italics.\",\n \"The phaseolin terminator corresponds to nucleotide sequence 2664-3884.SEQ ID NO:22 and 23 set forth the nucleotide sequence of the phaseolin promoter-thioredoxin-reductase-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequence.\",\n \"The thioredoxin-reductase coding sequence and its deduced amino acid sequence is indicated in SEQ ID NO:22.The phaseolin promoter corresponds to nucleotide 6-1554.The thioredoxin-reductase coding sequence corresponds to nt 1555-2556 and the deduced amino acid is set forth in SEQ ID NO:23.The phaseolin terminator corresponds to nucleotide sequence 2563-3782.SEQ ID NOs:24, 25 and 26 show the nucleotide sequence of the phaseolin promoter-oleosin thioredoxin-reductase-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.\",\n \"The oleosin-thioredoxin-reductase coding sequence and its deduced amino acid sequence is indicated.\",\n \"The phaseolin promoter corresponds to nucleotide 6-1554.The sequence encoding oleosin corresponds to nt 1555-2313, the intron in this sequence (nt 1980-2147) is indicated in italics.\",\n \"The thioredoxin-reductase coding sequence corresponds to nt 2314-3315.The phaseolin terminator corresponds to nucleotide sequence 3321-4540.SEQ ID NOs:27, 28 and 29 show the nucleotide sequence of the phaseolin promoter-thioredoxin-reductase oleosin-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.\",\n \"The thioredoxin-reductase coding sequence and its deduced amino acid sequence is indicated.\",\n \"The phaseolin promoter corresponds to nucleotide 6-1554.The thioredoxin-reductase coding sequence corresponds to nt 1555-2553.The sequence encoding oleosin corresponds to nt 2554-3315, the intron in this sequence (nt 2751-3146) is indicated in italics.\",\n \"The phaseolin terminator corresponds to nucleotide sequence 3321-4540.SEQ ID NO:30, 31 and 32 show the sequence of the phaseolin promoter-oleosin-Mlep thioredoxin-reductase/thioredoxin-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.\",\n \"The oleosin-Mlep thioredoxin-reductase/thioredoxin coding sequence and its deduced amino acid sequence is indicated.\",\n \"The phaseolin promoter corresponds to nucleotide 6-1554.The sequence encoding oleosin corresponds to nt 1555-2313, the intron in this sequence (nt) is indicated in italics.\",\n \"The Mlep thioredoxin-reductase/thioredoxin coding sequence corresponds to nt 2314-3690.The phaseolin terminator corresponds to nucleotide sequence 3698-4917.SEQ ID NOs:33, 34 and 35 set forth the nucleotide sequence of the phaseolin promoter-oleosin-thioredoxin-reductase-linker-thioredoxin-phaseolin terminator region of pSBS2542, and the deduced amino acid sequences.\",\n \"The deduced amino acid sequence of oleosin-thioredoxin-reductase-linker-thioredoxin is also shown in SEQ ID NO:33.Amino acids representing oleosin are set forth at positions 1-173, those amino acids representing thioredoxin-reductase are set forth at positions 174-501, those amino acids representing the linker or spacer peptide are set forth at positions 501-524, and those representing thioredoxin are set forth at positions 525-636.SEQ ID NOs:38 and 39 set forth the nucleotide sequence of Arabidopsis Thaliana Thioredoxin h (Trx h 1) and the encoded protein, respectively.\",\n \"SEQ ID NOs:40 and 41 set forth the nucleotide sequence of Arabidopsis Thaliana Thioredoxin Reductase (NTR1) and the encoded protein, respectively.\",\n \"SEQ ID NOs:42 and 43 set forth the nucleotide sequence of E. Coli Thioredoxin (TrxA) and the encoded protein, respectively.\",\n \"SEQ ID NOs:44 and 45, set forth the nucleotide sequence of E. Coli Thioredoxin Reductase and the encoded protein, respectively.\",\n \"SEQ ID NOs:46 and 47 set forth the nucleotide sequence of Human Thioredoxin and the encoded protein, respectively.\",\n \"SEQ ID NOs:48 and 49, set forth the nucleotide sequence of Human Thioredoxin Reductase and the encoded protein, respectively.\",\n \"SEQ ID NOs:50 and 51, respectively, set forth the nucleotide sequence of Mycobacterium leprae Thioredoxin-Thioredoxin Reductase and the encoded protein, respectively.\",\n \"SEQ ID NOs:52-313 are described in Table 5.TABLE 5 EXAMPLES OF REDOX PROTEINS SWISS PROTEIN IDENTIFIER SEQ.\",\n \"ID NO.\",\n \"(in parenthesis) PLANT THIOREDOXINS Thioredoxin f-type 52 (Q9XFH8) Thioredoxin F-type 1, chloroplast precursor (TRX- F1).\",\n \"- Arabidopsis thaliana (Mouse-ear cress) 53 (Q9XFH9) Thioredoxin F-type 2, chloroplast precursor (TRX- F2).\",\n \"{GENE: AT5G16400 OR MQK4.13} - Arabidopsis thaliana (Mouse-ear cress) 54 (O48897) Thioredoxin F-type, chloroplast precursor (TRX-F).\",\n \"{GENE: TRXF} - Brassica napus (Rape) 55 (O81332) Thioredoxin F-type, chloroplast precursor (TRX-F).\",\n \"- Mesembryanthemum crystallinum (Common ice plant) 56 (P29450) Thioredoxin F-type, chloroplast precursor (TRX-F).\",\n \"- Pisum sativum (Garden pea) 57 (P09856) Thioredoxin F-type, chloroplast precursor (TRX-F).\",\n \"- Spinacia oleracea (Spinach) Thioredoxin m-type 58 (P06544) Thioredoxin 1 (TRX-1) (Thioredoxin M).\",\n \"{GENE: TRXA} - Anabaena sp.\",\n \"(strain PCC 7119) 59 (O48737) Thioredoxin M-type 1, chloroplast precursor (TRX- M1).\",\n \"{GENE: AT1G03680 OR F21B7_7 OR F21B7.28} - Arabidopsis thaliana (Mouse-ear cress) 60 (Q9SEU8) Thioredoxin M-type 2, chloroplast precursor (TRX- M2).\",\n \"{GENE: AT4G03520 OR F9H3.15 OR T5L23.1} - Arabidopsis thaliana (Mouse-ear cress) 61 (Q9SEU7) Thioredoxin M-type 3, chloroplast precursor (TRX- M3).\",\n \"{GENE: AT2G15570 OR F9O13.12} - Arabidopsis thaliana (Mouse-ear cress) 62 (Q9SEU6) Thioredoxin M-type 4, chloroplast precursor (TRX- M4).\",\n \"- Arabidopsis thaliana (Mouse-ear cress) 63 (Q9XGS0) Thioredoxin M-type, chloroplast precursor (TRX-M).\",\n \"- Brassica napus (Rape) 64 (P23400) Thioredoxin M-type, chloroplast precursor (TRX-M) (Thioredoxin CH2).\",\n \"{GENE: TRXM} - Chlamydomonas reinhardtii 65 (Q41864) Thioredoxin M-type, chloroplast precursor (TRX-M).\",\n \"{GENE: TRM1} - Zea mays (Maize) 66 (Q9ZP20) Thioredoxin M-type, chloroplast precursor (TRX-M).\",\n \"- Oryza sativa (Rice) 67 (P48384) Thioredoxin M-type, chloroplast precursor (TRX-M).\",\n \"- Pisum sativum (Garden pea) 68 (P07591) Thioredoxin M-type, chloroplast precursor (TRX-M).\",\n \"- Spinacia oleracea (Spinach) 69 (Q9ZP21) Thioredoxin M-type, chloroplast precursor (TRX-M).\",\n \"- Triticum aestivum (Wheat) 70 (P12243) Thioredoxin 1 (TRX-1) (Thioredoxin M).\",\n \"{GENE: TRXA OR TRXM} - Synechococcus sp.\",\n \"(strain PCC 7942) (Anacystis nidulans R2) 71 (P37395) Thioredoxin.\",\n \"{GENE: TRXA OR TRX} - Cyanidium caldarium [Chloroplast] 72 (O22022) Thioredoxin.\",\n \"{GENE: TRXA OR TRXM} - Cyanidioschyzon merolae [Chloroplast] 73 (P50338) Thioredoxin.\",\n \"{GENE: TRXA} - Griffithsia pacifica [Chloroplast] 74 (P50254) Thioredoxin.\",\n \"{GENE: TRXA} - Porphyra yezoensis [Chloroplast] 75 (P51225) Thioredoxin.\",\n \"{GENE: TRXA} - Porphyra purpurea [Chloroplast] Thioredoxin h-type 76 (P29448) Thioredoxin H-type 1 (TRX-H-1).\",\n \"{GENE: TRX1 OR AT3G51030 OR F24M12.70} - Arabidopsis thaliana (Mouse- ear cress) 77 (P20857) Thioredoxin 2 (TRX-2).\",\n \"{GENE: TRXB} - Anabaena sp.\",\n \"(strain PCC 7120) 78 (Q42388) Thioredoxin H-type 1 (TRX-H-1) (Pollen coat protein).\",\n \"{GENE: THL-1 OR BOPC17} - Brassica napus (Rape), Brassica oleracea (Cauliflower) 79 (P29449) Thioredoxin H-type 1 (TRX-H1).\",\n \"- Nicotiana tabacum (Common tobacco) 80 (Q38879) Thioredoxin H-type 2 (TRX-H-2).\",\n \"{GENE: TRX2 OR AT5G39950 OR MYH19.14} - Arabidopsis thaliana (Mouse-ear cress) 81 (Q39362) Thioredoxin H-type 2 (TRX-H-2).\",\n \"{GENE: THL-2} - Brassica napus (Rape) 82 (Q07090) Thioredoxin H-type 2 (TRX-H2).\",\n \"- Nicotiana tabacum (Common tobacco) 83 (Q42403) Thioredoxin H-type 3 (TRX-H-3).\",\n \"{GENE: TRX3 OR AT5G42980 OR MBD2.18} - Arabidopsis thaliana (Mouse-ear cress) 84 (Q39239) Thioredoxin H-type 4 (TRX-H-4).\",\n \"{GENE: TRX4} - Arabidopsis thaliana (Mouse-ear cress) 85 (Q39241) Thioredoxin H-type 5 (TRX-H-5).\",\n \"{GENE: TRX5} - Arabidopsis thaliana (Mouse-ear cress) 86 (O64432) Thioredoxin H-type (TRX-H).\",\n \"{GENE: PEC-2} - Brassica rapa (Turnip) 87 (P80028) Thioredoxin H-type (TRX-H) (Thioredoxin CH1).\",\n \"{GENE: TRXH} - Chlamydomonas reinhardtii 88 (Q96419) Thioredoxin H-type (TRX-H).\",\n \"- Fagopyrum esculentum (Common buckwheat) 89 (Q42443) Thioredoxin H-type (TRX-H) (Phloem sap 13 kDa protein-1).\",\n \"- Oryza sativa (Rice) 90 (O65049) Thioredoxin H-type (TRX-H).\",\n \"{GENE: SB09} - Picea mariana (Black spruce) 91 (Q43636) Thioredoxin H-type (TRX-H).\",\n \"- Ricinus communis (Castor bean) 92 (O64394) Thioredoxin H-type (TRX-H) (TrxTa).\",\n \"- Triticum aestivum (Wheat) 93 (P29429) Thioredoxin.\",\n \"- Emericella nidulans (Aspergillus nidulans) VIRUSES, BACTERIA AND FUNGI THIOREDOXINS 94 (P80579) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Alicyclobacillus acidocaldarius (Bacillus acidocaldarius) 95 (O28137) Thioredoxin.\",\n \"{GENE: AF2145} - Archaeoglobus fulgidus 96 (P14949) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR TRX} - Bacillus subtilis 97 (P00276) Thioredoxin.\",\n \"{GENE: NRDC} - Bacteriophage T4 98 (O51088) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR BB0061} - Borrelia burgdorferi (Lyme disease spirochete) 99 (P57653) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR BU597} - Buchnera aphidicola (subsp.\",\n \"Acyrthosiphon pisum) (Acyrthosiphon pisum symbiotic bacterium) 100 (O51890) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Buchnera aphidicola (subsp.\",\n \"Schizaphis graminum) 101 (P10472) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Chlorobium limicola f.sp.\",\n \"thiosulfatophilum 102 (Q9PJK3) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR TC0826} - Chlamydia muridarum 103 (Q9Z7P5) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR CPN0659 OR CP0088} - Chlamydia pneumoniae (Chlamydophila pneumoniae) 104 (P52227) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Chlamydia psittaci (Chlamydophila psittaci) 105 (O84544) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR CT539} - Chlamydia trachomatis 106 (P00275) Thioredoxin C-1.- Corynebacterium nephridii 107 (P07887) Thioredoxin C-2.- Corynebacterium nephridii 108 (P52228) Thioredoxin C-3.- Corynebacterium nephridii 109 (P09857) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Chromatium vinosum 110 (P21609) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Clostridium litorale (Bacterium W6) 111 (P81108) Thioredoxin (TRX) (Fragment).\",\n \"{GENE: TRXA} - Clostridium sporogenes 112 (P81109) Thioredoxin (TRX) (Fragment).\",\n \"{GENE: TRXA} - Clostridium sticklandii 113 (Q9UW02) Thioredoxin (Allergen Cop c 2).\",\n \"- Coprinus comatus (Shaggy mane) 114 (P29445) Thioredoxin 1.\",\n \"{GENE: TRXA OR TRX1} - Dictyostelium discoideum (Slime mold) 115 (P29446) Thioredoxin 2 (Fragment).\",\n \"{GENE: TRXB OR TRX2} - Dictyostelium discoideum (Slime mold) 116 (P29447) Thioredoxin 3.\",\n \"{GENE: TRXC OR TRX3} - Dictyostelium discoideum (Slime mold) 117 (P00274) Thioredoxin 1 (TRX1) (TRX).\",\n \"{GENE: TRXA OR TSNC OR FIPA OR B3781} - Escherichia coli, Salmonella typhimurium 118 (P52232) Thioredoxin-like protein SLR0233.\",\n \"{GENE: SLR0233} - Synechocystis sp.\",\n \"(strain PCC 6803) 119 (P33636) Thioredoxin 2 (Trx2).\",\n \"{GENE: TRXC OR B2582 OR Z3867 OR ECS3448} - Escherichia coli, Escherichia coli O157:H7 120 (P21610) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Eubacterium acidaminophilum 121 (P43785) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR TRXM OR HI0084} - Haemophilus influenzae 122 (P43787) Thioredoxin-like protein HI1115.\",\n \"{GENE: HI1115} - Haemophilus influenzae 123 (P56430) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR HP0824 OR JHP0763} - Helicobacter pylori (Campylobacter pylori), Helicobacter pylori J99 (Campylobacter pylori J99) 124 (Q9S386) Thioredoxin (EC 1.6.4.5) {GENE: TRXA} - Listeria monocytogenes 125 (Q57755) Thioredoxin.\",\n \"{GENE: TRX OR MJ0307} - Methanococcus jannaschii 126 (P47370) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR TRX OR MG124} - Mycoplasma genitalium 127 (P46843) Bifunctional thioredoxin-reductase/thioredoxin [Includes: Thioredoxin-reductase (EC 1.6.4.5) (TRXR); Thioredoxin].\",\n \"{GENE: TRXB/A OR TRX OR ML2703} - Mycobacterium leprae 128 (P75512) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR TRX OR MPN263 OR MP570} - Mycoplasma pneumoniae 129 (O30974) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Mycobacterium smegmatis 130 (P52229) Thioredoxin (TRX) (MPT46).\",\n \"{GENE: TRXA OR TRX OR TRXC OR RV3914 OR MT4033 OR MTV028.05} - Mycobacterium tuberculosis 131 (P42115) Thioredoxin.\",\n \"{GENE: TRX} - Neurospora crassa 132 (P34723) Thioredoxin.\",\n \"{GENE: TRXA} - Penicillium chrysogenum 133 (Q9X2T1) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR TRX OR PA5240} - Pseudomonas aeruginosa 134 (P10473) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Rhodospirillum rubrum 135 (P08058) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Rhodobacter sphaeroides (Rhodopseudomonas sphaeroides) 136 (Q9ZEE0) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR RP002} - Rickettsia prowazekii 137 (P33791) Thioredoxin (TRX) (Fragment).\",\n \"{GENE: TRXA} - Streptomyces aureofaciens 138 (P52230) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR SCH24.11C} - Streptomyces coelicolor 139 (Q05739) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Streptomyces clavuligerus 140 (P52231) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR SLR0623} - Synechocystis sp.\",\n \"(strain PCC 6803) 141 (P73263) Thioredoxin-like protein SLR1139.\",\n \"{GENE: SLR1139} - Synechocystis sp.\",\n \"(strain PCC 6803) 142 (P52233) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Thiobacillus ferrooxidans 143 (P96132) Thioredoxin (TRX) (Fragment).\",\n \"{GENE: TRXA} - Thiocapsa roseopersicina 144 (P81110) Thioredoxin (TRX) (Fragment).\",\n \"{GENE: TRXA} - Tissierella creatinophila 145 (O83889) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR TP0919} - Treponema pallidum ANIMAL THIOREDOXIN 146 (O97680) Thioredoxin.\",\n \"{GENE: TXN} - Bos taurus (Bovine) 147 (Q95108) Thioredoxin, mitochondrial precursor (MT-TRX).\",\n \"{GENE: TXN2} - Bos taurus (Bovine) 148 (Q09433) Thioredoxin.\",\n \"{GENE: B0228.5} - Caenorhabditis elegans 149 (P99505) Thioredoxin (Fragment).\",\n \"{GENE: TXN} - Canis familiaris (Dog 150 (P08629) Thioredoxin.\",\n \"{GENE: TXN} - Gallus gallus (Chicken) 151 (P47938) Thioredoxin (Deadhead protein).\",\n \"{GENE: DHD OR CG4193} - Drosophila melanogaster (Fruit fly) 152 (P10599) Thioredoxin (ATL-derived factor) (ADF) (Surface associated sulphydryl protein) (SASP).\",\n \"{GENE: TXN OR TRDX OR TRX} - Homo sapiens (Human) 153 (Q99757) Thioredoxin, mitochondrial precursor (MT-TRX).\",\n \"{GENE: TXN2} - Homo sapiens (Human) 154 (P29451) Thioredoxin.\",\n \"{GENE: TXN} - Macaca mulatta (Rhesus macaque) 155 (P10639) Thioredoxin (ATL-derived factor) (ADF).\",\n \"{GENE: TXN} - Mus musculus (Mouse) 156 (P97493) Thioredoxin, mitochondrial precursor (MT-TRX).\",\n \"{GENE: TXN2} - Mus musculus (Mouse) 157 (P82460) Thioredoxin (Fragment).\",\n \"{GENE: TXN} - Sus scrofa (Pig) 158 (P08628) Thioredoxin.\",\n \"{GENE: TXN} - Oryctolagus cuniculus (Rabbit) 159 (P11232) Thioredoxin.\",\n \"{GENE: TXN} - Rattus norvegicus (Rat) 160 (P97615) Thioredoxin, mitochondrial precursor (MT-TRX).\",\n \"{GENE: TXN2 OR TRX2} - Rattus norvegicus (Rat) 161 (P50413) Thioredoxin.\",\n \"{GENE: TXN} - Ovis aries (Sheep) PLANTS THIOREDOXIN-LIKE PROTEINS 162 (O23166) THIOL-DISULFIDE INTERCHANGE LIKE PROTEIN (THIOREDOXIN-LIKE PROTEIN) {GENE: C7A10.160 OR AT4G37200 OR HCF164} - Arabidopsis thaliana (Mouse-ear cress) 163 (Q9C9Y6) Thioredoxin-like protein {GENE: F17O14.18} - Arabidopsis thaliana (Mouse-ear cress) 164 (Q9FYD5) Thioredoxin-like protein {GENE: F21E1_180} - Arabidopsis thaliana (Mouse-ear cress) 165 (Q38878) THIOREDOXIN-LIKE PROTEIN {GENE: TRX6 OR T7D17.3} - Arabidopsis thaliana (Mouse-ear cress) 166 (Q9LVI2) Thioredoxin-like protein - Arabidopsis thaliana (Mouse-ear cress) 167 (Q9SCN9) Thioredoxin-like protein {GENE: T4D2.150} - Arabidopsis thaliana (Mouse-ear cress) 168 (Q9SRD7) Thioredoxin-like protein, 49720-48645 {GENE: F28O16.13} - Arabidopsis thaliana (Mouse-ear cress) 169 (Q9SU84) THIOREDOXIN-LIKE PROTEIN {GENE: T16L4.180 OR AT4G29670} - Arabidopsis thaliana (Mouse-ear cress) 170 (Q9SWG6) Thioredoxin-like protein {GENE: TRX} - Hordeum bulbosum 171 (Q9SWG4) Thioredoxin-like protein {GENE: TRX} - Lolium perenne (Perennial ryegrass) 172 (Q9AS75) Thioredoxin-like protein {GENE: P0028E10.17} - Oryza sativa (Rice) 173 (O04002) CDSP32 protein (Chloroplast Drought-induced Stress Protein of 32 kDa) - Solanum tuberosum (Potato) 174 (Q9SWG5) Thioredoxin-like protein {GENE: TRX} - Secale cereale (Rye) 175 (Q9SP36) Thioredoxin-like protein (Fragment) {GENE: TRX} - Secale cereale (Rye) 176 (Q9U515) Thioredoxin-like protein - Manduca sexta (Tobacco hawkmoth) (Tobacco hornworm) VIRUSES, BACTERIA AND FUNGI THIOREDOXIN-LIKE PROTEINS 177 (P43221) Thiol: disulfide interchange protein tlpA (Cytochrome c biogenesis protein tIpA).\",\n \"{GENE: TLPA} - Bradyrhizobium japonicum 178 (P43787) Thioredoxin-like protein HI1115.\",\n \"{GENE: HI1115} - Haemophilus influenzae 179 (Q9GUP7) Thioredoxin-like protein {GENE: TRXLP} - Leishmania major 180 (Q9UVH0) Thioredoxin-like protein - Mortierella alpina 181 (P95355) Thioredoxin-like protein - Neisseria gonorrhoeae 182 (Q98G37) Thioredoxin-like protein {GENE: MLL3505} - Rhizobium loti (Mesorhizobium loti) 183 (P36893) Thiol: disulfide interchange protein helX precursor (Cytochrome c biogenesis protein helX).\",\n \"{GENE: HELX} - Rhodobacter capsulatus (Rhodopseudomonas capsulata) 184 (P52232) Thioredoxin-like protein SLR0233.\",\n \"{GENE: SLR0233} - Synechocystis sp.\",\n \"(strain PCC 6803) 185 (P73263) Thioredoxin-like protein SLR1139.\",\n \"{GENE: SLR1139} - Synechocystis sp.\",\n \"(strain PCC 6803) 186 (Q9USR1) Thioredoxin-like protein {GENE: SPBC577.08C} - Schizosaccharomyces pombe (Fission yeast) 187 (Q9R788) Thioredoxin {GENE: TPTRX} - Treponema pallidum ANIMALS THIOREDOXIN-LIKE PROTEINS 188 (Q9UAV4) F46E10.9 PROTEIN (THIOREDOXIN-LIKE PROTEIN DPY-11) {GENE: F46E10.9 OR DPY-11} - Caenorhabditis elegans 189 (Q9N2K6) Thioredoxin-like protein (Y54E10A.3 protein) (Thioredoxin-like protein TXL) {GENE: TXL OR Y54E10A.3} - Caenorhabditis elegans 190 (Q9VRP3) THIOREDOXIN-LIKE PROTEIN TXL (CG5495 PROTEIN) {GENE: TXL OR CG5495} - Drosophila melanogaster (Fruit fly) 191 (O43396) Thioredoxin-like protein (32 kDa thioredoxin-related protein).\",\n \"{GENE: TXNL OR TRP32 OR TXL} - Homo sapiens (Human) 192 (O76003) Thioredoxin-like protein - Homo sapiens (Human) 193 (Q9S753) THIOREDOXIN-LIKE PROTEIN {GENE: TRX} - Phalaris coerulescens 194 (O77404) TRYPAREDOXIN - Trypanosoma brucei brucei PLANT THIOREDOXIN-REDUCTASES 195 (Q39243) Thioredoxin-reductase 1 (EC 1.6.4.5) (NADPH- dependent thioredoxin-reductase 1) (NTR 1).\",\n \"{GENE: NTR1 OR AT4G35460 OR F15J1.30} - Arabidopsis thaliana (Mouse-ear cress) 196 (Q39242) Thioredoxin-reductase 2 (EC 1.6.4.5) (NADPH- dependent thioredoxin-reductase 2) (NTR 2).\",\n \"{GENE: NTR2 OR AT2G17420 OR F5J6.18} - Arabidopsis thaliana (Mouse-ear cress) VIRUSES, BACTERIA AND FUNGI THIOREDOXIN-REDUCTASES 197 (O66790) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR AQ_500} - Aquifex aeolicus 198 (P80880) Thioredoxin-reductase (EC 1.6.4.5) (TRXR) (General stress protein 35) (GSP35).\",\n \"{GENE: TRXB} - Bacillus subtilis 199 (P94284) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR BB0515} - Borrelia burgdorferi (Lyme disease spirochete) 200 (P57399) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR BU314} - Buchnera aphidicola (subsp.\",\n \"Acyrthosiphon pisum) (Acyrthosiphon pisum symbiotic bacterium) 201 (P81433) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB} - Buchnera aphidicola (subsp.\",\n \"Schizaphis graminum) 202 (Q9PKT7) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR TC0375} - Chlamydia muridarum 203 (Q9Z8M4) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR CPN0314 OR CP0444} - Chiamydia pneumoniae (Chlamydophila pneumoniae) 204 (O84101) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR CT099} - Chlamydia trachomatis 205 (P52213) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB} - Clostridium litorale (Bacterium W6) 206 (P39916) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB} - Coxiella burnetii 207 (P09625) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR B0888 OR Z1232 OR ECSO973} - Escherichia coli, Escherichia coli O157: H7 208 (P50971) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB} - Eubacterium acidaminophilum 209 (P43788) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR HI1158} - Haemophilus influenzae 210 (Q9ZL18) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR JHP0764} - Helicobacter pylori J99 (Campylobacter pylori J99) 211 (P56431) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR HP0825} - Helicobacter pylori (Campylobacter pylori) 212 (O32823) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR LMO2478} - Listeria monocytogenes 213 (P47348) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR MG102} - Mycoplasma genitalium 214 (P46843) Bifunctional thioredoxin-reductase/thioredoxin [Includes: Thioredoxin-reductase (EC 1.6.4.5) (TRXR); Thioredoxin].\",\n \"{GENE: TRXB/A OR TRX OR ML2703} - Mycobacterium leprae 215 (P75531) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR MPN240 OR MP591} - Mycoplasma pneumoniae 216 (O30973) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB} - Mycobacterium smegmatis 217 (P52214) Thioredoxin-reductase (EC 1.6.4.5) (TRXR) (TR).\",\n \"{GENE: TRXB OR RV3913 OR MT4032 OR MTV028.04} - Mycobacterium tuberculosis 218 (P51978) Thioredoxin-reductase (EC 1.6.4.5).\",\n \"{GENE: CYS-9} - Neurospora crassa 219 (P43496) Thioredoxin-reductase (EC 1.6.4.5).\",\n \"{GENE: TRXB} - Penicillium chrysogenum 220 (Q9ZD97) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR RP445} - Rickettsia prowazekii 221 (Q92375) Thioredoxin-reductase (EC 1.6.4.5).\",\n \"{GENE: SPBC3F6.03} - Schizosaccharomyces pombe (Fission yeast) 222 (Q05741) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB} - Streptomyces clavuligerus 223 (P52215) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR SCH24.12} - Streptomyces coelicolor 224 (O83790) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR TP0814} - Treponema pallidum 225 (P80892) Thioredoxin-reductase (EC 1.6.4.5) (TRXR) (Fragment).\",\n \"{GENE: TRXB} - Vibrio fischeri 226 (P29509) Thioredoxin-reductase 1 (EC 1.6.4.5).\",\n \"{GENE: TRR1 OR YDR353W OR D9476.5} - Saccharomyces cerevisiae (Baker's yeast) 227 (P38816) Thioredoxin-reductase 2, mitochondrial precursor (EC 1.6.4.5).\",\n \"{GENE: TRR2 OR YHR106W} - Saccharomyces cerevisiae (Baker's yeast) ANIMAL THIOREDOXIN-REDUCTASES 228 (O62768) Thioredoxin-reductase (EC 1.6.4.5).\",\n \"{GENE: TXNRD1} - Bos taurus (Bovine) 229 (Q17745) Thioredoxin-reductase (EC 1.6.4.5).\",\n \"{GENE: C06G3.7} - Caenorhabditis elegans 230 (Q16881) Thioredoxin-reductase (EC 1.6.4.5).\",\n \"{GENE: TXNRD1} - Homo sapiens (Human) 231 (Q25861) Thioredoxin-reductase (EC 1.6.4.5) (TrxR).\",\n \"{GENE: TR OR GR} - Plasmodium falciparum (isolate FCH-5) Other thioredoxin-reductases PLANTS THIOREDOXIN-REDUCTASES 232 (O22229) Thioredoxin-reductase {GENE: AT2G41680} - Arabidopsis thaliana (Mouse-ear cress) 233 (Q39951) NADPH thioredoxin-reductase (Fragment} - Helianthus annuus (Common sunflower) VIRUSES, BACTERIA AND FUNGI THIOREDOXIN-REDUCTASES 234 (O28718) THioredoxin-reductase (TRXB) {GENE: AF1554} - Archaeoglobus fulgidus 235 (Q9K703) Thioredoxin-reductase (NADPH) (EC 1.6.4.5) {GENE: TRXB OR BH3571} - Bacillus halodurans 236 (Q9K7F3) Thioredoxin-reductase {GENE: BH3408} - Bacillus halodurans 237 (Q9KCZ0) Thioredoxin-reductase {GENE: BH1429} - Bacillus halodurans 238 (Q9KCZ1) Thioredoxin-reductase {GENE: BH1428} - Bacillus halodurans 239 (Q9PIY1) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB OR CJ0146} - Campylobacter jejuni 240 (Q9A4G3) Thioredoxin-reductase {GENE: CC2871} - Caulobacter crescentus 241 (Q97EM8) Thioredoxin-reductase {GENE: CAC3082} - Clostridium acetobutylicum 242 (Q97IU2) Thioredoxin-reductase {GENE: CAC1548} - Clostridium acetobutylicum 243 (Q9EV96) Thioredoxin-reductase {GENE: TRXB} - Clostridium sticklandii 244 (Q9RSY7) THioredoxin-reductase {GENE: DR1982} - Deinococcus radiodurans 245 (O30739) Thioredoxin-reductase (Fragment} - Enterococcus faecalis (Streptococcus faecalis) 246 (O54535) Thioredoxin-reductase {GENE: TRXB OR TRXB1_2 OR VNG6452G OR TRXB1_1 OR VNG6074G} - Halobacterium sp.\",\n \"(strain NRC-1) [Plasmid pNRC100, and Plasmid pNRC200] 247 (P82854) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB2} - Halobacterium sp.\",\n \"(strain NRC-1) 248 (Q9HN08) Thioredoxin-reductase {GENE: TXRB3 OR VNG2301G} - Halobacterium sp.\",\n \"(strain NRC-1) 249 (O25779) THioredoxin-reductase (TRXB) {GENE: HP1164} - Helicobacter pylori (Campylobacter pylori) 250 (O86255) Thioredoxin-reductase {GENE: TRXB} - Klebsiella oxytoca 251 (Q9AEV9) Thioredoxin-reductase (Fragment) {GENE: TRXB} - Lactococcus lactis (subsp.\",\n \"lactis) (Streptococcus lactis) 252 (Q9CF34) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB2} - Lactococcus lactis (subsp.\",\n \"lactis) (Streptococcus lactis) 253 (Q9CH02) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB1} - Lactococcus lactis (subsp.\",\n \"lactis) (Streptococcus lactis) 254 (Q9ZFC8) Thioredoxin-reductase (Fragment) {GENE: TRXB} - Lactococcus lactis 255 (O32822) Hypothetical 39.7 kDa protein (Fragment) - Listeria monocytogenes 256 (O26804) THioredoxin-reductase {GENE: MTH708} - Methanothermobacter thermautotrophicus 257 (P94397) Homologue of thioredoxin-reductase of Mycoplama genitalium {GENE: YCGT} - Bacillus subtilis 258 (Q98PK9) THioredoxin-reductase (EC 1.6.4.5) {GENE: MYPU_7130} - Mycoplasma pulmonis 259 (Q9JU23) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB OR NMA1538} - Neisseria meningitidis (serogroup A) 260 (Q9JZ28) Thioredoxin-reductase {GENE: NMB1324} - Neisseria meningitidis (serogroup B) 261 (Q9I0M2) Thioredoxin-reductase 1 {GENE: TRXB1 OR PA2616} - Pseudomonas aeruginosa 262 (Q9I592) Thioredoxin-reductase 2 {GENE: TRXB2 OR PA0849} - Pseudomonas aeruginosa 263 (Q9V0Q8) THioredoxin-reductase (TRXB) {GENE: TRXB OR PAB0500} - Pyrococcus abyssi 264 (Q9ZD33) THioredoxin-reductase (TRXB2) {GENE: RP514} - Rickettsia prowazekii 265 (O54079) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB} - Staphylococcus aureus 266 (Q9RIS2) Thioredoxin-reductase {GENE: TRXB OR TRXB2} - Streptomyces coelicolor 267 (Q9K4L6) Thioredoxin-reductase {GENE: SC5F8.08C} - Streptomyces coelicolor 268 (Q97PY2) Thioredoxin-reductase {GENE: SP1458} - Streptococcus pneumoniae 269 (Q9A0B5) Thioredoxin-reductase {GENE: SPY0850} - Streptococcus pyogenes 270 (Q97V69) Thioredoxin-reductase (trxB-2) (EC 1.6.4.5) {GENE: TRXB-2} - Sulfolobus solfataricus 271 (Q97W27) Thioredoxin-reductase (trxB-3) (EC 1.6.4.5) {GENE: TRXB-3} - Sulfolobus solfataricus 272 (Q97WJ5) Thioredoxin-reductase (trxB-1) (EC 1.6.4.5) {GENE: TRXB-1} - Sulfolobus solfataricus 273 (Q98I59) Thioredoxin-reductase {GENE: MLL2552} - Rhizobium loti (Mesorhizobium loti) 274 (Q98M06) Thioredoxin-reductase {GENE: MLL0792} - Rhizobium loti (Mesorhizobium loti) 275 (Q9UR80) 35 kDa THioredoxin-reductase HOMOLOG (FRAGMENT) {GENE: TRR1 AND YDR353W} - Saccharomyces cerevisiae (Baker's yeast) 276 (Q9ZEH4) THIOREDOXIN {GENE: TRXA OR SA0992} - Staphylococcus aureus, Staphylococcus aureus subsp.\",\n \"aureus N315 277 (Q9S1H1) Thioredoxin-reductase (Fragment) {GENE: TRXB} - Staphylococcus xylosus 278 (Q9HJI4) Thioredoxin-reductase {GENE: TA0984} - Thermoplasma acidophilum 279 (Q9WZX3) THioredoxin-reductase {GENE: TM0869} - Thermotoga maritima 280 (Q979K8) Thioredoxin-reductase {GENE: TVG1183005} - Thermoplasma volcanium 281 (Q9PR71) Thioredoxin-reductase {GENE: TRXB OR UU074} - Ureaplasma parvum (Ureaplasma urealyticum biotype 1) 282 (Q9KSS4) Thioredoxin-reductase {GENE: VC1182} - Vibrio cholerae 283 (Q9PDD1) Thioredoxin-reductase {GENE: XF1448} - Xylella fastidiosa 284 (Q9X5F7) Thioredoxin-reductase {GENE: TRXB1} - Zymomonas mobilis ANIMAL THIOREDOXIN-REDUCTASES 285 (Q9GKW9) Thioredoxin-reductase 3 (Fragment) {GENE: TRXR3} - Bos taurus (Bovine) 286 (Q9N2I8) Thioredoxin-reductase (EC 1.6.4.5) - Bos taurus (Bovine) 287 (Q9N2K1) Thioredoxin-reductase homolog - Caenorhabditis elegans 288 (Q9NJH3) Thioredoxin-reductase - Caenorhabditis elegans 289 (Q9VNT5) CG11401 PROTEIN (THioredoxin-reductase 2) {GENE: TRXR-2 OR CG11401} - Drosophila melanogaster (Fruit fly) 290 (O95840) Thioredoxin-reductase - Homo sapiens (Human) 291 (Q9UES8) Thioredoxin-reductase GRIM-12 - Homo sapiens (Human) 292 (Q9UH79) Thioredoxin-reductase {GENE: TR} - Homo sapiens (Human) 293 (Q9UQU8) Thioredoxin-reductase - Homo sapiens (Human) 294 (Q9NNW6) Thioredoxin-reductase TR2 (Fragment) - Homo sapiens (Human) 295 (Q9NNW7) Thioredoxin-reductase TR3 - Homo sapiens (Human) 296 (Q9P101) Thioredoxin-reductase 3 (Fragment) {GENE: TRXR3} - Homo sapiens (Human) 297 (Q9P2Y0) Thioredoxin-reductase II beta (EC 1.6.4.5) - Homo sapiens (Human) 298 (Q9H2Z5) Mitochondrial thioredoxin-reductase {GENE: TRXR2A} - Homo sapiens (Human) 299 (Q99475) KM-102-DERIVED REDUCTASE-LIKE FACTOR (THioredoxin-reductase) - Homo sapiens (Human) 300 (Q99P49) Thioredoxin-reductase 1 {GENE: TXNRD1} - Mus musculus (Mouse) 301 (Q9CSV5) Thioredoxin-reductase 1 (Fragment) {GENE: TXNRD1} - Mus musculus (Mouse) 302 (Q9CZE5) Thioredoxin-reductase 1 {GENE: TXNRD1} - Mus musculus (Mouse) 303 (Q9JHA7) Thioredoxin-reductase TR3 {GENE: TXNRD2 OR TR3} - Mus musculus (Mouse) 304 (Q9JLT4) Thioredoxin-reductase {GENE: TXNRD2 OR TRXR2} - Mus musculus (Mouse) 305 (Q9JMH5) Thioredoxin-reductase 2 {GENE: TXNRD2 OR TXNRD2} - Mus musculus (Mouse) 306 (Q9JMH6) Thioredoxin-reductase 1 {GENE: TXNRD1 OR TXNRD1} - Mus musculus (Mouse) 307 (O89049) Thioredoxin-reductase - Rattus norvegicus (Rat) 308 (Q9JKZ3) Thioredoxin-reductase 1 (Fragment) - Rattus norvegicus (Rat) 309 (Q9JKZ4) Thioredoxin-reductase 1 - Rattus norvegicus (Rat) 310 (Q9JLE6) Thioredoxin-reductase (Fragment) - Rattus norvegicus (Rat) 311 (Q9R1I3) NADPH-dependent thioredoxin-reductase {GENE: TRR1} - Rattus norvegicus (Rat) 312 (Q9Z0J5) Thioredoxin-reductase precursor {GENE: TRXR2} - Rattus norvegicus (Rat) 313 (Q9MYY8) Redox enzyme thioredoxin-reductase - Sus scrofa (Pig)\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10450903"}}},{"rowIdx":287,"cells":{"text":{"kind":"list like","value":[["Adaptive comparator circuit and acoustic distance sensor comprising said circuit","The invention relates to an adaptive comparator circuit comprising a comparator with a threshold value input which receives a threshold voltage and a signal input which receives a voltage signal.","The threshold value input is connected to one pole by means of a switch and connected to the other pole of a first voltage source by means of a capacitor.","The threshold value input is also connected to the signal input of the comparator by means of a diode or a second voltage source.","The switch is controlled by the control signal of a signal transmitter.","When the switch is in a closed position, the capacitor is charged with the voltage of the first voltage source.","When the switch is in an open position, the capacitor is discharged in such a way that the threshold voltage of the signal voltage is corrected at a given time interval."],["1.An adaptive comparator circuit, particularly for an acoustic distance sensor, comprising a first comparator (1), having a first threshold voltage input (1a) to which a first threshold voltage (Uref1) is applied and a first signal input (1b) to which a voltage signal (Usignal) is applied and, when a voltage signal (Usignal) is present that is greater than the first threshold voltage (Uref1), said comparator emits a first switching signal (S1), characterized by a signal transmitter (2), especially a microcontroller or microprocessor, that at times emits a control signal (SSt), a first switch (3, 103) that has a control means (3a, 103a), a switch input (3b, 103b) and a switch output (3c, 103c), and that can be regulated or activated through the effect of the control signal (SSt) on the control means (3a, 103a) in such a way that it is in the closed state when the signal transmitter (2) emits a control signal (SSt), and otherwise it is in the open state, or vice versa, whereby the switch input (3b, 103b) is connected to the positive or negative first pole (4a) of a first direct voltage source and the switch output (3c, 103c) is connected to the first threshold voltage input (1a), so that the first threshold voltage (Uref1) is equal to the voltage present at the switch output (3c, 103c), a capacitor (6) via which the switch output is connected to the negative or positive pole (4b) of the first direct voltage source, and a diode (5) via which the switch output (3c, 103c) is connected to the first signal input (1b) in such a way that the anode (5a) or the cathode (5b) of the diode (5) is connected to the switch output (3c, 103c).","2.The adaptive comparator according to claim 1, characterized in that, instead of the diode (5), a second direct voltage source (7) is used whose positive pole (7a) is in the place of the anode (5a) of the diode (5) and whose negative pole (7b) is in the place of the cathode (5b) of the diode (5).","3.The adaptive comparator according to claim 2, characterized in that the voltage supplied by the second voltage source (7) can be regulated or adjusted.","4.The adaptive comparator according to claim 1, characterized in that the signal transmitter (2) emits an electric control signal (SSt) at regular intervals.","5.The adaptive comparator according to claim 1, characterized in that the first switch (3, 103) is a switch that can be electrically or electronically controlled by the control means (3a, 103a) and the control signal (SSt) is an electric signal that is transmitted by the signal transmitter (2) via a control output (2a) and fed to the control input (3a, 103a).","6.The adaptive comparator according to claim 5, characterized in that the first switch (3, 103) is a transistor (103) and the control-current terminal (3a, 103a) is the base (103) or the gate terminal of the transistor (103).","7.The adaptive comparator according to claim 1, characterized in that a first resistor (11) is connected between the first pole (4a) and the first threshold voltage input (1a), and a second resistor (12) is connected between the first threshold voltage input (1a) and the second pole (4b), so that the first resistor (11) and the second resistor (12) form a first voltage divider (11, 12).","8.The adaptive comparator according to claim 7, characterized in that first voltage divider (11, 12) is an adjustable potentiometer whose pick-up is connected to the switch output (3c, 103c).","9.The adaptive comparator according to claim 1, characterized by a second comparator (20) that has a second threshold voltage input (20a) and a second signal input (20b), and that emits a second switching signal S2 when a greater voltage is present at the second signal input (20b) than at the second threshold voltage input (20a), a third resistor (13) via which the first pole (4a) is connected to the second threshold voltage input (20a), a fourth resistor (14) that, on the one hand, is connected to the second threshold voltage input (20a) and, on the other hand, to the second pole (4b) of the first voltage source, so that the third resistor (13) and the fourth resistor (14) form a second voltage divider (13, 14), a second switch (23) that is connected in series to the diode (5) and that can be regulated or activated by the second switching signal S2 in such a way that it is in the closed state when the second comparator (20) emits the second switching signal S2, and otherwise it is in the open state.","10.The adaptive comparator according to claim 9, characterized in that a fifth resistor (15) is connected between the switch output (3c, 103c) and the second threshold voltage input (20a).","11.The adaptive comparator according to claim 9, characterized in that the second voltage divider (13, 14) is a potentiometer.","12.The adaptive comparator according to claim 1, characterized in that a sixth resistor (16) is connected between the control output (2a) and the control-current terminal (3a, 103a).","13.The adaptive comparator according to claim 6, characterized in that a seventh resistor (17) is connected between the control-current terminal (3a, 103a) and the first pole (4a).","14.The adaptive comparator according to claim 1, characterized in that the diode (5) or the second voltage source (7) is connected in series to an eighth resistor (18).","15.The adaptive comparator according to claim 1, characterized in that, before reaching the first signal input (1b), the voltage signal Usignal passes through a voltage follower or impedance transformer (21).","16.The adaptive comparator according to claim 1, characterized in that the voltage supplied by the first voltage source (4a, 4b) is greater in magnitude than the voltage of the maximum of the voltage signal Usignal.","17.The adaptive comparator according to claim 1, characterized in that the diode (5) is connected to at least another diode codirectionally in series.","18.An acoustic distance sensor, comprising a control unit (31) that at times transmits signal pulses (41) to an oscillator (32) which, during the presence of a signal pulse (41), transmits an alternating voltage to a sound transducer (34) that is excited by the alternating voltage to emit sound waves and that is capable of receiving sound waves coming back to the sound transducer (34) as an echo as a result of reflection, and of converting them into an electric received signal, and also comprising an envelope curve shaper (37) to which the received signal is fed and which forms the envelope curve of the received signal and emits it as a voltage signal Usignal, characterized in that the voltage signal is fed to an adaptive comparator circuit comprising a first comparator (1) having a first threshold voltage input (1a) to which a first threshold voltage (Uref1) is applied and a first signal input (1b) to which a voltage signal (Usignal) is applied and, when a voltage signal (Usignal) is present that is greater than the first threshold voltage Uref1, said comparator (1) emits a first switching signal (S1), a signal transmitter (2), especially a microcontroller or microprocessor, that at times emits a control signal (SSt), a first switch (3, 103) that has a control means (3a, 103a), a switch input (3b, 103b) and a switch output (3c, 103c), and that can be regulated or activated through the effect of the control signal (SSt) on the control means (3a, 103a) in such a way that it is in the closed state when the signal transmitter (2) emits a control signal (SSt), and otherwise it is in the open state, or vice versa, whereby the switch input (3b, 103b) is connected to the positive or negative first pole (4a) of a first direct voltage source and the switch output (3c, 103c) is connected to the first threshold voltage input (1a), so that the first threshold voltage (Uref1) is equal to the voltage present at the switch output (3c, 103c), a capacitor (6) via which the switch output (3c, 103c) is connected to the negative or positive pole (4b) of the first direct voltage source, and a diode (5) via which the switch output (3c, 103c) is connected to the first signal input (1b) in such a way that the anode (5a) or the cathode (5b) of the diode (5) is connected to the switch output (3c, 103c).","19.The acoustic distance sensor according to claim 18, characterized in that, instead of the diode (5), a second direct voltage source (7) is used whose positive pole (7a) is in the place of the anode (5a) of the diode (5) and whose negative pole (7b) is in the place of the cathode (5b) of the diode (5).","20.The acoustic distance sensor according to claim 18 characterized in that the alternating voltage emitted by the oscillator (32) reaches the sound transducer (34) after passing through a driver stage (33).","21.The acoustic distance sensor according to claim 18, characterized in that, before entering the envelope curve shaper (37), the received signal passes through an amplifier (36c).","22.The acoustic distance sensor according to claim 21, characterized in that the amplifier (36c) is a logarithmic amplifier.","23.The acoustic distance sensor according to claim 18, characterized in that, before entering the envelope curve shaper (37), the received signal passes through a voltage limiter (35) that limits the amplitude of the received signal to a maximum value.","24.The acoustic distance sensor according to claim 18, characterized in that the signal transmitter (2) is integrated into the control unit (31) or is a component of the control unit (31).","25.The acoustic distance sensor according to claim 18, characterized in that the first switching signal (S1) is transmitted to the control unit (31) for evaluation purposes.","26.The acoustic distance sensor according to claim 18, characterized in that the signal pulses (41) are synchronized with the control signals (SSt) in such a way that the control signals (SSt) each begin before the signal pulses (41).","27.The acoustic distance sensor according to claim 18, characterized in that the signal pulses 41 are synchronized with the control signals SSt in such a way that the control signals SSt each begin before or simultaneously with the end of the signal pulses (41).","28.An adaptive comparator circuit for an acoustic distance sensor, comprising a first comparator (1) having a first signal input and a first threshold voltage input; a first direct voltage source (4) for delivering a threshold voltage to the first threshold voltage input, wherein the first direct voltage source comprises a first and a second pole; a first switch (3), wherein the first switch is connected to the first direct voltage source and to the first threshold voltage input; a signal transmitter (2), wherein the signal transmitter controls the first switch through an input of the switch; a capacitor (6), wherein a first plate of the capacitor is connected to the a first threshold voltage input and wherein a second plate of the capacitor is connected to second pole the first direct voltage source; a diode (5) connected the first signal input and to the first threshold voltage input of the first comparator.","29.The adaptive comparator circuit according to claim 28, wherein the diode is connected in series to a first resistor.","30.The adaptive comparator circuit according to claim 1, wherein the signal transmitter emits an electric control signal at regular intervals.","31.The adaptive comparator circuit according to claim 28 further comprising a second resistor (11) connected between the first pole of the first direct voltage source and the first threshold voltage input, and a third resistor (12) connected between the first threshold voltage input and the second pole of the first direct voltage source, so that the second resistor (11) and the third resistor (12) form a first voltage divider (11, 12).","32.The adaptive comparator circuit according to claim 31, wherein the first voltage divider (11, 12) is an adjustable potentiometer whose pick-up is connected to the first threshold voltage input.","33.The adaptive comparator circuit according to claim 28 further comprising a fourth resistor (17) connected between the first pole of the first direct voltage source and the input of the first switch; a fifth resistor (16) connected between the input of the first switch and the output of the signal transmitter (2)."],["<SOH> BRIEF DESCRIPTION OF THE DRAWING IN WHICH THE FOLLOWING IS SHOWN <EOH>FIG.","1 —a schematic block diagram of a variant of an acoustic distance sensor according to the state of the art, FIG.","2 —a schematic representation of a typical course over time of a) a signal pulse, b) an amplification factor, c) an envelope curve and d) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.","1 , FIG.","3 —a schematic block diagram of another variant of an acoustic distance sensor according to the state of the art, FIG.","4 —a schematic representation of a typical course over time of a) a timing pulse, b) an envelope curve and c) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.","3 , FIG.","5 —a circuit diagram of an embodiment of a comparator circuit according to the invention, FIG.","6 —a circuit diagram of an alternative embodiment of a comparator circuit according to the invention, FIGS.","7 and 8 —a schematic block diagram of embodiments of an acoustic distance sensor according to the invention, FIG.","9 —a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) an envelope curve and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.","8 , whereby an echo occurs during the quiescent phase, FIG.","10 —a schematic representation of a typical course over time of a) an envelope curve and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.","8 , whereby an echo additionally occurs during the relaxation phase, and FIGS.","11 to 13 —circuit diagrams of further embodiments of comparator circuits according to the invention.","detailed-description description=\"Detailed Description\" end=\"lead\"?","FIGS.","1 to 4 serve to further illustrate the state of the art.","FIG.","1 shows a schematic block diagram of a variant of an acoustic distance sensor 30 a with which a measure according to the state of the art has been taken in order to reduce the short range.","The distance sensor 30 a shown in FIG.","1 comprises a control unit 31 , an oscillator 32 , a driver 33 , a sound transducer 34 , a voltage limiter 35 , an amplifier 36 a with regulatable amplification factor, an envelope curve shaper 37 , a comparator 1 as well as an amplification factor actuation stage 38 a.","The control unit 31 transmits short signal pulses 41 to the oscillator 32 .","The latter oscillates when a signal pulse is applied, thus emitting an alternating voltage of, for example, 400 kHz, which is then amplified by the driver 33 and subsequently used to excite the sound transducer 34 so that the latter is excited so as to oscillate, i.e.","to emit sound waves.","Corresponding to the short signal pulses, the sound transducer 34 emits short sound wave packets that constitute the transmittal pulse of the distance sensor 30 a.","During the transmitting phase, the sound transducer 34 oscillates at a high oscillation amplitude.","This period of time is followed by a relaxation time in which the oscillation amplitude dies out—usually exponentially.","In the subsequent quiescent phase, the oscillation amplitude generally remains at a constant mean disturbance level.","The signal emitted by the sound transducer and thus also echoes returning after the distance-proportional propagation time are fed via the voltage limiter 35 to the amplifier 36 a with a regulatable amplification factor.","The voltage limiter keeps the high voltage used for exciting the sound transducer away from the amplifier 36 a Using, for example, an internal rectifier circuit, the envelope curve shaper 37 forms the envelope curve of the voltage limiter 35 on the basis of the alternating voltage signal emitted by said voltage limiter 35 and transmits to the comparator 1 an envelope curve voltage signal whose course over time corresponds to that of the envelope curve.","The comparator 1 transmits a switching signal to the control unit 31 for evaluation purposes when the envelope curve voltage signal is greater than a first steadily supplied threshold voltage.","In particular, the comparator then emits a switching signal when an echo signal is received whose magnitude exceeds that of the threshold voltage.","The amplification factor of the amplifier 36 a is regulated by the amplification factor actuation stage 38 a in a time-dependent manner such that the amplification factor is minimal during and immediately after the transmittal pulse and grows as soon as the oscillation amplitude of the sound transducer 34 has died out to such an extent that the magnitude of the received signal is no longer limited by the voltage limiter 35 .","Therefore, the growth of the amplification factor does not begin completely simultaneously with the end of the signal pulse, but rather delayed by a certain lag time dt with respect thereto ( FIG.","2 ).","The amplification factor actuation stage 38 a receives the time information necessary for synchronizing the time behavior of the amplification factor with the rhythm of the transmittal pulse from the control unit 31 .","In this manner a reduction of the short range can be achieved.","The course over time can advantageously be selected in such a way that the distance-dependent attenuation of the echo is countered and the system sensitivity remains constant.","FIG.","2 shows a schematic representation of a course over time of a) a signal pulse, b) an amplification factor of the amplifier 36 a , c) an envelope curve and threshold voltage as well as d) a comparator output signal, that can typically occur during the operation of the distance sensor shown in FIG.","1 .","The time axes of the curves a) to d) are selected identically in order to illustrate the courses over time.","As was already explained with reference to FIG.","1 , during the signal pulse 41 , the amplification factor 42 reaches a minimum 42 a that lasts longer than the signal pulse 41 by a certain lag time dt, and then it grows monotonously over time.","During the signal pulse 41 , the envelope curve voltage 43 reaches a maximum (envelope curve segment 43 a ) at which it still remains during the lag time dt after the end of the signal pulse 41 .","Then the envelope curve voltage 43 drops during the relaxation phase (envelope curve segment 43 b ), whereby the course of the envelope curve 43 b results from the multiplication of, for example, the exponential decay of the sound transducer amplitude by the growing amplification factor.","The temporary lowering of the amplification factor during and after the signal pulse causes the envelope curve voltage 43 to fall below the threshold voltage 44 earlier than would be the case if the amplification factor were not lowered.","The advantageous result of this is a reduction of the short range.","In the quiescent phase, the envelope curve voltage 43 finally reaches a low constant level (envelope curve segment 43 b ).","The threshold voltage 44 is constant over time.","Since the comparator 1 emits a switching signal, as soon as the envelope curve voltage 43 exceeds the threshold voltage 44 , in a disadvantageous manner, at the beginning of the signal pulse 41 , a dummy switching signal 45 is always triggered that lasts until the envelope curve voltage 43 has dropped below the threshold value 44 .","An echo signal (envelope curve segment 43 d ) causes a useful switching signal 46 so that the control unit has to be capable of distinguishing a useful switching signal 46 from a dummy switching signal 45 which, without the presence of an echo signal, was caused exclusively by the signal pulse 41 .","The capability to make such a distinction requires complex hardware or software resources and thus entails disadvantages.","Further drawbacks associated with a distance sensor of the type illustrated in FIG.","1 were already explained above.","As already explained above, another method of reducing the short range lies in providing a threshold voltage that can be changed over time.","For purposes of further illustrating the state of the art, FIG.","3 shows a schematic block diagram of a corresponding variant of an acoustic distance sensor 30 b .","The distance sensor 30 b shown in FIG.","3 comprises a control unit 31 , an oscillator 32 , a driver 33 , a sound transducer 34 , a voltage limiter 35 , a preferably logarithmic amplifier 36 b , an envelope curve shaper 37 , a comparator 1 as well as a threshold value actuation stage 38 b which emits a threshold voltage that can be changed over time and transmits it to the comparator 1 .","The essential difference from the distance sensor 30 a illustrated in FIG.","1 lies in the fact that the short range reduction is not achieved by an amplification factor that can be changed over time, but rather by a threshold voltage that can be changed over time.","FIG.","4 shows a schematic representation of a course over time of a) a signal pulse, b) an envelope curve and threshold value as well as c) a comparator output signal, that can typically occur during the operation of the distance sensor 30 b shown in FIG.","3 .","The time axes of the curves a) to c) are selected identically in order to illustrate the courses over time.","During a signal pulse 41 , the envelope curve voltage 53 reaches a maximum (envelope curve segment 53 a ) at which it still remains during the lag time dt after the end of the signal pulse 41 .","Then the envelope curve voltage 53 drops linearly during the relaxation phase (envelope curve segment 53 b ), since the amplifier 36 b is a logarithmic amplifier.","Finally, in the quiescent phase, the envelope curve voltage 53 reaches a low constant level (envelope curve segment 53 c ).","The threshold voltage 54 is not constant over time but rather is regulated by the threshold value actuation stage 38 b in such a way that it rises before every signal pulse 41 , remains at a maximum value for a certain period of time (envelope curve segment 54 a ), then drops (curve segment 54 b ) and finally reaches a constant level (envelope curve segment 54 c ) during the quiescent phase, whereby the threshold voltage 54 remains greater than the envelope curve voltage 53 , as long as no echo is received.","The threshold value actuation stage 38 b receives the necessary time information for synchronizing the time behavior of the threshold voltage 54 with the rhythm of the signal pulse 41 from the control unit 31 .","The advantageous result is a reduction of the short range.","Moreover, advantageously, no dummy switching signal is triggered by the signal pulse 41 .","Rather, a switching signal 56 is triggered only by the rise in the envelope curve 53 (envelope curve segment 53 d ) associated with an echo, so that the switching signal 56 is a useful switching signal 56 .","Drawbacks associated with a distance sensor of the type illustrated in FIG.","3 were already explained above.","Now reference is made to FIG.","5 , which illustrates a schematic circuit diagram of an embodiment 10 a of an adaptive comparator circuit according to the invention It comprises a signal transmitter 2 , a first regulatable switch 3 , a capacitor 6 , a diode 5 and a first comparator 1 , that has a first threshold value input 1 a and a first signal input 1 b.","At the first threshold value input 1 a , a threshold voltage U ref1 , is present that is emitted internally in the adaptive comparator circuit according to the invention.","At the signal input 1 b , a voltage signal U signal is present that can be, for example, an envelope curve of the received signal of a sound transducer and that is fed via a first electric terminal 8 to the adaptive comparator circuit according to the invention.","The first comparator 1 emits a first switching signal S 1 when the voltage signal U signal is greater than the first threshold voltage U ref1 .","The first switching signal S 1 can be tapped, for example; via a second electric terminal 9 and transmitted for evaluation purposes.","Via a control output 2 a , the signal transmitter 2 emits an electric control signal S St at times, for example, at regular intervals.","In the simplest case, the signal transmitter 2 can be, for instance, a manually operated signaling key.","Moreover, the signal transmitter can be, for example, an electronic square-wave timing generator circuit with a suitable duty cycle.","Preferably, the signal transmitter 2 is a microcontroller or microprocessor that is programmed in such a way that, at regular intervals, it emits an electric control signal S St having a time duration of typically, for example, 20 microseconds.","The first switch 3 has a control means 3 a , a switch input 3 b and a switch output 3 c .","The control means 3 a is connected to the control output 2 a and can be regulated by the control signal S St in such a way that it is in the closed state when the signal transmitter 2 emits a control signal S St , and otherwise it is in the open state.","The first switch 3 can be, for example, a transistor.","The switch input 3 b is connected to the positive first pole 4 a of a first direct voltage source 4 a , 4 b (not shown here).","The switch output 3 c is connected to the first threshold value input 1 a of the comparator 1 , so that the first threshold voltage U ref1 is equal to the voltage present at the switch output 3 c .","Thus, when the switch 3 is closed, the potential of the positive first pole 4 a is present at the first threshold value input 1 a of the comparator 1 .","The switch output 3 c is also applied via a capacitor 6 to the negative second pole 4 b of the first direct voltage source so that the capacitor 6 is charged with the voltage supplied by the first voltage source when the switch 3 is closed.","The switch output 3 c is also connected to the anode 5 a of a diode 5 .","The cathode 5 b of the diode 5 is connected to the first signal input 1 b of the comparator.","As soon as the signal transmitter 2 emits a control signal S St , the first switch 3 is in the closed state.","Therefore, the capacitor 6 is charged with the voltage supplied by the first voltage source.","When the switch 3 is closed, this voltage is also present at the threshold value input 1 a.","After the end of the control signal S St , the switch 3 is in the open state.","The capacitor 6 can now discharge according to the invention via the diode 5 to such an extent until the first U ref1 reaches a value that corresponds to the sum of the voltage signal U signal and the diode flow voltage dU.","This means that the voltage that is established at the capacitor 6 depends on the magnitude of the voltage signal U signal : the smaller the voltage signal U signal , the more the voltage and thus the threshold voltage U ref1 drop according to the invention at the capacitor.","Hence, a threshold voltage U ref1 is established that is greater than the voltage signal U signal by the fundamental voltage distance dU.","According to the invention, the task of the diode 5 is to allow the discharging of the capacitor 6 and thus a drop in the threshold voltage U ref1 for as long as and only until the difference of U ref1 −U signal has dropped to the fundamental voltage distance dU, but to prevent a recharging of the capacitor and thus an increase in the threshold voltage if the voltage signal U signal increases.","If the voltage signal U signal drops, then the threshold voltage U ref1 follows the voltage signal U signal at the distance dU.","In contrast, if the voltage signal U signal increases, then the threshold voltage U ref1 remains constant because of the blocking effect of the diode 5 .","Of course, the polarity of the first direct voltage source can also be the opposite from the one in FIG.","1 , so that the first pole 4 a is negative and the second pole 4 b is positive.","In this case, the diode 5 in the circuit shown in FIG.","1 has to be arranged in the reverse direction, so that its cathode is connected to the switch output 3 c and its anode to the second pole 4 b.","In another embodiment of the invention, the fundamental voltage distance dU is augmented by a codirectional series connection of a plurality of diodes 5 or second direct voltage sources 7 .","According to the invention, as an alternative, the function of the diode 5 can be assumed by a second direct voltage source whose positive pole is in the place of the anode 5 a of the diode 5 and whose negative pole is in the place of the cathode 5 b of the diode 5 .","FIG.","6 shows an embodiment 10 b of an adaptive comparator circuit according to the invention, which can be used as an alternative to that shown in FIG.","1 .","The diode shown in FIG.","5 has been replaced by a second voltage source 7 , whereby its positive pole 7 a has been put in the place of the anode 5 a and the negative pole 7 b has been put in the place of the cathode 5 b .","The fundamental voltage distance dU shown in FIG.","1 has now been replaced by the voltage of the voltage source 7 , that is to say, the fundamental voltage distance dU between U ref1 and U signal is now defined by the voltage supplied by the second direct voltage source.","If the voltage supplied by the second voltage source 7 can be regulated or adjusted, then the fundamental voltage distance dU can be varied as needed.","FIG.","7 shows a schematic block diagram of an embodiment of an acoustic distance sensor according to the invention in which the adaptive comparator circuit 10 a shown in FIG.","5 is used.","A control unit 31 transmits short signal pulses at times, preferably at regular intervals, each lasting, for example, 25 microseconds, to an oscillator 32 .","When a signal pulse is present, the oscillator 32 transmits an alternating voltage to a sound transducer 34 that is excited by the latter to emit sound waves.","The sound transducer 34 is capable of receiving sound waves reflected, for example, from an object and coming back to the sound transducer 34 as an echo, and of converting them into an electric received signal, whereby the propagation time of the sound waves is a measure of the distance from the object.","The received signal is fed to an envelope curve shaper 37 which forms the envelope curve of the received signal and transmits it via an electric terminal 8 to the adaptive comparator circuit 10 a shown in FIG.","5 .","In a preferred embodiment of the invention, the signal pulses 41 that the control unit 31 transmits to the oscillator 32 are synchronized with the control signals S St that the control unit 2 transmits to the switch 3 and this is done in such a way that the control signals S St each begin shortly before the signal pulses 41 as will still be explained below with reference to FIGS.","9 a and 9 b .","In this manner, it is ensured that, already at the beginning of the transmittal phase, the threshold voltage U ref1 is greater than the voltage signal U signal , so that no dummy switching signal is triggered.","In another preferred embodiment of the invention, the signal pulses 41 are advantageously synchronized with the control signals S St in such a way that the control signals S St each begin before or simultaneously with the end of the signal pulses 41 .","This ensures that, without any time lag, the threshold voltage U ref1 is supplied to the dropping voltage signal U signal at the fundamental voltage distance dU already at the beginning of the relaxation phase.","The sensitivity of the distance sensor is defined by the fundamental voltage distance dU.","FIG.","8 shows a schematic block diagram of another embodiment of an acoustic distance sensor according to the invention, in which the adaptive comparator circuit 10 a shown in FIG.","5 is likewise used.","The distance sensor shown in FIG.","8 —in comparison to that shown in FIG.","7 —additionally has a driver 33 that amplifies the alternating voltage supplied by the oscillator 32 and transmits it to the sound transducer 34 .","Moreover, before the received signal is fed into the envelope curve shaper 37 , it is passed through a voltage limiter 35 and through a logarithmic amplifier 36 c. Among other things, the voltage limiter 35 counters an overmodulation of the amplifier.","The amplifier 36 c serves to adapt the low input level of just a few microvolts to the higher input level of the subsequent evaluation circuit.","In FIG.","8 , in order to facilitate the synchronization, the signal transmitter 2 is accommodated in the control unit 31 .","For example, the signal transmitter 2 and the control unit 31 can be combined in one single unit, for example, in a microprocessor, that concurrently fulfills the function of the signal transmitter 2 as well as that of the control unit 31 .","In FIG.","8 , the first switching signal S 1 of the control unit 31 is also supplied for evaluation purposes, especially for determining the propagation time and calculating the distance.","FIGS.","9 and 10 serve to further explain the advantageous effects of the invention, whereby the time axes of all of the curves have been selected identically in order to illustrate the course over time.","FIG.","9 shows a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) a voltage signal and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.","8 , whereby an echo occurs during the quiescent phase.","The control unit 31 transmits short signal pulses 41 to the oscillator 32 , as a result of which the sound transducer 34 is excited so as to emit a threshold voltage packet ( FIG.","9 , Curve a).","Moreover, the signal transmitter 2 transmits a control signal S St to the switch 3 , which is synchronized with the signal pulse 41 in such a way that the control signal S St begins shortly before the signal pulse 41 and ends at the same time as or during the presence of the former ( FIG.","9 , Curve b).","FIG.","9 , Curve c, shows the course of the voltage signal U signal (solid curve) that is identical to the envelope curve, and the course of the threshold voltage U ref1 (dotted curve).","The course of the U signal during the transmittal and relaxation phase and at the beginning of the quiescent phase corresponds essentially to that of the envelope curve 53 , which was explained with reference to FIG.","4 .","While the control signal S St is present at the switch 3 , the latter is opened, so that a threshold voltage U ref1 is established which is identical to the voltage supplied by the first direct voltage source 4 a , 4 b ( FIG.","9 , Curve c).","Therefore, this has been advantageously selected in such a way that it is greater than the maximum of the voltage signal, so that the adaptation of the threshold voltage to the voltage signal begins immediately after the opening of the switch 3 .","In the present example, the maximum possible magnitude of the voltage signal is supplied by the voltage limiter 35 or by the overmodulation limit of the amplifier 36 c ( FIG.","8 ).","According to the invention, after the end of the control signal S St , a threshold voltage U ref1 is established that is greater than the voltage signal U signal by the voltage distance dU.","Therefore, the threshold voltage U ref1 drops immediately after the end of the control signal by a certain quantity.","From this point in time on, the threshold voltage proceeds parallel-offset by a fundamental voltage distance dU with respect to that of the voltage signal U signal as long as the latter does not increase.","A dummy switching signal is not triggered.","If, however, the voltage signal U signal increases, the threshold voltage remains constant so that a first echo peak 60 of the voltage signal U signal leads to a triggering of the comparator 1 as soon as the voltage signal U signal exceeds the threshold voltage U ref1 .","In this case, the comparator 1 emits a first switching signal S 1 ( FIG.","9 , Curve d).","FIG.","10 shows a schematic representation of a typical course over time of a) a voltage signal and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.","8 , whereby an echo occurs during the relaxation phase and another echo occurs during the quiescent phase.","The courses over time of the signal pulse 41 and of the control signal S St are identical to those shown in FIG.","9 .","During the relaxation time, an echo signal occurs, which manifests itself in a second echo peak 61 of the voltage signal U signal ( FIG.","10 , Curve a).","Up until the beginning of the second echo peak 61 , the curves correspond to those shown in FIG.","9 , Curve c. According to the invention, however, the decrease of the threshold voltage U ref1 comes to a halt as soon as the voltage signal U signal is no longer dropping, and a first switching signal S 1a is triggered by the second echo peak 61 .","After the second echo peak 61 that occurs during the relaxation time, the fundamental voltage distance dU is once again established between the threshold voltage U ref1 and the voltage signal U signal .","During the quiescent phase, another, third echo peak 62 of the voltage signal U signal occurs, as a result of which another switching signal S 1b is triggered.","Now reference will be made to FIGS.","11 to 13 , which show additional advantageous embodiments of an adaptive comparator circuit according to the invention.","FIG.","11 shows a preferred embodiment 10 c of the invention which differs from the adaptive comparator circuit 10 a shown in FIG.","6 as follows: a) The first switch is a transistor 103 .","The base 103 a of the transistor 103 serves as the control-current terminal, the emitter 103 b serves as the switch input and the collector 103 c as the switch output.","b) A first resistor 11 is connected between the first pole 4 a and the first threshold value input 1 a , and a second resistor 12 is connected between the first threshold value input 1 a and the second pole 4 b , so that the first resistor 11 and the second resistor 12 form a first voltage divider 11 , 12 .","Therefore, the magnitude of the first threshold voltage U ref1 counters the voltage that is supplied by the first voltage divider.","Thus, through the use of the first voltage divider 11 , 12 , the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal after the voltage signal U signal has died out can advantageously be changed by the fundamental voltage distance dU, to a value dU a .","When this embodiment 10 c of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be changed and, in particular increased.","This is advantageous for many applications such as, for example, for the detection of small objects.","The first voltage divider can be an adjustable potentiometer whose pick-up is connected to the switch output.","c) A sixth resistor 16 is connected between the control output 2 a of the signal transmitter 2 and the base 103 a , and a seventh resistor 17 is connected between the base 103 a and the first pole 4 a , which is a plus pole in the example shown.","The purpose of these resistors was already explained above.","d) The diode 5 is connected in series to an eighth resistor 18 which serves to slow down the discharging of the capacitor 6 and thus to reduce the speed with which the threshold voltage U ref1 is adapted to the voltage signal U signal .","The eighth resistor also serves to smooth the course of the threshold voltage U ref1 with respect to very rapid fluctuations of the voltage signal U signal .","FIG.","12 shows another preferred embodiment 10 d of the invention, which differs from the adaptive comparator circuit 10 c shown in FIG.","11 in that, before the voltage signal U signal reaches the first signal input 1 b , it passes through an impedance transformer.","This embodiment is especially advantageous when the voltage signal arriving at the electric terminal 8 is high-ohmic, whereby said voltage signal can be the envelope curve voltage generated, for example, by an envelope curve shaper.","FIG.","13 shows another preferred embodiment 10 e of the invention which differs from the adaptive comparator circuit 10 a shown in FIG.","6 as follows: a) The first switch is a transistor 103 .","The base 103 a of the transistor 103 serves as the control-current terminal, the emitter 103 b serves as the switch input and the collector 103 c as the switch output.","b) A second comparator 20 is used that has a second threshold value input 20 a and a second signal input 20 b , and that emits a second switching signal S 2 when a greater voltage is present at the second signal input 20 b than at the second threshold value input 20 a. c) The first pole 4 a of the first voltage source 4 a , 4 b is connected to the second threshold value input 20 a via a third resistor 13 .","The second threshold value input 20 a is connected to the second pole 4 b via a fourth resistor 14 , so that the third and the fourth resistors 13 , 14 form a second voltage divider which can be configured as a potentiometer.","d) The collector 103 c that serves as the switch output and the second threshold value input 20 a are connected via a fifth resistor 15 .","e) A second switch 23 is connected in series to the diode 5 and the former can be regulated or activated by the second switching signal S 2 in such a way that it is in the closed state when the second comparator 20 emits the second switching signal S 2 , and otherwise it is in the open state.","The second switch 23 can be a transistor.","f) A sixth resistor 16 is connected between the control output 2 a of the signal transmitter 2 and the base 103 a , and a seventh resistor 17 is connected between the base 103 a and the first pole 4 a , which is a plus pole in the example shown.","The purpose of these resistors was already explained above.","g) The diode 5 is connected in series to an eighth resistor 18 .","The purpose of this resistor was already explained with reference to FIG.","11 .","h) Before reaching the first signal input 1 b , the voltage signal passes through an impedance transformer 21 whose purpose was already explained with reference to FIG.","12 .","Due to the second voltage divider 13 , 14 , a voltage U ref2 is present at the second threshold value input 20 a .","As long as the second switch 23 is closed, the first threshold voltage U ref1 follows the dropping voltage signal U signal , parallel-offset by the fundamental voltage distance dU, as explained above.","According to the invention, however, the second switch opens as soon as the voltage signal U signal exceeds the second threshold voltage U ref2 .","As a result, the discharge of the capacitor 6 and thus the dropping of the first threshold voltage U ref1 are ended.","Hence, in this embodiment of the invention, the capacitor 6 can only discharge until the first threshold voltage U ref1 has dropped to the magnitude of the second threshold voltage U ref2 supplied by the second potentiometer 13 , 14 .","Thus, through the use of the second voltage divider 13 , 14 , the voltage distance by which the first threshold voltage is advantageously changed parallel-offset after the decay of the voltage signal U signal with respect to voltage signal U signal , from the fundamental voltage distance dU to a value dU b .","The second voltage divider 13 , 14 can be an adjustable potentiometer whose pick-up is connected to the second threshold value input 20 a.","The fifth resistor 15 is connected between the switch output 103 c and the second threshold value input 20 a .","Therefore, the magnitude of the first threshold voltage U ref1 counters the voltage that is supplied by the second voltage divider 13 , 14 .","In particular, through the use of the fifth resistor 15 , it can additionally be achieved that the voltage distance by which the first threshold voltage is parallel-offset with respect to the voltage signal U signal strives towards a value dU b that—depending on the voltage supplied by the second voltage divider 13 , 14 —can advantageously be not only larger but also smaller than the fundamental voltage distance dU.","When this embodiment 10 e of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be either increased or decreased by means of the second voltage divider 13 , 14 .","All of the embodiments of an adaptive comparator circuit according to the invention and thus also the embodiments 10 b , 10 c , 10 d and 10 e illustrated in FIG.","6 and FIGS.","11 to 13 can be used according to the invention in the acoustic distance sensor shown in FIG.","8 instead of the adaptive comparator circuit 10 a shown there by way of an example."],["TECHNICAL AREA The invention relates to an adaptive comparator circuit that can be used particularly for reducing the short range of an acoustic distance sensor, as well as to an acoustic distance sensor with such a circuit.","STATE OF THE ART Acoustic distance sensors are used to measure distances by means of the pulse-propagation time method.","In this process, a sound transducer emits a short sound pulse that propagates in a medium (gas, liquid or solid).","This pulse is reflected, for example, on an object and comes back as an echo.","The distance to the object can be determined on the basis of the propagation time of the sound pulse and the sound velocity in the medium.","As a rule, the frequency of the sound used for such measurements lies in the ultra-sound range, typically in the range between 20 kHz and 500 kHz, for example, at 400 kHz.","The sound transducer, which in particular can be a piezoelectric ceramic element, is normally used to transmit the sound pulse as well as to receive the echo.","Typically, sound pulses of about 3 to 50 oscillations periods each are emitted.","This results in time durations of typically about 6 to 2050 μs for the emitted sound pulses.","During this time, i.e.","the transmitting phase, the sound transducer oscillates at a high oscillation amplitude.","This time duration is followed by a relaxation time in which the oscillation amplitude dies out—usually exponentially.","The relaxation time is typically, for example, 300 μs to 3 ms.","The time interval between the end of the relaxation time and the emission of the next sound pulse will be called “quiescent phase” below.","Due to noise and other interferences, however, the oscillation amplitude does not drop completely to zero even during the quiescent phase, but rather remains at a certain constant mean disturbance level.","Therefore, during the receiving phase that follows the transmitting phase, even in the absence of a useful echo, the oscillation amplitude that dies out during the relaxation time and the mean disturbance level consistently form a finite background signal that is very large immediately after the end of the transmitting phase, that generally decreases exponentially within the relaxation time and that finally remains at the disturbance level.","A useful echo signal is superimposed on this background and, as a rule, is detected by means of a comparator that emits a switching signal when the received signal is greater than a certain reference value that will be called “threshold voltage” hereinafter.","On the one hand, the threshold voltage should be as low as possible so that even weak useful echo signals, e.g.","of small objects, can be detected, which translates into high sensitivity.","On the other hand, the threshold voltage has to lie above the disturbance level by a certain safety margin so as to avoid erroneous triggering of the comparator during the quiescent phase.","When the threshold voltage is constant over time, useful echo signals can be detected as soon as the background signal has dropped below the threshold voltage as a result of the relaxation of the sound transducer.","Before this time, however, that is to say, during the transmitting phase and during part of the relaxation time, the background signal is greater than the threshold voltage so that the comparator—irrespective of the presence of an echo—is triggered in any case during this time.","Thus, during this time, called dead time, the system is fundamentally insensitive to the reception of echoes.","Therefore, the measurement of distances below a certain minimum distance is impossible.","This minimum distance is called the “short range” and is typically a few centimeters.","The existence of the short range often has a detrimental effect, for example, wherever due to space constraints, the sensor is to be situated as close as possible to the object to be detected.","Various methods are known for reducing the short range.","The simplest method is to increase the threshold voltage.","This causes the background signal, which dies out after the transmitting phase, to drop below the threshold voltage at an earlier point in time within the relaxation time.","However, this method entails the serious drawback that an increase in the threshold voltage translates into a reduction of the system sensitivity, as a result of which weak echoes can no longer be detected.","Another method lies in conveying the received signal through an amplifier whose amplification factor can be changed over time.","The amplification factor is minimal immediately after the transmittal pulse and is then boosted, for example, by means of a suitable electronic circuit as a function of the time.","In this manner, despite a constant threshold voltage over time, a reduction of the short range can be combined with a high sensitivity during the quiescent phase.","Of course, for this purpose, the course over time of the amplification factor has to be adapted to the relaxation time.","The drawback is that a one-time optimal adaptation is not sufficient here, since the relaxation time of the sound transducer is not constant but rather, it depends on the medium on the temperature, on the age of the sound transducer and on other influencing factors.","Therefore, in actual practice, either complex compensation measures have to be undertaken or re-adaptations have to be made repeatedly, or else a large safety margin has to be maintained right from the start for purposes of optimal adaptation, which diminishes the system sensitivity, at least during the relaxation time.","Another method is to provide a threshold voltage that can be changed over time.","In the ideal case, this threshold voltage drops at a gradient that is similar to the decaying background signal and, at the beginning of the quiescent phase, turns into a constant value, whereby the threshold voltage nevertheless always remains sufficiently greater than the background signal.","This method can be additionally improved by subjecting the background signal to a logarithmic amplification so that the decrease is linear instead of exponential.","The drawback here, however, is that a one-time adaptation of the time behavior of the threshold voltage is not sufficient to ensure optimal operation over the long run, since here, too, the individual dying-out behavior of the sound transducer is not taken into account on a case-to-case basis.","Another method for reducing the short range lies in the propagation time-dependent control of the length of the emitted sound pulses.","In the case of a short propagation time, that is to say, in the case of an object that is close, very short sound pulses are used to that the oscillation amplitude of the sound transducer does not fully develop and thus the short range is reduced.","Another method lies in the use of threshold voltage that is dynamically changed by means of a microprocessor and a D/A transducer.","The microprocessor regulates the D/A transducer in such a way that a dynamic threshold is created that always lies above the dying-out signal.","Here, an A/D transducer measures the decaying transmission signal.","These methods, however, call for considerable hardware and software resources.","Therefore, the invention is based on the objective of simply and inexpensively providing a comparator circuit with which, in every single case, the course over time of the threshold voltage is automatically adapted to the individual decay behavior of the sound transducer and the magnitude of the disturbance level.","This objective is achieved according to the invention by an adaptive comparator circuit, especially for an acoustic distance sensor, comprising a first comparator having a first threshold value input to which a first threshold voltage is applied and having a first signal input to which a voltage signal (Usignal) is applied and, when a voltage signal is present that is greater than the first threshold voltage, said comparator emits a first switching signal, characterized by a signal transmitter, especially a microcontroller or microprocessor, that at times emits a control signal, a first switch that has a control means, a switch input and a switch output, and that can be regulated or activated through the effect of the control signal on the control means in such a way that it is in the closed state when the signal transmitter emits a control signal, and otherwise it is in the open state, or vice versa, whereby the switch input is connected to the positive or negative first pole of a first direct voltage source and the switch output is connected to the first threshold value input, so that the first threshold voltage is equal to the voltage present at the switch output, a capacitor via which the switch output is connected to the negative or positive pole of the first direct voltage source, and a diode via which the switch output is connected to the first signal input in such a way that the anode or the cathode of the diode is connected to the switch output.","This is the first basic form of an adaptive comparator circuit according to the invention.","According to the invention, instead of the diode, a second direct voltage source can be used whose positive pole is in the place of the anode of the diode and whose negative pole is in the place of the cathode of the diode.","This is the second basic form of an adaptive comparator circuit according to the invention.","These two basic forms can be augmented advantageously as will still be explained below.","An acoustic distance sensor according to the invention comprises a control unit that at times transmits signal pulses to an oscillator which, during the presence of a signal pulse, transmits an alternating voltage to a sound transducer that is excited by the alternating voltage to emit sound waves and that is capable of receiving sound waves coming back to the sound transducer in the form of an echo as a result of reflection, and of converting them into an electric received signal, and it also comprises an envelope curve shaper to which the received signal is fed and which forms the envelope curve of the received signal and emits it as a signal voltage, characterized in that the signal voltage is fed to an adaptive comparator circuit comprising a first comparator having a first threshold value input to which a first threshold voltage is applied and a first signal input to which a voltage signal is applied and, when a voltage signal is present that is greater than the first threshold voltage, said comparator emits a first switching signal, a signal transmitter, especially a microcontroller or microprocessor, that at times emits a control signal, a first switch that has a control means, a switch input and a switch output, and that can be regulated or activated through the effect of the control signal on the control means in such a way that it is in the closed state when the signal transmitter emits a control signal, and otherwise it is in the open state, or vice versa, whereby the switch input is connected to the positive or negative first pole of a first direct voltage source and the switch output is connected to the first threshold value input, so that the first threshold voltage is equal to the voltage present at the switch output, a capacitor via which the switch output is connected to the negative or positive pole of the first direct voltage source, and a diode via which the switch output is connected to the first signal input in such a way that the anode or the cathode of the diode is connected to the switch output.","An acoustic distance sensor according to the invention thus comprises the adaptive comparator circuit according to the invention.","Preferably, the control signal emitted by the signal transmitter is an electric control signal and the first switch is an electrically or electronically controlled switch, for example, a transistor, whose base terminal or gate terminal function as a control-current terminal or, for example, a relay.","In another embodiment of the invention, the control signal emitted by the signal transmitter is an optical control signal that is emitted, for example, by a light diode, and the first switch is an optically controlled switch, e.g.","a photoresistor.","As soon as the signal transmitter emits a control signal, the first switch is in the closed state, so that the capacitor is charged with the voltage supplied by the first voltage source.","After the end of the control signal, the switch is in the open state.","The capacitor voltage and thus the first threshold voltage present at the first threshold value input now assumes a value that corresponds to the sum of the voltage signal and the diode flow voltage or else to the sum of the voltage signal and the voltage of the second direct voltage source.","The diode flow voltage or the voltage of the second direct voltage source are referred to below as “fundamental voltage distance” dU.","The task of the diode or of the second direct voltage source is to allow the discharging of the capacitor and thus a drop in the threshold voltage for as long as and only until the difference of the threshold voltage minus the voltage signal has dropped to the fundamental voltage distance dU, but to prevent a recharging of the capacitor and thus an increase in the threshold voltage if the voltage signal increases.","Therefore, the time behavior of the threshold voltage proceeds parallel-offset by a fundamental voltage distance dU with respect to that of the voltage signal as long as the voltage signal does not increase.","In contrast, if the voltage signal increases, the threshold voltage remains constant.","Consequently, a maximum, e.g.","a peak, of the voltage signal leads to the triggering of the comparator as soon as the voltage signal exceeds the threshold voltage.","If the voltage signal drops again after having increased, then the threshold voltage follows the voltage signal, once again parallel-offset, as soon as the fundamental, voltage distance dU has been reached again.","Hence, according to the invention, the threshold voltage is specified by the magnitude of the voltage signal at every point in time of the receiving phase.","If the course over time of the voltage signal is changed, then there is automatically a correspondingly changed course over time of the threshold voltage.","Thus, the course over time of the threshold voltage is automatically adapted to that of the voltage signal.","The diode flow voltage is temperature-dependent.","It decreases as the temperature rises.","Consequently, an acoustic distance sensor according to the invention becomes more sensitive as the temperature rises.","On the other hand, the attenuation of ultrasound waves generally increases as the temperature rises.","Therefore, the resultant sensitivity loss is advantageously compensated for partially with the use of a diode to create the fundamental voltage distance dU.","As already explained above, according to the invention, instead of the diode, a second direct voltage source can be used whose positive pole is in the place of the anode of the diode and whose negative pole is in the place of the cathode of the diode.","In this case, the fundamental voltage distance dU is defined by the voltage supplied by the second direct voltage source.","The basic forms described above of adaptive comparator circuits according to the invention can be advantageously augmented as will be explained below.","In a preferred embodiment of the invention, one of the above-described basic forms has been augmented by a first resistor that is connected between the first pole and the first threshold value input, and by a second resistor that is connected between the first threshold value input and the second pole, so that the first resistor and the second resistor form a first voltage divider.","Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the first voltage divider.","Thus, through the use of the first voltage divider, the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal after the voltage signal Usignal has died out can advantageously be changed by the fundamental voltage distance dU, to a value dUa.","When this embodiment of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be changed and, in particular increased.","The first voltage divider can be an adjustable potentiometer whose pick-up is connected to the switch output.","Another preferred embodiment of an adaptive comparator circuit according to the invention comprises the following in addition to the basic forms described above: a second comparator that has a second threshold value input and a second signal input, and that emits a second switching signal when a greater voltage is present at the second signal input than at the second threshold value input, a third resistor via which the first pole of the first direct voltage source is connected to the second threshold value input, a fourth resistor that, on the one hand, is connected to the second threshold value input and, on the other hand, to the second pole of the first voltage source, so that the third resistor and the fourth resistor form a second voltage divider, and a second switch that is connected in series to the diode and that can be regulated or activated by the second switching signal in such a way that it is in the closed state when the second comparator emits the second switching signal, and otherwise it is in the open state.","Due to the second voltage divider, a voltage is present at the second threshold value input that will be called “second threshold voltage” Uref2 below.","As long as the second switch is closed, the first threshold voltage follows the dropping voltage signal Usignal with the fundamental voltage distance dU, as explained above.","According to the invention, however, the second switch opens as soon as the voltage signal Usignal exceeds the second threshold voltage Uref2.As a result, the discharge of the capacitor and thus the dropping of the first threshold voltage are ended.","Hence, in this embodiment of the invention, the capacitor can only discharge until the first threshold voltage Uref1 has dropped to the value of the second threshold voltage Uref2.Thus, through the use of the second voltage divider, the voltage distance by which the first threshold voltage is changed parallel-offset with respect to voltage signal Usignal after the decay of the voltage signal Usignal, advantageously by the fundamental voltage distance dU to a value dUb.","When this embodiment of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor according to the invention, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be reduced.","The second voltage divider can be an adjustable potentiometer whose pick-up is connected to the second threshold value input.","In another configuration of this embodiment of the invention, a fifth resistor is connected between the switch output and the second threshold value input.","Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the second voltage divider.","In particular, through the use of the fifth resistor, it can be additionally achieved that the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal strives towards a value dUb that—depending on the voltage supplied by the second voltage divider—can advantageously be not only larger but also smaller than the fundamental voltage distance dU.","When this embodiment of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be either increased or decreased.","In another advantageous embodiment of the invention, the voltage signal passes through a voltage follower or impedance transformer before reaching the first signal input.","In another embodiment of the invention, a sixth resistor is connected between the control output and the control-current terminal for purposes of limiting the current flow.","In another embodiment of the invention, the first switch is a transistor whose base functions as a control-current terminal, whereby a seventh resistor is connected between the base and the first pole of the first direct voltage source.","In another embodiment of the invention, the diode or the second direct voltage source is connected in series with an eighth resistor.","This brings about a slowing down of the discharging of the capacitor as well as a smoothing of the threshold voltage, which is advantageous for many applications.","The voltage supplied by the first voltage source is preferably selected such that it is greater in magnitude than the value of the maximum of the voltage signal.","In this manner, it is prevented that the first comparator is triggered while the first switch is still closed, that is to say, while the threshold voltage cannot yet adapt to the voltage signal.","Moreover, in this manner, it is advantageously achieved that, in any case, the adaptation of the threshold voltage to the voltage signal can begin immediately after the opening of the first switch.","An adaptive comparator circuit according to the invention can be used especially advantageously in an acoustic distance sensor.","In this case, the voltage signal is advantageously formed by the envelope curve of the received signal supplied by the sound transducer.","This received signal consists of a superimposition of the background signal with the useful echo signal.","The formation of the envelope curve, for example, by means of a rectifier circuit, is known from the state of the art and will thus not be elaborated upon in greater depth here.","In the absence of an echo, the envelope curve dies out exponentially during the relaxation phase and, at the beginning of the quiescent phase, turns into a horizontal straight line.","Such an envelope curve will be called “background envelope curve” below.","The threshold voltage adapts according to the invention to the individual course of the background envelope curve in that it follows said background envelope curve parallel-offset by a voltage distance dU or dUa or dUb.","An echo causes a peak to be superimposed on the background envelope curve so that the voltage signal increases.","According to the invention, the threshold voltage does not follow this increase, so that the comparator is triggered and the echo can thus be detected.","As already mentioned above, the voltage supplied by the first voltage source is preferably selected such that it is greater in magnitude than the value of the maximum of the voltage signal.","Since in this case, the threshold voltage already follows the background envelope curve at the voltage distance dU at the beginning of the relaxation phase, the voltage signal in the absence of an echo is not greater than the threshold voltage at any point in time, especially not during the entire relaxation phase.","Therefore, throughout the entire receiving phase, the comparator can only be triggered by an echo, but not by the decay behavior of the sound transducer.","Thus, an acoustic distance sensor according to the invention is fundamentally able to detect an echo during the quiescent phase as well as especially during the entire relaxation phase.","As a result, the undesired short range of the sensor is considerably reduced according to the invention.","BRIEF DESCRIPTION OF THE DRAWING IN WHICH THE FOLLOWING IS SHOWN FIG.","1—a schematic block diagram of a variant of an acoustic distance sensor according to the state of the art, FIG.","2—a schematic representation of a typical course over time of a) a signal pulse, b) an amplification factor, c) an envelope curve and d) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.","1, FIG.","3—a schematic block diagram of another variant of an acoustic distance sensor according to the state of the art, FIG.","4—a schematic representation of a typical course over time of a) a timing pulse, b) an envelope curve and c) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.","3, FIG.","5—a circuit diagram of an embodiment of a comparator circuit according to the invention, FIG.","6—a circuit diagram of an alternative embodiment of a comparator circuit according to the invention, FIGS.","7 and 8—a schematic block diagram of embodiments of an acoustic distance sensor according to the invention, FIG.","9—a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) an envelope curve and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.","8, whereby an echo occurs during the quiescent phase, FIG.","10—a schematic representation of a typical course over time of a) an envelope curve and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.","8, whereby an echo additionally occurs during the relaxation phase, and FIGS.","11 to 13—circuit diagrams of further embodiments of comparator circuits according to the invention.","FIGS.","1 to 4 serve to further illustrate the state of the art.","FIG.","1 shows a schematic block diagram of a variant of an acoustic distance sensor 30a with which a measure according to the state of the art has been taken in order to reduce the short range.","The distance sensor 30a shown in FIG.","1 comprises a control unit 31, an oscillator 32, a driver 33, a sound transducer 34, a voltage limiter 35, an amplifier 36a with regulatable amplification factor, an envelope curve shaper 37, a comparator 1 as well as an amplification factor actuation stage 38a.","The control unit 31 transmits short signal pulses 41 to the oscillator 32.The latter oscillates when a signal pulse is applied, thus emitting an alternating voltage of, for example, 400 kHz, which is then amplified by the driver 33 and subsequently used to excite the sound transducer 34 so that the latter is excited so as to oscillate, i.e.","to emit sound waves.","Corresponding to the short signal pulses, the sound transducer 34 emits short sound wave packets that constitute the transmittal pulse of the distance sensor 30a.","During the transmitting phase, the sound transducer 34 oscillates at a high oscillation amplitude.","This period of time is followed by a relaxation time in which the oscillation amplitude dies out—usually exponentially.","In the subsequent quiescent phase, the oscillation amplitude generally remains at a constant mean disturbance level.","The signal emitted by the sound transducer and thus also echoes returning after the distance-proportional propagation time are fed via the voltage limiter 35 to the amplifier 36a with a regulatable amplification factor.","The voltage limiter keeps the high voltage used for exciting the sound transducer away from the amplifier 36a Using, for example, an internal rectifier circuit, the envelope curve shaper 37 forms the envelope curve of the voltage limiter 35 on the basis of the alternating voltage signal emitted by said voltage limiter 35 and transmits to the comparator 1 an envelope curve voltage signal whose course over time corresponds to that of the envelope curve.","The comparator 1 transmits a switching signal to the control unit 31 for evaluation purposes when the envelope curve voltage signal is greater than a first steadily supplied threshold voltage.","In particular, the comparator then emits a switching signal when an echo signal is received whose magnitude exceeds that of the threshold voltage.","The amplification factor of the amplifier 36a is regulated by the amplification factor actuation stage 38a in a time-dependent manner such that the amplification factor is minimal during and immediately after the transmittal pulse and grows as soon as the oscillation amplitude of the sound transducer 34 has died out to such an extent that the magnitude of the received signal is no longer limited by the voltage limiter 35.Therefore, the growth of the amplification factor does not begin completely simultaneously with the end of the signal pulse, but rather delayed by a certain lag time dt with respect thereto (FIG.","2).","The amplification factor actuation stage 38a receives the time information necessary for synchronizing the time behavior of the amplification factor with the rhythm of the transmittal pulse from the control unit 31.In this manner a reduction of the short range can be achieved.","The course over time can advantageously be selected in such a way that the distance-dependent attenuation of the echo is countered and the system sensitivity remains constant.","FIG.","2 shows a schematic representation of a course over time of a) a signal pulse, b) an amplification factor of the amplifier 36a, c) an envelope curve and threshold voltage as well as d) a comparator output signal, that can typically occur during the operation of the distance sensor shown in FIG.","1.The time axes of the curves a) to d) are selected identically in order to illustrate the courses over time.","As was already explained with reference to FIG.","1, during the signal pulse 41, the amplification factor 42 reaches a minimum 42a that lasts longer than the signal pulse 41 by a certain lag time dt, and then it grows monotonously over time.","During the signal pulse 41, the envelope curve voltage 43 reaches a maximum (envelope curve segment 43a) at which it still remains during the lag time dt after the end of the signal pulse 41.Then the envelope curve voltage 43 drops during the relaxation phase (envelope curve segment 43b), whereby the course of the envelope curve 43b results from the multiplication of, for example, the exponential decay of the sound transducer amplitude by the growing amplification factor.","The temporary lowering of the amplification factor during and after the signal pulse causes the envelope curve voltage 43 to fall below the threshold voltage 44 earlier than would be the case if the amplification factor were not lowered.","The advantageous result of this is a reduction of the short range.","In the quiescent phase, the envelope curve voltage 43 finally reaches a low constant level (envelope curve segment 43b).","The threshold voltage 44 is constant over time.","Since the comparator 1 emits a switching signal, as soon as the envelope curve voltage 43 exceeds the threshold voltage 44, in a disadvantageous manner, at the beginning of the signal pulse 41, a dummy switching signal 45 is always triggered that lasts until the envelope curve voltage 43 has dropped below the threshold value 44.An echo signal (envelope curve segment 43d) causes a useful switching signal 46 so that the control unit has to be capable of distinguishing a useful switching signal 46 from a dummy switching signal 45 which, without the presence of an echo signal, was caused exclusively by the signal pulse 41.The capability to make such a distinction requires complex hardware or software resources and thus entails disadvantages.","Further drawbacks associated with a distance sensor of the type illustrated in FIG.","1 were already explained above.","As already explained above, another method of reducing the short range lies in providing a threshold voltage that can be changed over time.","For purposes of further illustrating the state of the art, FIG.","3 shows a schematic block diagram of a corresponding variant of an acoustic distance sensor 30b.","The distance sensor 30b shown in FIG.","3 comprises a control unit 31, an oscillator 32, a driver 33, a sound transducer 34, a voltage limiter 35, a preferably logarithmic amplifier 36b, an envelope curve shaper 37, a comparator 1 as well as a threshold value actuation stage 38b which emits a threshold voltage that can be changed over time and transmits it to the comparator 1.The essential difference from the distance sensor 30a illustrated in FIG.","1 lies in the fact that the short range reduction is not achieved by an amplification factor that can be changed over time, but rather by a threshold voltage that can be changed over time.","FIG.","4 shows a schematic representation of a course over time of a) a signal pulse, b) an envelope curve and threshold value as well as c) a comparator output signal, that can typically occur during the operation of the distance sensor 30b shown in FIG.","3.The time axes of the curves a) to c) are selected identically in order to illustrate the courses over time.","During a signal pulse 41, the envelope curve voltage 53 reaches a maximum (envelope curve segment 53a) at which it still remains during the lag time dt after the end of the signal pulse 41.Then the envelope curve voltage 53 drops linearly during the relaxation phase (envelope curve segment 53b), since the amplifier 36b is a logarithmic amplifier.","Finally, in the quiescent phase, the envelope curve voltage 53 reaches a low constant level (envelope curve segment 53c).","The threshold voltage 54 is not constant over time but rather is regulated by the threshold value actuation stage 38b in such a way that it rises before every signal pulse 41, remains at a maximum value for a certain period of time (envelope curve segment 54a), then drops (curve segment 54b) and finally reaches a constant level (envelope curve segment 54c) during the quiescent phase, whereby the threshold voltage 54 remains greater than the envelope curve voltage 53, as long as no echo is received.","The threshold value actuation stage 38b receives the necessary time information for synchronizing the time behavior of the threshold voltage 54 with the rhythm of the signal pulse 41 from the control unit 31.The advantageous result is a reduction of the short range.","Moreover, advantageously, no dummy switching signal is triggered by the signal pulse 41.Rather, a switching signal 56 is triggered only by the rise in the envelope curve 53 (envelope curve segment 53d) associated with an echo, so that the switching signal 56 is a useful switching signal 56.Drawbacks associated with a distance sensor of the type illustrated in FIG.","3 were already explained above.","Now reference is made to FIG.","5, which illustrates a schematic circuit diagram of an embodiment 10a of an adaptive comparator circuit according to the invention It comprises a signal transmitter 2, a first regulatable switch 3, a capacitor 6, a diode 5 and a first comparator 1, that has a first threshold value input 1a and a first signal input 1b.","At the first threshold value input 1a, a threshold voltage Uref1, is present that is emitted internally in the adaptive comparator circuit according to the invention.","At the signal input 1b, a voltage signal Usignal is present that can be, for example, an envelope curve of the received signal of a sound transducer and that is fed via a first electric terminal 8 to the adaptive comparator circuit according to the invention.","The first comparator 1 emits a first switching signal S1 when the voltage signal Usignal is greater than the first threshold voltage Uref1.The first switching signal S1 can be tapped, for example; via a second electric terminal 9 and transmitted for evaluation purposes.","Via a control output 2a, the signal transmitter 2 emits an electric control signal SSt at times, for example, at regular intervals.","In the simplest case, the signal transmitter 2 can be, for instance, a manually operated signaling key.","Moreover, the signal transmitter can be, for example, an electronic square-wave timing generator circuit with a suitable duty cycle.","Preferably, the signal transmitter 2 is a microcontroller or microprocessor that is programmed in such a way that, at regular intervals, it emits an electric control signal SSt having a time duration of typically, for example, 20 microseconds.","The first switch 3 has a control means 3a, a switch input 3b and a switch output 3c.","The control means 3a is connected to the control output 2a and can be regulated by the control signal SSt in such a way that it is in the closed state when the signal transmitter 2 emits a control signal SSt, and otherwise it is in the open state.","The first switch 3 can be, for example, a transistor.","The switch input 3b is connected to the positive first pole 4a of a first direct voltage source 4a, 4b (not shown here).","The switch output 3c is connected to the first threshold value input 1a of the comparator 1, so that the first threshold voltage Uref1 is equal to the voltage present at the switch output 3c.","Thus, when the switch 3 is closed, the potential of the positive first pole 4a is present at the first threshold value input 1a of the comparator 1.The switch output 3c is also applied via a capacitor 6 to the negative second pole 4b of the first direct voltage source so that the capacitor 6 is charged with the voltage supplied by the first voltage source when the switch 3 is closed.","The switch output 3c is also connected to the anode 5a of a diode 5.The cathode 5b of the diode 5 is connected to the first signal input 1b of the comparator.","As soon as the signal transmitter 2 emits a control signal SSt, the first switch 3 is in the closed state.","Therefore, the capacitor 6 is charged with the voltage supplied by the first voltage source.","When the switch 3 is closed, this voltage is also present at the threshold value input 1a.","After the end of the control signal SSt, the switch 3 is in the open state.","The capacitor 6 can now discharge according to the invention via the diode 5 to such an extent until the first Uref1 reaches a value that corresponds to the sum of the voltage signal Usignal and the diode flow voltage dU.","This means that the voltage that is established at the capacitor 6 depends on the magnitude of the voltage signal Usignal: the smaller the voltage signal Usignal, the more the voltage and thus the threshold voltage Uref1 drop according to the invention at the capacitor.","Hence, a threshold voltage Uref1 is established that is greater than the voltage signal Usignal by the fundamental voltage distance dU.","According to the invention, the task of the diode 5 is to allow the discharging of the capacitor 6 and thus a drop in the threshold voltage Uref1 for as long as and only until the difference of Uref1−Usignal has dropped to the fundamental voltage distance dU, but to prevent a recharging of the capacitor and thus an increase in the threshold voltage if the voltage signal Usignal increases.","If the voltage signal Usignal drops, then the threshold voltage Uref1 follows the voltage signal Usignal at the distance dU.","In contrast, if the voltage signal Usignal increases, then the threshold voltage Uref1 remains constant because of the blocking effect of the diode 5.Of course, the polarity of the first direct voltage source can also be the opposite from the one in FIG.","1, so that the first pole 4a is negative and the second pole 4b is positive.","In this case, the diode 5 in the circuit shown in FIG.","1 has to be arranged in the reverse direction, so that its cathode is connected to the switch output 3c and its anode to the second pole 4b.","In another embodiment of the invention, the fundamental voltage distance dU is augmented by a codirectional series connection of a plurality of diodes 5 or second direct voltage sources 7.According to the invention, as an alternative, the function of the diode 5 can be assumed by a second direct voltage source whose positive pole is in the place of the anode 5a of the diode 5 and whose negative pole is in the place of the cathode 5b of the diode 5.FIG.","6 shows an embodiment 10b of an adaptive comparator circuit according to the invention, which can be used as an alternative to that shown in FIG.","1.The diode shown in FIG.","5 has been replaced by a second voltage source 7, whereby its positive pole 7a has been put in the place of the anode 5a and the negative pole 7b has been put in the place of the cathode 5b.","The fundamental voltage distance dU shown in FIG.","1 has now been replaced by the voltage of the voltage source 7, that is to say, the fundamental voltage distance dU between Uref1 and Usignal is now defined by the voltage supplied by the second direct voltage source.","If the voltage supplied by the second voltage source 7 can be regulated or adjusted, then the fundamental voltage distance dU can be varied as needed.","FIG.","7 shows a schematic block diagram of an embodiment of an acoustic distance sensor according to the invention in which the adaptive comparator circuit 10a shown in FIG.","5 is used.","A control unit 31 transmits short signal pulses at times, preferably at regular intervals, each lasting, for example, 25 microseconds, to an oscillator 32.When a signal pulse is present, the oscillator 32 transmits an alternating voltage to a sound transducer 34 that is excited by the latter to emit sound waves.","The sound transducer 34 is capable of receiving sound waves reflected, for example, from an object and coming back to the sound transducer 34 as an echo, and of converting them into an electric received signal, whereby the propagation time of the sound waves is a measure of the distance from the object.","The received signal is fed to an envelope curve shaper 37 which forms the envelope curve of the received signal and transmits it via an electric terminal 8 to the adaptive comparator circuit 10a shown in FIG.","5.In a preferred embodiment of the invention, the signal pulses 41 that the control unit 31 transmits to the oscillator 32 are synchronized with the control signals SSt that the control unit 2 transmits to the switch 3 and this is done in such a way that the control signals SSt each begin shortly before the signal pulses 41 as will still be explained below with reference to FIGS.","9a and 9b.","In this manner, it is ensured that, already at the beginning of the transmittal phase, the threshold voltage Uref1 is greater than the voltage signal Usignal, so that no dummy switching signal is triggered.","In another preferred embodiment of the invention, the signal pulses 41 are advantageously synchronized with the control signals SSt in such a way that the control signals SSt each begin before or simultaneously with the end of the signal pulses 41.This ensures that, without any time lag, the threshold voltage Uref1 is supplied to the dropping voltage signal Usignal at the fundamental voltage distance dU already at the beginning of the relaxation phase.","The sensitivity of the distance sensor is defined by the fundamental voltage distance dU.","FIG.","8 shows a schematic block diagram of another embodiment of an acoustic distance sensor according to the invention, in which the adaptive comparator circuit 10a shown in FIG.","5 is likewise used.","The distance sensor shown in FIG.","8—in comparison to that shown in FIG.","7—additionally has a driver 33 that amplifies the alternating voltage supplied by the oscillator 32 and transmits it to the sound transducer 34.Moreover, before the received signal is fed into the envelope curve shaper 37, it is passed through a voltage limiter 35 and through a logarithmic amplifier 36c.","Among other things, the voltage limiter 35 counters an overmodulation of the amplifier.","The amplifier 36c serves to adapt the low input level of just a few microvolts to the higher input level of the subsequent evaluation circuit.","In FIG.","8, in order to facilitate the synchronization, the signal transmitter 2 is accommodated in the control unit 31.For example, the signal transmitter 2 and the control unit 31 can be combined in one single unit, for example, in a microprocessor, that concurrently fulfills the function of the signal transmitter 2 as well as that of the control unit 31.In FIG.","8, the first switching signal S1 of the control unit 31 is also supplied for evaluation purposes, especially for determining the propagation time and calculating the distance.","FIGS.","9 and 10 serve to further explain the advantageous effects of the invention, whereby the time axes of all of the curves have been selected identically in order to illustrate the course over time.","FIG.","9 shows a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) a voltage signal and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.","8, whereby an echo occurs during the quiescent phase.","The control unit 31 transmits short signal pulses 41 to the oscillator 32, as a result of which the sound transducer 34 is excited so as to emit a threshold voltage packet (FIG.","9, Curve a).","Moreover, the signal transmitter 2 transmits a control signal SSt to the switch 3, which is synchronized with the signal pulse 41 in such a way that the control signal SSt begins shortly before the signal pulse 41 and ends at the same time as or during the presence of the former (FIG.","9, Curve b).","FIG.","9, Curve c, shows the course of the voltage signal Usignal (solid curve) that is identical to the envelope curve, and the course of the threshold voltage Uref1 (dotted curve).","The course of the Usignal during the transmittal and relaxation phase and at the beginning of the quiescent phase corresponds essentially to that of the envelope curve 53, which was explained with reference to FIG.","4.While the control signal SSt is present at the switch 3, the latter is opened, so that a threshold voltage Uref1 is established which is identical to the voltage supplied by the first direct voltage source 4a, 4b (FIG.","9, Curve c).","Therefore, this has been advantageously selected in such a way that it is greater than the maximum of the voltage signal, so that the adaptation of the threshold voltage to the voltage signal begins immediately after the opening of the switch 3.In the present example, the maximum possible magnitude of the voltage signal is supplied by the voltage limiter 35 or by the overmodulation limit of the amplifier 36c (FIG.","8).","According to the invention, after the end of the control signal SSt, a threshold voltage Uref1 is established that is greater than the voltage signal Usignal by the voltage distance dU.","Therefore, the threshold voltage Uref1 drops immediately after the end of the control signal by a certain quantity.","From this point in time on, the threshold voltage proceeds parallel-offset by a fundamental voltage distance dU with respect to that of the voltage signal Usignal as long as the latter does not increase.","A dummy switching signal is not triggered.","If, however, the voltage signal Usignal increases, the threshold voltage remains constant so that a first echo peak 60 of the voltage signal Usignal leads to a triggering of the comparator 1 as soon as the voltage signal Usignal exceeds the threshold voltage Uref1.In this case, the comparator 1 emits a first switching signal S1 (FIG.","9, Curve d).","FIG.","10 shows a schematic representation of a typical course over time of a) a voltage signal and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.","8, whereby an echo occurs during the relaxation phase and another echo occurs during the quiescent phase.","The courses over time of the signal pulse 41 and of the control signal SSt are identical to those shown in FIG.","9.During the relaxation time, an echo signal occurs, which manifests itself in a second echo peak 61 of the voltage signal Usignal (FIG.","10, Curve a).","Up until the beginning of the second echo peak 61, the curves correspond to those shown in FIG.","9, Curve c. According to the invention, however, the decrease of the threshold voltage Uref1 comes to a halt as soon as the voltage signal Usignal is no longer dropping, and a first switching signal S1a is triggered by the second echo peak 61.After the second echo peak 61 that occurs during the relaxation time, the fundamental voltage distance dU is once again established between the threshold voltage Uref1 and the voltage signal Usignal.","During the quiescent phase, another, third echo peak 62 of the voltage signal Usignal occurs, as a result of which another switching signal S1b is triggered.","Now reference will be made to FIGS.","11 to 13, which show additional advantageous embodiments of an adaptive comparator circuit according to the invention.","FIG.","11 shows a preferred embodiment 10c of the invention which differs from the adaptive comparator circuit 10a shown in FIG.","6 as follows: a) The first switch is a transistor 103.The base 103a of the transistor 103 serves as the control-current terminal, the emitter 103b serves as the switch input and the collector 103c as the switch output.","b) A first resistor 11 is connected between the first pole 4a and the first threshold value input 1a, and a second resistor 12 is connected between the first threshold value input 1a and the second pole 4b, so that the first resistor 11 and the second resistor 12 form a first voltage divider 11, 12.Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the first voltage divider.","Thus, through the use of the first voltage divider 11, 12, the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal after the voltage signal Usignal has died out can advantageously be changed by the fundamental voltage distance dU, to a value dUa.","When this embodiment 10c of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be changed and, in particular increased.","This is advantageous for many applications such as, for example, for the detection of small objects.","The first voltage divider can be an adjustable potentiometer whose pick-up is connected to the switch output.","c) A sixth resistor 16 is connected between the control output 2a of the signal transmitter 2 and the base 103a, and a seventh resistor 17 is connected between the base 103a and the first pole 4a, which is a plus pole in the example shown.","The purpose of these resistors was already explained above.","d) The diode 5 is connected in series to an eighth resistor 18 which serves to slow down the discharging of the capacitor 6 and thus to reduce the speed with which the threshold voltage Uref1 is adapted to the voltage signal Usignal.","The eighth resistor also serves to smooth the course of the threshold voltage Uref1 with respect to very rapid fluctuations of the voltage signal Usignal.","FIG.","12 shows another preferred embodiment 10d of the invention, which differs from the adaptive comparator circuit 10c shown in FIG.","11 in that, before the voltage signal Usignal reaches the first signal input 1b, it passes through an impedance transformer.","This embodiment is especially advantageous when the voltage signal arriving at the electric terminal 8 is high-ohmic, whereby said voltage signal can be the envelope curve voltage generated, for example, by an envelope curve shaper.","FIG.","13 shows another preferred embodiment 10e of the invention which differs from the adaptive comparator circuit 10a shown in FIG.","6 as follows: a) The first switch is a transistor 103.The base 103a of the transistor 103 serves as the control-current terminal, the emitter 103b serves as the switch input and the collector 103c as the switch output.","b) A second comparator 20 is used that has a second threshold value input 20a and a second signal input 20b, and that emits a second switching signal S2 when a greater voltage is present at the second signal input 20b than at the second threshold value input 20a.","c) The first pole 4a of the first voltage source 4a, 4b is connected to the second threshold value input 20a via a third resistor 13.The second threshold value input 20a is connected to the second pole 4b via a fourth resistor 14, so that the third and the fourth resistors 13, 14 form a second voltage divider which can be configured as a potentiometer.","d) The collector 103c that serves as the switch output and the second threshold value input 20a are connected via a fifth resistor 15.e) A second switch 23 is connected in series to the diode 5 and the former can be regulated or activated by the second switching signal S2 in such a way that it is in the closed state when the second comparator 20 emits the second switching signal S2, and otherwise it is in the open state.","The second switch 23 can be a transistor.","f) A sixth resistor 16 is connected between the control output 2a of the signal transmitter 2 and the base 103a, and a seventh resistor 17 is connected between the base 103a and the first pole 4a, which is a plus pole in the example shown.","The purpose of these resistors was already explained above.","g) The diode 5 is connected in series to an eighth resistor 18.The purpose of this resistor was already explained with reference to FIG.","11.h) Before reaching the first signal input 1b, the voltage signal passes through an impedance transformer 21 whose purpose was already explained with reference to FIG.","12.Due to the second voltage divider 13, 14, a voltage Uref2 is present at the second threshold value input 20a.","As long as the second switch 23 is closed, the first threshold voltage Uref1 follows the dropping voltage signal Usignal, parallel-offset by the fundamental voltage distance dU, as explained above.","According to the invention, however, the second switch opens as soon as the voltage signal Usignal exceeds the second threshold voltage Uref2.As a result, the discharge of the capacitor 6 and thus the dropping of the first threshold voltage Uref1 are ended.","Hence, in this embodiment of the invention, the capacitor 6 can only discharge until the first threshold voltage Uref1 has dropped to the magnitude of the second threshold voltage Uref2 supplied by the second potentiometer 13, 14.Thus, through the use of the second voltage divider 13, 14, the voltage distance by which the first threshold voltage is advantageously changed parallel-offset after the decay of the voltage signal Usignal with respect to voltage signal Usignal, from the fundamental voltage distance dU to a value dUb.","The second voltage divider 13, 14 can be an adjustable potentiometer whose pick-up is connected to the second threshold value input 20a.","The fifth resistor 15 is connected between the switch output 103c and the second threshold value input 20a.","Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the second voltage divider 13, 14.In particular, through the use of the fifth resistor 15, it can additionally be achieved that the voltage distance by which the first threshold voltage is parallel-offset with respect to the voltage signal Usignal strives towards a value dUb that—depending on the voltage supplied by the second voltage divider 13, 14—can advantageously be not only larger but also smaller than the fundamental voltage distance dU.","When this embodiment 10e of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be either increased or decreased by means of the second voltage divider 13, 14.All of the embodiments of an adaptive comparator circuit according to the invention and thus also the embodiments 10b, 10c, 10d and 10e illustrated in FIG.","6 and FIGS.","11 to 13 can be used according to the invention in the acoustic distance sensor shown in FIG.","8 instead of the adaptive comparator circuit 10a shown there by way of an example.","Commercial Utilization The invention can be utilized commercially, for example, in distance measuring technology, in filling level measuring technology and in automation technology.","The main figure is FIG.","5.List of Reference Numerals and Letters 1 first comparator 1a first threshold value input 1b first signal input 2 signal transmitter 2a control output of 2 2b input of 2 3 first switch 3a control-current terminal of 3 3b switch input of 3 3c switch output of 3 4a, 4b first, second pole of the first direct voltage source 5 diode 5a, 5b anode, cathode of 5 6 capacitor 7a, 7b positive, negative pole of 7 7 second direct voltage source 8, 9 first, second electric terminal 10a,b,c,d,e adaptive comparator circuits 11-18 first to eighth resistor 20 second comparator 20a second threshold value input 20b second signal input 21 voltage follower 23 second switch 30a distance sensor with constant threshold voltage 30b distance sensor with variable threshold voltage 31 control unit 32 oscillator 33 driver 34 sound transducer 35 voltage limiter 36a amplifier with regulatable amplification factor 36b, c logarithmic amplifier 37 envelope curve shaper 38a amplification factor actuation stage 38b threshold value actuation stage 41 signal pulses 42 course over time of the amplification factor of 36a 42a minimum of 42 43, 53 courses over time of the envelope curve voltage 43a, 53a segments of 43, 53 during the signal pulses 41, 51 43b, 53b segments of 43, 53 during the relaxation time of 34 43c, 53c segments of 43, 53 during the quiescent phase 43d, 53d segments of 43, 53 during an echo 44, 54 threshold values of 30a, 30b 45 dummy switching signal 46, 56 useful switching signals 60 first echo peak 61 second echo peak 62 third echo peak 103 transistor 103a base of 103 103b emitter of 103 103c collector of 103 S1, S1a, S1b switching signals of 1 S2 switching signal of 20 dU fundamental voltage distance Uref1 first threshold voltage Usignal voltage signal"]],"string":"[\n [\n \"Adaptive comparator circuit and acoustic distance sensor comprising said circuit\",\n \"The invention relates to an adaptive comparator circuit comprising a comparator with a threshold value input which receives a threshold voltage and a signal input which receives a voltage signal.\",\n \"The threshold value input is connected to one pole by means of a switch and connected to the other pole of a first voltage source by means of a capacitor.\",\n \"The threshold value input is also connected to the signal input of the comparator by means of a diode or a second voltage source.\",\n \"The switch is controlled by the control signal of a signal transmitter.\",\n \"When the switch is in a closed position, the capacitor is charged with the voltage of the first voltage source.\",\n \"When the switch is in an open position, the capacitor is discharged in such a way that the threshold voltage of the signal voltage is corrected at a given time interval.\"\n ],\n [\n \"1.An adaptive comparator circuit, particularly for an acoustic distance sensor, comprising a first comparator (1), having a first threshold voltage input (1a) to which a first threshold voltage (Uref1) is applied and a first signal input (1b) to which a voltage signal (Usignal) is applied and, when a voltage signal (Usignal) is present that is greater than the first threshold voltage (Uref1), said comparator emits a first switching signal (S1), characterized by a signal transmitter (2), especially a microcontroller or microprocessor, that at times emits a control signal (SSt), a first switch (3, 103) that has a control means (3a, 103a), a switch input (3b, 103b) and a switch output (3c, 103c), and that can be regulated or activated through the effect of the control signal (SSt) on the control means (3a, 103a) in such a way that it is in the closed state when the signal transmitter (2) emits a control signal (SSt), and otherwise it is in the open state, or vice versa, whereby the switch input (3b, 103b) is connected to the positive or negative first pole (4a) of a first direct voltage source and the switch output (3c, 103c) is connected to the first threshold voltage input (1a), so that the first threshold voltage (Uref1) is equal to the voltage present at the switch output (3c, 103c), a capacitor (6) via which the switch output is connected to the negative or positive pole (4b) of the first direct voltage source, and a diode (5) via which the switch output (3c, 103c) is connected to the first signal input (1b) in such a way that the anode (5a) or the cathode (5b) of the diode (5) is connected to the switch output (3c, 103c).\",\n \"2.The adaptive comparator according to claim 1, characterized in that, instead of the diode (5), a second direct voltage source (7) is used whose positive pole (7a) is in the place of the anode (5a) of the diode (5) and whose negative pole (7b) is in the place of the cathode (5b) of the diode (5).\",\n \"3.The adaptive comparator according to claim 2, characterized in that the voltage supplied by the second voltage source (7) can be regulated or adjusted.\",\n \"4.The adaptive comparator according to claim 1, characterized in that the signal transmitter (2) emits an electric control signal (SSt) at regular intervals.\",\n \"5.The adaptive comparator according to claim 1, characterized in that the first switch (3, 103) is a switch that can be electrically or electronically controlled by the control means (3a, 103a) and the control signal (SSt) is an electric signal that is transmitted by the signal transmitter (2) via a control output (2a) and fed to the control input (3a, 103a).\",\n \"6.The adaptive comparator according to claim 5, characterized in that the first switch (3, 103) is a transistor (103) and the control-current terminal (3a, 103a) is the base (103) or the gate terminal of the transistor (103).\",\n \"7.The adaptive comparator according to claim 1, characterized in that a first resistor (11) is connected between the first pole (4a) and the first threshold voltage input (1a), and a second resistor (12) is connected between the first threshold voltage input (1a) and the second pole (4b), so that the first resistor (11) and the second resistor (12) form a first voltage divider (11, 12).\",\n \"8.The adaptive comparator according to claim 7, characterized in that first voltage divider (11, 12) is an adjustable potentiometer whose pick-up is connected to the switch output (3c, 103c).\",\n \"9.The adaptive comparator according to claim 1, characterized by a second comparator (20) that has a second threshold voltage input (20a) and a second signal input (20b), and that emits a second switching signal S2 when a greater voltage is present at the second signal input (20b) than at the second threshold voltage input (20a), a third resistor (13) via which the first pole (4a) is connected to the second threshold voltage input (20a), a fourth resistor (14) that, on the one hand, is connected to the second threshold voltage input (20a) and, on the other hand, to the second pole (4b) of the first voltage source, so that the third resistor (13) and the fourth resistor (14) form a second voltage divider (13, 14), a second switch (23) that is connected in series to the diode (5) and that can be regulated or activated by the second switching signal S2 in such a way that it is in the closed state when the second comparator (20) emits the second switching signal S2, and otherwise it is in the open state.\",\n \"10.The adaptive comparator according to claim 9, characterized in that a fifth resistor (15) is connected between the switch output (3c, 103c) and the second threshold voltage input (20a).\",\n \"11.The adaptive comparator according to claim 9, characterized in that the second voltage divider (13, 14) is a potentiometer.\",\n \"12.The adaptive comparator according to claim 1, characterized in that a sixth resistor (16) is connected between the control output (2a) and the control-current terminal (3a, 103a).\",\n \"13.The adaptive comparator according to claim 6, characterized in that a seventh resistor (17) is connected between the control-current terminal (3a, 103a) and the first pole (4a).\",\n \"14.The adaptive comparator according to claim 1, characterized in that the diode (5) or the second voltage source (7) is connected in series to an eighth resistor (18).\",\n \"15.The adaptive comparator according to claim 1, characterized in that, before reaching the first signal input (1b), the voltage signal Usignal passes through a voltage follower or impedance transformer (21).\",\n \"16.The adaptive comparator according to claim 1, characterized in that the voltage supplied by the first voltage source (4a, 4b) is greater in magnitude than the voltage of the maximum of the voltage signal Usignal.\",\n \"17.The adaptive comparator according to claim 1, characterized in that the diode (5) is connected to at least another diode codirectionally in series.\",\n \"18.An acoustic distance sensor, comprising a control unit (31) that at times transmits signal pulses (41) to an oscillator (32) which, during the presence of a signal pulse (41), transmits an alternating voltage to a sound transducer (34) that is excited by the alternating voltage to emit sound waves and that is capable of receiving sound waves coming back to the sound transducer (34) as an echo as a result of reflection, and of converting them into an electric received signal, and also comprising an envelope curve shaper (37) to which the received signal is fed and which forms the envelope curve of the received signal and emits it as a voltage signal Usignal, characterized in that the voltage signal is fed to an adaptive comparator circuit comprising a first comparator (1) having a first threshold voltage input (1a) to which a first threshold voltage (Uref1) is applied and a first signal input (1b) to which a voltage signal (Usignal) is applied and, when a voltage signal (Usignal) is present that is greater than the first threshold voltage Uref1, said comparator (1) emits a first switching signal (S1), a signal transmitter (2), especially a microcontroller or microprocessor, that at times emits a control signal (SSt), a first switch (3, 103) that has a control means (3a, 103a), a switch input (3b, 103b) and a switch output (3c, 103c), and that can be regulated or activated through the effect of the control signal (SSt) on the control means (3a, 103a) in such a way that it is in the closed state when the signal transmitter (2) emits a control signal (SSt), and otherwise it is in the open state, or vice versa, whereby the switch input (3b, 103b) is connected to the positive or negative first pole (4a) of a first direct voltage source and the switch output (3c, 103c) is connected to the first threshold voltage input (1a), so that the first threshold voltage (Uref1) is equal to the voltage present at the switch output (3c, 103c), a capacitor (6) via which the switch output (3c, 103c) is connected to the negative or positive pole (4b) of the first direct voltage source, and a diode (5) via which the switch output (3c, 103c) is connected to the first signal input (1b) in such a way that the anode (5a) or the cathode (5b) of the diode (5) is connected to the switch output (3c, 103c).\",\n \"19.The acoustic distance sensor according to claim 18, characterized in that, instead of the diode (5), a second direct voltage source (7) is used whose positive pole (7a) is in the place of the anode (5a) of the diode (5) and whose negative pole (7b) is in the place of the cathode (5b) of the diode (5).\",\n \"20.The acoustic distance sensor according to claim 18 characterized in that the alternating voltage emitted by the oscillator (32) reaches the sound transducer (34) after passing through a driver stage (33).\",\n \"21.The acoustic distance sensor according to claim 18, characterized in that, before entering the envelope curve shaper (37), the received signal passes through an amplifier (36c).\",\n \"22.The acoustic distance sensor according to claim 21, characterized in that the amplifier (36c) is a logarithmic amplifier.\",\n \"23.The acoustic distance sensor according to claim 18, characterized in that, before entering the envelope curve shaper (37), the received signal passes through a voltage limiter (35) that limits the amplitude of the received signal to a maximum value.\",\n \"24.The acoustic distance sensor according to claim 18, characterized in that the signal transmitter (2) is integrated into the control unit (31) or is a component of the control unit (31).\",\n \"25.The acoustic distance sensor according to claim 18, characterized in that the first switching signal (S1) is transmitted to the control unit (31) for evaluation purposes.\",\n \"26.The acoustic distance sensor according to claim 18, characterized in that the signal pulses (41) are synchronized with the control signals (SSt) in such a way that the control signals (SSt) each begin before the signal pulses (41).\",\n \"27.The acoustic distance sensor according to claim 18, characterized in that the signal pulses 41 are synchronized with the control signals SSt in such a way that the control signals SSt each begin before or simultaneously with the end of the signal pulses (41).\",\n \"28.An adaptive comparator circuit for an acoustic distance sensor, comprising a first comparator (1) having a first signal input and a first threshold voltage input; a first direct voltage source (4) for delivering a threshold voltage to the first threshold voltage input, wherein the first direct voltage source comprises a first and a second pole; a first switch (3), wherein the first switch is connected to the first direct voltage source and to the first threshold voltage input; a signal transmitter (2), wherein the signal transmitter controls the first switch through an input of the switch; a capacitor (6), wherein a first plate of the capacitor is connected to the a first threshold voltage input and wherein a second plate of the capacitor is connected to second pole the first direct voltage source; a diode (5) connected the first signal input and to the first threshold voltage input of the first comparator.\",\n \"29.The adaptive comparator circuit according to claim 28, wherein the diode is connected in series to a first resistor.\",\n \"30.The adaptive comparator circuit according to claim 1, wherein the signal transmitter emits an electric control signal at regular intervals.\",\n \"31.The adaptive comparator circuit according to claim 28 further comprising a second resistor (11) connected between the first pole of the first direct voltage source and the first threshold voltage input, and a third resistor (12) connected between the first threshold voltage input and the second pole of the first direct voltage source, so that the second resistor (11) and the third resistor (12) form a first voltage divider (11, 12).\",\n \"32.The adaptive comparator circuit according to claim 31, wherein the first voltage divider (11, 12) is an adjustable potentiometer whose pick-up is connected to the first threshold voltage input.\",\n \"33.The adaptive comparator circuit according to claim 28 further comprising a fourth resistor (17) connected between the first pole of the first direct voltage source and the input of the first switch; a fifth resistor (16) connected between the input of the first switch and the output of the signal transmitter (2).\"\n ],\n [\n \"<SOH> BRIEF DESCRIPTION OF THE DRAWING IN WHICH THE FOLLOWING IS SHOWN <EOH>FIG.\",\n \"1 —a schematic block diagram of a variant of an acoustic distance sensor according to the state of the art, FIG.\",\n \"2 —a schematic representation of a typical course over time of a) a signal pulse, b) an amplification factor, c) an envelope curve and d) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.\",\n \"1 , FIG.\",\n \"3 —a schematic block diagram of another variant of an acoustic distance sensor according to the state of the art, FIG.\",\n \"4 —a schematic representation of a typical course over time of a) a timing pulse, b) an envelope curve and c) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.\",\n \"3 , FIG.\",\n \"5 —a circuit diagram of an embodiment of a comparator circuit according to the invention, FIG.\",\n \"6 —a circuit diagram of an alternative embodiment of a comparator circuit according to the invention, FIGS.\",\n \"7 and 8 —a schematic block diagram of embodiments of an acoustic distance sensor according to the invention, FIG.\",\n \"9 —a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) an envelope curve and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.\",\n \"8 , whereby an echo occurs during the quiescent phase, FIG.\",\n \"10 —a schematic representation of a typical course over time of a) an envelope curve and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.\",\n \"8 , whereby an echo additionally occurs during the relaxation phase, and FIGS.\",\n \"11 to 13 —circuit diagrams of further embodiments of comparator circuits according to the invention.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\",\n \"FIGS.\",\n \"1 to 4 serve to further illustrate the state of the art.\",\n \"FIG.\",\n \"1 shows a schematic block diagram of a variant of an acoustic distance sensor 30 a with which a measure according to the state of the art has been taken in order to reduce the short range.\",\n \"The distance sensor 30 a shown in FIG.\",\n \"1 comprises a control unit 31 , an oscillator 32 , a driver 33 , a sound transducer 34 , a voltage limiter 35 , an amplifier 36 a with regulatable amplification factor, an envelope curve shaper 37 , a comparator 1 as well as an amplification factor actuation stage 38 a.\",\n \"The control unit 31 transmits short signal pulses 41 to the oscillator 32 .\",\n \"The latter oscillates when a signal pulse is applied, thus emitting an alternating voltage of, for example, 400 kHz, which is then amplified by the driver 33 and subsequently used to excite the sound transducer 34 so that the latter is excited so as to oscillate, i.e.\",\n \"to emit sound waves.\",\n \"Corresponding to the short signal pulses, the sound transducer 34 emits short sound wave packets that constitute the transmittal pulse of the distance sensor 30 a.\",\n \"During the transmitting phase, the sound transducer 34 oscillates at a high oscillation amplitude.\",\n \"This period of time is followed by a relaxation time in which the oscillation amplitude dies out—usually exponentially.\",\n \"In the subsequent quiescent phase, the oscillation amplitude generally remains at a constant mean disturbance level.\",\n \"The signal emitted by the sound transducer and thus also echoes returning after the distance-proportional propagation time are fed via the voltage limiter 35 to the amplifier 36 a with a regulatable amplification factor.\",\n \"The voltage limiter keeps the high voltage used for exciting the sound transducer away from the amplifier 36 a Using, for example, an internal rectifier circuit, the envelope curve shaper 37 forms the envelope curve of the voltage limiter 35 on the basis of the alternating voltage signal emitted by said voltage limiter 35 and transmits to the comparator 1 an envelope curve voltage signal whose course over time corresponds to that of the envelope curve.\",\n \"The comparator 1 transmits a switching signal to the control unit 31 for evaluation purposes when the envelope curve voltage signal is greater than a first steadily supplied threshold voltage.\",\n \"In particular, the comparator then emits a switching signal when an echo signal is received whose magnitude exceeds that of the threshold voltage.\",\n \"The amplification factor of the amplifier 36 a is regulated by the amplification factor actuation stage 38 a in a time-dependent manner such that the amplification factor is minimal during and immediately after the transmittal pulse and grows as soon as the oscillation amplitude of the sound transducer 34 has died out to such an extent that the magnitude of the received signal is no longer limited by the voltage limiter 35 .\",\n \"Therefore, the growth of the amplification factor does not begin completely simultaneously with the end of the signal pulse, but rather delayed by a certain lag time dt with respect thereto ( FIG.\",\n \"2 ).\",\n \"The amplification factor actuation stage 38 a receives the time information necessary for synchronizing the time behavior of the amplification factor with the rhythm of the transmittal pulse from the control unit 31 .\",\n \"In this manner a reduction of the short range can be achieved.\",\n \"The course over time can advantageously be selected in such a way that the distance-dependent attenuation of the echo is countered and the system sensitivity remains constant.\",\n \"FIG.\",\n \"2 shows a schematic representation of a course over time of a) a signal pulse, b) an amplification factor of the amplifier 36 a , c) an envelope curve and threshold voltage as well as d) a comparator output signal, that can typically occur during the operation of the distance sensor shown in FIG.\",\n \"1 .\",\n \"The time axes of the curves a) to d) are selected identically in order to illustrate the courses over time.\",\n \"As was already explained with reference to FIG.\",\n \"1 , during the signal pulse 41 , the amplification factor 42 reaches a minimum 42 a that lasts longer than the signal pulse 41 by a certain lag time dt, and then it grows monotonously over time.\",\n \"During the signal pulse 41 , the envelope curve voltage 43 reaches a maximum (envelope curve segment 43 a ) at which it still remains during the lag time dt after the end of the signal pulse 41 .\",\n \"Then the envelope curve voltage 43 drops during the relaxation phase (envelope curve segment 43 b ), whereby the course of the envelope curve 43 b results from the multiplication of, for example, the exponential decay of the sound transducer amplitude by the growing amplification factor.\",\n \"The temporary lowering of the amplification factor during and after the signal pulse causes the envelope curve voltage 43 to fall below the threshold voltage 44 earlier than would be the case if the amplification factor were not lowered.\",\n \"The advantageous result of this is a reduction of the short range.\",\n \"In the quiescent phase, the envelope curve voltage 43 finally reaches a low constant level (envelope curve segment 43 b ).\",\n \"The threshold voltage 44 is constant over time.\",\n \"Since the comparator 1 emits a switching signal, as soon as the envelope curve voltage 43 exceeds the threshold voltage 44 , in a disadvantageous manner, at the beginning of the signal pulse 41 , a dummy switching signal 45 is always triggered that lasts until the envelope curve voltage 43 has dropped below the threshold value 44 .\",\n \"An echo signal (envelope curve segment 43 d ) causes a useful switching signal 46 so that the control unit has to be capable of distinguishing a useful switching signal 46 from a dummy switching signal 45 which, without the presence of an echo signal, was caused exclusively by the signal pulse 41 .\",\n \"The capability to make such a distinction requires complex hardware or software resources and thus entails disadvantages.\",\n \"Further drawbacks associated with a distance sensor of the type illustrated in FIG.\",\n \"1 were already explained above.\",\n \"As already explained above, another method of reducing the short range lies in providing a threshold voltage that can be changed over time.\",\n \"For purposes of further illustrating the state of the art, FIG.\",\n \"3 shows a schematic block diagram of a corresponding variant of an acoustic distance sensor 30 b .\",\n \"The distance sensor 30 b shown in FIG.\",\n \"3 comprises a control unit 31 , an oscillator 32 , a driver 33 , a sound transducer 34 , a voltage limiter 35 , a preferably logarithmic amplifier 36 b , an envelope curve shaper 37 , a comparator 1 as well as a threshold value actuation stage 38 b which emits a threshold voltage that can be changed over time and transmits it to the comparator 1 .\",\n \"The essential difference from the distance sensor 30 a illustrated in FIG.\",\n \"1 lies in the fact that the short range reduction is not achieved by an amplification factor that can be changed over time, but rather by a threshold voltage that can be changed over time.\",\n \"FIG.\",\n \"4 shows a schematic representation of a course over time of a) a signal pulse, b) an envelope curve and threshold value as well as c) a comparator output signal, that can typically occur during the operation of the distance sensor 30 b shown in FIG.\",\n \"3 .\",\n \"The time axes of the curves a) to c) are selected identically in order to illustrate the courses over time.\",\n \"During a signal pulse 41 , the envelope curve voltage 53 reaches a maximum (envelope curve segment 53 a ) at which it still remains during the lag time dt after the end of the signal pulse 41 .\",\n \"Then the envelope curve voltage 53 drops linearly during the relaxation phase (envelope curve segment 53 b ), since the amplifier 36 b is a logarithmic amplifier.\",\n \"Finally, in the quiescent phase, the envelope curve voltage 53 reaches a low constant level (envelope curve segment 53 c ).\",\n \"The threshold voltage 54 is not constant over time but rather is regulated by the threshold value actuation stage 38 b in such a way that it rises before every signal pulse 41 , remains at a maximum value for a certain period of time (envelope curve segment 54 a ), then drops (curve segment 54 b ) and finally reaches a constant level (envelope curve segment 54 c ) during the quiescent phase, whereby the threshold voltage 54 remains greater than the envelope curve voltage 53 , as long as no echo is received.\",\n \"The threshold value actuation stage 38 b receives the necessary time information for synchronizing the time behavior of the threshold voltage 54 with the rhythm of the signal pulse 41 from the control unit 31 .\",\n \"The advantageous result is a reduction of the short range.\",\n \"Moreover, advantageously, no dummy switching signal is triggered by the signal pulse 41 .\",\n \"Rather, a switching signal 56 is triggered only by the rise in the envelope curve 53 (envelope curve segment 53 d ) associated with an echo, so that the switching signal 56 is a useful switching signal 56 .\",\n \"Drawbacks associated with a distance sensor of the type illustrated in FIG.\",\n \"3 were already explained above.\",\n \"Now reference is made to FIG.\",\n \"5 , which illustrates a schematic circuit diagram of an embodiment 10 a of an adaptive comparator circuit according to the invention It comprises a signal transmitter 2 , a first regulatable switch 3 , a capacitor 6 , a diode 5 and a first comparator 1 , that has a first threshold value input 1 a and a first signal input 1 b.\",\n \"At the first threshold value input 1 a , a threshold voltage U ref1 , is present that is emitted internally in the adaptive comparator circuit according to the invention.\",\n \"At the signal input 1 b , a voltage signal U signal is present that can be, for example, an envelope curve of the received signal of a sound transducer and that is fed via a first electric terminal 8 to the adaptive comparator circuit according to the invention.\",\n \"The first comparator 1 emits a first switching signal S 1 when the voltage signal U signal is greater than the first threshold voltage U ref1 .\",\n \"The first switching signal S 1 can be tapped, for example; via a second electric terminal 9 and transmitted for evaluation purposes.\",\n \"Via a control output 2 a , the signal transmitter 2 emits an electric control signal S St at times, for example, at regular intervals.\",\n \"In the simplest case, the signal transmitter 2 can be, for instance, a manually operated signaling key.\",\n \"Moreover, the signal transmitter can be, for example, an electronic square-wave timing generator circuit with a suitable duty cycle.\",\n \"Preferably, the signal transmitter 2 is a microcontroller or microprocessor that is programmed in such a way that, at regular intervals, it emits an electric control signal S St having a time duration of typically, for example, 20 microseconds.\",\n \"The first switch 3 has a control means 3 a , a switch input 3 b and a switch output 3 c .\",\n \"The control means 3 a is connected to the control output 2 a and can be regulated by the control signal S St in such a way that it is in the closed state when the signal transmitter 2 emits a control signal S St , and otherwise it is in the open state.\",\n \"The first switch 3 can be, for example, a transistor.\",\n \"The switch input 3 b is connected to the positive first pole 4 a of a first direct voltage source 4 a , 4 b (not shown here).\",\n \"The switch output 3 c is connected to the first threshold value input 1 a of the comparator 1 , so that the first threshold voltage U ref1 is equal to the voltage present at the switch output 3 c .\",\n \"Thus, when the switch 3 is closed, the potential of the positive first pole 4 a is present at the first threshold value input 1 a of the comparator 1 .\",\n \"The switch output 3 c is also applied via a capacitor 6 to the negative second pole 4 b of the first direct voltage source so that the capacitor 6 is charged with the voltage supplied by the first voltage source when the switch 3 is closed.\",\n \"The switch output 3 c is also connected to the anode 5 a of a diode 5 .\",\n \"The cathode 5 b of the diode 5 is connected to the first signal input 1 b of the comparator.\",\n \"As soon as the signal transmitter 2 emits a control signal S St , the first switch 3 is in the closed state.\",\n \"Therefore, the capacitor 6 is charged with the voltage supplied by the first voltage source.\",\n \"When the switch 3 is closed, this voltage is also present at the threshold value input 1 a.\",\n \"After the end of the control signal S St , the switch 3 is in the open state.\",\n \"The capacitor 6 can now discharge according to the invention via the diode 5 to such an extent until the first U ref1 reaches a value that corresponds to the sum of the voltage signal U signal and the diode flow voltage dU.\",\n \"This means that the voltage that is established at the capacitor 6 depends on the magnitude of the voltage signal U signal : the smaller the voltage signal U signal , the more the voltage and thus the threshold voltage U ref1 drop according to the invention at the capacitor.\",\n \"Hence, a threshold voltage U ref1 is established that is greater than the voltage signal U signal by the fundamental voltage distance dU.\",\n \"According to the invention, the task of the diode 5 is to allow the discharging of the capacitor 6 and thus a drop in the threshold voltage U ref1 for as long as and only until the difference of U ref1 −U signal has dropped to the fundamental voltage distance dU, but to prevent a recharging of the capacitor and thus an increase in the threshold voltage if the voltage signal U signal increases.\",\n \"If the voltage signal U signal drops, then the threshold voltage U ref1 follows the voltage signal U signal at the distance dU.\",\n \"In contrast, if the voltage signal U signal increases, then the threshold voltage U ref1 remains constant because of the blocking effect of the diode 5 .\",\n \"Of course, the polarity of the first direct voltage source can also be the opposite from the one in FIG.\",\n \"1 , so that the first pole 4 a is negative and the second pole 4 b is positive.\",\n \"In this case, the diode 5 in the circuit shown in FIG.\",\n \"1 has to be arranged in the reverse direction, so that its cathode is connected to the switch output 3 c and its anode to the second pole 4 b.\",\n \"In another embodiment of the invention, the fundamental voltage distance dU is augmented by a codirectional series connection of a plurality of diodes 5 or second direct voltage sources 7 .\",\n \"According to the invention, as an alternative, the function of the diode 5 can be assumed by a second direct voltage source whose positive pole is in the place of the anode 5 a of the diode 5 and whose negative pole is in the place of the cathode 5 b of the diode 5 .\",\n \"FIG.\",\n \"6 shows an embodiment 10 b of an adaptive comparator circuit according to the invention, which can be used as an alternative to that shown in FIG.\",\n \"1 .\",\n \"The diode shown in FIG.\",\n \"5 has been replaced by a second voltage source 7 , whereby its positive pole 7 a has been put in the place of the anode 5 a and the negative pole 7 b has been put in the place of the cathode 5 b .\",\n \"The fundamental voltage distance dU shown in FIG.\",\n \"1 has now been replaced by the voltage of the voltage source 7 , that is to say, the fundamental voltage distance dU between U ref1 and U signal is now defined by the voltage supplied by the second direct voltage source.\",\n \"If the voltage supplied by the second voltage source 7 can be regulated or adjusted, then the fundamental voltage distance dU can be varied as needed.\",\n \"FIG.\",\n \"7 shows a schematic block diagram of an embodiment of an acoustic distance sensor according to the invention in which the adaptive comparator circuit 10 a shown in FIG.\",\n \"5 is used.\",\n \"A control unit 31 transmits short signal pulses at times, preferably at regular intervals, each lasting, for example, 25 microseconds, to an oscillator 32 .\",\n \"When a signal pulse is present, the oscillator 32 transmits an alternating voltage to a sound transducer 34 that is excited by the latter to emit sound waves.\",\n \"The sound transducer 34 is capable of receiving sound waves reflected, for example, from an object and coming back to the sound transducer 34 as an echo, and of converting them into an electric received signal, whereby the propagation time of the sound waves is a measure of the distance from the object.\",\n \"The received signal is fed to an envelope curve shaper 37 which forms the envelope curve of the received signal and transmits it via an electric terminal 8 to the adaptive comparator circuit 10 a shown in FIG.\",\n \"5 .\",\n \"In a preferred embodiment of the invention, the signal pulses 41 that the control unit 31 transmits to the oscillator 32 are synchronized with the control signals S St that the control unit 2 transmits to the switch 3 and this is done in such a way that the control signals S St each begin shortly before the signal pulses 41 as will still be explained below with reference to FIGS.\",\n \"9 a and 9 b .\",\n \"In this manner, it is ensured that, already at the beginning of the transmittal phase, the threshold voltage U ref1 is greater than the voltage signal U signal , so that no dummy switching signal is triggered.\",\n \"In another preferred embodiment of the invention, the signal pulses 41 are advantageously synchronized with the control signals S St in such a way that the control signals S St each begin before or simultaneously with the end of the signal pulses 41 .\",\n \"This ensures that, without any time lag, the threshold voltage U ref1 is supplied to the dropping voltage signal U signal at the fundamental voltage distance dU already at the beginning of the relaxation phase.\",\n \"The sensitivity of the distance sensor is defined by the fundamental voltage distance dU.\",\n \"FIG.\",\n \"8 shows a schematic block diagram of another embodiment of an acoustic distance sensor according to the invention, in which the adaptive comparator circuit 10 a shown in FIG.\",\n \"5 is likewise used.\",\n \"The distance sensor shown in FIG.\",\n \"8 —in comparison to that shown in FIG.\",\n \"7 —additionally has a driver 33 that amplifies the alternating voltage supplied by the oscillator 32 and transmits it to the sound transducer 34 .\",\n \"Moreover, before the received signal is fed into the envelope curve shaper 37 , it is passed through a voltage limiter 35 and through a logarithmic amplifier 36 c. Among other things, the voltage limiter 35 counters an overmodulation of the amplifier.\",\n \"The amplifier 36 c serves to adapt the low input level of just a few microvolts to the higher input level of the subsequent evaluation circuit.\",\n \"In FIG.\",\n \"8 , in order to facilitate the synchronization, the signal transmitter 2 is accommodated in the control unit 31 .\",\n \"For example, the signal transmitter 2 and the control unit 31 can be combined in one single unit, for example, in a microprocessor, that concurrently fulfills the function of the signal transmitter 2 as well as that of the control unit 31 .\",\n \"In FIG.\",\n \"8 , the first switching signal S 1 of the control unit 31 is also supplied for evaluation purposes, especially for determining the propagation time and calculating the distance.\",\n \"FIGS.\",\n \"9 and 10 serve to further explain the advantageous effects of the invention, whereby the time axes of all of the curves have been selected identically in order to illustrate the course over time.\",\n \"FIG.\",\n \"9 shows a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) a voltage signal and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.\",\n \"8 , whereby an echo occurs during the quiescent phase.\",\n \"The control unit 31 transmits short signal pulses 41 to the oscillator 32 , as a result of which the sound transducer 34 is excited so as to emit a threshold voltage packet ( FIG.\",\n \"9 , Curve a).\",\n \"Moreover, the signal transmitter 2 transmits a control signal S St to the switch 3 , which is synchronized with the signal pulse 41 in such a way that the control signal S St begins shortly before the signal pulse 41 and ends at the same time as or during the presence of the former ( FIG.\",\n \"9 , Curve b).\",\n \"FIG.\",\n \"9 , Curve c, shows the course of the voltage signal U signal (solid curve) that is identical to the envelope curve, and the course of the threshold voltage U ref1 (dotted curve).\",\n \"The course of the U signal during the transmittal and relaxation phase and at the beginning of the quiescent phase corresponds essentially to that of the envelope curve 53 , which was explained with reference to FIG.\",\n \"4 .\",\n \"While the control signal S St is present at the switch 3 , the latter is opened, so that a threshold voltage U ref1 is established which is identical to the voltage supplied by the first direct voltage source 4 a , 4 b ( FIG.\",\n \"9 , Curve c).\",\n \"Therefore, this has been advantageously selected in such a way that it is greater than the maximum of the voltage signal, so that the adaptation of the threshold voltage to the voltage signal begins immediately after the opening of the switch 3 .\",\n \"In the present example, the maximum possible magnitude of the voltage signal is supplied by the voltage limiter 35 or by the overmodulation limit of the amplifier 36 c ( FIG.\",\n \"8 ).\",\n \"According to the invention, after the end of the control signal S St , a threshold voltage U ref1 is established that is greater than the voltage signal U signal by the voltage distance dU.\",\n \"Therefore, the threshold voltage U ref1 drops immediately after the end of the control signal by a certain quantity.\",\n \"From this point in time on, the threshold voltage proceeds parallel-offset by a fundamental voltage distance dU with respect to that of the voltage signal U signal as long as the latter does not increase.\",\n \"A dummy switching signal is not triggered.\",\n \"If, however, the voltage signal U signal increases, the threshold voltage remains constant so that a first echo peak 60 of the voltage signal U signal leads to a triggering of the comparator 1 as soon as the voltage signal U signal exceeds the threshold voltage U ref1 .\",\n \"In this case, the comparator 1 emits a first switching signal S 1 ( FIG.\",\n \"9 , Curve d).\",\n \"FIG.\",\n \"10 shows a schematic representation of a typical course over time of a) a voltage signal and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.\",\n \"8 , whereby an echo occurs during the relaxation phase and another echo occurs during the quiescent phase.\",\n \"The courses over time of the signal pulse 41 and of the control signal S St are identical to those shown in FIG.\",\n \"9 .\",\n \"During the relaxation time, an echo signal occurs, which manifests itself in a second echo peak 61 of the voltage signal U signal ( FIG.\",\n \"10 , Curve a).\",\n \"Up until the beginning of the second echo peak 61 , the curves correspond to those shown in FIG.\",\n \"9 , Curve c. According to the invention, however, the decrease of the threshold voltage U ref1 comes to a halt as soon as the voltage signal U signal is no longer dropping, and a first switching signal S 1a is triggered by the second echo peak 61 .\",\n \"After the second echo peak 61 that occurs during the relaxation time, the fundamental voltage distance dU is once again established between the threshold voltage U ref1 and the voltage signal U signal .\",\n \"During the quiescent phase, another, third echo peak 62 of the voltage signal U signal occurs, as a result of which another switching signal S 1b is triggered.\",\n \"Now reference will be made to FIGS.\",\n \"11 to 13 , which show additional advantageous embodiments of an adaptive comparator circuit according to the invention.\",\n \"FIG.\",\n \"11 shows a preferred embodiment 10 c of the invention which differs from the adaptive comparator circuit 10 a shown in FIG.\",\n \"6 as follows: a) The first switch is a transistor 103 .\",\n \"The base 103 a of the transistor 103 serves as the control-current terminal, the emitter 103 b serves as the switch input and the collector 103 c as the switch output.\",\n \"b) A first resistor 11 is connected between the first pole 4 a and the first threshold value input 1 a , and a second resistor 12 is connected between the first threshold value input 1 a and the second pole 4 b , so that the first resistor 11 and the second resistor 12 form a first voltage divider 11 , 12 .\",\n \"Therefore, the magnitude of the first threshold voltage U ref1 counters the voltage that is supplied by the first voltage divider.\",\n \"Thus, through the use of the first voltage divider 11 , 12 , the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal after the voltage signal U signal has died out can advantageously be changed by the fundamental voltage distance dU, to a value dU a .\",\n \"When this embodiment 10 c of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be changed and, in particular increased.\",\n \"This is advantageous for many applications such as, for example, for the detection of small objects.\",\n \"The first voltage divider can be an adjustable potentiometer whose pick-up is connected to the switch output.\",\n \"c) A sixth resistor 16 is connected between the control output 2 a of the signal transmitter 2 and the base 103 a , and a seventh resistor 17 is connected between the base 103 a and the first pole 4 a , which is a plus pole in the example shown.\",\n \"The purpose of these resistors was already explained above.\",\n \"d) The diode 5 is connected in series to an eighth resistor 18 which serves to slow down the discharging of the capacitor 6 and thus to reduce the speed with which the threshold voltage U ref1 is adapted to the voltage signal U signal .\",\n \"The eighth resistor also serves to smooth the course of the threshold voltage U ref1 with respect to very rapid fluctuations of the voltage signal U signal .\",\n \"FIG.\",\n \"12 shows another preferred embodiment 10 d of the invention, which differs from the adaptive comparator circuit 10 c shown in FIG.\",\n \"11 in that, before the voltage signal U signal reaches the first signal input 1 b , it passes through an impedance transformer.\",\n \"This embodiment is especially advantageous when the voltage signal arriving at the electric terminal 8 is high-ohmic, whereby said voltage signal can be the envelope curve voltage generated, for example, by an envelope curve shaper.\",\n \"FIG.\",\n \"13 shows another preferred embodiment 10 e of the invention which differs from the adaptive comparator circuit 10 a shown in FIG.\",\n \"6 as follows: a) The first switch is a transistor 103 .\",\n \"The base 103 a of the transistor 103 serves as the control-current terminal, the emitter 103 b serves as the switch input and the collector 103 c as the switch output.\",\n \"b) A second comparator 20 is used that has a second threshold value input 20 a and a second signal input 20 b , and that emits a second switching signal S 2 when a greater voltage is present at the second signal input 20 b than at the second threshold value input 20 a. c) The first pole 4 a of the first voltage source 4 a , 4 b is connected to the second threshold value input 20 a via a third resistor 13 .\",\n \"The second threshold value input 20 a is connected to the second pole 4 b via a fourth resistor 14 , so that the third and the fourth resistors 13 , 14 form a second voltage divider which can be configured as a potentiometer.\",\n \"d) The collector 103 c that serves as the switch output and the second threshold value input 20 a are connected via a fifth resistor 15 .\",\n \"e) A second switch 23 is connected in series to the diode 5 and the former can be regulated or activated by the second switching signal S 2 in such a way that it is in the closed state when the second comparator 20 emits the second switching signal S 2 , and otherwise it is in the open state.\",\n \"The second switch 23 can be a transistor.\",\n \"f) A sixth resistor 16 is connected between the control output 2 a of the signal transmitter 2 and the base 103 a , and a seventh resistor 17 is connected between the base 103 a and the first pole 4 a , which is a plus pole in the example shown.\",\n \"The purpose of these resistors was already explained above.\",\n \"g) The diode 5 is connected in series to an eighth resistor 18 .\",\n \"The purpose of this resistor was already explained with reference to FIG.\",\n \"11 .\",\n \"h) Before reaching the first signal input 1 b , the voltage signal passes through an impedance transformer 21 whose purpose was already explained with reference to FIG.\",\n \"12 .\",\n \"Due to the second voltage divider 13 , 14 , a voltage U ref2 is present at the second threshold value input 20 a .\",\n \"As long as the second switch 23 is closed, the first threshold voltage U ref1 follows the dropping voltage signal U signal , parallel-offset by the fundamental voltage distance dU, as explained above.\",\n \"According to the invention, however, the second switch opens as soon as the voltage signal U signal exceeds the second threshold voltage U ref2 .\",\n \"As a result, the discharge of the capacitor 6 and thus the dropping of the first threshold voltage U ref1 are ended.\",\n \"Hence, in this embodiment of the invention, the capacitor 6 can only discharge until the first threshold voltage U ref1 has dropped to the magnitude of the second threshold voltage U ref2 supplied by the second potentiometer 13 , 14 .\",\n \"Thus, through the use of the second voltage divider 13 , 14 , the voltage distance by which the first threshold voltage is advantageously changed parallel-offset after the decay of the voltage signal U signal with respect to voltage signal U signal , from the fundamental voltage distance dU to a value dU b .\",\n \"The second voltage divider 13 , 14 can be an adjustable potentiometer whose pick-up is connected to the second threshold value input 20 a.\",\n \"The fifth resistor 15 is connected between the switch output 103 c and the second threshold value input 20 a .\",\n \"Therefore, the magnitude of the first threshold voltage U ref1 counters the voltage that is supplied by the second voltage divider 13 , 14 .\",\n \"In particular, through the use of the fifth resistor 15 , it can additionally be achieved that the voltage distance by which the first threshold voltage is parallel-offset with respect to the voltage signal U signal strives towards a value dU b that—depending on the voltage supplied by the second voltage divider 13 , 14 —can advantageously be not only larger but also smaller than the fundamental voltage distance dU.\",\n \"When this embodiment 10 e of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be either increased or decreased by means of the second voltage divider 13 , 14 .\",\n \"All of the embodiments of an adaptive comparator circuit according to the invention and thus also the embodiments 10 b , 10 c , 10 d and 10 e illustrated in FIG.\",\n \"6 and FIGS.\",\n \"11 to 13 can be used according to the invention in the acoustic distance sensor shown in FIG.\",\n \"8 instead of the adaptive comparator circuit 10 a shown there by way of an example.\"\n ],\n [\n \"TECHNICAL AREA The invention relates to an adaptive comparator circuit that can be used particularly for reducing the short range of an acoustic distance sensor, as well as to an acoustic distance sensor with such a circuit.\",\n \"STATE OF THE ART Acoustic distance sensors are used to measure distances by means of the pulse-propagation time method.\",\n \"In this process, a sound transducer emits a short sound pulse that propagates in a medium (gas, liquid or solid).\",\n \"This pulse is reflected, for example, on an object and comes back as an echo.\",\n \"The distance to the object can be determined on the basis of the propagation time of the sound pulse and the sound velocity in the medium.\",\n \"As a rule, the frequency of the sound used for such measurements lies in the ultra-sound range, typically in the range between 20 kHz and 500 kHz, for example, at 400 kHz.\",\n \"The sound transducer, which in particular can be a piezoelectric ceramic element, is normally used to transmit the sound pulse as well as to receive the echo.\",\n \"Typically, sound pulses of about 3 to 50 oscillations periods each are emitted.\",\n \"This results in time durations of typically about 6 to 2050 μs for the emitted sound pulses.\",\n \"During this time, i.e.\",\n \"the transmitting phase, the sound transducer oscillates at a high oscillation amplitude.\",\n \"This time duration is followed by a relaxation time in which the oscillation amplitude dies out—usually exponentially.\",\n \"The relaxation time is typically, for example, 300 μs to 3 ms.\",\n \"The time interval between the end of the relaxation time and the emission of the next sound pulse will be called “quiescent phase” below.\",\n \"Due to noise and other interferences, however, the oscillation amplitude does not drop completely to zero even during the quiescent phase, but rather remains at a certain constant mean disturbance level.\",\n \"Therefore, during the receiving phase that follows the transmitting phase, even in the absence of a useful echo, the oscillation amplitude that dies out during the relaxation time and the mean disturbance level consistently form a finite background signal that is very large immediately after the end of the transmitting phase, that generally decreases exponentially within the relaxation time and that finally remains at the disturbance level.\",\n \"A useful echo signal is superimposed on this background and, as a rule, is detected by means of a comparator that emits a switching signal when the received signal is greater than a certain reference value that will be called “threshold voltage” hereinafter.\",\n \"On the one hand, the threshold voltage should be as low as possible so that even weak useful echo signals, e.g.\",\n \"of small objects, can be detected, which translates into high sensitivity.\",\n \"On the other hand, the threshold voltage has to lie above the disturbance level by a certain safety margin so as to avoid erroneous triggering of the comparator during the quiescent phase.\",\n \"When the threshold voltage is constant over time, useful echo signals can be detected as soon as the background signal has dropped below the threshold voltage as a result of the relaxation of the sound transducer.\",\n \"Before this time, however, that is to say, during the transmitting phase and during part of the relaxation time, the background signal is greater than the threshold voltage so that the comparator—irrespective of the presence of an echo—is triggered in any case during this time.\",\n \"Thus, during this time, called dead time, the system is fundamentally insensitive to the reception of echoes.\",\n \"Therefore, the measurement of distances below a certain minimum distance is impossible.\",\n \"This minimum distance is called the “short range” and is typically a few centimeters.\",\n \"The existence of the short range often has a detrimental effect, for example, wherever due to space constraints, the sensor is to be situated as close as possible to the object to be detected.\",\n \"Various methods are known for reducing the short range.\",\n \"The simplest method is to increase the threshold voltage.\",\n \"This causes the background signal, which dies out after the transmitting phase, to drop below the threshold voltage at an earlier point in time within the relaxation time.\",\n \"However, this method entails the serious drawback that an increase in the threshold voltage translates into a reduction of the system sensitivity, as a result of which weak echoes can no longer be detected.\",\n \"Another method lies in conveying the received signal through an amplifier whose amplification factor can be changed over time.\",\n \"The amplification factor is minimal immediately after the transmittal pulse and is then boosted, for example, by means of a suitable electronic circuit as a function of the time.\",\n \"In this manner, despite a constant threshold voltage over time, a reduction of the short range can be combined with a high sensitivity during the quiescent phase.\",\n \"Of course, for this purpose, the course over time of the amplification factor has to be adapted to the relaxation time.\",\n \"The drawback is that a one-time optimal adaptation is not sufficient here, since the relaxation time of the sound transducer is not constant but rather, it depends on the medium on the temperature, on the age of the sound transducer and on other influencing factors.\",\n \"Therefore, in actual practice, either complex compensation measures have to be undertaken or re-adaptations have to be made repeatedly, or else a large safety margin has to be maintained right from the start for purposes of optimal adaptation, which diminishes the system sensitivity, at least during the relaxation time.\",\n \"Another method is to provide a threshold voltage that can be changed over time.\",\n \"In the ideal case, this threshold voltage drops at a gradient that is similar to the decaying background signal and, at the beginning of the quiescent phase, turns into a constant value, whereby the threshold voltage nevertheless always remains sufficiently greater than the background signal.\",\n \"This method can be additionally improved by subjecting the background signal to a logarithmic amplification so that the decrease is linear instead of exponential.\",\n \"The drawback here, however, is that a one-time adaptation of the time behavior of the threshold voltage is not sufficient to ensure optimal operation over the long run, since here, too, the individual dying-out behavior of the sound transducer is not taken into account on a case-to-case basis.\",\n \"Another method for reducing the short range lies in the propagation time-dependent control of the length of the emitted sound pulses.\",\n \"In the case of a short propagation time, that is to say, in the case of an object that is close, very short sound pulses are used to that the oscillation amplitude of the sound transducer does not fully develop and thus the short range is reduced.\",\n \"Another method lies in the use of threshold voltage that is dynamically changed by means of a microprocessor and a D/A transducer.\",\n \"The microprocessor regulates the D/A transducer in such a way that a dynamic threshold is created that always lies above the dying-out signal.\",\n \"Here, an A/D transducer measures the decaying transmission signal.\",\n \"These methods, however, call for considerable hardware and software resources.\",\n \"Therefore, the invention is based on the objective of simply and inexpensively providing a comparator circuit with which, in every single case, the course over time of the threshold voltage is automatically adapted to the individual decay behavior of the sound transducer and the magnitude of the disturbance level.\",\n \"This objective is achieved according to the invention by an adaptive comparator circuit, especially for an acoustic distance sensor, comprising a first comparator having a first threshold value input to which a first threshold voltage is applied and having a first signal input to which a voltage signal (Usignal) is applied and, when a voltage signal is present that is greater than the first threshold voltage, said comparator emits a first switching signal, characterized by a signal transmitter, especially a microcontroller or microprocessor, that at times emits a control signal, a first switch that has a control means, a switch input and a switch output, and that can be regulated or activated through the effect of the control signal on the control means in such a way that it is in the closed state when the signal transmitter emits a control signal, and otherwise it is in the open state, or vice versa, whereby the switch input is connected to the positive or negative first pole of a first direct voltage source and the switch output is connected to the first threshold value input, so that the first threshold voltage is equal to the voltage present at the switch output, a capacitor via which the switch output is connected to the negative or positive pole of the first direct voltage source, and a diode via which the switch output is connected to the first signal input in such a way that the anode or the cathode of the diode is connected to the switch output.\",\n \"This is the first basic form of an adaptive comparator circuit according to the invention.\",\n \"According to the invention, instead of the diode, a second direct voltage source can be used whose positive pole is in the place of the anode of the diode and whose negative pole is in the place of the cathode of the diode.\",\n \"This is the second basic form of an adaptive comparator circuit according to the invention.\",\n \"These two basic forms can be augmented advantageously as will still be explained below.\",\n \"An acoustic distance sensor according to the invention comprises a control unit that at times transmits signal pulses to an oscillator which, during the presence of a signal pulse, transmits an alternating voltage to a sound transducer that is excited by the alternating voltage to emit sound waves and that is capable of receiving sound waves coming back to the sound transducer in the form of an echo as a result of reflection, and of converting them into an electric received signal, and it also comprises an envelope curve shaper to which the received signal is fed and which forms the envelope curve of the received signal and emits it as a signal voltage, characterized in that the signal voltage is fed to an adaptive comparator circuit comprising a first comparator having a first threshold value input to which a first threshold voltage is applied and a first signal input to which a voltage signal is applied and, when a voltage signal is present that is greater than the first threshold voltage, said comparator emits a first switching signal, a signal transmitter, especially a microcontroller or microprocessor, that at times emits a control signal, a first switch that has a control means, a switch input and a switch output, and that can be regulated or activated through the effect of the control signal on the control means in such a way that it is in the closed state when the signal transmitter emits a control signal, and otherwise it is in the open state, or vice versa, whereby the switch input is connected to the positive or negative first pole of a first direct voltage source and the switch output is connected to the first threshold value input, so that the first threshold voltage is equal to the voltage present at the switch output, a capacitor via which the switch output is connected to the negative or positive pole of the first direct voltage source, and a diode via which the switch output is connected to the first signal input in such a way that the anode or the cathode of the diode is connected to the switch output.\",\n \"An acoustic distance sensor according to the invention thus comprises the adaptive comparator circuit according to the invention.\",\n \"Preferably, the control signal emitted by the signal transmitter is an electric control signal and the first switch is an electrically or electronically controlled switch, for example, a transistor, whose base terminal or gate terminal function as a control-current terminal or, for example, a relay.\",\n \"In another embodiment of the invention, the control signal emitted by the signal transmitter is an optical control signal that is emitted, for example, by a light diode, and the first switch is an optically controlled switch, e.g.\",\n \"a photoresistor.\",\n \"As soon as the signal transmitter emits a control signal, the first switch is in the closed state, so that the capacitor is charged with the voltage supplied by the first voltage source.\",\n \"After the end of the control signal, the switch is in the open state.\",\n \"The capacitor voltage and thus the first threshold voltage present at the first threshold value input now assumes a value that corresponds to the sum of the voltage signal and the diode flow voltage or else to the sum of the voltage signal and the voltage of the second direct voltage source.\",\n \"The diode flow voltage or the voltage of the second direct voltage source are referred to below as “fundamental voltage distance” dU.\",\n \"The task of the diode or of the second direct voltage source is to allow the discharging of the capacitor and thus a drop in the threshold voltage for as long as and only until the difference of the threshold voltage minus the voltage signal has dropped to the fundamental voltage distance dU, but to prevent a recharging of the capacitor and thus an increase in the threshold voltage if the voltage signal increases.\",\n \"Therefore, the time behavior of the threshold voltage proceeds parallel-offset by a fundamental voltage distance dU with respect to that of the voltage signal as long as the voltage signal does not increase.\",\n \"In contrast, if the voltage signal increases, the threshold voltage remains constant.\",\n \"Consequently, a maximum, e.g.\",\n \"a peak, of the voltage signal leads to the triggering of the comparator as soon as the voltage signal exceeds the threshold voltage.\",\n \"If the voltage signal drops again after having increased, then the threshold voltage follows the voltage signal, once again parallel-offset, as soon as the fundamental, voltage distance dU has been reached again.\",\n \"Hence, according to the invention, the threshold voltage is specified by the magnitude of the voltage signal at every point in time of the receiving phase.\",\n \"If the course over time of the voltage signal is changed, then there is automatically a correspondingly changed course over time of the threshold voltage.\",\n \"Thus, the course over time of the threshold voltage is automatically adapted to that of the voltage signal.\",\n \"The diode flow voltage is temperature-dependent.\",\n \"It decreases as the temperature rises.\",\n \"Consequently, an acoustic distance sensor according to the invention becomes more sensitive as the temperature rises.\",\n \"On the other hand, the attenuation of ultrasound waves generally increases as the temperature rises.\",\n \"Therefore, the resultant sensitivity loss is advantageously compensated for partially with the use of a diode to create the fundamental voltage distance dU.\",\n \"As already explained above, according to the invention, instead of the diode, a second direct voltage source can be used whose positive pole is in the place of the anode of the diode and whose negative pole is in the place of the cathode of the diode.\",\n \"In this case, the fundamental voltage distance dU is defined by the voltage supplied by the second direct voltage source.\",\n \"The basic forms described above of adaptive comparator circuits according to the invention can be advantageously augmented as will be explained below.\",\n \"In a preferred embodiment of the invention, one of the above-described basic forms has been augmented by a first resistor that is connected between the first pole and the first threshold value input, and by a second resistor that is connected between the first threshold value input and the second pole, so that the first resistor and the second resistor form a first voltage divider.\",\n \"Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the first voltage divider.\",\n \"Thus, through the use of the first voltage divider, the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal after the voltage signal Usignal has died out can advantageously be changed by the fundamental voltage distance dU, to a value dUa.\",\n \"When this embodiment of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be changed and, in particular increased.\",\n \"The first voltage divider can be an adjustable potentiometer whose pick-up is connected to the switch output.\",\n \"Another preferred embodiment of an adaptive comparator circuit according to the invention comprises the following in addition to the basic forms described above: a second comparator that has a second threshold value input and a second signal input, and that emits a second switching signal when a greater voltage is present at the second signal input than at the second threshold value input, a third resistor via which the first pole of the first direct voltage source is connected to the second threshold value input, a fourth resistor that, on the one hand, is connected to the second threshold value input and, on the other hand, to the second pole of the first voltage source, so that the third resistor and the fourth resistor form a second voltage divider, and a second switch that is connected in series to the diode and that can be regulated or activated by the second switching signal in such a way that it is in the closed state when the second comparator emits the second switching signal, and otherwise it is in the open state.\",\n \"Due to the second voltage divider, a voltage is present at the second threshold value input that will be called “second threshold voltage” Uref2 below.\",\n \"As long as the second switch is closed, the first threshold voltage follows the dropping voltage signal Usignal with the fundamental voltage distance dU, as explained above.\",\n \"According to the invention, however, the second switch opens as soon as the voltage signal Usignal exceeds the second threshold voltage Uref2.As a result, the discharge of the capacitor and thus the dropping of the first threshold voltage are ended.\",\n \"Hence, in this embodiment of the invention, the capacitor can only discharge until the first threshold voltage Uref1 has dropped to the value of the second threshold voltage Uref2.Thus, through the use of the second voltage divider, the voltage distance by which the first threshold voltage is changed parallel-offset with respect to voltage signal Usignal after the decay of the voltage signal Usignal, advantageously by the fundamental voltage distance dU to a value dUb.\",\n \"When this embodiment of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor according to the invention, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be reduced.\",\n \"The second voltage divider can be an adjustable potentiometer whose pick-up is connected to the second threshold value input.\",\n \"In another configuration of this embodiment of the invention, a fifth resistor is connected between the switch output and the second threshold value input.\",\n \"Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the second voltage divider.\",\n \"In particular, through the use of the fifth resistor, it can be additionally achieved that the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal strives towards a value dUb that—depending on the voltage supplied by the second voltage divider—can advantageously be not only larger but also smaller than the fundamental voltage distance dU.\",\n \"When this embodiment of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be either increased or decreased.\",\n \"In another advantageous embodiment of the invention, the voltage signal passes through a voltage follower or impedance transformer before reaching the first signal input.\",\n \"In another embodiment of the invention, a sixth resistor is connected between the control output and the control-current terminal for purposes of limiting the current flow.\",\n \"In another embodiment of the invention, the first switch is a transistor whose base functions as a control-current terminal, whereby a seventh resistor is connected between the base and the first pole of the first direct voltage source.\",\n \"In another embodiment of the invention, the diode or the second direct voltage source is connected in series with an eighth resistor.\",\n \"This brings about a slowing down of the discharging of the capacitor as well as a smoothing of the threshold voltage, which is advantageous for many applications.\",\n \"The voltage supplied by the first voltage source is preferably selected such that it is greater in magnitude than the value of the maximum of the voltage signal.\",\n \"In this manner, it is prevented that the first comparator is triggered while the first switch is still closed, that is to say, while the threshold voltage cannot yet adapt to the voltage signal.\",\n \"Moreover, in this manner, it is advantageously achieved that, in any case, the adaptation of the threshold voltage to the voltage signal can begin immediately after the opening of the first switch.\",\n \"An adaptive comparator circuit according to the invention can be used especially advantageously in an acoustic distance sensor.\",\n \"In this case, the voltage signal is advantageously formed by the envelope curve of the received signal supplied by the sound transducer.\",\n \"This received signal consists of a superimposition of the background signal with the useful echo signal.\",\n \"The formation of the envelope curve, for example, by means of a rectifier circuit, is known from the state of the art and will thus not be elaborated upon in greater depth here.\",\n \"In the absence of an echo, the envelope curve dies out exponentially during the relaxation phase and, at the beginning of the quiescent phase, turns into a horizontal straight line.\",\n \"Such an envelope curve will be called “background envelope curve” below.\",\n \"The threshold voltage adapts according to the invention to the individual course of the background envelope curve in that it follows said background envelope curve parallel-offset by a voltage distance dU or dUa or dUb.\",\n \"An echo causes a peak to be superimposed on the background envelope curve so that the voltage signal increases.\",\n \"According to the invention, the threshold voltage does not follow this increase, so that the comparator is triggered and the echo can thus be detected.\",\n \"As already mentioned above, the voltage supplied by the first voltage source is preferably selected such that it is greater in magnitude than the value of the maximum of the voltage signal.\",\n \"Since in this case, the threshold voltage already follows the background envelope curve at the voltage distance dU at the beginning of the relaxation phase, the voltage signal in the absence of an echo is not greater than the threshold voltage at any point in time, especially not during the entire relaxation phase.\",\n \"Therefore, throughout the entire receiving phase, the comparator can only be triggered by an echo, but not by the decay behavior of the sound transducer.\",\n \"Thus, an acoustic distance sensor according to the invention is fundamentally able to detect an echo during the quiescent phase as well as especially during the entire relaxation phase.\",\n \"As a result, the undesired short range of the sensor is considerably reduced according to the invention.\",\n \"BRIEF DESCRIPTION OF THE DRAWING IN WHICH THE FOLLOWING IS SHOWN FIG.\",\n \"1—a schematic block diagram of a variant of an acoustic distance sensor according to the state of the art, FIG.\",\n \"2—a schematic representation of a typical course over time of a) a signal pulse, b) an amplification factor, c) an envelope curve and d) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.\",\n \"1, FIG.\",\n \"3—a schematic block diagram of another variant of an acoustic distance sensor according to the state of the art, FIG.\",\n \"4—a schematic representation of a typical course over time of a) a timing pulse, b) an envelope curve and c) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.\",\n \"3, FIG.\",\n \"5—a circuit diagram of an embodiment of a comparator circuit according to the invention, FIG.\",\n \"6—a circuit diagram of an alternative embodiment of a comparator circuit according to the invention, FIGS.\",\n \"7 and 8—a schematic block diagram of embodiments of an acoustic distance sensor according to the invention, FIG.\",\n \"9—a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) an envelope curve and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.\",\n \"8, whereby an echo occurs during the quiescent phase, FIG.\",\n \"10—a schematic representation of a typical course over time of a) an envelope curve and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.\",\n \"8, whereby an echo additionally occurs during the relaxation phase, and FIGS.\",\n \"11 to 13—circuit diagrams of further embodiments of comparator circuits according to the invention.\",\n \"FIGS.\",\n \"1 to 4 serve to further illustrate the state of the art.\",\n \"FIG.\",\n \"1 shows a schematic block diagram of a variant of an acoustic distance sensor 30a with which a measure according to the state of the art has been taken in order to reduce the short range.\",\n \"The distance sensor 30a shown in FIG.\",\n \"1 comprises a control unit 31, an oscillator 32, a driver 33, a sound transducer 34, a voltage limiter 35, an amplifier 36a with regulatable amplification factor, an envelope curve shaper 37, a comparator 1 as well as an amplification factor actuation stage 38a.\",\n \"The control unit 31 transmits short signal pulses 41 to the oscillator 32.The latter oscillates when a signal pulse is applied, thus emitting an alternating voltage of, for example, 400 kHz, which is then amplified by the driver 33 and subsequently used to excite the sound transducer 34 so that the latter is excited so as to oscillate, i.e.\",\n \"to emit sound waves.\",\n \"Corresponding to the short signal pulses, the sound transducer 34 emits short sound wave packets that constitute the transmittal pulse of the distance sensor 30a.\",\n \"During the transmitting phase, the sound transducer 34 oscillates at a high oscillation amplitude.\",\n \"This period of time is followed by a relaxation time in which the oscillation amplitude dies out—usually exponentially.\",\n \"In the subsequent quiescent phase, the oscillation amplitude generally remains at a constant mean disturbance level.\",\n \"The signal emitted by the sound transducer and thus also echoes returning after the distance-proportional propagation time are fed via the voltage limiter 35 to the amplifier 36a with a regulatable amplification factor.\",\n \"The voltage limiter keeps the high voltage used for exciting the sound transducer away from the amplifier 36a Using, for example, an internal rectifier circuit, the envelope curve shaper 37 forms the envelope curve of the voltage limiter 35 on the basis of the alternating voltage signal emitted by said voltage limiter 35 and transmits to the comparator 1 an envelope curve voltage signal whose course over time corresponds to that of the envelope curve.\",\n \"The comparator 1 transmits a switching signal to the control unit 31 for evaluation purposes when the envelope curve voltage signal is greater than a first steadily supplied threshold voltage.\",\n \"In particular, the comparator then emits a switching signal when an echo signal is received whose magnitude exceeds that of the threshold voltage.\",\n \"The amplification factor of the amplifier 36a is regulated by the amplification factor actuation stage 38a in a time-dependent manner such that the amplification factor is minimal during and immediately after the transmittal pulse and grows as soon as the oscillation amplitude of the sound transducer 34 has died out to such an extent that the magnitude of the received signal is no longer limited by the voltage limiter 35.Therefore, the growth of the amplification factor does not begin completely simultaneously with the end of the signal pulse, but rather delayed by a certain lag time dt with respect thereto (FIG.\",\n \"2).\",\n \"The amplification factor actuation stage 38a receives the time information necessary for synchronizing the time behavior of the amplification factor with the rhythm of the transmittal pulse from the control unit 31.In this manner a reduction of the short range can be achieved.\",\n \"The course over time can advantageously be selected in such a way that the distance-dependent attenuation of the echo is countered and the system sensitivity remains constant.\",\n \"FIG.\",\n \"2 shows a schematic representation of a course over time of a) a signal pulse, b) an amplification factor of the amplifier 36a, c) an envelope curve and threshold voltage as well as d) a comparator output signal, that can typically occur during the operation of the distance sensor shown in FIG.\",\n \"1.The time axes of the curves a) to d) are selected identically in order to illustrate the courses over time.\",\n \"As was already explained with reference to FIG.\",\n \"1, during the signal pulse 41, the amplification factor 42 reaches a minimum 42a that lasts longer than the signal pulse 41 by a certain lag time dt, and then it grows monotonously over time.\",\n \"During the signal pulse 41, the envelope curve voltage 43 reaches a maximum (envelope curve segment 43a) at which it still remains during the lag time dt after the end of the signal pulse 41.Then the envelope curve voltage 43 drops during the relaxation phase (envelope curve segment 43b), whereby the course of the envelope curve 43b results from the multiplication of, for example, the exponential decay of the sound transducer amplitude by the growing amplification factor.\",\n \"The temporary lowering of the amplification factor during and after the signal pulse causes the envelope curve voltage 43 to fall below the threshold voltage 44 earlier than would be the case if the amplification factor were not lowered.\",\n \"The advantageous result of this is a reduction of the short range.\",\n \"In the quiescent phase, the envelope curve voltage 43 finally reaches a low constant level (envelope curve segment 43b).\",\n \"The threshold voltage 44 is constant over time.\",\n \"Since the comparator 1 emits a switching signal, as soon as the envelope curve voltage 43 exceeds the threshold voltage 44, in a disadvantageous manner, at the beginning of the signal pulse 41, a dummy switching signal 45 is always triggered that lasts until the envelope curve voltage 43 has dropped below the threshold value 44.An echo signal (envelope curve segment 43d) causes a useful switching signal 46 so that the control unit has to be capable of distinguishing a useful switching signal 46 from a dummy switching signal 45 which, without the presence of an echo signal, was caused exclusively by the signal pulse 41.The capability to make such a distinction requires complex hardware or software resources and thus entails disadvantages.\",\n \"Further drawbacks associated with a distance sensor of the type illustrated in FIG.\",\n \"1 were already explained above.\",\n \"As already explained above, another method of reducing the short range lies in providing a threshold voltage that can be changed over time.\",\n \"For purposes of further illustrating the state of the art, FIG.\",\n \"3 shows a schematic block diagram of a corresponding variant of an acoustic distance sensor 30b.\",\n \"The distance sensor 30b shown in FIG.\",\n \"3 comprises a control unit 31, an oscillator 32, a driver 33, a sound transducer 34, a voltage limiter 35, a preferably logarithmic amplifier 36b, an envelope curve shaper 37, a comparator 1 as well as a threshold value actuation stage 38b which emits a threshold voltage that can be changed over time and transmits it to the comparator 1.The essential difference from the distance sensor 30a illustrated in FIG.\",\n \"1 lies in the fact that the short range reduction is not achieved by an amplification factor that can be changed over time, but rather by a threshold voltage that can be changed over time.\",\n \"FIG.\",\n \"4 shows a schematic representation of a course over time of a) a signal pulse, b) an envelope curve and threshold value as well as c) a comparator output signal, that can typically occur during the operation of the distance sensor 30b shown in FIG.\",\n \"3.The time axes of the curves a) to c) are selected identically in order to illustrate the courses over time.\",\n \"During a signal pulse 41, the envelope curve voltage 53 reaches a maximum (envelope curve segment 53a) at which it still remains during the lag time dt after the end of the signal pulse 41.Then the envelope curve voltage 53 drops linearly during the relaxation phase (envelope curve segment 53b), since the amplifier 36b is a logarithmic amplifier.\",\n \"Finally, in the quiescent phase, the envelope curve voltage 53 reaches a low constant level (envelope curve segment 53c).\",\n \"The threshold voltage 54 is not constant over time but rather is regulated by the threshold value actuation stage 38b in such a way that it rises before every signal pulse 41, remains at a maximum value for a certain period of time (envelope curve segment 54a), then drops (curve segment 54b) and finally reaches a constant level (envelope curve segment 54c) during the quiescent phase, whereby the threshold voltage 54 remains greater than the envelope curve voltage 53, as long as no echo is received.\",\n \"The threshold value actuation stage 38b receives the necessary time information for synchronizing the time behavior of the threshold voltage 54 with the rhythm of the signal pulse 41 from the control unit 31.The advantageous result is a reduction of the short range.\",\n \"Moreover, advantageously, no dummy switching signal is triggered by the signal pulse 41.Rather, a switching signal 56 is triggered only by the rise in the envelope curve 53 (envelope curve segment 53d) associated with an echo, so that the switching signal 56 is a useful switching signal 56.Drawbacks associated with a distance sensor of the type illustrated in FIG.\",\n \"3 were already explained above.\",\n \"Now reference is made to FIG.\",\n \"5, which illustrates a schematic circuit diagram of an embodiment 10a of an adaptive comparator circuit according to the invention It comprises a signal transmitter 2, a first regulatable switch 3, a capacitor 6, a diode 5 and a first comparator 1, that has a first threshold value input 1a and a first signal input 1b.\",\n \"At the first threshold value input 1a, a threshold voltage Uref1, is present that is emitted internally in the adaptive comparator circuit according to the invention.\",\n \"At the signal input 1b, a voltage signal Usignal is present that can be, for example, an envelope curve of the received signal of a sound transducer and that is fed via a first electric terminal 8 to the adaptive comparator circuit according to the invention.\",\n \"The first comparator 1 emits a first switching signal S1 when the voltage signal Usignal is greater than the first threshold voltage Uref1.The first switching signal S1 can be tapped, for example; via a second electric terminal 9 and transmitted for evaluation purposes.\",\n \"Via a control output 2a, the signal transmitter 2 emits an electric control signal SSt at times, for example, at regular intervals.\",\n \"In the simplest case, the signal transmitter 2 can be, for instance, a manually operated signaling key.\",\n \"Moreover, the signal transmitter can be, for example, an electronic square-wave timing generator circuit with a suitable duty cycle.\",\n \"Preferably, the signal transmitter 2 is a microcontroller or microprocessor that is programmed in such a way that, at regular intervals, it emits an electric control signal SSt having a time duration of typically, for example, 20 microseconds.\",\n \"The first switch 3 has a control means 3a, a switch input 3b and a switch output 3c.\",\n \"The control means 3a is connected to the control output 2a and can be regulated by the control signal SSt in such a way that it is in the closed state when the signal transmitter 2 emits a control signal SSt, and otherwise it is in the open state.\",\n \"The first switch 3 can be, for example, a transistor.\",\n \"The switch input 3b is connected to the positive first pole 4a of a first direct voltage source 4a, 4b (not shown here).\",\n \"The switch output 3c is connected to the first threshold value input 1a of the comparator 1, so that the first threshold voltage Uref1 is equal to the voltage present at the switch output 3c.\",\n \"Thus, when the switch 3 is closed, the potential of the positive first pole 4a is present at the first threshold value input 1a of the comparator 1.The switch output 3c is also applied via a capacitor 6 to the negative second pole 4b of the first direct voltage source so that the capacitor 6 is charged with the voltage supplied by the first voltage source when the switch 3 is closed.\",\n \"The switch output 3c is also connected to the anode 5a of a diode 5.The cathode 5b of the diode 5 is connected to the first signal input 1b of the comparator.\",\n \"As soon as the signal transmitter 2 emits a control signal SSt, the first switch 3 is in the closed state.\",\n \"Therefore, the capacitor 6 is charged with the voltage supplied by the first voltage source.\",\n \"When the switch 3 is closed, this voltage is also present at the threshold value input 1a.\",\n \"After the end of the control signal SSt, the switch 3 is in the open state.\",\n \"The capacitor 6 can now discharge according to the invention via the diode 5 to such an extent until the first Uref1 reaches a value that corresponds to the sum of the voltage signal Usignal and the diode flow voltage dU.\",\n \"This means that the voltage that is established at the capacitor 6 depends on the magnitude of the voltage signal Usignal: the smaller the voltage signal Usignal, the more the voltage and thus the threshold voltage Uref1 drop according to the invention at the capacitor.\",\n \"Hence, a threshold voltage Uref1 is established that is greater than the voltage signal Usignal by the fundamental voltage distance dU.\",\n \"According to the invention, the task of the diode 5 is to allow the discharging of the capacitor 6 and thus a drop in the threshold voltage Uref1 for as long as and only until the difference of Uref1−Usignal has dropped to the fundamental voltage distance dU, but to prevent a recharging of the capacitor and thus an increase in the threshold voltage if the voltage signal Usignal increases.\",\n \"If the voltage signal Usignal drops, then the threshold voltage Uref1 follows the voltage signal Usignal at the distance dU.\",\n \"In contrast, if the voltage signal Usignal increases, then the threshold voltage Uref1 remains constant because of the blocking effect of the diode 5.Of course, the polarity of the first direct voltage source can also be the opposite from the one in FIG.\",\n \"1, so that the first pole 4a is negative and the second pole 4b is positive.\",\n \"In this case, the diode 5 in the circuit shown in FIG.\",\n \"1 has to be arranged in the reverse direction, so that its cathode is connected to the switch output 3c and its anode to the second pole 4b.\",\n \"In another embodiment of the invention, the fundamental voltage distance dU is augmented by a codirectional series connection of a plurality of diodes 5 or second direct voltage sources 7.According to the invention, as an alternative, the function of the diode 5 can be assumed by a second direct voltage source whose positive pole is in the place of the anode 5a of the diode 5 and whose negative pole is in the place of the cathode 5b of the diode 5.FIG.\",\n \"6 shows an embodiment 10b of an adaptive comparator circuit according to the invention, which can be used as an alternative to that shown in FIG.\",\n \"1.The diode shown in FIG.\",\n \"5 has been replaced by a second voltage source 7, whereby its positive pole 7a has been put in the place of the anode 5a and the negative pole 7b has been put in the place of the cathode 5b.\",\n \"The fundamental voltage distance dU shown in FIG.\",\n \"1 has now been replaced by the voltage of the voltage source 7, that is to say, the fundamental voltage distance dU between Uref1 and Usignal is now defined by the voltage supplied by the second direct voltage source.\",\n \"If the voltage supplied by the second voltage source 7 can be regulated or adjusted, then the fundamental voltage distance dU can be varied as needed.\",\n \"FIG.\",\n \"7 shows a schematic block diagram of an embodiment of an acoustic distance sensor according to the invention in which the adaptive comparator circuit 10a shown in FIG.\",\n \"5 is used.\",\n \"A control unit 31 transmits short signal pulses at times, preferably at regular intervals, each lasting, for example, 25 microseconds, to an oscillator 32.When a signal pulse is present, the oscillator 32 transmits an alternating voltage to a sound transducer 34 that is excited by the latter to emit sound waves.\",\n \"The sound transducer 34 is capable of receiving sound waves reflected, for example, from an object and coming back to the sound transducer 34 as an echo, and of converting them into an electric received signal, whereby the propagation time of the sound waves is a measure of the distance from the object.\",\n \"The received signal is fed to an envelope curve shaper 37 which forms the envelope curve of the received signal and transmits it via an electric terminal 8 to the adaptive comparator circuit 10a shown in FIG.\",\n \"5.In a preferred embodiment of the invention, the signal pulses 41 that the control unit 31 transmits to the oscillator 32 are synchronized with the control signals SSt that the control unit 2 transmits to the switch 3 and this is done in such a way that the control signals SSt each begin shortly before the signal pulses 41 as will still be explained below with reference to FIGS.\",\n \"9a and 9b.\",\n \"In this manner, it is ensured that, already at the beginning of the transmittal phase, the threshold voltage Uref1 is greater than the voltage signal Usignal, so that no dummy switching signal is triggered.\",\n \"In another preferred embodiment of the invention, the signal pulses 41 are advantageously synchronized with the control signals SSt in such a way that the control signals SSt each begin before or simultaneously with the end of the signal pulses 41.This ensures that, without any time lag, the threshold voltage Uref1 is supplied to the dropping voltage signal Usignal at the fundamental voltage distance dU already at the beginning of the relaxation phase.\",\n \"The sensitivity of the distance sensor is defined by the fundamental voltage distance dU.\",\n \"FIG.\",\n \"8 shows a schematic block diagram of another embodiment of an acoustic distance sensor according to the invention, in which the adaptive comparator circuit 10a shown in FIG.\",\n \"5 is likewise used.\",\n \"The distance sensor shown in FIG.\",\n \"8—in comparison to that shown in FIG.\",\n \"7—additionally has a driver 33 that amplifies the alternating voltage supplied by the oscillator 32 and transmits it to the sound transducer 34.Moreover, before the received signal is fed into the envelope curve shaper 37, it is passed through a voltage limiter 35 and through a logarithmic amplifier 36c.\",\n \"Among other things, the voltage limiter 35 counters an overmodulation of the amplifier.\",\n \"The amplifier 36c serves to adapt the low input level of just a few microvolts to the higher input level of the subsequent evaluation circuit.\",\n \"In FIG.\",\n \"8, in order to facilitate the synchronization, the signal transmitter 2 is accommodated in the control unit 31.For example, the signal transmitter 2 and the control unit 31 can be combined in one single unit, for example, in a microprocessor, that concurrently fulfills the function of the signal transmitter 2 as well as that of the control unit 31.In FIG.\",\n \"8, the first switching signal S1 of the control unit 31 is also supplied for evaluation purposes, especially for determining the propagation time and calculating the distance.\",\n \"FIGS.\",\n \"9 and 10 serve to further explain the advantageous effects of the invention, whereby the time axes of all of the curves have been selected identically in order to illustrate the course over time.\",\n \"FIG.\",\n \"9 shows a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) a voltage signal and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.\",\n \"8, whereby an echo occurs during the quiescent phase.\",\n \"The control unit 31 transmits short signal pulses 41 to the oscillator 32, as a result of which the sound transducer 34 is excited so as to emit a threshold voltage packet (FIG.\",\n \"9, Curve a).\",\n \"Moreover, the signal transmitter 2 transmits a control signal SSt to the switch 3, which is synchronized with the signal pulse 41 in such a way that the control signal SSt begins shortly before the signal pulse 41 and ends at the same time as or during the presence of the former (FIG.\",\n \"9, Curve b).\",\n \"FIG.\",\n \"9, Curve c, shows the course of the voltage signal Usignal (solid curve) that is identical to the envelope curve, and the course of the threshold voltage Uref1 (dotted curve).\",\n \"The course of the Usignal during the transmittal and relaxation phase and at the beginning of the quiescent phase corresponds essentially to that of the envelope curve 53, which was explained with reference to FIG.\",\n \"4.While the control signal SSt is present at the switch 3, the latter is opened, so that a threshold voltage Uref1 is established which is identical to the voltage supplied by the first direct voltage source 4a, 4b (FIG.\",\n \"9, Curve c).\",\n \"Therefore, this has been advantageously selected in such a way that it is greater than the maximum of the voltage signal, so that the adaptation of the threshold voltage to the voltage signal begins immediately after the opening of the switch 3.In the present example, the maximum possible magnitude of the voltage signal is supplied by the voltage limiter 35 or by the overmodulation limit of the amplifier 36c (FIG.\",\n \"8).\",\n \"According to the invention, after the end of the control signal SSt, a threshold voltage Uref1 is established that is greater than the voltage signal Usignal by the voltage distance dU.\",\n \"Therefore, the threshold voltage Uref1 drops immediately after the end of the control signal by a certain quantity.\",\n \"From this point in time on, the threshold voltage proceeds parallel-offset by a fundamental voltage distance dU with respect to that of the voltage signal Usignal as long as the latter does not increase.\",\n \"A dummy switching signal is not triggered.\",\n \"If, however, the voltage signal Usignal increases, the threshold voltage remains constant so that a first echo peak 60 of the voltage signal Usignal leads to a triggering of the comparator 1 as soon as the voltage signal Usignal exceeds the threshold voltage Uref1.In this case, the comparator 1 emits a first switching signal S1 (FIG.\",\n \"9, Curve d).\",\n \"FIG.\",\n \"10 shows a schematic representation of a typical course over time of a) a voltage signal and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.\",\n \"8, whereby an echo occurs during the relaxation phase and another echo occurs during the quiescent phase.\",\n \"The courses over time of the signal pulse 41 and of the control signal SSt are identical to those shown in FIG.\",\n \"9.During the relaxation time, an echo signal occurs, which manifests itself in a second echo peak 61 of the voltage signal Usignal (FIG.\",\n \"10, Curve a).\",\n \"Up until the beginning of the second echo peak 61, the curves correspond to those shown in FIG.\",\n \"9, Curve c. According to the invention, however, the decrease of the threshold voltage Uref1 comes to a halt as soon as the voltage signal Usignal is no longer dropping, and a first switching signal S1a is triggered by the second echo peak 61.After the second echo peak 61 that occurs during the relaxation time, the fundamental voltage distance dU is once again established between the threshold voltage Uref1 and the voltage signal Usignal.\",\n \"During the quiescent phase, another, third echo peak 62 of the voltage signal Usignal occurs, as a result of which another switching signal S1b is triggered.\",\n \"Now reference will be made to FIGS.\",\n \"11 to 13, which show additional advantageous embodiments of an adaptive comparator circuit according to the invention.\",\n \"FIG.\",\n \"11 shows a preferred embodiment 10c of the invention which differs from the adaptive comparator circuit 10a shown in FIG.\",\n \"6 as follows: a) The first switch is a transistor 103.The base 103a of the transistor 103 serves as the control-current terminal, the emitter 103b serves as the switch input and the collector 103c as the switch output.\",\n \"b) A first resistor 11 is connected between the first pole 4a and the first threshold value input 1a, and a second resistor 12 is connected between the first threshold value input 1a and the second pole 4b, so that the first resistor 11 and the second resistor 12 form a first voltage divider 11, 12.Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the first voltage divider.\",\n \"Thus, through the use of the first voltage divider 11, 12, the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal after the voltage signal Usignal has died out can advantageously be changed by the fundamental voltage distance dU, to a value dUa.\",\n \"When this embodiment 10c of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be changed and, in particular increased.\",\n \"This is advantageous for many applications such as, for example, for the detection of small objects.\",\n \"The first voltage divider can be an adjustable potentiometer whose pick-up is connected to the switch output.\",\n \"c) A sixth resistor 16 is connected between the control output 2a of the signal transmitter 2 and the base 103a, and a seventh resistor 17 is connected between the base 103a and the first pole 4a, which is a plus pole in the example shown.\",\n \"The purpose of these resistors was already explained above.\",\n \"d) The diode 5 is connected in series to an eighth resistor 18 which serves to slow down the discharging of the capacitor 6 and thus to reduce the speed with which the threshold voltage Uref1 is adapted to the voltage signal Usignal.\",\n \"The eighth resistor also serves to smooth the course of the threshold voltage Uref1 with respect to very rapid fluctuations of the voltage signal Usignal.\",\n \"FIG.\",\n \"12 shows another preferred embodiment 10d of the invention, which differs from the adaptive comparator circuit 10c shown in FIG.\",\n \"11 in that, before the voltage signal Usignal reaches the first signal input 1b, it passes through an impedance transformer.\",\n \"This embodiment is especially advantageous when the voltage signal arriving at the electric terminal 8 is high-ohmic, whereby said voltage signal can be the envelope curve voltage generated, for example, by an envelope curve shaper.\",\n \"FIG.\",\n \"13 shows another preferred embodiment 10e of the invention which differs from the adaptive comparator circuit 10a shown in FIG.\",\n \"6 as follows: a) The first switch is a transistor 103.The base 103a of the transistor 103 serves as the control-current terminal, the emitter 103b serves as the switch input and the collector 103c as the switch output.\",\n \"b) A second comparator 20 is used that has a second threshold value input 20a and a second signal input 20b, and that emits a second switching signal S2 when a greater voltage is present at the second signal input 20b than at the second threshold value input 20a.\",\n \"c) The first pole 4a of the first voltage source 4a, 4b is connected to the second threshold value input 20a via a third resistor 13.The second threshold value input 20a is connected to the second pole 4b via a fourth resistor 14, so that the third and the fourth resistors 13, 14 form a second voltage divider which can be configured as a potentiometer.\",\n \"d) The collector 103c that serves as the switch output and the second threshold value input 20a are connected via a fifth resistor 15.e) A second switch 23 is connected in series to the diode 5 and the former can be regulated or activated by the second switching signal S2 in such a way that it is in the closed state when the second comparator 20 emits the second switching signal S2, and otherwise it is in the open state.\",\n \"The second switch 23 can be a transistor.\",\n \"f) A sixth resistor 16 is connected between the control output 2a of the signal transmitter 2 and the base 103a, and a seventh resistor 17 is connected between the base 103a and the first pole 4a, which is a plus pole in the example shown.\",\n \"The purpose of these resistors was already explained above.\",\n \"g) The diode 5 is connected in series to an eighth resistor 18.The purpose of this resistor was already explained with reference to FIG.\",\n \"11.h) Before reaching the first signal input 1b, the voltage signal passes through an impedance transformer 21 whose purpose was already explained with reference to FIG.\",\n \"12.Due to the second voltage divider 13, 14, a voltage Uref2 is present at the second threshold value input 20a.\",\n \"As long as the second switch 23 is closed, the first threshold voltage Uref1 follows the dropping voltage signal Usignal, parallel-offset by the fundamental voltage distance dU, as explained above.\",\n \"According to the invention, however, the second switch opens as soon as the voltage signal Usignal exceeds the second threshold voltage Uref2.As a result, the discharge of the capacitor 6 and thus the dropping of the first threshold voltage Uref1 are ended.\",\n \"Hence, in this embodiment of the invention, the capacitor 6 can only discharge until the first threshold voltage Uref1 has dropped to the magnitude of the second threshold voltage Uref2 supplied by the second potentiometer 13, 14.Thus, through the use of the second voltage divider 13, 14, the voltage distance by which the first threshold voltage is advantageously changed parallel-offset after the decay of the voltage signal Usignal with respect to voltage signal Usignal, from the fundamental voltage distance dU to a value dUb.\",\n \"The second voltage divider 13, 14 can be an adjustable potentiometer whose pick-up is connected to the second threshold value input 20a.\",\n \"The fifth resistor 15 is connected between the switch output 103c and the second threshold value input 20a.\",\n \"Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the second voltage divider 13, 14.In particular, through the use of the fifth resistor 15, it can additionally be achieved that the voltage distance by which the first threshold voltage is parallel-offset with respect to the voltage signal Usignal strives towards a value dUb that—depending on the voltage supplied by the second voltage divider 13, 14—can advantageously be not only larger but also smaller than the fundamental voltage distance dU.\",\n \"When this embodiment 10e of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be either increased or decreased by means of the second voltage divider 13, 14.All of the embodiments of an adaptive comparator circuit according to the invention and thus also the embodiments 10b, 10c, 10d and 10e illustrated in FIG.\",\n \"6 and FIGS.\",\n \"11 to 13 can be used according to the invention in the acoustic distance sensor shown in FIG.\",\n \"8 instead of the adaptive comparator circuit 10a shown there by way of an example.\",\n \"Commercial Utilization The invention can be utilized commercially, for example, in distance measuring technology, in filling level measuring technology and in automation technology.\",\n \"The main figure is FIG.\",\n \"5.List of Reference Numerals and Letters 1 first comparator 1a first threshold value input 1b first signal input 2 signal transmitter 2a control output of 2 2b input of 2 3 first switch 3a control-current terminal of 3 3b switch input of 3 3c switch output of 3 4a, 4b first, second pole of the first direct voltage source 5 diode 5a, 5b anode, cathode of 5 6 capacitor 7a, 7b positive, negative pole of 7 7 second direct voltage source 8, 9 first, second electric terminal 10a,b,c,d,e adaptive comparator circuits 11-18 first to eighth resistor 20 second comparator 20a second threshold value input 20b second signal input 21 voltage follower 23 second switch 30a distance sensor with constant threshold voltage 30b distance sensor with variable threshold voltage 31 control unit 32 oscillator 33 driver 34 sound transducer 35 voltage limiter 36a amplifier with regulatable amplification factor 36b, c logarithmic amplifier 37 envelope curve shaper 38a amplification factor actuation stage 38b threshold value actuation stage 41 signal pulses 42 course over time of the amplification factor of 36a 42a minimum of 42 43, 53 courses over time of the envelope curve voltage 43a, 53a segments of 43, 53 during the signal pulses 41, 51 43b, 53b segments of 43, 53 during the relaxation time of 34 43c, 53c segments of 43, 53 during the quiescent phase 43d, 53d segments of 43, 53 during an echo 44, 54 threshold values of 30a, 30b 45 dummy switching signal 46, 56 useful switching signals 60 first echo peak 61 second echo peak 62 third echo peak 103 transistor 103a base of 103 103b emitter of 103 103c collector of 103 S1, S1a, S1b switching signals of 1 S2 switching signal of 20 dU fundamental voltage distance Uref1 first threshold voltage Usignal voltage signal\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10450932"}}},{"rowIdx":288,"cells":{"text":{"kind":"list like","value":[["Fuel injector and method for installing a fuel injector in a valve seat","A fuel injector, which, at its inflow-side end, has an intake connector at whose outer circumference at least two sealing rings are provided, which seal the fuel injector from a valve seat.","Due to an intermediate ring, the two sealing rings are axially set apart from one another in a respective ring chamber.","The intermediate ring is embodied independently of the intake connector and is made up of two parts.","A flexible outer ring is locked in place on an inner extruded support ring, the outer ring only being mounted once the first sealing ring has already been moved across the support ring and has been stripped off.","The fuel injector is particularly suited for the use in mixture-compressing internal combustion engines having external ignition."],["1-13.","(canceled) 14.A fuel injector for injecting fuel into a combustion chamber of a mixture-compressing internal combustion engine having external ignition, the fuel injector having a longitudinal valve axis, the fuel injector comprising: an intake connector situated at an inflow-side end, and having at least two sealing rings provided at an outer periphery of the intake connector to seal the fuel injector from a valve seat; and an intermediate ring, wherein, via the intermediate ring, the two sealing rings are axially set apart from one another, being situated in each case in a ring chamber, the intermediate ring being embodied independently of the intake connector and including two parts.","15.The fuel injector of claim 14, wherein the intermediate ring includes an inner support ring and an outer ring.","16.The fuel injector of claim 15, wherein the inner support ring, via an inwardly projecting lip, engages in a groove of the intake connector.","17.The fuel injector of claim 15, wherein the inner support ring is made of plastic.","18.The fuel injector of claim 17, wherein the inner support ring is made of the same plastic as a plastic extrusion coat partially surrounding the fuel injector.","19.The fuel injector of claim 14, further comprising: an outwardly projecting locking bead, at an outer diameter of the support ring, at which the outer ring is able to lock into place.","20.The fuel injector of claim 19, wherein an upper locking surface of the locking bead extends largely perpendicularly to the longitudinal valve axis and a lower locking surface has a slanted arrangement.","21.The fuel injector of claim 15, wherein the outer ring is made of flexible plastic.","22.The fuel injector of claim 14, wherein the intermediate ring forms a support shoulder for at least one of the at least two sealing rings.","23.A method for installing a fuel injector in a valve seat, the fuel injector, at the inflow-side end, having an intake connector at whose outer circumference at least two sealing rings are provided, which are axially set apart from one another by an intermediate ring and which seal the fuel injector from the valve seat, the method comprising: affixing an inner support ring of the intermediate ring on a circumference of the intake connector; spreading open a first one of the at least two sealing rings, mounting it on the intake connector, moving it across the inner support ring and stripping it into a ring chamber, and mounting an outer ring on the inner support ring to complete the intermediate ring; spreading open a second sealing ring, mounting it on the intake connector and stripping it into an additional ring chamber; and subsequently inserting the fuel injector in the valve seat together with a sealing system thus mounted on the intake connector.","24.The method of claim 23, wherein the inner support ring of the intermediate ring is extruded on a circumference of the intake connector in a same extrusion procedure as a plastic extrusion coat at least partially surrounding the fuel injector.","25.The method of claim 23, wherein the outer ring of the intermediate ring is locked into place on a locking bead of the inner support ring.","26.The method of claim 23, wherein the fuel injector is secured after having been inserted in the valve seat with corresponding affixation elements provided on the valve seat and on the fuel injector."],["<SOH> BACKGROUND INFORMATION <EOH>At the outer periphery of various fuel injectors, a plurality of sealing elements in the form of sealing rings may be arranged in succession so as to ensure an effective sealing of the fuel injector from a valve seat.","For instance, German Patent Application No.","28 27 789 refers to providing two sealing rings at a stepped inflow connector of the fuel injector.","These may be embodied as whole ring stacks, which then include a stuffing-box ring, an o-ring, an asbestos ring cord, and a plastic fusion ring arranged in immediate succession.","German Patent Application No.","28 27 878 refers to fuel injectors whose sealing is accomplished via sealing stacks, additional rings, having so-called emergency-sealing properties, being provided in addition to the actual sealing rings in o-form.","Furthermore, German Patent Application No.","28 27 850 refers to spacing two sealing rings at the periphery of the fuel injector by using a shoulder that radially projects beyond the outer circumference of the inflow-connecting piece.","Furthermore, a sealing system on a fuel injector is referred to in German Patent Application No.","37 03 615, in which two sealing rings in each case surround one of two connecting pieces independently from one another and appropriately seal it from a fuel-supply line.","Moreover, German Patent Application No.","197 35 665 refers to a fuel-injection system in which a fuel injector is connected via an intermediate piece to a valve-seat connector of a fuel-supply line and sealed therefrom.","In this arrangement, both the inflow connector of the fuel injector and also the intermediate pieces each have a sealing ring at the outer circumference, which spatially are thus clearly located at a distance from one another."],["<SOH> SUMMARY OF THE INVENTION <EOH>The exemplary fuel injector according to the present invention provides a sealing system having two sealing rings at its outer periphery, which may be especially easy to install, avoid excessive expansion and excessive spread of the sealing rings and may therefore basically avoid damage of the sealing rings.","Nevertheless, it may guarantee sufficiently large support shoulders for the sealing rings and completely prevent the release of hydrocarbons into the environment, in compliance with ever stricter environmental demands.","Of particular advantage may be the simple handling of the two partial components of the intermediate ring.","While the inner support ring may be installed in a very simple manner in a groove on the intake connector of the fuel injector by way of an inwardly projecting lip, the outer ring may be snapped into place on the support ring in an uncomplicated and reliable manner.","The locking bead on the support ring used for locking is arranged such that the high compression forces of the one sealing ring during the installation of the fuel injector in the receiving socket are absorbed by an upper horizontal locking surface.","The lower axial demounting forces of the other sealing ring are sufficiently absorbed by a lower locking surface of the locking bead, which has a slanted configuration.","The exemplary method according to the present invention for installing a fuel injector in a valve seat may have the advantage that the overall installation may be performed in a fully automatic manner.","In an especially advantageous manner, the sealing rings are handled very gently during the mounting, so that damage to the sealing rings is excluded.","It may be especially advantageous that the sealing rings, despite being inserted in separate ring chambers, are barely spread open, so that there is no need to stretch them to a dangerous extent.","Because the intermediate ring is arranged or configured in two parts, it is ensured that the first sealing ring is able to be installed without being overstretched, yet sufficiently large support shoulders are subsequently available for both sealing rings."],["FIELD OF THE INVENTION The present invention relates to a fuel injector, and a method for installing a fuel injector in a valve seat.","BACKGROUND INFORMATION At the outer periphery of various fuel injectors, a plurality of sealing elements in the form of sealing rings may be arranged in succession so as to ensure an effective sealing of the fuel injector from a valve seat.","For instance, German Patent Application No.","28 27 789 refers to providing two sealing rings at a stepped inflow connector of the fuel injector.","These may be embodied as whole ring stacks, which then include a stuffing-box ring, an o-ring, an asbestos ring cord, and a plastic fusion ring arranged in immediate succession.","German Patent Application No.","28 27 878 refers to fuel injectors whose sealing is accomplished via sealing stacks, additional rings, having so-called emergency-sealing properties, being provided in addition to the actual sealing rings in o-form.","Furthermore, German Patent Application No.","28 27 850 refers to spacing two sealing rings at the periphery of the fuel injector by using a shoulder that radially projects beyond the outer circumference of the inflow-connecting piece.","Furthermore, a sealing system on a fuel injector is referred to in German Patent Application No.","37 03 615, in which two sealing rings in each case surround one of two connecting pieces independently from one another and appropriately seal it from a fuel-supply line.","Moreover, German Patent Application No.","197 35 665 refers to a fuel-injection system in which a fuel injector is connected via an intermediate piece to a valve-seat connector of a fuel-supply line and sealed therefrom.","In this arrangement, both the inflow connector of the fuel injector and also the intermediate pieces each have a sealing ring at the outer circumference, which spatially are thus clearly located at a distance from one another.","SUMMARY OF THE INVENTION The exemplary fuel injector according to the present invention provides a sealing system having two sealing rings at its outer periphery, which may be especially easy to install, avoid excessive expansion and excessive spread of the sealing rings and may therefore basically avoid damage of the sealing rings.","Nevertheless, it may guarantee sufficiently large support shoulders for the sealing rings and completely prevent the release of hydrocarbons into the environment, in compliance with ever stricter environmental demands.","Of particular advantage may be the simple handling of the two partial components of the intermediate ring.","While the inner support ring may be installed in a very simple manner in a groove on the intake connector of the fuel injector by way of an inwardly projecting lip, the outer ring may be snapped into place on the support ring in an uncomplicated and reliable manner.","The locking bead on the support ring used for locking is arranged such that the high compression forces of the one sealing ring during the installation of the fuel injector in the receiving socket are absorbed by an upper horizontal locking surface.","The lower axial demounting forces of the other sealing ring are sufficiently absorbed by a lower locking surface of the locking bead, which has a slanted configuration.","The exemplary method according to the present invention for installing a fuel injector in a valve seat may have the advantage that the overall installation may be performed in a fully automatic manner.","In an especially advantageous manner, the sealing rings are handled very gently during the mounting, so that damage to the sealing rings is excluded.","It may be especially advantageous that the sealing rings, despite being inserted in separate ring chambers, are barely spread open, so that there is no need to stretch them to a dangerous extent.","Because the intermediate ring is arranged or configured in two parts, it is ensured that the first sealing ring is able to be installed without being overstretched, yet sufficiently large support shoulders are subsequently available for both sealing rings.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 shows an inflow-side end of a fuel injector having a sealing system configured according to the present invention for sealing the fuel injector from a valve seat.","FIG.","2 shows an enlargement of the cut-out section II in FIG.","1.DETAILED DESCRIPTION FIG.","1 shows a partial representation of a fuel injector 1, which, for instance, is embodied as a fuel injector for the direct injection of fuel into the combustion chamber of a mixture-compressing internal combustion engine having external ignition.","By way of an inflow-side end 2, fuel injector 1 projects into a corresponding valve seat 3.Valve seat 3 is embodied, for instance, as a cup-shaped intake connecting piece 4, which constitutes part of a fuel-supply line (not shown further), such as a fuel rail.","The fuel-supply line usually has a plurality of intake connecting pieces 4, so that the fuel conveyed via the fuel-supply line may be distributed to a plurality of fuel injectors 1.Intake connecting piece 4 has a stepped arrangement or configuration, for example, and, at its end facing away from the fuel-supply line, has one or a plurality of affixation elements 5 projecting in the manner of a lip.","These affixation elements 5 engage in openings 6 of an securing clamp 7, which is used to reliably secure fuel injector 1 in valve seat 3.Securing clamp 7 is inserted, for instance, in a circumferential groove 8 at the outer periphery of a plastic extrusion coat 9 of fuel injector 1.Fuel injector 1 is largely surrounded by plastic extrusion coat 9, for example.","However, at inflow-side end 2 of fuel injector 1, a metal intake connector 11 for the supply of fuel to the interior of fuel injector 1 projects beyond plastic extrusion coat 9.Above plastic extrusion coat 9, two sealing rings 13, 14, configured as O-rings, are situated at the outer periphery of intake connector 11.Due to an intermediate ring 15 configured according to the exemplary embodiment of the present invention, the two sealing rings 13, 14, which follow each other closely in the axial direction, are positioned at a spatial distance from one another.","Forming the upstream end of fuel injector 1 is an upper retaining ring 16, which is made of plastic and mounted on intake connector 11.In this manner, both sealing rings 13, 14 are embedded between retaining ring 16 and upper end face 17 of plastic extrusion coat 9, intermediate ring 15 forming two ring chambers 18, 19 for sealing rings 13, 14.In contrast to the sealing rings having so-called emergency-sealing properties known in part from the related art, the two sealing rings 13, 14 are two “full-fledged” sealing rings made of known elastomer sealing material.","The necessity of two sealing rings immediately following one another at the periphery of fuel injector 1 results from the composition of certain fuels.","In long-term use of sealing rings it has become apparent that hydrocarbons permeate through the elastomers normally used for sealing rings and may even permeate them completely.","In order to fully prevent the emission of hydrocarbons into the environment in accordance with ever stricter environmental regulations, it may be advantageous to situate an additional sealing ring immediately behind a first sealing ring.","In this manner, the second sealing ring following a sealing ring permeated in the manner described above, offers a substantially improved sealing reliability.","Intermediate ring 15, which partitions the sealing chamber into two ring chambers 18, 19, is made up of two sub-components.","An inner support ring 22 engages, for instance, in a circumferential groove 23 at the periphery of intake connector 11.Ideally, support ring 22 is extruded (injected) on the metal valve body, together with plastic extrusion coat 9 and retaining ring 16, in one plastic-extrusion procedure.","On its inner side, support ring 22 has a slanted lip 24, which projects toward the inside and is able to be inserted in groove 23 with form accuracy.","Provided at the outer diameter of support ring 22 is a locking bead 25, which projects radially outward and at which an outer ring 26, which also belongs to intermediate ring 15, is able to be snapped into place.","The region of locking bead 25 is shown in FIG.","2 in an enlarged view.","Locking bead 25 at support ring 22 is arranged or configured such that the high compression forces of second sealing ring 13 during the installation of fuel injector 1 in intake connecting piece 4 are absorbed by an upper locking surface 28, which mostly extends perpendicularly to a longitudinal valve axis 30.The lower axial demounting forces of first sealing ring 14 are sufficiently absorbed by a lower locking surface 29 of locking bead 25, locking surface 29 having a slanted arrangement or configuration.","Outer ring 26 is made of flexible plastic.","When outer ring 26 is mounted on inner support ring 22, this plastic expands and then locks into place in the limit position above locking bead 25.Intermediate ring 15, made up of outer ring 26 and support ring 22, is used as support shoulder for second sealing ring 13.The mounting of the sealing system on intake connector 11 of fuel injector 1 is briefly explained in the following.","As already described above, plastic extrusion coat 9, inner support ring 22 of intermediate ring 15, and retaining ring 16 are, for instance, simultaneously extruded on the metal valve body in one extrusion procedure.","Subsequently, first sealing ring 14 is spread open, mounted on intake connector 11, moved across support ring 22 and stripped into ring chamber 19.Then, outer ring 26 is clipped onto inner support ring 22 to complete intermediate ring 15.Second sealing ring 13 is then spread open, mounted on intake connector 11 and stripped into ring chamber 18.With the aid of the sealing system mounted in this manner on intake connector 11, fuel injector 1 may be inserted in valve seat 3.The exemplary embodiment and/or method of the present invention is not limited to the described exemplary embodiment or method.","Thus, intake connector 11, for instance, may have a stepped arrangement or configuration in the region of sealing rings 13, 14 and/or sealing rings 13, 14 may have different cross sections or diameters."]],"string":"[\n [\n \"Fuel injector and method for installing a fuel injector in a valve seat\",\n \"A fuel injector, which, at its inflow-side end, has an intake connector at whose outer circumference at least two sealing rings are provided, which seal the fuel injector from a valve seat.\",\n \"Due to an intermediate ring, the two sealing rings are axially set apart from one another in a respective ring chamber.\",\n \"The intermediate ring is embodied independently of the intake connector and is made up of two parts.\",\n \"A flexible outer ring is locked in place on an inner extruded support ring, the outer ring only being mounted once the first sealing ring has already been moved across the support ring and has been stripped off.\",\n \"The fuel injector is particularly suited for the use in mixture-compressing internal combustion engines having external ignition.\"\n ],\n [\n \"1-13.\",\n \"(canceled) 14.A fuel injector for injecting fuel into a combustion chamber of a mixture-compressing internal combustion engine having external ignition, the fuel injector having a longitudinal valve axis, the fuel injector comprising: an intake connector situated at an inflow-side end, and having at least two sealing rings provided at an outer periphery of the intake connector to seal the fuel injector from a valve seat; and an intermediate ring, wherein, via the intermediate ring, the two sealing rings are axially set apart from one another, being situated in each case in a ring chamber, the intermediate ring being embodied independently of the intake connector and including two parts.\",\n \"15.The fuel injector of claim 14, wherein the intermediate ring includes an inner support ring and an outer ring.\",\n \"16.The fuel injector of claim 15, wherein the inner support ring, via an inwardly projecting lip, engages in a groove of the intake connector.\",\n \"17.The fuel injector of claim 15, wherein the inner support ring is made of plastic.\",\n \"18.The fuel injector of claim 17, wherein the inner support ring is made of the same plastic as a plastic extrusion coat partially surrounding the fuel injector.\",\n \"19.The fuel injector of claim 14, further comprising: an outwardly projecting locking bead, at an outer diameter of the support ring, at which the outer ring is able to lock into place.\",\n \"20.The fuel injector of claim 19, wherein an upper locking surface of the locking bead extends largely perpendicularly to the longitudinal valve axis and a lower locking surface has a slanted arrangement.\",\n \"21.The fuel injector of claim 15, wherein the outer ring is made of flexible plastic.\",\n \"22.The fuel injector of claim 14, wherein the intermediate ring forms a support shoulder for at least one of the at least two sealing rings.\",\n \"23.A method for installing a fuel injector in a valve seat, the fuel injector, at the inflow-side end, having an intake connector at whose outer circumference at least two sealing rings are provided, which are axially set apart from one another by an intermediate ring and which seal the fuel injector from the valve seat, the method comprising: affixing an inner support ring of the intermediate ring on a circumference of the intake connector; spreading open a first one of the at least two sealing rings, mounting it on the intake connector, moving it across the inner support ring and stripping it into a ring chamber, and mounting an outer ring on the inner support ring to complete the intermediate ring; spreading open a second sealing ring, mounting it on the intake connector and stripping it into an additional ring chamber; and subsequently inserting the fuel injector in the valve seat together with a sealing system thus mounted on the intake connector.\",\n \"24.The method of claim 23, wherein the inner support ring of the intermediate ring is extruded on a circumference of the intake connector in a same extrusion procedure as a plastic extrusion coat at least partially surrounding the fuel injector.\",\n \"25.The method of claim 23, wherein the outer ring of the intermediate ring is locked into place on a locking bead of the inner support ring.\",\n \"26.The method of claim 23, wherein the fuel injector is secured after having been inserted in the valve seat with corresponding affixation elements provided on the valve seat and on the fuel injector.\"\n ],\n [\n \"<SOH> BACKGROUND INFORMATION <EOH>At the outer periphery of various fuel injectors, a plurality of sealing elements in the form of sealing rings may be arranged in succession so as to ensure an effective sealing of the fuel injector from a valve seat.\",\n \"For instance, German Patent Application No.\",\n \"28 27 789 refers to providing two sealing rings at a stepped inflow connector of the fuel injector.\",\n \"These may be embodied as whole ring stacks, which then include a stuffing-box ring, an o-ring, an asbestos ring cord, and a plastic fusion ring arranged in immediate succession.\",\n \"German Patent Application No.\",\n \"28 27 878 refers to fuel injectors whose sealing is accomplished via sealing stacks, additional rings, having so-called emergency-sealing properties, being provided in addition to the actual sealing rings in o-form.\",\n \"Furthermore, German Patent Application No.\",\n \"28 27 850 refers to spacing two sealing rings at the periphery of the fuel injector by using a shoulder that radially projects beyond the outer circumference of the inflow-connecting piece.\",\n \"Furthermore, a sealing system on a fuel injector is referred to in German Patent Application No.\",\n \"37 03 615, in which two sealing rings in each case surround one of two connecting pieces independently from one another and appropriately seal it from a fuel-supply line.\",\n \"Moreover, German Patent Application No.\",\n \"197 35 665 refers to a fuel-injection system in which a fuel injector is connected via an intermediate piece to a valve-seat connector of a fuel-supply line and sealed therefrom.\",\n \"In this arrangement, both the inflow connector of the fuel injector and also the intermediate pieces each have a sealing ring at the outer circumference, which spatially are thus clearly located at a distance from one another.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The exemplary fuel injector according to the present invention provides a sealing system having two sealing rings at its outer periphery, which may be especially easy to install, avoid excessive expansion and excessive spread of the sealing rings and may therefore basically avoid damage of the sealing rings.\",\n \"Nevertheless, it may guarantee sufficiently large support shoulders for the sealing rings and completely prevent the release of hydrocarbons into the environment, in compliance with ever stricter environmental demands.\",\n \"Of particular advantage may be the simple handling of the two partial components of the intermediate ring.\",\n \"While the inner support ring may be installed in a very simple manner in a groove on the intake connector of the fuel injector by way of an inwardly projecting lip, the outer ring may be snapped into place on the support ring in an uncomplicated and reliable manner.\",\n \"The locking bead on the support ring used for locking is arranged such that the high compression forces of the one sealing ring during the installation of the fuel injector in the receiving socket are absorbed by an upper horizontal locking surface.\",\n \"The lower axial demounting forces of the other sealing ring are sufficiently absorbed by a lower locking surface of the locking bead, which has a slanted configuration.\",\n \"The exemplary method according to the present invention for installing a fuel injector in a valve seat may have the advantage that the overall installation may be performed in a fully automatic manner.\",\n \"In an especially advantageous manner, the sealing rings are handled very gently during the mounting, so that damage to the sealing rings is excluded.\",\n \"It may be especially advantageous that the sealing rings, despite being inserted in separate ring chambers, are barely spread open, so that there is no need to stretch them to a dangerous extent.\",\n \"Because the intermediate ring is arranged or configured in two parts, it is ensured that the first sealing ring is able to be installed without being overstretched, yet sufficiently large support shoulders are subsequently available for both sealing rings.\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention relates to a fuel injector, and a method for installing a fuel injector in a valve seat.\",\n \"BACKGROUND INFORMATION At the outer periphery of various fuel injectors, a plurality of sealing elements in the form of sealing rings may be arranged in succession so as to ensure an effective sealing of the fuel injector from a valve seat.\",\n \"For instance, German Patent Application No.\",\n \"28 27 789 refers to providing two sealing rings at a stepped inflow connector of the fuel injector.\",\n \"These may be embodied as whole ring stacks, which then include a stuffing-box ring, an o-ring, an asbestos ring cord, and a plastic fusion ring arranged in immediate succession.\",\n \"German Patent Application No.\",\n \"28 27 878 refers to fuel injectors whose sealing is accomplished via sealing stacks, additional rings, having so-called emergency-sealing properties, being provided in addition to the actual sealing rings in o-form.\",\n \"Furthermore, German Patent Application No.\",\n \"28 27 850 refers to spacing two sealing rings at the periphery of the fuel injector by using a shoulder that radially projects beyond the outer circumference of the inflow-connecting piece.\",\n \"Furthermore, a sealing system on a fuel injector is referred to in German Patent Application No.\",\n \"37 03 615, in which two sealing rings in each case surround one of two connecting pieces independently from one another and appropriately seal it from a fuel-supply line.\",\n \"Moreover, German Patent Application No.\",\n \"197 35 665 refers to a fuel-injection system in which a fuel injector is connected via an intermediate piece to a valve-seat connector of a fuel-supply line and sealed therefrom.\",\n \"In this arrangement, both the inflow connector of the fuel injector and also the intermediate pieces each have a sealing ring at the outer circumference, which spatially are thus clearly located at a distance from one another.\",\n \"SUMMARY OF THE INVENTION The exemplary fuel injector according to the present invention provides a sealing system having two sealing rings at its outer periphery, which may be especially easy to install, avoid excessive expansion and excessive spread of the sealing rings and may therefore basically avoid damage of the sealing rings.\",\n \"Nevertheless, it may guarantee sufficiently large support shoulders for the sealing rings and completely prevent the release of hydrocarbons into the environment, in compliance with ever stricter environmental demands.\",\n \"Of particular advantage may be the simple handling of the two partial components of the intermediate ring.\",\n \"While the inner support ring may be installed in a very simple manner in a groove on the intake connector of the fuel injector by way of an inwardly projecting lip, the outer ring may be snapped into place on the support ring in an uncomplicated and reliable manner.\",\n \"The locking bead on the support ring used for locking is arranged such that the high compression forces of the one sealing ring during the installation of the fuel injector in the receiving socket are absorbed by an upper horizontal locking surface.\",\n \"The lower axial demounting forces of the other sealing ring are sufficiently absorbed by a lower locking surface of the locking bead, which has a slanted configuration.\",\n \"The exemplary method according to the present invention for installing a fuel injector in a valve seat may have the advantage that the overall installation may be performed in a fully automatic manner.\",\n \"In an especially advantageous manner, the sealing rings are handled very gently during the mounting, so that damage to the sealing rings is excluded.\",\n \"It may be especially advantageous that the sealing rings, despite being inserted in separate ring chambers, are barely spread open, so that there is no need to stretch them to a dangerous extent.\",\n \"Because the intermediate ring is arranged or configured in two parts, it is ensured that the first sealing ring is able to be installed without being overstretched, yet sufficiently large support shoulders are subsequently available for both sealing rings.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 shows an inflow-side end of a fuel injector having a sealing system configured according to the present invention for sealing the fuel injector from a valve seat.\",\n \"FIG.\",\n \"2 shows an enlargement of the cut-out section II in FIG.\",\n \"1.DETAILED DESCRIPTION FIG.\",\n \"1 shows a partial representation of a fuel injector 1, which, for instance, is embodied as a fuel injector for the direct injection of fuel into the combustion chamber of a mixture-compressing internal combustion engine having external ignition.\",\n \"By way of an inflow-side end 2, fuel injector 1 projects into a corresponding valve seat 3.Valve seat 3 is embodied, for instance, as a cup-shaped intake connecting piece 4, which constitutes part of a fuel-supply line (not shown further), such as a fuel rail.\",\n \"The fuel-supply line usually has a plurality of intake connecting pieces 4, so that the fuel conveyed via the fuel-supply line may be distributed to a plurality of fuel injectors 1.Intake connecting piece 4 has a stepped arrangement or configuration, for example, and, at its end facing away from the fuel-supply line, has one or a plurality of affixation elements 5 projecting in the manner of a lip.\",\n \"These affixation elements 5 engage in openings 6 of an securing clamp 7, which is used to reliably secure fuel injector 1 in valve seat 3.Securing clamp 7 is inserted, for instance, in a circumferential groove 8 at the outer periphery of a plastic extrusion coat 9 of fuel injector 1.Fuel injector 1 is largely surrounded by plastic extrusion coat 9, for example.\",\n \"However, at inflow-side end 2 of fuel injector 1, a metal intake connector 11 for the supply of fuel to the interior of fuel injector 1 projects beyond plastic extrusion coat 9.Above plastic extrusion coat 9, two sealing rings 13, 14, configured as O-rings, are situated at the outer periphery of intake connector 11.Due to an intermediate ring 15 configured according to the exemplary embodiment of the present invention, the two sealing rings 13, 14, which follow each other closely in the axial direction, are positioned at a spatial distance from one another.\",\n \"Forming the upstream end of fuel injector 1 is an upper retaining ring 16, which is made of plastic and mounted on intake connector 11.In this manner, both sealing rings 13, 14 are embedded between retaining ring 16 and upper end face 17 of plastic extrusion coat 9, intermediate ring 15 forming two ring chambers 18, 19 for sealing rings 13, 14.In contrast to the sealing rings having so-called emergency-sealing properties known in part from the related art, the two sealing rings 13, 14 are two “full-fledged” sealing rings made of known elastomer sealing material.\",\n \"The necessity of two sealing rings immediately following one another at the periphery of fuel injector 1 results from the composition of certain fuels.\",\n \"In long-term use of sealing rings it has become apparent that hydrocarbons permeate through the elastomers normally used for sealing rings and may even permeate them completely.\",\n \"In order to fully prevent the emission of hydrocarbons into the environment in accordance with ever stricter environmental regulations, it may be advantageous to situate an additional sealing ring immediately behind a first sealing ring.\",\n \"In this manner, the second sealing ring following a sealing ring permeated in the manner described above, offers a substantially improved sealing reliability.\",\n \"Intermediate ring 15, which partitions the sealing chamber into two ring chambers 18, 19, is made up of two sub-components.\",\n \"An inner support ring 22 engages, for instance, in a circumferential groove 23 at the periphery of intake connector 11.Ideally, support ring 22 is extruded (injected) on the metal valve body, together with plastic extrusion coat 9 and retaining ring 16, in one plastic-extrusion procedure.\",\n \"On its inner side, support ring 22 has a slanted lip 24, which projects toward the inside and is able to be inserted in groove 23 with form accuracy.\",\n \"Provided at the outer diameter of support ring 22 is a locking bead 25, which projects radially outward and at which an outer ring 26, which also belongs to intermediate ring 15, is able to be snapped into place.\",\n \"The region of locking bead 25 is shown in FIG.\",\n \"2 in an enlarged view.\",\n \"Locking bead 25 at support ring 22 is arranged or configured such that the high compression forces of second sealing ring 13 during the installation of fuel injector 1 in intake connecting piece 4 are absorbed by an upper locking surface 28, which mostly extends perpendicularly to a longitudinal valve axis 30.The lower axial demounting forces of first sealing ring 14 are sufficiently absorbed by a lower locking surface 29 of locking bead 25, locking surface 29 having a slanted arrangement or configuration.\",\n \"Outer ring 26 is made of flexible plastic.\",\n \"When outer ring 26 is mounted on inner support ring 22, this plastic expands and then locks into place in the limit position above locking bead 25.Intermediate ring 15, made up of outer ring 26 and support ring 22, is used as support shoulder for second sealing ring 13.The mounting of the sealing system on intake connector 11 of fuel injector 1 is briefly explained in the following.\",\n \"As already described above, plastic extrusion coat 9, inner support ring 22 of intermediate ring 15, and retaining ring 16 are, for instance, simultaneously extruded on the metal valve body in one extrusion procedure.\",\n \"Subsequently, first sealing ring 14 is spread open, mounted on intake connector 11, moved across support ring 22 and stripped into ring chamber 19.Then, outer ring 26 is clipped onto inner support ring 22 to complete intermediate ring 15.Second sealing ring 13 is then spread open, mounted on intake connector 11 and stripped into ring chamber 18.With the aid of the sealing system mounted in this manner on intake connector 11, fuel injector 1 may be inserted in valve seat 3.The exemplary embodiment and/or method of the present invention is not limited to the described exemplary embodiment or method.\",\n \"Thus, intake connector 11, for instance, may have a stepped arrangement or configuration in the region of sealing rings 13, 14 and/or sealing rings 13, 14 may have different cross sections or diameters.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10450961"}}},{"rowIdx":289,"cells":{"text":{"kind":"list like","value":[["PROCESS FOR THE PRODUCTION OF GRAIN ORIENTED ELECTRICAL STEEL STRIPS","A process for the production of grain oriented electrical Fe—Si strips in which a Si-containing alloy is directly cast as a strip between 2.5-5.0 mm thick and cold rolled in one stage, or in more stages with intermediate annealing, to a final thickness of between 0.15-1.0 mm.","The strip is then continuously annealed to carry out the primary recrystallization and then annealed to carry out the oriented secondary recrystallization.","The process further includes that after solidification of the strip, and before its coiling, a phase transformation from Ferrite to Austenite is induced into the metal matrix for a volume fraction between 25-60%, obtained by controlling the alloy composition so that the Austenite fraction is allowed within the stability equilibrium between the two phases.","The strip is then deformed by rolling in-line with the casting step to obtain a deformation higher than 20% in the temperature interval 1000-1300° C."],["1-8.","(canceled) 9.A process for the production of electrical grain oriented Fe—Si strips in which a Si-containing molten alloy composition is directly cast as continuous strips 2.5 to 5 mm thick, cold rolled in one step or more steps with intermediate annealing to a final thickness of between 1 and 0.15 mm, the strip being then continuously annealed to carry out oriented secondary recrystallisation, characterised in that after the strip solidification and before coiling of said strip in a coiling phase, a ferrite to austenite transformation is induced in the metal matrix via deformation, by rolling said strip between two cooled rolls to obtain a deformation over 20% in a temperature range of 1000-1300° C., thereby producing a volume fraction of austenite to be between 25 and 60%, said molten alloy composition being chosen such that said volume fraction of austenite is stable in a temperature interval of between 1100 and 1200° C. 10.The process according to claim 9, in which between the rolling phase and the coiling one, the strip is held between 1100 and 1200° C. for at least 5 s. 11.The process according to claim 9, in which the as-solidified strip thickness is comprised between 1.5 and 4.0 mm and after the rolling phase the strip is quenched to obtain a volume fraction of martensite comprised between 5 and 15%.","12.The process according to claim 9, in which before cold rolling the strip is annealed at a maximum temperature of 1200° C. 13.The process according to claim 12, in which after said annealing the strip is continuously quenched from a temperature comprised between 750 and 950° C. down to 400° C. in less than 12 s. 14.The process according to claim 9, in which the cast alloy comprises 2.5-5.0 wt % Si, 200-1000 ppm C, 0.05-0.5 wt % Mn, 0.07-0.5 wt % Cu, less than 2 wt % Cr+Ni+Mo.","Less than 30 ppm O, less than 500 ppm S+Se, 50-400 ppm Al, less than 100 ppm N. 15.The process according to claim 9, in which in the alloy at least an element is added chosen in the group consisting of Zr, Ti, Ce, B, Ta, Nb, V, Co. 16.The process according to claim 9, in which in the alloy at least an element is added chosen between Sn, Sb, P, Bi.","17.A process for the production of grain oriented electrical Fe—Si strip, in which a Si-containing alloy is directly cast as continuous strip 2.5 to 5 mm thick, in-line hot-rolled and then cold-rolled in one step or more steps, with intermediate annealing, to a final thickness of between 1 and 0.15 mm, the cold-rolled strip being, then continuously annealed to carry out primary recrystallisation and subsequently again annealed to carry out secondary recrystallisation, characterized in that the Si-containing alloy composition is selected to induce in the alloy, during the hot-rolling step in which a deformation rate of at least 20% is utilized in a temperature interval of between 1000 and 1300° C., a ferrite to austenite phase transformation with an austenite volume fraction of between 25 to 60% stable in a temperature interval of between 1100 to 1200° C. 18.The process according to claim 17, in which between the hot-rolling and the coiling steps the strip is held in the temperature range of 1100 to 1200° C. for at least 5 s. 19.The process according to claim 17, in which an as-cast strip 2.5 to 4 mm thick is in-line hot-rolled and the thus obtained hot-rolled strip is quenched to obtain a volume fraction of martensite comprised between 5 and 15%.","20.The process according to claim 17, in which before cold-rolling the strip is annealed at a maximum temperature of 1200° C. 21.The process according to claim 20, in which, after said annealing, the strip is continuously quenched from a temperature of between 750 and 950° C. down to 400° C. in less than 12 s. 22.The process according to claim 17, in which the cast alloy comprises 2.5-5.0 wt % Si, 200-1000 ppm C, 0.05-0.5 wt % Mn, 0.07-0.5% Cu, less than 2.0% Cr+Ni+Mo, less than 30 ppm O, less than 500 ppm S+Se, 50-400 ppm Al, less than 100 ppm N. 23.The process according to claim 22, in which in the alloy at least an element is added chosen in the group consisting of Zr, Ti, Ce, B, Ta, Nb, V, Co. 24.The process according to claim 22, in which in the allot at least an element is added chosen between Sn, Sb, P, Bi."],["<SOH> FIELD OF THE INVENTION <EOH>Present invention refers to the production of grain oriented electrical steel strips havig excellent magnetic characteristics, dedicated to the production of transformer cores.","More precisely, rhe invention refers to a process in which a Fe—Si alloy is continuously cast directly as strip and, before coiling, the strip itself is continuously deformed by rolling to induce the formation in the metal matrix of a given fraction of Austenite, controlled as amount and distribution, thus obtaining a strip micro-structure stably and uniformly recrystallised before cold rolling."],["<SOH> SUMMARY OF THE INVENTION <EOH>The aim of present invention is to solve the inconveniences due to the quality of electrical steel strips deriving from strip casting.","Thus, it is an object of present invention a process for the production of electrical steel strips in which, through an in-line thickness reduction of the strip between casting and coiling stations, a significant level of recrystallisation by means of phase transformation is induced, thus normalising the crystalline structure before cold rolling, so that possible fluctuations in the process conditions are substantially non-influent with respect to the quality of the final product.","Another object of present invention is to make it possible to industrially produce grain oriented electrical steel strips having excellent magnetic characteristics and constant quality, the process being stable and simplyfied with respect to the conventional processes currently utilised.","Further objects of present invention will be evident from the following description of the invention.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["FIELD OF THE INVENTION Present invention refers to the production of grain oriented electrical steel strips havig excellent magnetic characteristics, dedicated to the production of transformer cores.","More precisely, rhe invention refers to a process in which a Fe—Si alloy is continuously cast directly as strip and, before coiling, the strip itself is continuously deformed by rolling to induce the formation in the metal matrix of a given fraction of Austenite, controlled as amount and distribution, thus obtaining a strip micro-structure stably and uniformly recrystallised before cold rolling.","STATE OF THE ART Grain oriented electrical steel strips (Fe—Si) are typically industrially produced as strips having a thickness comprised between 0,18 and 0,50 mm and are characterised by magnetic properties variable according to the specific product class.","Said classification substantially refers to the specific power losses of the strip subjected to given electromagnetic work conditions (e.g.","P50 Hz at 1,7 Tesla, in W/kg), evaluated along a specific reference direction (rolling direction).","The main utilisation of said strips is the production of transformer cores.","Good magnetic properties (strongly anisotropic) are obtained controlling the final crystalline structure of the strips to obtain all, or almost all, the grains oriented to have their easiest magnetization direction (the <001> axis) aligned in the most perfect way with the rolling direction.","In practice, final products are obtained having the grains mean diameter generally comprised between 1 and 20 mm having an orientation centred around the Goss orientation ({110} <001>).","The minor the angular dispersion around the Goss one, the better the product magnetic permeability and hence the lesser the magnetic losses.","The final products having low magnetic losses (core losses) and high permeability have interesting advantages in terms of design, dimensions and yield of the transformers.","The first industrial production of the above materials was described by the U.S. Firm ARMCO at the beginning of the thirties (U.S. Pat.","No.","1,956,559).","Many important improvements have been since introduced in the production technology of grain oriented electrical strips, in terms both of magnetic and physical quality of products and of transformation costs and cycles rationalisation.","All existing technologies exploit the same metallurgical strategy to obtain a very strong Goss structure in the final products, i.e.","the process of oriented secondary recrystallisation guided by uniformly distributed second phases and/or segregating elements.","The, non metallic, second phases and the segregating elements play a fundamental role in controlling (slowing down) the movement of grain boundaries doing the final annealing which actuates the selective secondary recrystallisation process.","In the original ARMCO technology, utilising MnS as inhibitor of the grain boundaries movement, and in the subsequent technology developed by NSC, in which the inhibitors are mainly aluminium nitrides (AIN+MnS) (EP 8.385, EP 17.830, EP 202.339), a very important binding step common to both production processes is the heating of the continuously cast slabs (ingots, in old times), immediately before the hot rolling, at very high temperatures (around 1400° C.) for a time sufficient to guarantee a complete dissolution of sulphides and/or nitrides coarsely precipitated during the slab cooling after casting, to re-precipitate them in a very fine and uniformly distributed form throughout the metallic matrix of the hot rolled strips.","Such a fine re-precipitation can be started and completed, as well as the precipitates dimensions adjusted, during the process, in any case, however, before the cold rolling.","The slab heating to said temperatures requires using special furnaces (pushing furnaces, liquid-slag walking-beam furnaces, induction furnaces) due to the ductility at high temperatures of the Fe-3% Si alloys and to formation of liquid slags.","New casting technologies of the liquid steel are intended to simplify the production processes to make them more compact and flexible and to reduce costs.","One of said technologies is the “thin slab” casting, consisting in the continuous casting of slabs having the typical thickness of conventional already roughened slabs, apt to a direct hot rolling, through a sequence of slabs continuous casting, treating in continuous tunnel-furnaces to rise/maintain the temperature of slabs and finishing-rolling, down to coiled strip.","The problems connected to the utilisation of said technique for grain oriented products mainly consist in the difficulty to maintain and control the high temperatures necessary to keep in solution the elements forming the second phases, which have to be finely precipitated at the beginning of the finishing hot-rolling step, if desired best micro-structural and magnetic characteristics are to be obtained in the end products.","Such problems were dealt with in different ways, for instance utilising the low thickness of the cast slabs in connection to specific concentration intervals of the micro-alloying elements to stably control the second phases precipitation (grain growth inhibitors) during hot rolling, or drastically modifying the strategy of the inhibitors formation in the metal matrix.","The casting technique potentially offering the highest rationalisation level of the processes and the higher production flexibility is the one consisting in the direct production of strips from the liquid steel (Strip Casting), totally eliminating the hot rolling step.","Such an exaordinary innovation was conceived and patented long time ago, and since long time were also devised and patented process conditions to produce electrical steel strips, and more particularly grain oriented ones.","However, up to now there is not an industrial production in the world of grain oriented electrical steel according to the above technique, though the state of the art relating to the casting machines is ready for industrial applications, as shown by existing plants, producing only carbon steels and stainless steels.","The present inventors believe that to industrially produce grain oriented electrical steel strips from direct solidification of a strip (Strip Casting) it is necessary to have a strip micro-structure before cold rolling significantly different from the one obtained during the casting stage.","The high solidification speed of the cast strip makes it difficult to have a homogeneous and reproducible grain structure throughout the strip and between different castings, due to the high sensitivity of the solidification structure to the fluctuations of the casting conditions and to the alloy composition.","The micro-structure of the intermediate products starting from strip casting is much more influenced by the solidification structure, with respect to the ones derived from conventional slab casting, because of the lack of deformation in the strip during the typical hot rolling.","SUMMARY OF THE INVENTION The aim of present invention is to solve the inconveniences due to the quality of electrical steel strips deriving from strip casting.","Thus, it is an object of present invention a process for the production of electrical steel strips in which, through an in-line thickness reduction of the strip between casting and coiling stations, a significant level of recrystallisation by means of phase transformation is induced, thus normalising the crystalline structure before cold rolling, so that possible fluctuations in the process conditions are substantially non-influent with respect to the quality of the final product.","Another object of present invention is to make it possible to industrially produce grain oriented electrical steel strips having excellent magnetic characteristics and constant quality, the process being stable and simplyfied with respect to the conventional processes currently utilised.","Further objects of present invention will be evident from the following description of the invention.","DETAILED DESCRIPTION OF THE INVENTION A first important aspect of present invention resides in that a molten alloy containing silicon is directly solidified in the form of a strip, through the casting technology known as strip casting (casting between twin cooled and counter-rotating rolls), thus avoiding, with respect to currently utilised technologies, casting the alloy in slabs or ingots, subjecting said slabs to thermal treatment in special high-temperature furnaces for long times (to attain the necessary thermal homogeneity) and transforming said slabs into strips through hot rolling with total reductions which, according to the slab casting technologies, vary between 96 and 99%.","A second important aspect of present invention resides in that the chemical composition of the Silicon containing alloy is selected specifically to control the thermodynamic stability of the Austenite phase in the matrix (face-centered cubic lattice) in equilibrium with the Ferrite phase (body-centered cubic lattice).","More precisely, to obtain excellent final magnetic characteristics, it is convenient to adjust the chemistry of the alloy so that an Austenite fraction comprised between 25 and 60% is stable between 1100 and 1200° C. Consequently, to balance the strong tendency of silicon to stabilise the Ferrite phase, a number of elements are utilised, favouring the Austenite formation.","Aming those elements, Carbon is particularly important due to its intrinsic austenitising effect as well as to its particular mobility into the matrix, making it possible its easy elimination by means of solid-state decarburising processes which, in this field, are usually carried out by extraction from the strip surfaces utilising annealing atmospheres having a controlled oxidising potential.","The carbon is conveniently present in the steel composition in amount apt to control the desired Austenite fraction, in that in this way it is possible to rise again the stability of the Ferrite by means of a simple decarburisation process, and thus avoiding during the final secondary recrystallisation annealing important phase transition phenomena which would be detrimental for the final desired texture.","As known, however, in said materials itis necessary to reduce the carbon content in the final products at levels of under 50 ppm, to eliminate the adverse effect on the core losses due to formation of carbides.","The higher the carbon content of the alloy, the longer the time necessary to carry out the decarburisation.","For productivity reasons it is then convenient to keep the carbon content within a maximum of 0,1 wt %.","Present inventors eveluated the obtainable Austenite fractions according to different alloy compositions both experimentally and according to empirical relationships available in the literature.","A third aspect of the invention resides in that the Derrite to Austenite transformation in the metal matrix of the cast strip is induced, in a temperature interval centered around 1150° C., typically 1000-1300° C., by means of a sudden deformation higher than 20%, by rolling between cooled rolls, in-line with the continuous casting and before the coiling.","Said sudden and localised deformation imparts to the material the energy necessary to nucleation and formation of the Austenite phase in the matrix, which phase would not be obtained for kinetic reasons, though thermodynamically very stable.","In fact, to obtain equilibrium conditions between the two phases at the considered temperature very long times are necessary, while the working and cooling times are intrinsecally very short, particularly in the case of direct casting as strip (strip casting).","The phase transformation from Ferrite to Austenite is tunable, according to present invention, in quantity, according to selection of chemical composition, and consistently reproducible, as necessary in an industrial process.","As a consequence of the phase transformation induced in the temperature interval defined according to present invention, the grains distribution in the produced strip, in terms both of dimensions and of texture, is exrtremely homogeneous and reproducible through the whole geometrical profile of the strip.","This, in particular, solves the problem the drawback of microstructural etereogeneity, typical of the production of oriented grain steel strips, in that the selection process of the final texture is sensible even to small local differences in the structure and orientation of grains, and even more sensible in the case of strip-cast products.","In fact, in the traditional processes the strip structure before cold rolling is the result of a strong hot deformation of the cast slabs, which contributes to fragment, recrystallise and homogenise the solidification structure; on the contrary, in the strips obtained by direct solidification the structure directly depends on the solidification one, and due to the high solidification speed and to the strongly dynamic nature of the process any even small fluctuation of the casting conditions (such as strip thickness, casting speed, heat transfer to the casting rolls, etc.)","can induce local variations, periodic or random, in the solidification structure and therefore in the final strips micro-structure throughout its geometrical profile.","The process of the invention overcomes the drawbacks inherent in the directly cast steel strips, due to lack of high hot deformation levels refining and homogenising the micro-structure.","Said high deformation levels are typical of technologies based on conventional casting, and in present invention are very efficiently replaced by causing a controlled, as amount and distribution, phase transformation Ferrite to Austenute, able to refine and homogenise the micro-structure.","The high solification speeds proper of strip casting are also an important metallurgical opportunity to exploit in the best way the process according to present invention.","In fact, in the traditional technologies starting from slabs or ingots the Ferrite/Austenite transformation, if any, is localised in chemical segregation zones, in which austenitising elements are concentrated, particularly in the semi-products core.","Thus, in said zones the austenitic transformation can occur, due to local concentration of austenitising elements, even if the mean chemical composition of the steel would not consent it.","On the contrary, in the strip casting the high solidifation speeds strongly limit the segregating phenomena, thus making homogeneous in the matrix the distribution of austenitising elements.","In said conditions, by hot rolling in the prescribed temperature field, it is obtained in a stable and reproducible way the volumetric fraction of Austenite, defined by chosing the steel composition, throughout the whole geometrical profile of the strip.","A further element of present invention if the definition of a process utilising a controlled volumetric fraction of Austenite, induced within the strip as above defined, to obtain a controlled distribution of hard phases (Carbides, Cementite, Pearlite, Bainite) and to control the formation of some Martensite (tetragonal lattice) within the metal matrix, by quenching the strip between the in-line hot rolling and the coiling steps.","The presence of homogeneously distributed hard phases (quenching phases) permits the cold rolling to control the adequate deformation texture, clearly because of the different deformation models and of the higher hardening levels obtained by cold rolling when hard phases are present with reference to the case in which a quenching structure is not present.","This permits to reduce the thickness of the strip to be cold rolled (for the same final thickness) and consequently to reduce the thickness of the cast strip, with important advantages on the casting productivity.","In fact, the thinner the cast strip, the higher the casting productivity, in that the strip becomes longer in direct proportion to the thickness reduction, while the casting speed rises with the square of the thickness reduction.","A further element of present invention is a process in which the strip, after in-line deformation, is kept at a temperature around 1150° C., typically 1100-1200° C., for at least 5 s, utilising a continuous heating apparatus between the in-line rolling mill and the coiler.","This can be obtained for instance a heating chamber provided with burners, or with electric heating, or with infrared lamps, or with an induction-heating apparatus; however, any active or passive system apt to obtain the desired strip temperature in the prescribed interval and for at least 5 s. In this case, the optional quenching step will be carried out at the exit from said chamber.","Another aspect of present invention is a process in which the strip is annealed, before cold rolling, at temperature not exceeding 1200° C., preferably not exceeding 1170° C. Such an annealing can be advantegeous for the grain oriented electrical steel strip production process, for a number of reasons, particularly with respect to the magnetic characteristics control of the final products.","Some useful phenomena for the process are, for instance, the precipitation of non-metallic second phases, necessary in present products to the control of the oriented secondary recrystallisation, or the possibility to carry out a controlled surface decarburisation of the strips before the cold rolling, which can have positive effects on the texture of the cold rolled strip.","Moreover, this annealing can offer the possibility to shift to this process step the formation of quenching phases, instead of forming them before coiling the strip after the casting process.","In this case, at the end of the annealing furnace a suitable cooling device must be present able to reach the necessary cooling speed.","For instance, the strip cooling can be usefully obtained with respect to the teaching of present invention, by means of a group of lances provided with nuzzles to spray on the strip surface a mixture of water and steam, at a controlled pressure.","Typically, after the in-line rolling the strip is quanched to obtain a Martensite volume fraction comprised between 5 and 15%.","The quenching device operate starting from a temperature of between 750 and 950° C., to cool down the strip down to 400° C. in less than 12 s. A last element of present invention is a process in which the chemical composition requires the presence of elements chosen between two distinct classes: (i) elements useful to control the desired equilibtium bewteen Austenite anf Ferrite in the metal matrix and (ii) elements useful to control a second phases distribution, such as sulphides, selenides, nitrides, carbo-nitrides etc., necessary for the grain growth control and of grain orientation during the primary and secondary recrystallisation steps.","Typically, the cast steel composition comprises 2,5-5 wt % Si; 200-1000 ppm C, 0,05-0,5 wt % Mn, 0,07-0,5 wt % Cu, less than 2 wt % Cr+Ni+Mo, less than 30 ppm O, less than 500 ppm S+Se, 50-400 ppm Al, less than 100 ppm N. To this composition at least an element can be added chosen in the group consisting of Zr, Ti, Ce, B, Ta, Nb, V and Co, and at least an element chosen in the group consisting of Sn, Sb, P, Bi.","Many are the elements useful to the equilibrium control berween Austenite and Ferrite phases and there are no specific choice limitations, but cost and yield convenience.","However, and specifically in electric-furnace steel shops utilising steel scraps as raw material, it can be convenient to balance the content of silicon as well as of chromium, nickel, molybdenum, niobium, copper, manganese and tin.","Many are also the elements useful to control the distribution of second phases particles for the grain growth inhibition.","It is convenient to chose said elements among the ones able to form suphides, selenides, carbonitrides, nitrides, to obtain a mix of second phases having different composition in which co-exist compounds thermally stable, as solubility, at different temperatures.","As a consequence of this choice, the drag force of the grain boundaries movement due to second phases particles gradually diminishes as temperature rises, in that during the heat treatments the more soluble particles will dissolve and/or grow before the less soluble ones.","This permits a better control of grain growth, with respect to the utilisation of inhibitors of a single composition type chacarterised by a narrower solubilisation temperatures interval.","The following Examples are intended solely to illustration purposes not limiting the scope of present invention.","EXAMPLE 1 A number of steels having the compositions shown in Table 1 were cast as a strip 3,5 mm thick in a strip casting machine provided with twin counter-rotating rolls.","The cast strips were then in-line hot rolled at the temperature of 1150° C. to a 2,0 mm thickness.","During the casting operation of each steel composition and at about mid casting time, the cast strip thickness was reduced to 2,0 mm and the in-line rolling suspended.","The hot rolled strips were then annealed at 1100° C. and single-stage cold rolled to 0,30 mm.","TABLE 1 C Si Mn S Steel (ppm) (%) (%) (ppm) Cr (ppm) Ni (ppm) Al (ppm) Cu (ppm) A 500 3.1 0.2 75 300 100 250 0.1 B 300 3.1 0.1 68 350 120 270 0.15 C 350 3.2 0.4 70 320 110 230 0.3 D 400 3.1 0.3 80 290 150 280 0.25 E 500 3.1 0.4 50 400 100 280 0.2 The cold rolled strips were then decarburised, coated with an MgO based annealing separator, box annealed with an heating rate of 15° C./h up to 1200° C., held at this temperature for 20 h, and then received an insulating and tensioning coating.","On the as-cast strips the austenite (γ phase) content at 1150° C. was calculated by means of dilatometric measures; data obtained are shown in Table 2.TABLE 2 Steel γ(1150) (%) A 27 B 11 C 15 D 19 E 25 The magnetic characteristics measured on the final product for the different steel composition are shown in Table 3.TABLE 3 In-line hot rolled Not in-line rolled Steel B800 (mT) B800 (Mt) A 1950 1700 B 1720 1650 C 1730 1630 D 1900 1680 E 1945 1710 EXAMPLE 2 A number of steels having different compositions as shown in Table 4 were directy cast as strips 2,1 mm thick in a strip-casting machine provided with twin counter-rotating rolls.","TABLE 4 C Si Mn S Steel (ppm) (%) (%) (ppm) Cr (ppm) Ni (ppm) Al (ppm) Cu (ppm) A 550 3.3 0.3 80 450 200 280 0.15 B 300 3.1 0.2 68 350 120 270 0.2 C 350 3.2 0.4 70 320 130 230 0.3 D 400 3.0 0.3 80 290 180 280 0.25 E 400 3.1 0.4 75 250 200 290 0.25 The cast strips were then in-line hot rolled at 1170° C. to a thickness of 1,0 mm, quenched by means of water and steam at high pressure down to a temperature of 150° C. and then coiled.","After casting about half of the steel the quenching was stopped and the strips wound at 700° C. Table 5 shows the Martensite fractions metallographically measured on the strip after coiling.","TABLE 5 Quenched strip Not-quenched strip Steel Martensite (%) Martensite (%) A 19 0 B 3 0 C 5 0 D 13 0 E 15 0 The strips were then divided into lesser coils, part of which were cold rolled to 0,3 mm (the casting A did show fragility problems during cold rolling and was not transformed into finished product), decarburised, coated with an MgO based annealing separator, then box annealed with a heating rate of 20° C./h up to 1200° C. and then held at this temperature for 20 h. Table 6 shows the magnetic characteristics (induction at 800 A/m) measured on the finished product.","TABLE 6 Quenched strip Not-quenched strip Steel B800 (mT) B800 (mT) A — 1830 B 1790 1650 C 1890 1630 D 1920 1820 E 1950 1830 EXAMPLE 3 The other lesser rolls of Example 2 without quenching and coiled at 700° C. were annealed at 1150° C. for 60 s, quenchecd by means of water and steam at high pressure down to 150° C., pickled and colied at room temperature.","The strips were then transformed into finished product as in preceding Example.","Table 7 shows the Martensite fractions measured on the coiled strips and relevant magnetic characteristics.","TABLE 7 Martensite B800 Steel (%) (mT) A 12 1950 B 2 1700 C 5 1740 D 8 1920 E 9 1920 EXAMPLE 4 Five different alloys of composition (in ppm) shown in Table 8 were cast directly as strips 2,2-2,4 mm thick in a casting machine with twin counter-rotating rolls.","TABLE 8 Si C Mn Cu Sn Cr Mo Nb Ni P Al Ce N S A 3.2 0.07 0.40 0.25 0.1 0.03 0.1 0.03 0.02 — 0.030 0.01 0.01 0.010 B 3.3 0.06 0.06 0.07 0.09 0.03 — 0.03 — 0.004 — 0.007 0.025 C 3.0 0.03 0.95 0.40 0.06 0.30 0.02 0.02 0.20 0.02 0.015 — 0.007 0.015 D 3.1 0.05 0.15 0.25 — 0.02 0.03 — 0.02 — 0.028 — 0.008 0.007 E 3.4 0.07 0.40 0.35 — 0.03 0.05 0.01 0.03 0.01 0.030 — 0.008 0.006 TABLE 9 Decarburation.","T (° C.) A1 B1 C1 D1 E1 A2 B2 C2 D2 E2 830 1890 1800 1920 1930 1910 1690 1520 1730 1640 1580 850 1930 1750 1940 1910 1920 1730 1540 1780 1540 1630 870 1940 1590 1890 1900 1890 1780 1530 1690 1520 1540 The cast steels were in-line hot rolled at 1150° C. to a thickness of 1,2 mm.","From said coiled strips were obtained lesser coils.","For each condition a strip was then double-stage annealed with quick heating to 1170° C., cooling at 1100° C. and quenched to room temperature with water plus steam jets (strips A1, B1, C1, D1, E1).","A second group of strips, similar the the previous one was annealed with a similar thermal cycle, without however the quenching step (strips A2, B2, C2, D2, E2).","All the strips were then single-stage cold rolled to a final thickness of 0,29 mm.","The strips were then treated in a continuous pilot line for primary recrystallisation, nitriding, secondary recrystallisation.","Each strip was then treated as follows: in the first treating zone (primary recrystallisation) the temperatures of 830, 850 and 870° C. were adopted, in a wet Nitrogen-Hydrogen atmosphere with a pH2O/pH2 ratio of 0,60 and for 180 s (50 of which for the heating at treating temperature) in the second treating zone nitriding was carried out at 890° C. in wet Nitrogen-Hydrogen atmosphere with a pH2O/pH2 ratio of 0,09, with the addition of 30% vol of ammonia, for 50 s in the third zone, at 1100° C. in a wet Nitrogen/Hydrogen atmosphere with a pH2O/pH2 ratio of 0,01 for 50 s. After coating with an Mg/O based annealing separator the strips treated in the pilot line were then box annealed with a heating rate of about 60° C./h up to 1200° C. in a 50% Nitrogen-Hydrogen atmosphere, held at this temperature for 3 h in pure hydrogen and cooled down to 800° C. in hydrogen and subsequently to room temperature in nitrogen.","The magnetic characteristics measured on samples of each of said strips were measured as induction mean value B800 in mT, and are shown in Table 9."]],"string":"[\n [\n \"PROCESS FOR THE PRODUCTION OF GRAIN ORIENTED ELECTRICAL STEEL STRIPS\",\n \"A process for the production of grain oriented electrical Fe—Si strips in which a Si-containing alloy is directly cast as a strip between 2.5-5.0 mm thick and cold rolled in one stage, or in more stages with intermediate annealing, to a final thickness of between 0.15-1.0 mm.\",\n \"The strip is then continuously annealed to carry out the primary recrystallization and then annealed to carry out the oriented secondary recrystallization.\",\n \"The process further includes that after solidification of the strip, and before its coiling, a phase transformation from Ferrite to Austenite is induced into the metal matrix for a volume fraction between 25-60%, obtained by controlling the alloy composition so that the Austenite fraction is allowed within the stability equilibrium between the two phases.\",\n \"The strip is then deformed by rolling in-line with the casting step to obtain a deformation higher than 20% in the temperature interval 1000-1300° C.\"\n ],\n [\n \"1-8.\",\n \"(canceled) 9.A process for the production of electrical grain oriented Fe—Si strips in which a Si-containing molten alloy composition is directly cast as continuous strips 2.5 to 5 mm thick, cold rolled in one step or more steps with intermediate annealing to a final thickness of between 1 and 0.15 mm, the strip being then continuously annealed to carry out oriented secondary recrystallisation, characterised in that after the strip solidification and before coiling of said strip in a coiling phase, a ferrite to austenite transformation is induced in the metal matrix via deformation, by rolling said strip between two cooled rolls to obtain a deformation over 20% in a temperature range of 1000-1300° C., thereby producing a volume fraction of austenite to be between 25 and 60%, said molten alloy composition being chosen such that said volume fraction of austenite is stable in a temperature interval of between 1100 and 1200° C. 10.The process according to claim 9, in which between the rolling phase and the coiling one, the strip is held between 1100 and 1200° C. for at least 5 s. 11.The process according to claim 9, in which the as-solidified strip thickness is comprised between 1.5 and 4.0 mm and after the rolling phase the strip is quenched to obtain a volume fraction of martensite comprised between 5 and 15%.\",\n \"12.The process according to claim 9, in which before cold rolling the strip is annealed at a maximum temperature of 1200° C. 13.The process according to claim 12, in which after said annealing the strip is continuously quenched from a temperature comprised between 750 and 950° C. down to 400° C. in less than 12 s. 14.The process according to claim 9, in which the cast alloy comprises 2.5-5.0 wt % Si, 200-1000 ppm C, 0.05-0.5 wt % Mn, 0.07-0.5 wt % Cu, less than 2 wt % Cr+Ni+Mo.\",\n \"Less than 30 ppm O, less than 500 ppm S+Se, 50-400 ppm Al, less than 100 ppm N. 15.The process according to claim 9, in which in the alloy at least an element is added chosen in the group consisting of Zr, Ti, Ce, B, Ta, Nb, V, Co. 16.The process according to claim 9, in which in the alloy at least an element is added chosen between Sn, Sb, P, Bi.\",\n \"17.A process for the production of grain oriented electrical Fe—Si strip, in which a Si-containing alloy is directly cast as continuous strip 2.5 to 5 mm thick, in-line hot-rolled and then cold-rolled in one step or more steps, with intermediate annealing, to a final thickness of between 1 and 0.15 mm, the cold-rolled strip being, then continuously annealed to carry out primary recrystallisation and subsequently again annealed to carry out secondary recrystallisation, characterized in that the Si-containing alloy composition is selected to induce in the alloy, during the hot-rolling step in which a deformation rate of at least 20% is utilized in a temperature interval of between 1000 and 1300° C., a ferrite to austenite phase transformation with an austenite volume fraction of between 25 to 60% stable in a temperature interval of between 1100 to 1200° C. 18.The process according to claim 17, in which between the hot-rolling and the coiling steps the strip is held in the temperature range of 1100 to 1200° C. for at least 5 s. 19.The process according to claim 17, in which an as-cast strip 2.5 to 4 mm thick is in-line hot-rolled and the thus obtained hot-rolled strip is quenched to obtain a volume fraction of martensite comprised between 5 and 15%.\",\n \"20.The process according to claim 17, in which before cold-rolling the strip is annealed at a maximum temperature of 1200° C. 21.The process according to claim 20, in which, after said annealing, the strip is continuously quenched from a temperature of between 750 and 950° C. down to 400° C. in less than 12 s. 22.The process according to claim 17, in which the cast alloy comprises 2.5-5.0 wt % Si, 200-1000 ppm C, 0.05-0.5 wt % Mn, 0.07-0.5% Cu, less than 2.0% Cr+Ni+Mo, less than 30 ppm O, less than 500 ppm S+Se, 50-400 ppm Al, less than 100 ppm N. 23.The process according to claim 22, in which in the alloy at least an element is added chosen in the group consisting of Zr, Ti, Ce, B, Ta, Nb, V, Co. 24.The process according to claim 22, in which in the allot at least an element is added chosen between Sn, Sb, P, Bi.\"\n ],\n [\n \"<SOH> FIELD OF THE INVENTION <EOH>Present invention refers to the production of grain oriented electrical steel strips havig excellent magnetic characteristics, dedicated to the production of transformer cores.\",\n \"More precisely, rhe invention refers to a process in which a Fe—Si alloy is continuously cast directly as strip and, before coiling, the strip itself is continuously deformed by rolling to induce the formation in the metal matrix of a given fraction of Austenite, controlled as amount and distribution, thus obtaining a strip micro-structure stably and uniformly recrystallised before cold rolling.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The aim of present invention is to solve the inconveniences due to the quality of electrical steel strips deriving from strip casting.\",\n \"Thus, it is an object of present invention a process for the production of electrical steel strips in which, through an in-line thickness reduction of the strip between casting and coiling stations, a significant level of recrystallisation by means of phase transformation is induced, thus normalising the crystalline structure before cold rolling, so that possible fluctuations in the process conditions are substantially non-influent with respect to the quality of the final product.\",\n \"Another object of present invention is to make it possible to industrially produce grain oriented electrical steel strips having excellent magnetic characteristics and constant quality, the process being stable and simplyfied with respect to the conventional processes currently utilised.\",\n \"Further objects of present invention will be evident from the following description of the invention.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"FIELD OF THE INVENTION Present invention refers to the production of grain oriented electrical steel strips havig excellent magnetic characteristics, dedicated to the production of transformer cores.\",\n \"More precisely, rhe invention refers to a process in which a Fe—Si alloy is continuously cast directly as strip and, before coiling, the strip itself is continuously deformed by rolling to induce the formation in the metal matrix of a given fraction of Austenite, controlled as amount and distribution, thus obtaining a strip micro-structure stably and uniformly recrystallised before cold rolling.\",\n \"STATE OF THE ART Grain oriented electrical steel strips (Fe—Si) are typically industrially produced as strips having a thickness comprised between 0,18 and 0,50 mm and are characterised by magnetic properties variable according to the specific product class.\",\n \"Said classification substantially refers to the specific power losses of the strip subjected to given electromagnetic work conditions (e.g.\",\n \"P50 Hz at 1,7 Tesla, in W/kg), evaluated along a specific reference direction (rolling direction).\",\n \"The main utilisation of said strips is the production of transformer cores.\",\n \"Good magnetic properties (strongly anisotropic) are obtained controlling the final crystalline structure of the strips to obtain all, or almost all, the grains oriented to have their easiest magnetization direction (the <001> axis) aligned in the most perfect way with the rolling direction.\",\n \"In practice, final products are obtained having the grains mean diameter generally comprised between 1 and 20 mm having an orientation centred around the Goss orientation ({110} <001>).\",\n \"The minor the angular dispersion around the Goss one, the better the product magnetic permeability and hence the lesser the magnetic losses.\",\n \"The final products having low magnetic losses (core losses) and high permeability have interesting advantages in terms of design, dimensions and yield of the transformers.\",\n \"The first industrial production of the above materials was described by the U.S. Firm ARMCO at the beginning of the thirties (U.S. Pat.\",\n \"No.\",\n \"1,956,559).\",\n \"Many important improvements have been since introduced in the production technology of grain oriented electrical strips, in terms both of magnetic and physical quality of products and of transformation costs and cycles rationalisation.\",\n \"All existing technologies exploit the same metallurgical strategy to obtain a very strong Goss structure in the final products, i.e.\",\n \"the process of oriented secondary recrystallisation guided by uniformly distributed second phases and/or segregating elements.\",\n \"The, non metallic, second phases and the segregating elements play a fundamental role in controlling (slowing down) the movement of grain boundaries doing the final annealing which actuates the selective secondary recrystallisation process.\",\n \"In the original ARMCO technology, utilising MnS as inhibitor of the grain boundaries movement, and in the subsequent technology developed by NSC, in which the inhibitors are mainly aluminium nitrides (AIN+MnS) (EP 8.385, EP 17.830, EP 202.339), a very important binding step common to both production processes is the heating of the continuously cast slabs (ingots, in old times), immediately before the hot rolling, at very high temperatures (around 1400° C.) for a time sufficient to guarantee a complete dissolution of sulphides and/or nitrides coarsely precipitated during the slab cooling after casting, to re-precipitate them in a very fine and uniformly distributed form throughout the metallic matrix of the hot rolled strips.\",\n \"Such a fine re-precipitation can be started and completed, as well as the precipitates dimensions adjusted, during the process, in any case, however, before the cold rolling.\",\n \"The slab heating to said temperatures requires using special furnaces (pushing furnaces, liquid-slag walking-beam furnaces, induction furnaces) due to the ductility at high temperatures of the Fe-3% Si alloys and to formation of liquid slags.\",\n \"New casting technologies of the liquid steel are intended to simplify the production processes to make them more compact and flexible and to reduce costs.\",\n \"One of said technologies is the “thin slab” casting, consisting in the continuous casting of slabs having the typical thickness of conventional already roughened slabs, apt to a direct hot rolling, through a sequence of slabs continuous casting, treating in continuous tunnel-furnaces to rise/maintain the temperature of slabs and finishing-rolling, down to coiled strip.\",\n \"The problems connected to the utilisation of said technique for grain oriented products mainly consist in the difficulty to maintain and control the high temperatures necessary to keep in solution the elements forming the second phases, which have to be finely precipitated at the beginning of the finishing hot-rolling step, if desired best micro-structural and magnetic characteristics are to be obtained in the end products.\",\n \"Such problems were dealt with in different ways, for instance utilising the low thickness of the cast slabs in connection to specific concentration intervals of the micro-alloying elements to stably control the second phases precipitation (grain growth inhibitors) during hot rolling, or drastically modifying the strategy of the inhibitors formation in the metal matrix.\",\n \"The casting technique potentially offering the highest rationalisation level of the processes and the higher production flexibility is the one consisting in the direct production of strips from the liquid steel (Strip Casting), totally eliminating the hot rolling step.\",\n \"Such an exaordinary innovation was conceived and patented long time ago, and since long time were also devised and patented process conditions to produce electrical steel strips, and more particularly grain oriented ones.\",\n \"However, up to now there is not an industrial production in the world of grain oriented electrical steel according to the above technique, though the state of the art relating to the casting machines is ready for industrial applications, as shown by existing plants, producing only carbon steels and stainless steels.\",\n \"The present inventors believe that to industrially produce grain oriented electrical steel strips from direct solidification of a strip (Strip Casting) it is necessary to have a strip micro-structure before cold rolling significantly different from the one obtained during the casting stage.\",\n \"The high solidification speed of the cast strip makes it difficult to have a homogeneous and reproducible grain structure throughout the strip and between different castings, due to the high sensitivity of the solidification structure to the fluctuations of the casting conditions and to the alloy composition.\",\n \"The micro-structure of the intermediate products starting from strip casting is much more influenced by the solidification structure, with respect to the ones derived from conventional slab casting, because of the lack of deformation in the strip during the typical hot rolling.\",\n \"SUMMARY OF THE INVENTION The aim of present invention is to solve the inconveniences due to the quality of electrical steel strips deriving from strip casting.\",\n \"Thus, it is an object of present invention a process for the production of electrical steel strips in which, through an in-line thickness reduction of the strip between casting and coiling stations, a significant level of recrystallisation by means of phase transformation is induced, thus normalising the crystalline structure before cold rolling, so that possible fluctuations in the process conditions are substantially non-influent with respect to the quality of the final product.\",\n \"Another object of present invention is to make it possible to industrially produce grain oriented electrical steel strips having excellent magnetic characteristics and constant quality, the process being stable and simplyfied with respect to the conventional processes currently utilised.\",\n \"Further objects of present invention will be evident from the following description of the invention.\",\n \"DETAILED DESCRIPTION OF THE INVENTION A first important aspect of present invention resides in that a molten alloy containing silicon is directly solidified in the form of a strip, through the casting technology known as strip casting (casting between twin cooled and counter-rotating rolls), thus avoiding, with respect to currently utilised technologies, casting the alloy in slabs or ingots, subjecting said slabs to thermal treatment in special high-temperature furnaces for long times (to attain the necessary thermal homogeneity) and transforming said slabs into strips through hot rolling with total reductions which, according to the slab casting technologies, vary between 96 and 99%.\",\n \"A second important aspect of present invention resides in that the chemical composition of the Silicon containing alloy is selected specifically to control the thermodynamic stability of the Austenite phase in the matrix (face-centered cubic lattice) in equilibrium with the Ferrite phase (body-centered cubic lattice).\",\n \"More precisely, to obtain excellent final magnetic characteristics, it is convenient to adjust the chemistry of the alloy so that an Austenite fraction comprised between 25 and 60% is stable between 1100 and 1200° C. Consequently, to balance the strong tendency of silicon to stabilise the Ferrite phase, a number of elements are utilised, favouring the Austenite formation.\",\n \"Aming those elements, Carbon is particularly important due to its intrinsic austenitising effect as well as to its particular mobility into the matrix, making it possible its easy elimination by means of solid-state decarburising processes which, in this field, are usually carried out by extraction from the strip surfaces utilising annealing atmospheres having a controlled oxidising potential.\",\n \"The carbon is conveniently present in the steel composition in amount apt to control the desired Austenite fraction, in that in this way it is possible to rise again the stability of the Ferrite by means of a simple decarburisation process, and thus avoiding during the final secondary recrystallisation annealing important phase transition phenomena which would be detrimental for the final desired texture.\",\n \"As known, however, in said materials itis necessary to reduce the carbon content in the final products at levels of under 50 ppm, to eliminate the adverse effect on the core losses due to formation of carbides.\",\n \"The higher the carbon content of the alloy, the longer the time necessary to carry out the decarburisation.\",\n \"For productivity reasons it is then convenient to keep the carbon content within a maximum of 0,1 wt %.\",\n \"Present inventors eveluated the obtainable Austenite fractions according to different alloy compositions both experimentally and according to empirical relationships available in the literature.\",\n \"A third aspect of the invention resides in that the Derrite to Austenite transformation in the metal matrix of the cast strip is induced, in a temperature interval centered around 1150° C., typically 1000-1300° C., by means of a sudden deformation higher than 20%, by rolling between cooled rolls, in-line with the continuous casting and before the coiling.\",\n \"Said sudden and localised deformation imparts to the material the energy necessary to nucleation and formation of the Austenite phase in the matrix, which phase would not be obtained for kinetic reasons, though thermodynamically very stable.\",\n \"In fact, to obtain equilibrium conditions between the two phases at the considered temperature very long times are necessary, while the working and cooling times are intrinsecally very short, particularly in the case of direct casting as strip (strip casting).\",\n \"The phase transformation from Ferrite to Austenite is tunable, according to present invention, in quantity, according to selection of chemical composition, and consistently reproducible, as necessary in an industrial process.\",\n \"As a consequence of the phase transformation induced in the temperature interval defined according to present invention, the grains distribution in the produced strip, in terms both of dimensions and of texture, is exrtremely homogeneous and reproducible through the whole geometrical profile of the strip.\",\n \"This, in particular, solves the problem the drawback of microstructural etereogeneity, typical of the production of oriented grain steel strips, in that the selection process of the final texture is sensible even to small local differences in the structure and orientation of grains, and even more sensible in the case of strip-cast products.\",\n \"In fact, in the traditional processes the strip structure before cold rolling is the result of a strong hot deformation of the cast slabs, which contributes to fragment, recrystallise and homogenise the solidification structure; on the contrary, in the strips obtained by direct solidification the structure directly depends on the solidification one, and due to the high solidification speed and to the strongly dynamic nature of the process any even small fluctuation of the casting conditions (such as strip thickness, casting speed, heat transfer to the casting rolls, etc.)\",\n \"can induce local variations, periodic or random, in the solidification structure and therefore in the final strips micro-structure throughout its geometrical profile.\",\n \"The process of the invention overcomes the drawbacks inherent in the directly cast steel strips, due to lack of high hot deformation levels refining and homogenising the micro-structure.\",\n \"Said high deformation levels are typical of technologies based on conventional casting, and in present invention are very efficiently replaced by causing a controlled, as amount and distribution, phase transformation Ferrite to Austenute, able to refine and homogenise the micro-structure.\",\n \"The high solification speeds proper of strip casting are also an important metallurgical opportunity to exploit in the best way the process according to present invention.\",\n \"In fact, in the traditional technologies starting from slabs or ingots the Ferrite/Austenite transformation, if any, is localised in chemical segregation zones, in which austenitising elements are concentrated, particularly in the semi-products core.\",\n \"Thus, in said zones the austenitic transformation can occur, due to local concentration of austenitising elements, even if the mean chemical composition of the steel would not consent it.\",\n \"On the contrary, in the strip casting the high solidifation speeds strongly limit the segregating phenomena, thus making homogeneous in the matrix the distribution of austenitising elements.\",\n \"In said conditions, by hot rolling in the prescribed temperature field, it is obtained in a stable and reproducible way the volumetric fraction of Austenite, defined by chosing the steel composition, throughout the whole geometrical profile of the strip.\",\n \"A further element of present invention if the definition of a process utilising a controlled volumetric fraction of Austenite, induced within the strip as above defined, to obtain a controlled distribution of hard phases (Carbides, Cementite, Pearlite, Bainite) and to control the formation of some Martensite (tetragonal lattice) within the metal matrix, by quenching the strip between the in-line hot rolling and the coiling steps.\",\n \"The presence of homogeneously distributed hard phases (quenching phases) permits the cold rolling to control the adequate deformation texture, clearly because of the different deformation models and of the higher hardening levels obtained by cold rolling when hard phases are present with reference to the case in which a quenching structure is not present.\",\n \"This permits to reduce the thickness of the strip to be cold rolled (for the same final thickness) and consequently to reduce the thickness of the cast strip, with important advantages on the casting productivity.\",\n \"In fact, the thinner the cast strip, the higher the casting productivity, in that the strip becomes longer in direct proportion to the thickness reduction, while the casting speed rises with the square of the thickness reduction.\",\n \"A further element of present invention is a process in which the strip, after in-line deformation, is kept at a temperature around 1150° C., typically 1100-1200° C., for at least 5 s, utilising a continuous heating apparatus between the in-line rolling mill and the coiler.\",\n \"This can be obtained for instance a heating chamber provided with burners, or with electric heating, or with infrared lamps, or with an induction-heating apparatus; however, any active or passive system apt to obtain the desired strip temperature in the prescribed interval and for at least 5 s. In this case, the optional quenching step will be carried out at the exit from said chamber.\",\n \"Another aspect of present invention is a process in which the strip is annealed, before cold rolling, at temperature not exceeding 1200° C., preferably not exceeding 1170° C. Such an annealing can be advantegeous for the grain oriented electrical steel strip production process, for a number of reasons, particularly with respect to the magnetic characteristics control of the final products.\",\n \"Some useful phenomena for the process are, for instance, the precipitation of non-metallic second phases, necessary in present products to the control of the oriented secondary recrystallisation, or the possibility to carry out a controlled surface decarburisation of the strips before the cold rolling, which can have positive effects on the texture of the cold rolled strip.\",\n \"Moreover, this annealing can offer the possibility to shift to this process step the formation of quenching phases, instead of forming them before coiling the strip after the casting process.\",\n \"In this case, at the end of the annealing furnace a suitable cooling device must be present able to reach the necessary cooling speed.\",\n \"For instance, the strip cooling can be usefully obtained with respect to the teaching of present invention, by means of a group of lances provided with nuzzles to spray on the strip surface a mixture of water and steam, at a controlled pressure.\",\n \"Typically, after the in-line rolling the strip is quanched to obtain a Martensite volume fraction comprised between 5 and 15%.\",\n \"The quenching device operate starting from a temperature of between 750 and 950° C., to cool down the strip down to 400° C. in less than 12 s. A last element of present invention is a process in which the chemical composition requires the presence of elements chosen between two distinct classes: (i) elements useful to control the desired equilibtium bewteen Austenite anf Ferrite in the metal matrix and (ii) elements useful to control a second phases distribution, such as sulphides, selenides, nitrides, carbo-nitrides etc., necessary for the grain growth control and of grain orientation during the primary and secondary recrystallisation steps.\",\n \"Typically, the cast steel composition comprises 2,5-5 wt % Si; 200-1000 ppm C, 0,05-0,5 wt % Mn, 0,07-0,5 wt % Cu, less than 2 wt % Cr+Ni+Mo, less than 30 ppm O, less than 500 ppm S+Se, 50-400 ppm Al, less than 100 ppm N. To this composition at least an element can be added chosen in the group consisting of Zr, Ti, Ce, B, Ta, Nb, V and Co, and at least an element chosen in the group consisting of Sn, Sb, P, Bi.\",\n \"Many are the elements useful to the equilibrium control berween Austenite and Ferrite phases and there are no specific choice limitations, but cost and yield convenience.\",\n \"However, and specifically in electric-furnace steel shops utilising steel scraps as raw material, it can be convenient to balance the content of silicon as well as of chromium, nickel, molybdenum, niobium, copper, manganese and tin.\",\n \"Many are also the elements useful to control the distribution of second phases particles for the grain growth inhibition.\",\n \"It is convenient to chose said elements among the ones able to form suphides, selenides, carbonitrides, nitrides, to obtain a mix of second phases having different composition in which co-exist compounds thermally stable, as solubility, at different temperatures.\",\n \"As a consequence of this choice, the drag force of the grain boundaries movement due to second phases particles gradually diminishes as temperature rises, in that during the heat treatments the more soluble particles will dissolve and/or grow before the less soluble ones.\",\n \"This permits a better control of grain growth, with respect to the utilisation of inhibitors of a single composition type chacarterised by a narrower solubilisation temperatures interval.\",\n \"The following Examples are intended solely to illustration purposes not limiting the scope of present invention.\",\n \"EXAMPLE 1 A number of steels having the compositions shown in Table 1 were cast as a strip 3,5 mm thick in a strip casting machine provided with twin counter-rotating rolls.\",\n \"The cast strips were then in-line hot rolled at the temperature of 1150° C. to a 2,0 mm thickness.\",\n \"During the casting operation of each steel composition and at about mid casting time, the cast strip thickness was reduced to 2,0 mm and the in-line rolling suspended.\",\n \"The hot rolled strips were then annealed at 1100° C. and single-stage cold rolled to 0,30 mm.\",\n \"TABLE 1 C Si Mn S Steel (ppm) (%) (%) (ppm) Cr (ppm) Ni (ppm) Al (ppm) Cu (ppm) A 500 3.1 0.2 75 300 100 250 0.1 B 300 3.1 0.1 68 350 120 270 0.15 C 350 3.2 0.4 70 320 110 230 0.3 D 400 3.1 0.3 80 290 150 280 0.25 E 500 3.1 0.4 50 400 100 280 0.2 The cold rolled strips were then decarburised, coated with an MgO based annealing separator, box annealed with an heating rate of 15° C./h up to 1200° C., held at this temperature for 20 h, and then received an insulating and tensioning coating.\",\n \"On the as-cast strips the austenite (γ phase) content at 1150° C. was calculated by means of dilatometric measures; data obtained are shown in Table 2.TABLE 2 Steel γ(1150) (%) A 27 B 11 C 15 D 19 E 25 The magnetic characteristics measured on the final product for the different steel composition are shown in Table 3.TABLE 3 In-line hot rolled Not in-line rolled Steel B800 (mT) B800 (Mt) A 1950 1700 B 1720 1650 C 1730 1630 D 1900 1680 E 1945 1710 EXAMPLE 2 A number of steels having different compositions as shown in Table 4 were directy cast as strips 2,1 mm thick in a strip-casting machine provided with twin counter-rotating rolls.\",\n \"TABLE 4 C Si Mn S Steel (ppm) (%) (%) (ppm) Cr (ppm) Ni (ppm) Al (ppm) Cu (ppm) A 550 3.3 0.3 80 450 200 280 0.15 B 300 3.1 0.2 68 350 120 270 0.2 C 350 3.2 0.4 70 320 130 230 0.3 D 400 3.0 0.3 80 290 180 280 0.25 E 400 3.1 0.4 75 250 200 290 0.25 The cast strips were then in-line hot rolled at 1170° C. to a thickness of 1,0 mm, quenched by means of water and steam at high pressure down to a temperature of 150° C. and then coiled.\",\n \"After casting about half of the steel the quenching was stopped and the strips wound at 700° C. Table 5 shows the Martensite fractions metallographically measured on the strip after coiling.\",\n \"TABLE 5 Quenched strip Not-quenched strip Steel Martensite (%) Martensite (%) A 19 0 B 3 0 C 5 0 D 13 0 E 15 0 The strips were then divided into lesser coils, part of which were cold rolled to 0,3 mm (the casting A did show fragility problems during cold rolling and was not transformed into finished product), decarburised, coated with an MgO based annealing separator, then box annealed with a heating rate of 20° C./h up to 1200° C. and then held at this temperature for 20 h. Table 6 shows the magnetic characteristics (induction at 800 A/m) measured on the finished product.\",\n \"TABLE 6 Quenched strip Not-quenched strip Steel B800 (mT) B800 (mT) A — 1830 B 1790 1650 C 1890 1630 D 1920 1820 E 1950 1830 EXAMPLE 3 The other lesser rolls of Example 2 without quenching and coiled at 700° C. were annealed at 1150° C. for 60 s, quenchecd by means of water and steam at high pressure down to 150° C., pickled and colied at room temperature.\",\n \"The strips were then transformed into finished product as in preceding Example.\",\n \"Table 7 shows the Martensite fractions measured on the coiled strips and relevant magnetic characteristics.\",\n \"TABLE 7 Martensite B800 Steel (%) (mT) A 12 1950 B 2 1700 C 5 1740 D 8 1920 E 9 1920 EXAMPLE 4 Five different alloys of composition (in ppm) shown in Table 8 were cast directly as strips 2,2-2,4 mm thick in a casting machine with twin counter-rotating rolls.\",\n \"TABLE 8 Si C Mn Cu Sn Cr Mo Nb Ni P Al Ce N S A 3.2 0.07 0.40 0.25 0.1 0.03 0.1 0.03 0.02 — 0.030 0.01 0.01 0.010 B 3.3 0.06 0.06 0.07 0.09 0.03 — 0.03 — 0.004 — 0.007 0.025 C 3.0 0.03 0.95 0.40 0.06 0.30 0.02 0.02 0.20 0.02 0.015 — 0.007 0.015 D 3.1 0.05 0.15 0.25 — 0.02 0.03 — 0.02 — 0.028 — 0.008 0.007 E 3.4 0.07 0.40 0.35 — 0.03 0.05 0.01 0.03 0.01 0.030 — 0.008 0.006 TABLE 9 Decarburation.\",\n \"T (° C.) A1 B1 C1 D1 E1 A2 B2 C2 D2 E2 830 1890 1800 1920 1930 1910 1690 1520 1730 1640 1580 850 1930 1750 1940 1910 1920 1730 1540 1780 1540 1630 870 1940 1590 1890 1900 1890 1780 1530 1690 1520 1540 The cast steels were in-line hot rolled at 1150° C. to a thickness of 1,2 mm.\",\n \"From said coiled strips were obtained lesser coils.\",\n \"For each condition a strip was then double-stage annealed with quick heating to 1170° C., cooling at 1100° C. and quenched to room temperature with water plus steam jets (strips A1, B1, C1, D1, E1).\",\n \"A second group of strips, similar the the previous one was annealed with a similar thermal cycle, without however the quenching step (strips A2, B2, C2, D2, E2).\",\n \"All the strips were then single-stage cold rolled to a final thickness of 0,29 mm.\",\n \"The strips were then treated in a continuous pilot line for primary recrystallisation, nitriding, secondary recrystallisation.\",\n \"Each strip was then treated as follows: in the first treating zone (primary recrystallisation) the temperatures of 830, 850 and 870° C. were adopted, in a wet Nitrogen-Hydrogen atmosphere with a pH2O/pH2 ratio of 0,60 and for 180 s (50 of which for the heating at treating temperature) in the second treating zone nitriding was carried out at 890° C. in wet Nitrogen-Hydrogen atmosphere with a pH2O/pH2 ratio of 0,09, with the addition of 30% vol of ammonia, for 50 s in the third zone, at 1100° C. in a wet Nitrogen/Hydrogen atmosphere with a pH2O/pH2 ratio of 0,01 for 50 s. After coating with an Mg/O based annealing separator the strips treated in the pilot line were then box annealed with a heating rate of about 60° C./h up to 1200° C. in a 50% Nitrogen-Hydrogen atmosphere, held at this temperature for 3 h in pure hydrogen and cooled down to 800° C. in hydrogen and subsequently to room temperature in nitrogen.\",\n \"The magnetic characteristics measured on samples of each of said strips were measured as induction mean value B800 in mT, and are shown in Table 9.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10450969"}}},{"rowIdx":290,"cells":{"text":{"kind":"list like","value":[["Generic architecture for adaptable software","This invention concerns adaptable software.","In particular it concerns a method for constructing an adaptable soft-ware application.","A first step involves instantiating a first set of knowledge elements (kitems) related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application and used to produce dynamic and flexible templates.","A second step involves instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.","In a further aspect it concerns an adaptable software application comprising at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations."],["1.A method for constructing an adaptable software application, comprising the steps of: instantiating a first set of kitems related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application; and, instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.","2.A method according to claim 1, where the step of instantiating the second set of kitems occurs dynamically, during the use of the application.","3.A method according to claim 1, where further instantiating steps produce further sets of kitems from the immediately preceding set.","4.A method according to claim 1, comprising the further step of directing enquiries against kitems at any level of instantiation using a consultation process.","5.A method according to claim 1, comprising the further step of accessing the business objects using a consultation process during use of the application.","6.A method according to claim 1, comprising the further step of accessing kitems related to the type of business objects for the purposes of inspecting or managing the design of the application.","7.An adaptable software application, comprising: at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.","8.An application comprising adaptable, intelligent modules or agents that communicate with each other, where each module has its business logic defined and implemented using the adaptable system of claim 7.9.An application according to claim 8, further comprising adaptable meta-module to define and implement the logic between the other modules.","10.An application according to claim 9, where the meta-module monitors a part of, or the whole chain of modules."],["<SOH> BACKGROUND ART <EOH>An application is a software package designed for the manipulation, management and processing of business elements or objects.","These are often, but not exclusively, documents.","Manipulation, management and processing covers operations such as definition, access, editing, display, distribution, etc.","Such applications are normally hard-coded using standard programming techniques.","The knowledge of the business requirements to be met by the application is designed into the architecture of the application and the code itself.","The consequence of this approach is that systems are difficult to design and develop.","They are also difficult to maintain and modify.","When change is required to software, due to changing business conditions for example, designers must carry out extensive redesign and coding.","In many cases, the effort is so difficult and time consuming that it is not attempted.","Another approach is to develop an expert system in which the business rules are separate from the hard-coded part of the application.","This has the advantage that the business rules can, in principle, be changed without having to change the application.","In practice, the process of entering and testing the rules is difficult and time consuming, and their reliability can only be ascertained through extensive testing.","Expert systems are used to build only these applications for which knowledge is explicit and available, and where the effort required to enter and maintain the rules is clearly offset by big savings during operations.","The difficulties associated with developing, implementing and maintaining expert systems are well documented in the expert system literature of the past twenty years.","Few applications use expert systems technology; it is a niche technology.","A further approach is to build knowledge applications as described in co-pending Australian patent application PR2152 entitled Generic Knowledge Agents, International patent application PCT/AU99/00501 entitled Generic Knowledge Management System, and International patent application PCT/AU01/01155 entitled Intelligent Courseware Development and Delivery.","The contents of these three applications are incorporated into this specification by reference.","A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.","The process for building knowledge applications starts in a knowledge application development environment where the business needs of the application determine the knowledge elements, or ‘kitems’, to be instantiated, or created, from contexts and templates.","The outcome is an application or knowledge base in which the kitems are regions, knowledge items etc in the application—one can think of these kitems as documents.","A knowledge application is typically characterised by a single instantiation process for all the kitems.","The kitems can also be used as contexts or templates from which further kitems can be instantiated.","Once the knowledge application is built the kitems can be accessed and managed, that is edited, displayed, distributed, etc.","This entails defining an enquiry in a consultation process that retrieves the knowledge elements of interest for perusal and further manipulation.","The enquiry is itself a kitem instantiated from a kitem template.","Although designed and implemented in a radically different way from traditional expert systems, the business role of these knowledge applications and the way knowledge is accessed are similar to that of expert systems."],["<SOH> SUMMARY OF THE INVENTION <EOH>The invention, in a first aspect, is a method for constructing an adaptable software application, comprising the steps of: Instantiating a first set of kitems related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application.","Instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.","The first level of knowledge and the corresponding instantiations in effect produce dynamic templates that work as dynamic adaptations to the needs of users.","Alternatively, these templates can be activated manually.","The business objects in an application are treated as knowledge elements that embody business knowledge, such as knowledge about events that took place, or are expected to take place, in the business.","The business objects are easily created, and can be easily manipulated.","Because business objects are knowledge elements, they can be easily managed, that is accessed, displayed, analysed, etc.","Instantiating the second set of kitems may take place dynamically, during the use of the application.","Applications can be built with a greater number of cascades or instantiations larger than two.","Enquiries can be directed against kitems at any level of instantiation using a consultation process.","The business objects may be accessed using a consultation process during use of the application.","The kitems related to the type of business objects may be accessed for inspecting and managing the design of the application.","The invention, in a second aspect, is an adaptable software application, comprising: at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.","The use of a two level instantiation, or cascading, design enables the first level to define the application without hard-coding, and the second to define the documents that are handled by the application.","So, adaptable software transforms the definition of the application into specifying a knowledge application, and treats its documents (or business objects) as knowledge items inside the application.","The application may involve adaptable, intelligent modules or agents that communicate with each other, where each module has its business logic defined and implemented using the adaptable architecture of the system.","This means that each module can be quickly developed and that its functionality can evolve with the business and with experience.","Each adaptable module implements the knowledge about the best way to run that module.","These modules are used to facilitate development and to increase the functionality and adaptability of the solution.","An adaptable meta-module may define and implement the logic between the other modules.","In addition the meta-module may monitor a part of, or the whole chain of modules.","This software is able to adapt itself automatically using the knowledge entered into it, without programming, to determine when and how it should adapt; that is, modify its behaviour.","It may also adapt dynamically to the needs of users as software is being used.","Adaptablability is a very important commercial feature for software.","It holds the promise of speeding up software development, implementation, customization and flexibility.","In addition to RAD (Rapid Application Development), adaptable software holds the promise of software that can be modified easily during all stages of its life-cycle, without major effort or redesign.","Adaptable software also holds the promise of software that can adapt itself, that is customize itself and evolve as needs change while it is being used.","Adaptable software has the potential to save, worldwide, enormous sums in development costs, and to reduce the total cost of ownership of software solutions over their lifetimes.","Using adaptable software generic applications can be quickly produced.","The frameworks for a variety of generic applications, for a variety of domains, can be quickly produced.","An application framework is a knowledge system in which the knowledge elements express the knowledge about what the application is meant to do.","Also client specific applications can be quickly produced.","Knowledge in generic framework can be easily modified or added to, so as to represent the knowledge about a client's specific requirements."],["TECHNICAL FIELD This invention concerns adaptable software.","In particular it concerns a method for constructing an adaptable software application.","In a further aspect it concerns an adaptable software application.","BACKGROUND ART An application is a software package designed for the manipulation, management and processing of business elements or objects.","These are often, but not exclusively, documents.","Manipulation, management and processing covers operations such as definition, access, editing, display, distribution, etc.","Such applications are normally hard-coded using standard programming techniques.","The knowledge of the business requirements to be met by the application is designed into the architecture of the application and the code itself.","The consequence of this approach is that systems are difficult to design and develop.","They are also difficult to maintain and modify.","When change is required to software, due to changing business conditions for example, designers must carry out extensive redesign and coding.","In many cases, the effort is so difficult and time consuming that it is not attempted.","Another approach is to develop an expert system in which the business rules are separate from the hard-coded part of the application.","This has the advantage that the business rules can, in principle, be changed without having to change the application.","In practice, the process of entering and testing the rules is difficult and time consuming, and their reliability can only be ascertained through extensive testing.","Expert systems are used to build only these applications for which knowledge is explicit and available, and where the effort required to enter and maintain the rules is clearly offset by big savings during operations.","The difficulties associated with developing, implementing and maintaining expert systems are well documented in the expert system literature of the past twenty years.","Few applications use expert systems technology; it is a niche technology.","A further approach is to build knowledge applications as described in co-pending Australian patent application PR2152 entitled Generic Knowledge Agents, International patent application PCT/AU99/00501 entitled Generic Knowledge Management System, and International patent application PCT/AU01/01155 entitled Intelligent Courseware Development and Delivery.","The contents of these three applications are incorporated into this specification by reference.","A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.","The process for building knowledge applications starts in a knowledge application development environment where the business needs of the application determine the knowledge elements, or ‘kitems’, to be instantiated, or created, from contexts and templates.","The outcome is an application or knowledge base in which the kitems are regions, knowledge items etc in the application—one can think of these kitems as documents.","A knowledge application is typically characterised by a single instantiation process for all the kitems.","The kitems can also be used as contexts or templates from which further kitems can be instantiated.","Once the knowledge application is built the kitems can be accessed and managed, that is edited, displayed, distributed, etc.","This entails defining an enquiry in a consultation process that retrieves the knowledge elements of interest for perusal and further manipulation.","The enquiry is itself a kitem instantiated from a kitem template.","Although designed and implemented in a radically different way from traditional expert systems, the business role of these knowledge applications and the way knowledge is accessed are similar to that of expert systems.","SUMMARY OF THE INVENTION The invention, in a first aspect, is a method for constructing an adaptable software application, comprising the steps of: Instantiating a first set of kitems related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application.","Instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.","The first level of knowledge and the corresponding instantiations in effect produce dynamic templates that work as dynamic adaptations to the needs of users.","Alternatively, these templates can be activated manually.","The business objects in an application are treated as knowledge elements that embody business knowledge, such as knowledge about events that took place, or are expected to take place, in the business.","The business objects are easily created, and can be easily manipulated.","Because business objects are knowledge elements, they can be easily managed, that is accessed, displayed, analysed, etc.","Instantiating the second set of kitems may take place dynamically, during the use of the application.","Applications can be built with a greater number of cascades or instantiations larger than two.","Enquiries can be directed against kitems at any level of instantiation using a consultation process.","The business objects may be accessed using a consultation process during use of the application.","The kitems related to the type of business objects may be accessed for inspecting and managing the design of the application.","The invention, in a second aspect, is an adaptable software application, comprising: at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.","The use of a two level instantiation, or cascading, design enables the first level to define the application without hard-coding, and the second to define the documents that are handled by the application.","So, adaptable software transforms the definition of the application into specifying a knowledge application, and treats its documents (or business objects) as knowledge items inside the application.","The application may involve adaptable, intelligent modules or agents that communicate with each other, where each module has its business logic defined and implemented using the adaptable architecture of the system.","This means that each module can be quickly developed and that its functionality can evolve with the business and with experience.","Each adaptable module implements the knowledge about the best way to run that module.","These modules are used to facilitate development and to increase the functionality and adaptability of the solution.","An adaptable meta-module may define and implement the logic between the other modules.","In addition the meta-module may monitor a part of, or the whole chain of modules.","This software is able to adapt itself automatically using the knowledge entered into it, without programming, to determine when and how it should adapt; that is, modify its behaviour.","It may also adapt dynamically to the needs of users as software is being used.","Adaptablability is a very important commercial feature for software.","It holds the promise of speeding up software development, implementation, customization and flexibility.","In addition to RAD (Rapid Application Development), adaptable software holds the promise of software that can be modified easily during all stages of its life-cycle, without major effort or redesign.","Adaptable software also holds the promise of software that can adapt itself, that is customize itself and evolve as needs change while it is being used.","Adaptable software has the potential to save, worldwide, enormous sums in development costs, and to reduce the total cost of ownership of software solutions over their lifetimes.","Using adaptable software generic applications can be quickly produced.","The frameworks for a variety of generic applications, for a variety of domains, can be quickly produced.","An application framework is a knowledge system in which the knowledge elements express the knowledge about what the application is meant to do.","Also client specific applications can be quickly produced.","Knowledge in generic framework can be easily modified or added to, so as to represent the knowledge about a client's specific requirements.","BRIEF DESCRIPTION OF THE DRAWINGS An example of the invention will now be described with reference to the accompanying drawings, in which: FIG.","1 is a block diagram showing the steps in the construction and use of an adaptable software application.","FIG.","2 is a block diagram showing the architecture of an adaptable software application.","BEST MODES OF THE INVENTION Adaptable software differs from the knowledge applications described in the background art in that there are at least two levels of instantiation instead of one; and the templates for the second level instantiations are the first level instantiations (they can be objects and not classes in object oriented programming).","Adaptable software extends the architecture of known knowledge applications.","The use of a two level instantiation, or cascading, design enables the first level to define the application (without hard-coding) and the second to define the documents that are handled by the application.","Adaptable software transforms the definition of the application (typically hardcoded) into specifying a knowledge application, and treats its documents (or business objects) as knowledge items inside the application.","The process for building an adaptable software application will now be described with reference to FIG.","1.The process starts in a knowledge application development environment 10 where the business needs of the application determine the knowledge to be instantiated, or created, from contexts and templates.","The first step 11 is the instantiation of this knowledge.","The outcome is an application module dynamic framework 12 in which the kitems relate to the type of business objects or operations which will be handled by the adaptable software application.","This first step is distinguished from the construction of an application knowledge base since it does not produce the documents or the knowledge to be accessed via a consultation.","Instead these kitems describe the application as a framework that will be used to generate the dynamic templates for the objects, documents, that relate to the application.","A second step 13 then involves populating the adaptable software application with business objects instantiated from contexts or templates produced in the first step.","The process for defining these business objects is similar to defining an enquiry in a knowledge application.","That is, parameters are specified that determine the features of the business object which is then created.","The outcome is an application module 14 populated with business objects.","The business objects so created can be managed 15, that is accessed, edited, displayed, distributed, etc.","These operations are simple to implement as they correspond to manipulating kitems, and they use standard kitem manipulation methods, and other specific methods if required.","For example, the business objects could be accessed using a question-answer session.","Specific applications (or modules) are implemented by customising the generic framework produced in the first step, and this corresponds to instantiating different kitems as business objects for each application or module.","This customizing can take place dynamically, during the use of the application.","This corresponds to dynamic adaptation of the application to the needs of users.","Enquiries can be directed against both levels of instantiated kitems via a consultation process.","Enquiries directed to the upper level of documents, produced by the first step, are typically used for inspecting and managing the design of the application.","To users, adaptable applications do not need to look different from normal applications.","For example, a dialogue could take place that enquires about the needs or intentions of a user.","Knowing the user's needs or intentions then enables the application to adapt or customize dynamically the template that is presented to the user; this templates defines the documents, or the type of documents, that the user enters into the application.","The two level instantiation process can also be described as cascading knowledge items.","That is, some knowledge items determine which other knowledge items will be activated or instantiated to define documents.","Applications can be built with a greater number of cascades or instantiations larger than two.","Most software applications are made of modules that communicate with each other.","For example, a typical business management application is made of a client module, a project module, a contact module, a resource module and an accounting module.","FIG.","2 shows adaptable, intelligent modules or agents.","In particular there are adaptable intelligent client 21, project 22, contact 23, resource 24 and accounting 25 modules.","Each module is adaptable.","For example, in the client module, a different set of questions could be asked based on the age or location or socio-economic profile.","Similarly for the other modules.","Each module has its business logic defined and implemented using an adaptable architecture.","This means that each module can be quickly developed and that its functionality can evolve with the business and with experience.","Each adaptable module implements the knowledge about the best way to run that module.","These modules are used to facilitate development and to increase the functionality and adaptability of the solution.","Adaptable meta-module(s) 30 are used to define and implement the logic between the other modules.","This could include workflow or project costing for example, where a client would be treated differently based on the number of projects commissioned in the past eighteen months and the success of these projects.","In addition the meta-modules are adaptable.","In FIG.","2, the meta-module monitors a part of, or the whole chain of modules.","For example, the accounting module may detect that some projects from some clients are not profitable.","That means that the project module would need to be modified (using its adaptable feature) to ensure that these projects are run differently.","It could also mean that additional information should be asked of these clients to perhaps rejects them as clients for a certain type of project.","In this case the client module is modified using its adaptable feature.","A specific example is an access control database.","All the operations possible with a database that needs to be controlled are listed as the context in a GKMS type application.","Access is to be controlled for each group of users and for users in the groups (control is determined by the group and, in some cases, by the user).","In this case, the first level of instantiation corresponds to defining the template to be used to give rights to each group of users.","Each user, when linked to a group get an access rights document based on the template for the group it belongs to.","This is the second level of instantiation.","This document represents the right of that user.","The administrator can then modify each document if desired to take account of the characteristics of the user within the group.","It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described.","The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive."]],"string":"[\n [\n \"Generic architecture for adaptable software\",\n \"This invention concerns adaptable software.\",\n \"In particular it concerns a method for constructing an adaptable soft-ware application.\",\n \"A first step involves instantiating a first set of knowledge elements (kitems) related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application and used to produce dynamic and flexible templates.\",\n \"A second step involves instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.\",\n \"In a further aspect it concerns an adaptable software application comprising at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.\"\n ],\n [\n \"1.A method for constructing an adaptable software application, comprising the steps of: instantiating a first set of kitems related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application; and, instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.\",\n \"2.A method according to claim 1, where the step of instantiating the second set of kitems occurs dynamically, during the use of the application.\",\n \"3.A method according to claim 1, where further instantiating steps produce further sets of kitems from the immediately preceding set.\",\n \"4.A method according to claim 1, comprising the further step of directing enquiries against kitems at any level of instantiation using a consultation process.\",\n \"5.A method according to claim 1, comprising the further step of accessing the business objects using a consultation process during use of the application.\",\n \"6.A method according to claim 1, comprising the further step of accessing kitems related to the type of business objects for the purposes of inspecting or managing the design of the application.\",\n \"7.An adaptable software application, comprising: at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.\",\n \"8.An application comprising adaptable, intelligent modules or agents that communicate with each other, where each module has its business logic defined and implemented using the adaptable system of claim 7.9.An application according to claim 8, further comprising adaptable meta-module to define and implement the logic between the other modules.\",\n \"10.An application according to claim 9, where the meta-module monitors a part of, or the whole chain of modules.\"\n ],\n [\n \"<SOH> BACKGROUND ART <EOH>An application is a software package designed for the manipulation, management and processing of business elements or objects.\",\n \"These are often, but not exclusively, documents.\",\n \"Manipulation, management and processing covers operations such as definition, access, editing, display, distribution, etc.\",\n \"Such applications are normally hard-coded using standard programming techniques.\",\n \"The knowledge of the business requirements to be met by the application is designed into the architecture of the application and the code itself.\",\n \"The consequence of this approach is that systems are difficult to design and develop.\",\n \"They are also difficult to maintain and modify.\",\n \"When change is required to software, due to changing business conditions for example, designers must carry out extensive redesign and coding.\",\n \"In many cases, the effort is so difficult and time consuming that it is not attempted.\",\n \"Another approach is to develop an expert system in which the business rules are separate from the hard-coded part of the application.\",\n \"This has the advantage that the business rules can, in principle, be changed without having to change the application.\",\n \"In practice, the process of entering and testing the rules is difficult and time consuming, and their reliability can only be ascertained through extensive testing.\",\n \"Expert systems are used to build only these applications for which knowledge is explicit and available, and where the effort required to enter and maintain the rules is clearly offset by big savings during operations.\",\n \"The difficulties associated with developing, implementing and maintaining expert systems are well documented in the expert system literature of the past twenty years.\",\n \"Few applications use expert systems technology; it is a niche technology.\",\n \"A further approach is to build knowledge applications as described in co-pending Australian patent application PR2152 entitled Generic Knowledge Agents, International patent application PCT/AU99/00501 entitled Generic Knowledge Management System, and International patent application PCT/AU01/01155 entitled Intelligent Courseware Development and Delivery.\",\n \"The contents of these three applications are incorporated into this specification by reference.\",\n \"A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.\",\n \"The process for building knowledge applications starts in a knowledge application development environment where the business needs of the application determine the knowledge elements, or ‘kitems’, to be instantiated, or created, from contexts and templates.\",\n \"The outcome is an application or knowledge base in which the kitems are regions, knowledge items etc in the application—one can think of these kitems as documents.\",\n \"A knowledge application is typically characterised by a single instantiation process for all the kitems.\",\n \"The kitems can also be used as contexts or templates from which further kitems can be instantiated.\",\n \"Once the knowledge application is built the kitems can be accessed and managed, that is edited, displayed, distributed, etc.\",\n \"This entails defining an enquiry in a consultation process that retrieves the knowledge elements of interest for perusal and further manipulation.\",\n \"The enquiry is itself a kitem instantiated from a kitem template.\",\n \"Although designed and implemented in a radically different way from traditional expert systems, the business role of these knowledge applications and the way knowledge is accessed are similar to that of expert systems.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The invention, in a first aspect, is a method for constructing an adaptable software application, comprising the steps of: Instantiating a first set of kitems related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application.\",\n \"Instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.\",\n \"The first level of knowledge and the corresponding instantiations in effect produce dynamic templates that work as dynamic adaptations to the needs of users.\",\n \"Alternatively, these templates can be activated manually.\",\n \"The business objects in an application are treated as knowledge elements that embody business knowledge, such as knowledge about events that took place, or are expected to take place, in the business.\",\n \"The business objects are easily created, and can be easily manipulated.\",\n \"Because business objects are knowledge elements, they can be easily managed, that is accessed, displayed, analysed, etc.\",\n \"Instantiating the second set of kitems may take place dynamically, during the use of the application.\",\n \"Applications can be built with a greater number of cascades or instantiations larger than two.\",\n \"Enquiries can be directed against kitems at any level of instantiation using a consultation process.\",\n \"The business objects may be accessed using a consultation process during use of the application.\",\n \"The kitems related to the type of business objects may be accessed for inspecting and managing the design of the application.\",\n \"The invention, in a second aspect, is an adaptable software application, comprising: at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.\",\n \"The use of a two level instantiation, or cascading, design enables the first level to define the application without hard-coding, and the second to define the documents that are handled by the application.\",\n \"So, adaptable software transforms the definition of the application into specifying a knowledge application, and treats its documents (or business objects) as knowledge items inside the application.\",\n \"The application may involve adaptable, intelligent modules or agents that communicate with each other, where each module has its business logic defined and implemented using the adaptable architecture of the system.\",\n \"This means that each module can be quickly developed and that its functionality can evolve with the business and with experience.\",\n \"Each adaptable module implements the knowledge about the best way to run that module.\",\n \"These modules are used to facilitate development and to increase the functionality and adaptability of the solution.\",\n \"An adaptable meta-module may define and implement the logic between the other modules.\",\n \"In addition the meta-module may monitor a part of, or the whole chain of modules.\",\n \"This software is able to adapt itself automatically using the knowledge entered into it, without programming, to determine when and how it should adapt; that is, modify its behaviour.\",\n \"It may also adapt dynamically to the needs of users as software is being used.\",\n \"Adaptablability is a very important commercial feature for software.\",\n \"It holds the promise of speeding up software development, implementation, customization and flexibility.\",\n \"In addition to RAD (Rapid Application Development), adaptable software holds the promise of software that can be modified easily during all stages of its life-cycle, without major effort or redesign.\",\n \"Adaptable software also holds the promise of software that can adapt itself, that is customize itself and evolve as needs change while it is being used.\",\n \"Adaptable software has the potential to save, worldwide, enormous sums in development costs, and to reduce the total cost of ownership of software solutions over their lifetimes.\",\n \"Using adaptable software generic applications can be quickly produced.\",\n \"The frameworks for a variety of generic applications, for a variety of domains, can be quickly produced.\",\n \"An application framework is a knowledge system in which the knowledge elements express the knowledge about what the application is meant to do.\",\n \"Also client specific applications can be quickly produced.\",\n \"Knowledge in generic framework can be easily modified or added to, so as to represent the knowledge about a client's specific requirements.\"\n ],\n [\n \"TECHNICAL FIELD This invention concerns adaptable software.\",\n \"In particular it concerns a method for constructing an adaptable software application.\",\n \"In a further aspect it concerns an adaptable software application.\",\n \"BACKGROUND ART An application is a software package designed for the manipulation, management and processing of business elements or objects.\",\n \"These are often, but not exclusively, documents.\",\n \"Manipulation, management and processing covers operations such as definition, access, editing, display, distribution, etc.\",\n \"Such applications are normally hard-coded using standard programming techniques.\",\n \"The knowledge of the business requirements to be met by the application is designed into the architecture of the application and the code itself.\",\n \"The consequence of this approach is that systems are difficult to design and develop.\",\n \"They are also difficult to maintain and modify.\",\n \"When change is required to software, due to changing business conditions for example, designers must carry out extensive redesign and coding.\",\n \"In many cases, the effort is so difficult and time consuming that it is not attempted.\",\n \"Another approach is to develop an expert system in which the business rules are separate from the hard-coded part of the application.\",\n \"This has the advantage that the business rules can, in principle, be changed without having to change the application.\",\n \"In practice, the process of entering and testing the rules is difficult and time consuming, and their reliability can only be ascertained through extensive testing.\",\n \"Expert systems are used to build only these applications for which knowledge is explicit and available, and where the effort required to enter and maintain the rules is clearly offset by big savings during operations.\",\n \"The difficulties associated with developing, implementing and maintaining expert systems are well documented in the expert system literature of the past twenty years.\",\n \"Few applications use expert systems technology; it is a niche technology.\",\n \"A further approach is to build knowledge applications as described in co-pending Australian patent application PR2152 entitled Generic Knowledge Agents, International patent application PCT/AU99/00501 entitled Generic Knowledge Management System, and International patent application PCT/AU01/01155 entitled Intelligent Courseware Development and Delivery.\",\n \"The contents of these three applications are incorporated into this specification by reference.\",\n \"A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.\",\n \"The process for building knowledge applications starts in a knowledge application development environment where the business needs of the application determine the knowledge elements, or ‘kitems’, to be instantiated, or created, from contexts and templates.\",\n \"The outcome is an application or knowledge base in which the kitems are regions, knowledge items etc in the application—one can think of these kitems as documents.\",\n \"A knowledge application is typically characterised by a single instantiation process for all the kitems.\",\n \"The kitems can also be used as contexts or templates from which further kitems can be instantiated.\",\n \"Once the knowledge application is built the kitems can be accessed and managed, that is edited, displayed, distributed, etc.\",\n \"This entails defining an enquiry in a consultation process that retrieves the knowledge elements of interest for perusal and further manipulation.\",\n \"The enquiry is itself a kitem instantiated from a kitem template.\",\n \"Although designed and implemented in a radically different way from traditional expert systems, the business role of these knowledge applications and the way knowledge is accessed are similar to that of expert systems.\",\n \"SUMMARY OF THE INVENTION The invention, in a first aspect, is a method for constructing an adaptable software application, comprising the steps of: Instantiating a first set of kitems related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application.\",\n \"Instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.\",\n \"The first level of knowledge and the corresponding instantiations in effect produce dynamic templates that work as dynamic adaptations to the needs of users.\",\n \"Alternatively, these templates can be activated manually.\",\n \"The business objects in an application are treated as knowledge elements that embody business knowledge, such as knowledge about events that took place, or are expected to take place, in the business.\",\n \"The business objects are easily created, and can be easily manipulated.\",\n \"Because business objects are knowledge elements, they can be easily managed, that is accessed, displayed, analysed, etc.\",\n \"Instantiating the second set of kitems may take place dynamically, during the use of the application.\",\n \"Applications can be built with a greater number of cascades or instantiations larger than two.\",\n \"Enquiries can be directed against kitems at any level of instantiation using a consultation process.\",\n \"The business objects may be accessed using a consultation process during use of the application.\",\n \"The kitems related to the type of business objects may be accessed for inspecting and managing the design of the application.\",\n \"The invention, in a second aspect, is an adaptable software application, comprising: at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.\",\n \"The use of a two level instantiation, or cascading, design enables the first level to define the application without hard-coding, and the second to define the documents that are handled by the application.\",\n \"So, adaptable software transforms the definition of the application into specifying a knowledge application, and treats its documents (or business objects) as knowledge items inside the application.\",\n \"The application may involve adaptable, intelligent modules or agents that communicate with each other, where each module has its business logic defined and implemented using the adaptable architecture of the system.\",\n \"This means that each module can be quickly developed and that its functionality can evolve with the business and with experience.\",\n \"Each adaptable module implements the knowledge about the best way to run that module.\",\n \"These modules are used to facilitate development and to increase the functionality and adaptability of the solution.\",\n \"An adaptable meta-module may define and implement the logic between the other modules.\",\n \"In addition the meta-module may monitor a part of, or the whole chain of modules.\",\n \"This software is able to adapt itself automatically using the knowledge entered into it, without programming, to determine when and how it should adapt; that is, modify its behaviour.\",\n \"It may also adapt dynamically to the needs of users as software is being used.\",\n \"Adaptablability is a very important commercial feature for software.\",\n \"It holds the promise of speeding up software development, implementation, customization and flexibility.\",\n \"In addition to RAD (Rapid Application Development), adaptable software holds the promise of software that can be modified easily during all stages of its life-cycle, without major effort or redesign.\",\n \"Adaptable software also holds the promise of software that can adapt itself, that is customize itself and evolve as needs change while it is being used.\",\n \"Adaptable software has the potential to save, worldwide, enormous sums in development costs, and to reduce the total cost of ownership of software solutions over their lifetimes.\",\n \"Using adaptable software generic applications can be quickly produced.\",\n \"The frameworks for a variety of generic applications, for a variety of domains, can be quickly produced.\",\n \"An application framework is a knowledge system in which the knowledge elements express the knowledge about what the application is meant to do.\",\n \"Also client specific applications can be quickly produced.\",\n \"Knowledge in generic framework can be easily modified or added to, so as to represent the knowledge about a client's specific requirements.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS An example of the invention will now be described with reference to the accompanying drawings, in which: FIG.\",\n \"1 is a block diagram showing the steps in the construction and use of an adaptable software application.\",\n \"FIG.\",\n \"2 is a block diagram showing the architecture of an adaptable software application.\",\n \"BEST MODES OF THE INVENTION Adaptable software differs from the knowledge applications described in the background art in that there are at least two levels of instantiation instead of one; and the templates for the second level instantiations are the first level instantiations (they can be objects and not classes in object oriented programming).\",\n \"Adaptable software extends the architecture of known knowledge applications.\",\n \"The use of a two level instantiation, or cascading, design enables the first level to define the application (without hard-coding) and the second to define the documents that are handled by the application.\",\n \"Adaptable software transforms the definition of the application (typically hardcoded) into specifying a knowledge application, and treats its documents (or business objects) as knowledge items inside the application.\",\n \"The process for building an adaptable software application will now be described with reference to FIG.\",\n \"1.The process starts in a knowledge application development environment 10 where the business needs of the application determine the knowledge to be instantiated, or created, from contexts and templates.\",\n \"The first step 11 is the instantiation of this knowledge.\",\n \"The outcome is an application module dynamic framework 12 in which the kitems relate to the type of business objects or operations which will be handled by the adaptable software application.\",\n \"This first step is distinguished from the construction of an application knowledge base since it does not produce the documents or the knowledge to be accessed via a consultation.\",\n \"Instead these kitems describe the application as a framework that will be used to generate the dynamic templates for the objects, documents, that relate to the application.\",\n \"A second step 13 then involves populating the adaptable software application with business objects instantiated from contexts or templates produced in the first step.\",\n \"The process for defining these business objects is similar to defining an enquiry in a knowledge application.\",\n \"That is, parameters are specified that determine the features of the business object which is then created.\",\n \"The outcome is an application module 14 populated with business objects.\",\n \"The business objects so created can be managed 15, that is accessed, edited, displayed, distributed, etc.\",\n \"These operations are simple to implement as they correspond to manipulating kitems, and they use standard kitem manipulation methods, and other specific methods if required.\",\n \"For example, the business objects could be accessed using a question-answer session.\",\n \"Specific applications (or modules) are implemented by customising the generic framework produced in the first step, and this corresponds to instantiating different kitems as business objects for each application or module.\",\n \"This customizing can take place dynamically, during the use of the application.\",\n \"This corresponds to dynamic adaptation of the application to the needs of users.\",\n \"Enquiries can be directed against both levels of instantiated kitems via a consultation process.\",\n \"Enquiries directed to the upper level of documents, produced by the first step, are typically used for inspecting and managing the design of the application.\",\n \"To users, adaptable applications do not need to look different from normal applications.\",\n \"For example, a dialogue could take place that enquires about the needs or intentions of a user.\",\n \"Knowing the user's needs or intentions then enables the application to adapt or customize dynamically the template that is presented to the user; this templates defines the documents, or the type of documents, that the user enters into the application.\",\n \"The two level instantiation process can also be described as cascading knowledge items.\",\n \"That is, some knowledge items determine which other knowledge items will be activated or instantiated to define documents.\",\n \"Applications can be built with a greater number of cascades or instantiations larger than two.\",\n \"Most software applications are made of modules that communicate with each other.\",\n \"For example, a typical business management application is made of a client module, a project module, a contact module, a resource module and an accounting module.\",\n \"FIG.\",\n \"2 shows adaptable, intelligent modules or agents.\",\n \"In particular there are adaptable intelligent client 21, project 22, contact 23, resource 24 and accounting 25 modules.\",\n \"Each module is adaptable.\",\n \"For example, in the client module, a different set of questions could be asked based on the age or location or socio-economic profile.\",\n \"Similarly for the other modules.\",\n \"Each module has its business logic defined and implemented using an adaptable architecture.\",\n \"This means that each module can be quickly developed and that its functionality can evolve with the business and with experience.\",\n \"Each adaptable module implements the knowledge about the best way to run that module.\",\n \"These modules are used to facilitate development and to increase the functionality and adaptability of the solution.\",\n \"Adaptable meta-module(s) 30 are used to define and implement the logic between the other modules.\",\n \"This could include workflow or project costing for example, where a client would be treated differently based on the number of projects commissioned in the past eighteen months and the success of these projects.\",\n \"In addition the meta-modules are adaptable.\",\n \"In FIG.\",\n \"2, the meta-module monitors a part of, or the whole chain of modules.\",\n \"For example, the accounting module may detect that some projects from some clients are not profitable.\",\n \"That means that the project module would need to be modified (using its adaptable feature) to ensure that these projects are run differently.\",\n \"It could also mean that additional information should be asked of these clients to perhaps rejects them as clients for a certain type of project.\",\n \"In this case the client module is modified using its adaptable feature.\",\n \"A specific example is an access control database.\",\n \"All the operations possible with a database that needs to be controlled are listed as the context in a GKMS type application.\",\n \"Access is to be controlled for each group of users and for users in the groups (control is determined by the group and, in some cases, by the user).\",\n \"In this case, the first level of instantiation corresponds to defining the template to be used to give rights to each group of users.\",\n \"Each user, when linked to a group get an access rights document based on the template for the group it belongs to.\",\n \"This is the second level of instantiation.\",\n \"This document represents the right of that user.\",\n \"The administrator can then modify each document if desired to take account of the characteristics of the user within the group.\",\n \"It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described.\",\n \"The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10450974"}}},{"rowIdx":291,"cells":{"text":{"kind":"list like","value":[["Generic knowledge agents","This invention concerns a generic knowledge software agent, or kuark, existing within a knowledge environment.","The kuark comprises: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark, and a destination context of information identifiable by the kuark.","The kuark has a defined source region, or pattern, within the and problems source context and a mapping between that source region and a defined destination region, or pattern, within the destination context.","The kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.","It also concerns systems or kitems, made up of kuarks and a process for constructing them.","It also concerns mult-kitem systems and searching processes for those systems.","Finally it also concerns software agents and a knowledge management system comprising kitems."],["1.A generic knowledge software agent, or kuark, existing within a knowledge environment, and comprising: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark and a destination context of information identifiable by the kuark; the kuark having a defined source region, or pattern, within the source context and a mapping between that source region and a defined destination region, or pattern, within the destination context; and the kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.","2.A software agent according to claim 1, and further comprising: a univeral id; a title; an explanation, which may be multimedia; a time and date created; an author; a time and date when last modified; a person who last modified agent; a version number; a status to an enquiry, such as available, candidate, rejected, definite; a description of the source context at time of last firing; a description of destination context after last firing; or, links to other kuarks.","3.A software agent according to claim 2, where the links are: the hierarchical parent's universal id, and more than one parents could be allowed; the id of the previous version kuark; the ids of the kuarks that are part of this kuark; or, the ids of the kuarks this kuark belongs to or is associated with.","4.A software agent according to claim 1, operating to: create a daughter/parent kuark; adopt an existing kuark as daughter or parent; display an internal state (contexts, regions, mapping); or make a copy each time it fires.","5.A software agent according to claim 1 where, depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping that determines the relevance of the environment.","6.A software agent according to claim 1 where, the mapping is a program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context, and.","the mapping executes when the environment is compatible with the source context and the region.","7.A software agent according to claim 1, implemented as classes and objects using an object oriented language.","8.A system, or kitem, comprising more than one software agent according to claim 1, where the kitem has its own properties and methods in addition to those of its components 9.A process for constructing a system, or kitem, according to claim 8 from its components, comprising the steps of: concatenating their source contexts; concatenating their destination contexts; concatenating their source regions; concatenating their destination regions; concatenating their mappings; concatenating their explanations.","10.A process according to claim 9, comprising the additional step of building a list of the component kuarks or kitems.","11.A system, or kitem, according to claim 8, where all its components (kuarks or kitems) keep their individual contexts.","12.A system, or kitem, according to claim 8, where all its components have their individual contexts concatenated.","13.A system, or kitem, according to claim 8, expressed as an object containing another object.","14.A multi-kitem system comprising two or more systems, or kitems, according to claim 8, where the kitems are linked to one another.","15.A multi-kitem system according to claim 14, comprising a serial arrangement of kitems in which the destination context of one kitem is used as at least part of the source context of another.","16.A multi-kitem system according to claim 14, comprising a parallel arrangement of kitems in which the kitems that have independent (or disjointed), but can also have overlapping, source contexts.","17.A kitem according to claim 8, including within itself a multi-kitem system and having access to all the properties and methods of kitems contained within it, in addition to the properties that belong only to that kitem.","18.A kitem according to claim 15, comprising two kuarks, where the first kuark, depending on the the state of the environment with respect to the source context and source region, fires a relevance mapping that determines the relevance of the environment, and the second kuark uses the output of the first kuark as its source context and region, and a has a mapping which is a program or procedure that produces material to be displayed as output when the relevance mapping fires.","19.A kitem according to claim 15, comprising two kuarks, where the first kuark, depending on the the state of the environment with respect to the source context and source region, fires a relevance mapping that determines the relevance of the environment, and the second kuark uses the output of the first kuark as part of its source context and its source region, and a has a mapping which is a program or procedure that produces a destination region as output when the relevance mapping fires.","20.A searching process for identifying those kitems in a multi-kitem system according to claim 14, that have source regions compatible with an enquiry, the process comprising the step of: determining the ‘relevance status’ of a kitem with respect to the enquiry or consultation so far, where an available status is the default status of a kitem before a search takes place, a candidate kitem is one that is not incompatible with the enquiry so far, and a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.","21.A searching process according to claim 20, where the source context is organised as a tree structure, and each kitem is indexed against the context tree so that each object in the context possesses a list of the kitems it is associated with, and during the search questions are context objects and all the indexed kitems to the questions asked and answered so far in the consultation have their relevance status checked and modified if appropriate.","22.A searching process according to claim 21, where the tree structure is a modified context space kitem, and the enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.","23.A searching process according to claim 20, where the source context is organised as a tree structure in which each element is a kitem and the search process is also a kitem which flags the kitems in the tree structurre that correspond to the questions answered so far.","24.A searching process according to claim 23, where context tree element determines whether all the indexed elements have taken into account the values of the context tree element, and if so, it passes the value(s) to the indexed kitems and waits until all the responses have been received before the next step in the enquiry can proceed.","25.A searching process according to claim 24, where the tree structure is a modified context space kitem, and the enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.","26.A searching process according to claim 20, where the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.","27.A searching process according to claim 20, where each kitem has a unique ID or address that determines its location in a distributed memory space or network, it also possess a set of applicability addresses that correspond to the kitems in its source region, and during processing, the enquiry process submits each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.","28.A searching process according to claim 27, where each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process; and the enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.","These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry, or enquiry step.","29.An agent comprising a kitem according to claim 15, and having the following structure: Beliefs, Desires and Intentions are parallel kitems and their respective components are also kitems; and the Beliefs, Desires and Intentions kitems are organised in a serial architecture, that is the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.","30.A knowledge management system, comprising: a context element is a display kitem according to claim 18; a source or destination context kitem is a parallel display kitem, and its members comprise the list of all the context elements defined in the domain of discourse; a context executable kitem to edit a context element, edit a context space or display a context space as a tree; a source or destination context tree as an executable kitem, where the display kitem display the elements that belong to the source and destination kitems, its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context; a knowledge editing kitem is an executable kitem that has: as source space: the source and destination contexts, as destination space: the source and destination contexts, as source region a subset of the source context that defines the applicability of the knowledge kitem, as mapping a link to a subset of the destination context, or a procedure or executable (sub-)kitem, as destination region a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure, and an explanation; a search kitem is an executable kitem with: as source context the source and destination contexts, as source region the enquiry as defined so far, as destination context the kitems in the domain of discourse (if known), as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry, as destination region the candidate and definite kitems ‘retrieved’ by the mapping; an enquiry kitem is an executable kitem with: as source space the enquiry (the consultation process, showing each step in the question-answer session), as destination space the status of the enquiry (answered, not answered, etc.","), the ids of the definite kitems; a display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way."],["<SOH> BACKGROUND ART <EOH>The model for the agents uses the inventions described in International Patent Application No.","PCT/AU99/00501 entitled ‘Generic Knowledge Management System’ (GKMS), and Australian provisional patent application no PR0852 entitled ‘Networked Knowledge Management And Learning’ (NKML) both of which are incorporated herein by reference.","A summary of these knowledge models will now be given with reference to FIGS.","1 to 4 .","A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.","Knowledge is mapped as regions (or patterns) in a source (or problem) space that are linked to regions (or patterns) in a destination (or solution) space, as illustrated in FIG.","1 .","The interactions for defining the problem and solution spaces, and for defining the mappings, and the interactions for accessing the knowledge take place in a network of client-servers, as illustrated in FIG.","2 .","FIGS.","3 and 4 show the decision-making process and the knowledge capture process, and how knowledge can be improved on an on-going basis.","FIG.","4 is an extension of FIG.","3 in which the consequences of the decisions taken by a GKMS are evaluated and used to qualify the knowledge in the GKMS.","Once again it involves signal exchanges between servers and clients.","The accepted architecture for agents is summarized in FIGS.","5 and 6 .","FIG.","5 illustrates the BDI model, that is agents have Beliefs, Desires and Intentions.","FIG.","5 shows that in each part of the BDI model, agents need knowledge to be able to operate.","They must have belief which is knowledge about what their role in existence is.","Their desires or goals represent knowledge about what they are capable of and their intentions represent the knowledge about implementing the goals.","Similarly, each of the DBI boxes in FIG.","5 is made of components, each representing a kind of knowledge in its category.","All these different types of knowledge can be easily and conveniently represented using the GKMS model for knowledge.","In addition, the use of that knowledge can be supported by the knowledge processing described in the GKMS patent.","This means that the advantages of the GKMS model for knowledge representation and processing can become available to agents.","It is also important to note that the use of the GKMS knowledge model for representation and processing is not restricted to the categories of knowledge shown in FIG.","5 , but to any knowledge that may be useful in agents.","FIG.","6 shows how an agent situated in an environment interacts with this environment.","Th interaction is very similar to FIGS.","3 and 4 which show how interaction with the environment and learning can take place in the GKMS model.","Therefore the GKMS model is also suitable for dealing with agents interacting with their environments and learning in the process."],["<SOH> SUMMARY OF THE INVENTION <EOH>The invention is a generic knowledge software agent, or kuark, existing within a knowledge environment, and comprising: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark and a destination context of information identifiable by the kuark; the kuark having a defined source region, or pattern, within the source context and a mapping between that source region and a defined destination region, or pattern, within the destination context; and the kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.","So the kuark makes predetermined information automatically available to a user when input criteria are met.","The kuark is only able to detect parameters that fall within its defined source context and when the environment satisfies the source region the mapping is triggered.","The source region may be identicle to the source context, or it may be a subset within it.","A kuark may comprise many other elements, such as: a univeral id; a title; an explanation, which may be multimedia; a time and date created; an author; a time and date when last modified; a person who last modified agent; a version number; a status to an enquiry, such as available, candidate, rejected, definite; a description of the source context at time of last firing; a description of destination context after last firing; and, links to other kuarks.","The links implement relationships to other kuarks.","The are four links: The hierarchical parent's universal id, and more than one parents could be allowed; The id of the previous version kuark; The ids of the kuarks that are part of this kuark; The ids of the kuarks this kuark belongs to or is associated with.","This last link may be implemented as separate kuark(s) designed to contain the IDs of the kuarks the kuark in question relates (or belongs) to.","The kuark may also operate to do the following: Create a daughter/parent kuark; Adopt an existing kuark as daughter or parent; Display an internal state (contexts, regions, mapping); and Make a copy each time it fires.","It is possible to implement these methods outside the current kuark, in a separate class or subroutine for example.","A first special kuark, known as a ‘relevance kuark’, determines the relevance of the environment, and depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping that determines (calculates) the relevance of the environment.","The relevance can be expressed as a probability.","The destination context may be made of the different relevance values that the environment can take with respect to the source context and region.","For example, the values could be ‘definitely relevant’, ‘possibly relevant’, ‘relevant’, ‘likely not relevant’, ‘not relevant’.","Another special kuark is known as a ‘procedure kuark’, and contains as a mapping a (usually small) program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context.","This kuark executes its mapping when the environment is compatible with the source context and the region.","That is, the input to the procedure is the source region in the source context.","The destination context describes the range of outputs the procedure can produce when run, and the destination region or pattern is the actual result of the execution of the mapping, using an instance of the environment.","Kuarks may be implemented as classes and objects using an object oriented language such as Java.","A kuark is a class in OOP and some of its properties are static, final, etc.","A kuark that is given values for its source and destination contexts and regions are objects or instances of the corresponding kuark class.","A system made of several kuarks is still a kuark with all the allowed properties and methods, but is known as a ‘kitem’.","In a kitem all the component kuark and kitems may keep their identities.","The new kitem may have its own properties and methods in addition to those of its component kitems.","The new kitem may have its own title and explanation, and may also have its own source and destination contexts and regions, and its own mapping.","A kitem may be constructed form kuarks and kitems by the following process: concatenating the source contexts; concatenating the destination contexts; concatenating the source regions; concatenating the destination regions; concatenating the mappings; concatenating the explanations.","Here concatenates means ‘take the union’ of the entities in question; for mappings, it is the juxtaposition of the individual mappings.","The kitem construction process may also build the list of kuarks or kitems that are part of the new kitem.","Typically, when a kitem is built, all its components (kuarks or kitems) keep their individual contexts.","Alternatively the kitem construction process may be set so that all components get the same source and destination contexts, that is the concatenation of the individual contexts of the kitem components.","A kuark or kitem can be represented as an object using an object oriented language such as Java.","A kitem can be expressed as an object containing another object (or a class with sub-classes) which preserves all the features for knowledge items disclosed in the GKMS patent.","This means that kitem objects can be generated without programming.","The other features disclosed in the GKMS patent also apply to kitems implemented as classes.","Kitems can be linked.","A link has a source or origin kitem and a destination kitem.","The links can be stored inside the kitem or kuark, as a property of the kitem.","An alternative is to store inside the kitem the address of the ‘link kitem’, that is, the kitem that is designed to contain the links that relate to a source kitem.","A multi-kuark or multi-kitem system may comprise a serial arrangement, in which the destination context of one kitem is used as the source context (or part of the source context) of another.","More than two kitems can be chained.","Alternatively, a parallel kitem is made of kitems that have independent (or disjointed), but can also have overlapping, source contexts.","Inside a parallel kitem, the component kitems are treated as peers, and links are defined between peers.","An application or knowledge base may comprise a set of knowledge items, kitems, defined with respect to a domain of discourse, where the kitems are arranged in parallel.","Some of the kitems may be linked in series or in parallel.","A kitem may be a class that supports the creation of objects, or knowledge elements, where the kitem has access to all the properties and methods of other kitems contained within it, in addition to the properties that belong only to that kitem.","Such a kitem is referred to as a ‘meta-kitem’ and is able to express meta-knowledge.","A display kitem is a serial kitem comprising two kuarks.","The first kuark is a relevance kuark.","It uses its region and the state of the environment to determines the relevance of the display kitem.","The second kuark is a procedure kuark and has the relevance (the output of the first kuark) as source context and region.","The material to be displayed comprises the destination region and the explanation.","An executable kitem is a serial kitem comprising two kuarks.","The first kuark is a relevance kuark.","It uses its region and the state of the environment to determines the relevance of the executable kitem.","The second kuark is a procudure kuark.","It has the relevance (the output of the first kuark) as region and as part of its source context.","When the relevance is not ‘null’, the second kuark executes its mapping to produce the destination region.","The inputs to the executable mapping are elements of the second kuark's source context that can be additional to the relevance context.","A knowledge item is a kitem comprising a source context, a source region, a destination context, a destination region, a mapping and an explanation.","Searching involves identifying those kitems that have source regions compatible with the enquiry.","It means determining the ‘relevance status’(available, candidate, rejected or definite) of a kitem with respect to the enquiry or consultation so far.","Searches may be synchronous or asynchronous.","An available status is the default status of a kitem before a search takes place; a candidate kitem is one that is not incompatible with the enquiry so far; a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.","The source context may be organised as a tree structure.","Each kitem may be indexed against the context tree.","This means that each object in the context possesses a list of the kitems it is associated with.","During processing, all the indexed kitems to the questions (context objects) asked and answered so far in the consultation have their relevance status checked and modified if appropriate.","Kitems which become definite can be presented to the user, candidates remain contenders for definite status depending on future answers, and rejected items will not be considered any longer.","At the implementation level, the context tree is a modified context space kitem.","The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.","These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.","The search process updates the status of the kitems according to the enquiry.","Alternatively, the search process (also a kitem) may flag the elements in the context tree that correspond to the questions answered so far.","Each context tree element is itself a kitem and its role is to determine whether all the indexed elements have taken into account the values of the context tree element.","If they do this, it passes the value(s) to the indexed kitems and waits until all the responses have been received.","The search process waits until all the tree elements corresponding to the enquiry so far have replied.","When all have replied, the enquiry has been processed (the knowledge elements have determined their status based on the enquiry so far).","Synchronisation is assured and the next step in the enquiry (presenting the results to the user and waiting for its input) can proceed.","At the implementation level, the context tree is a modified context space kitem.","The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.","These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.","Each kitem updates its status according to the enquiry and informs the searc process.","Alternatively, in asynchronous searches, the source region may determine whether the outcome of the kitem becomes applicable.","That is, the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.","The state of the relevance region determines what happens to the application region.","Where each kitem has a unique ID or address that determines its location in a distributed memory space or network, it may also possess a set of applicability addresses that correspond to the kitems in its source region.","During processing, the enquiry process may submit each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.","In this model, each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process.","The enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.","These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry (or enquiry step).","Each kitem can decide whether to answer more than once to an enquiry from the same origin or process.","It is up to the enquiry process to manage all the replies it gets from the network.","The solution outlined here is applicable to distributed processing.","Another model for asynchronous searches is to give even more responsibility to the distributed kitems, and each kitem has the address of each of the context objects in its context and in its region (a subset of the context).","The kitem interrogates the network at regular interval to determine whether the status of the region, and hence of the applicability of the kitem, has changed.","Cascading refers to a knowledge element having, as part of its destination region, an object that belongs to the source region of another knowledge element.","When the first knowledge element fires, it may change its source region in a way that impact on the applicability of the second knowledge element.","The second knowledge element may have to fire, not as a direct effect of the enquiry but as an indirect effect, that is, a consequence of another knowledge element having changed its state.","This process can extend to any arbitrary number of knowledge elements, called cascading knowledge elements.","In order to ensure that all the cascaded elements update their state if appropriate, it is necessary to a) link the cascaded elements, and b) have the search process update the relevance status of these cascaded knowledge elements (synchronous search) or c) to communicate the state of the environment (outputs of previously fired knowledge elements) to the linked cascaded knowledge elements (asynchronous search).","In a multi-kitem system, knowledge processing involves running a question-answer session.","The system asks questions, get answers and, on the basis of these answers, asks further questions until the correct answers are found or no answers exist.","The steps involved are: identifying the most important questions (the importance is determined by the ability of the answers to the question to lead to the correct answers as quickly as possible); asking these questions; on the basis of the answers to the questions, determining whether any answers can be found, and the next best questions to ask.","Identifying the most promising questions may involve a synchronous search through a context tree with processing delegated to a search agent.","As the context tree is updated each time a question is considered, the most important questions to ask next depend on the consultation so far; that is, on the state of the context tree after the previous enquiry has been processed.","Therefore the questions are selected dynamically.","Alternatively the search may have processing delegated to each kitem.","Context tree search with processing delegated to each kitem As explained above, the search process waits until all the tree elements corresponding to the enquiry so far have replied.","It is the responsibility of the enquiry process to collect these replies and to count the number that come from each of the tree context element: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.","In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relerance status), this process can also be asynchronous it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.","In an asynchronous searches the enquiry process (that sends the enquiry over the network for kitems to process it) may perform several tasks: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.","In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relevance status), this process can be asynchronous; it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.","In situations where the domain of discourse of a body of knowledge is known, one can identify the kitems with the largest number of indexed kitems with relevance status available or candidate in order to find hidden knowledge.","The process of producing a context tree is a compilation of the kitems in an application or domain of discourse.","Compilation keeps the identity of the kitems and all the data and information in these kitems that pertain to their execution.","Keeping the identity of these kitems enables the non-executable parts of the kitems to be retrieved at execution time on a ‘as needed’ basis.","There are two types of compilation: Just-in-time compilation imposes the compiled kitem to take the latest version of each kitem in its process, in particular the latest version of the source and destination regions; or Permanent compilation accepts that the kitems do not change (between compilations) and that their attributes and methods can be used without checking them.","Therefore they are included in the compiled kitem.","This is faster than the partial compilation.","An agent may be represented as a kitem with the following structure: Beliefs 61 , Desires 62 and Intentions 63 are parallel kitems (their respective components are also kitems); the Beliefs 61 , Desires 62 and Intentions 63 kitems are organised in a serial architecture.","That is, the agent kitem is a serial kitem; the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.","The kitems needed to produce a workable implementation for knowledge management are shown below: A context element is a display kitem (a serial kitem made of two kuarks).","A source or destination context kitem is a parallel display kitem.","Its members comprise the list of all the context elements defined in the domain of discourse.","Context executable kitems may edit a context element, edit a context space or display a context space as a tree.","A source or destination context tree is an executable kitem.","The display kitem may display the elements that belong to the source and destination kitems.","Its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context.","A knowledge editing kitem is an executable kitem that has: As source space: the source and destination contexts.","As destination space: the source and destination contexts.","As source region: a subset of the source context that defines the applicability of the knowledge kitem.","As mapping: a link to a subset of the destination context, or a procedure or executable (sub-)kitem.","As destination region: a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure.","An explanation.","A search kitem is an executable kitem with: as source context: the source and destination contexts; as source region: the enquiry as defined so far; as destination context: the kitems in the domain of discourse (if known); as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry; as destination region: the candidate and definite kitems ‘retrieved’ by the mapping.","An enquiry kitem is an executable kitem with: as source space: the enquiry (the consultation process, showing each step in the question-answer session); as destination space: the status of the enquiry (answered, not answered, etc.","), the ids of the definite kitems.","A display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.","This technology supports rapid application development (RAD).","RAD is a very important commercial feature for software.","It holds the promise of speeding up software development, implementation and customization.","RAD, or any significant progress in this area, has the potential to save, worldwide, enormous sums (billion of dollars) in development, and to reduce the total cost of ownership of software solutions over their lifetimes.","RAD is also a very serious technical challenge for designers.","Agents hold the promise of autonomous and intelligent software that is capable of co-operating with other agents (and human beings) towards achieving a goal.","In addition agents, with proper architecture, should scale up.","That is, they could provide a promising platform for building complex, adaptable systems that could have knoweldge and perhaps exhibit intelligent behaviour.","Currently agents are difficult to design and buid.","This is because agents draw on several disciplines, each one bringing its own theoretical and practical difficulties to design and development.","They are: control theory, cognitive psychology and artificial intelligence."],["TECHNICAL FIELD This invention concerns generic knowledge software agents, or kuarks, and their use as the building blocks for agent based applications.","It also concerns systems or kitems, made up of kuarks and a process for constructing them.","It also concerns mult-kitem systems and searching processes for those systems.","Finally it also cocerns software agents and a knowledge management system comprising kitems.","BACKGROUND ART The model for the agents uses the inventions described in International Patent Application No.","PCT/AU99/00501 entitled ‘Generic Knowledge Management System’ (GKMS), and Australian provisional patent application no PR0852 entitled ‘Networked Knowledge Management And Learning’ (NKML) both of which are incorporated herein by reference.","A summary of these knowledge models will now be given with reference to FIGS.","1 to 4.A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.","Knowledge is mapped as regions (or patterns) in a source (or problem) space that are linked to regions (or patterns) in a destination (or solution) space, as illustrated in FIG.","1.The interactions for defining the problem and solution spaces, and for defining the mappings, and the interactions for accessing the knowledge take place in a network of client-servers, as illustrated in FIG.","2.FIGS.","3 and 4 show the decision-making process and the knowledge capture process, and how knowledge can be improved on an on-going basis.","FIG.","4 is an extension of FIG.","3 in which the consequences of the decisions taken by a GKMS are evaluated and used to qualify the knowledge in the GKMS.","Once again it involves signal exchanges between servers and clients.","The accepted architecture for agents is summarized in FIGS.","5 and 6.FIG.","5 illustrates the BDI model, that is agents have Beliefs, Desires and Intentions.","FIG.","5 shows that in each part of the BDI model, agents need knowledge to be able to operate.","They must have belief which is knowledge about what their role in existence is.","Their desires or goals represent knowledge about what they are capable of and their intentions represent the knowledge about implementing the goals.","Similarly, each of the DBI boxes in FIG.","5 is made of components, each representing a kind of knowledge in its category.","All these different types of knowledge can be easily and conveniently represented using the GKMS model for knowledge.","In addition, the use of that knowledge can be supported by the knowledge processing described in the GKMS patent.","This means that the advantages of the GKMS model for knowledge representation and processing can become available to agents.","It is also important to note that the use of the GKMS knowledge model for representation and processing is not restricted to the categories of knowledge shown in FIG.","5, but to any knowledge that may be useful in agents.","FIG.","6 shows how an agent situated in an environment interacts with this environment.","Th interaction is very similar to FIGS.","3 and 4 which show how interaction with the environment and learning can take place in the GKMS model.","Therefore the GKMS model is also suitable for dealing with agents interacting with their environments and learning in the process.","SUMMARY OF THE INVENTION The invention is a generic knowledge software agent, or kuark, existing within a knowledge environment, and comprising: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark and a destination context of information identifiable by the kuark; the kuark having a defined source region, or pattern, within the source context and a mapping between that source region and a defined destination region, or pattern, within the destination context; and the kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.","So the kuark makes predetermined information automatically available to a user when input criteria are met.","The kuark is only able to detect parameters that fall within its defined source context and when the environment satisfies the source region the mapping is triggered.","The source region may be identicle to the source context, or it may be a subset within it.","A kuark may comprise many other elements, such as: a univeral id; a title; an explanation, which may be multimedia; a time and date created; an author; a time and date when last modified; a person who last modified agent; a version number; a status to an enquiry, such as available, candidate, rejected, definite; a description of the source context at time of last firing; a description of destination context after last firing; and, links to other kuarks.","The links implement relationships to other kuarks.","The are four links: The hierarchical parent's universal id, and more than one parents could be allowed; The id of the previous version kuark; The ids of the kuarks that are part of this kuark; The ids of the kuarks this kuark belongs to or is associated with.","This last link may be implemented as separate kuark(s) designed to contain the IDs of the kuarks the kuark in question relates (or belongs) to.","The kuark may also operate to do the following: Create a daughter/parent kuark; Adopt an existing kuark as daughter or parent; Display an internal state (contexts, regions, mapping); and Make a copy each time it fires.","It is possible to implement these methods outside the current kuark, in a separate class or subroutine for example.","A first special kuark, known as a ‘relevance kuark’, determines the relevance of the environment, and depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping that determines (calculates) the relevance of the environment.","The relevance can be expressed as a probability.","The destination context may be made of the different relevance values that the environment can take with respect to the source context and region.","For example, the values could be ‘definitely relevant’, ‘possibly relevant’, ‘relevant’, ‘likely not relevant’, ‘not relevant’.","Another special kuark is known as a ‘procedure kuark’, and contains as a mapping a (usually small) program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context.","This kuark executes its mapping when the environment is compatible with the source context and the region.","That is, the input to the procedure is the source region in the source context.","The destination context describes the range of outputs the procedure can produce when run, and the destination region or pattern is the actual result of the execution of the mapping, using an instance of the environment.","Kuarks may be implemented as classes and objects using an object oriented language such as Java.","A kuark is a class in OOP and some of its properties are static, final, etc.","A kuark that is given values for its source and destination contexts and regions are objects or instances of the corresponding kuark class.","A system made of several kuarks is still a kuark with all the allowed properties and methods, but is known as a ‘kitem’.","In a kitem all the component kuark and kitems may keep their identities.","The new kitem may have its own properties and methods in addition to those of its component kitems.","The new kitem may have its own title and explanation, and may also have its own source and destination contexts and regions, and its own mapping.","A kitem may be constructed form kuarks and kitems by the following process: concatenating the source contexts; concatenating the destination contexts; concatenating the source regions; concatenating the destination regions; concatenating the mappings; concatenating the explanations.","Here concatenates means ‘take the union’ of the entities in question; for mappings, it is the juxtaposition of the individual mappings.","The kitem construction process may also build the list of kuarks or kitems that are part of the new kitem.","Typically, when a kitem is built, all its components (kuarks or kitems) keep their individual contexts.","Alternatively the kitem construction process may be set so that all components get the same source and destination contexts, that is the concatenation of the individual contexts of the kitem components.","A kuark or kitem can be represented as an object using an object oriented language such as Java.","A kitem can be expressed as an object containing another object (or a class with sub-classes) which preserves all the features for knowledge items disclosed in the GKMS patent.","This means that kitem objects can be generated without programming.","The other features disclosed in the GKMS patent also apply to kitems implemented as classes.","Kitems can be linked.","A link has a source or origin kitem and a destination kitem.","The links can be stored inside the kitem or kuark, as a property of the kitem.","An alternative is to store inside the kitem the address of the ‘link kitem’, that is, the kitem that is designed to contain the links that relate to a source kitem.","A multi-kuark or multi-kitem system may comprise a serial arrangement, in which the destination context of one kitem is used as the source context (or part of the source context) of another.","More than two kitems can be chained.","Alternatively, a parallel kitem is made of kitems that have independent (or disjointed), but can also have overlapping, source contexts.","Inside a parallel kitem, the component kitems are treated as peers, and links are defined between peers.","An application or knowledge base may comprise a set of knowledge items, kitems, defined with respect to a domain of discourse, where the kitems are arranged in parallel.","Some of the kitems may be linked in series or in parallel.","A kitem may be a class that supports the creation of objects, or knowledge elements, where the kitem has access to all the properties and methods of other kitems contained within it, in addition to the properties that belong only to that kitem.","Such a kitem is referred to as a ‘meta-kitem’ and is able to express meta-knowledge.","A display kitem is a serial kitem comprising two kuarks.","The first kuark is a relevance kuark.","It uses its region and the state of the environment to determines the relevance of the display kitem.","The second kuark is a procedure kuark and has the relevance (the output of the first kuark) as source context and region.","The material to be displayed comprises the destination region and the explanation.","An executable kitem is a serial kitem comprising two kuarks.","The first kuark is a relevance kuark.","It uses its region and the state of the environment to determines the relevance of the executable kitem.","The second kuark is a procudure kuark.","It has the relevance (the output of the first kuark) as region and as part of its source context.","When the relevance is not ‘null’, the second kuark executes its mapping to produce the destination region.","The inputs to the executable mapping are elements of the second kuark's source context that can be additional to the relevance context.","A knowledge item is a kitem comprising a source context, a source region, a destination context, a destination region, a mapping and an explanation.","Searching involves identifying those kitems that have source regions compatible with the enquiry.","It means determining the ‘relevance status’(available, candidate, rejected or definite) of a kitem with respect to the enquiry or consultation so far.","Searches may be synchronous or asynchronous.","An available status is the default status of a kitem before a search takes place; a candidate kitem is one that is not incompatible with the enquiry so far; a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.","The source context may be organised as a tree structure.","Each kitem may be indexed against the context tree.","This means that each object in the context possesses a list of the kitems it is associated with.","During processing, all the indexed kitems to the questions (context objects) asked and answered so far in the consultation have their relevance status checked and modified if appropriate.","Kitems which become definite can be presented to the user, candidates remain contenders for definite status depending on future answers, and rejected items will not be considered any longer.","At the implementation level, the context tree is a modified context space kitem.","The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.","These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.","The search process updates the status of the kitems according to the enquiry.","Alternatively, the search process (also a kitem) may flag the elements in the context tree that correspond to the questions answered so far.","Each context tree element is itself a kitem and its role is to determine whether all the indexed elements have taken into account the values of the context tree element.","If they do this, it passes the value(s) to the indexed kitems and waits until all the responses have been received.","The search process waits until all the tree elements corresponding to the enquiry so far have replied.","When all have replied, the enquiry has been processed (the knowledge elements have determined their status based on the enquiry so far).","Synchronisation is assured and the next step in the enquiry (presenting the results to the user and waiting for its input) can proceed.","At the implementation level, the context tree is a modified context space kitem.","The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.","These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.","Each kitem updates its status according to the enquiry and informs the searc process.","Alternatively, in asynchronous searches, the source region may determine whether the outcome of the kitem becomes applicable.","That is, the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.","The state of the relevance region determines what happens to the application region.","Where each kitem has a unique ID or address that determines its location in a distributed memory space or network, it may also possess a set of applicability addresses that correspond to the kitems in its source region.","During processing, the enquiry process may submit each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.","In this model, each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process.","The enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.","These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry (or enquiry step).","Each kitem can decide whether to answer more than once to an enquiry from the same origin or process.","It is up to the enquiry process to manage all the replies it gets from the network.","The solution outlined here is applicable to distributed processing.","Another model for asynchronous searches is to give even more responsibility to the distributed kitems, and each kitem has the address of each of the context objects in its context and in its region (a subset of the context).","The kitem interrogates the network at regular interval to determine whether the status of the region, and hence of the applicability of the kitem, has changed.","Cascading refers to a knowledge element having, as part of its destination region, an object that belongs to the source region of another knowledge element.","When the first knowledge element fires, it may change its source region in a way that impact on the applicability of the second knowledge element.","The second knowledge element may have to fire, not as a direct effect of the enquiry but as an indirect effect, that is, a consequence of another knowledge element having changed its state.","This process can extend to any arbitrary number of knowledge elements, called cascading knowledge elements.","In order to ensure that all the cascaded elements update their state if appropriate, it is necessary to a) link the cascaded elements, and b) have the search process update the relevance status of these cascaded knowledge elements (synchronous search) or c) to communicate the state of the environment (outputs of previously fired knowledge elements) to the linked cascaded knowledge elements (asynchronous search).","In a multi-kitem system, knowledge processing involves running a question-answer session.","The system asks questions, get answers and, on the basis of these answers, asks further questions until the correct answers are found or no answers exist.","The steps involved are: identifying the most important questions (the importance is determined by the ability of the answers to the question to lead to the correct answers as quickly as possible); asking these questions; on the basis of the answers to the questions, determining whether any answers can be found, and the next best questions to ask.","Identifying the most promising questions may involve a synchronous search through a context tree with processing delegated to a search agent.","As the context tree is updated each time a question is considered, the most important questions to ask next depend on the consultation so far; that is, on the state of the context tree after the previous enquiry has been processed.","Therefore the questions are selected dynamically.","Alternatively the search may have processing delegated to each kitem.","Context tree search with processing delegated to each kitem As explained above, the search process waits until all the tree elements corresponding to the enquiry so far have replied.","It is the responsibility of the enquiry process to collect these replies and to count the number that come from each of the tree context element: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.","In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relerance status), this process can also be asynchronous it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.","In an asynchronous searches the enquiry process (that sends the enquiry over the network for kitems to process it) may perform several tasks: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.","In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relevance status), this process can be asynchronous; it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.","In situations where the domain of discourse of a body of knowledge is known, one can identify the kitems with the largest number of indexed kitems with relevance status available or candidate in order to find hidden knowledge.","The process of producing a context tree is a compilation of the kitems in an application or domain of discourse.","Compilation keeps the identity of the kitems and all the data and information in these kitems that pertain to their execution.","Keeping the identity of these kitems enables the non-executable parts of the kitems to be retrieved at execution time on a ‘as needed’ basis.","There are two types of compilation: Just-in-time compilation imposes the compiled kitem to take the latest version of each kitem in its process, in particular the latest version of the source and destination regions; or Permanent compilation accepts that the kitems do not change (between compilations) and that their attributes and methods can be used without checking them.","Therefore they are included in the compiled kitem.","This is faster than the partial compilation.","An agent may be represented as a kitem with the following structure: Beliefs 61, Desires 62 and Intentions 63 are parallel kitems (their respective components are also kitems); the Beliefs 61, Desires 62 and Intentions 63 kitems are organised in a serial architecture.","That is, the agent kitem is a serial kitem; the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.","The kitems needed to produce a workable implementation for knowledge management are shown below: A context element is a display kitem (a serial kitem made of two kuarks).","A source or destination context kitem is a parallel display kitem.","Its members comprise the list of all the context elements defined in the domain of discourse.","Context executable kitems may edit a context element, edit a context space or display a context space as a tree.","A source or destination context tree is an executable kitem.","The display kitem may display the elements that belong to the source and destination kitems.","Its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context.","A knowledge editing kitem is an executable kitem that has: As source space: the source and destination contexts.","As destination space: the source and destination contexts.","As source region: a subset of the source context that defines the applicability of the knowledge kitem.","As mapping: a link to a subset of the destination context, or a procedure or executable (sub-)kitem.","As destination region: a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure.","An explanation.","A search kitem is an executable kitem with: as source context: the source and destination contexts; as source region: the enquiry as defined so far; as destination context: the kitems in the domain of discourse (if known); as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry; as destination region: the candidate and definite kitems ‘retrieved’ by the mapping.","An enquiry kitem is an executable kitem with: as source space: the enquiry (the consultation process, showing each step in the question-answer session); as destination space: the status of the enquiry (answered, not answered, etc.","), the ids of the definite kitems.","A display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.","This technology supports rapid application development (RAD).","RAD is a very important commercial feature for software.","It holds the promise of speeding up software development, implementation and customization.","RAD, or any significant progress in this area, has the potential to save, worldwide, enormous sums (billion of dollars) in development, and to reduce the total cost of ownership of software solutions over their lifetimes.","RAD is also a very serious technical challenge for designers.","Agents hold the promise of autonomous and intelligent software that is capable of co-operating with other agents (and human beings) towards achieving a goal.","In addition agents, with proper architecture, should scale up.","That is, they could provide a promising platform for building complex, adaptable systems that could have knoweldge and perhaps exhibit intelligent behaviour.","Currently agents are difficult to design and buid.","This is because agents draw on several disciplines, each one bringing its own theoretical and practical difficulties to design and development.","They are: control theory, cognitive psychology and artificial intelligence.","BRIEF DESCRIPTION OF THE DRAWINGS The background to the invention has been described above with reference to the following drawings: FIG.","1 is a diagram illustrating knowledge as mappings between a problem space and a solution space.","FIG.","2 is a diagram illustrating a network of client-servers, each able to run agents.","FIG.","3 is a diagram illustrating a knowledge capture loop in the GKMS.","FIG.","4 is a diagram illustrating an expanded knowledge capture loop in an on-the-job problem-solving system.","FIG.","5 is a diagram illustrating the agent DBI model.","FIG.","6 is a diagram illustrating an agent situated in and interacting with its environment.","The invention will now be described with reference to the following drawings: FIG.","7 is a diagram illustrating a generic elementary cognitive element (kuark).","FIG.","8 is a diagram illustrating a relevance kuark.","FIG.","9 is a diagram illustrating a procedure kuark.","FIG.","10 is a diagram illustrating a serial architecture.","FIG.","11 is a diagram illustrating a parallel architecture.","FIG.","12 is a diagram illustrating the agent implementing the DBI model as a serial kitem.","BEST MODES OF THE INVENTION Generic Elementary Cognitive Agent (Kuark) Referring to FIG.","7, the generic elementary cognitivey agent (kuark) 10 is the building block of agent-based applications.","The kuark knowledge model implements and extends the GKMS model described in the GKMS patent.","The kuark 10 monitors the environment by detecting all environment parameters compatible with the source region 11 and source context 12.When the source region 11, which determines the applicability of the mapping 13, is satisfied, the mapping 13 fires.","The mapping determines the output region 14 in the destination context 15 and makes the region available to the output, and hence to the environment.","Regions are sometimes referred to as patterns.","EXAMPLE 1 The source context comprises two sets of integers and the destination context one set of integers.","The source region is the positive integers for the two sets, the mapping a multiplication of the two inputs accepted in the source region.","The destination region is the result of the multiplication of the two positive numbers.","EXAMPLE 2 The source context is the state of a logical variable and the region the value ‘true’ of that variable.","The destination context is the state of the same variable and the mapping the ‘negation’ of the original value, that is ‘false’.","EXAMPLE 3 The source context is the temperature and pressure inside a boiler and the destination context a list of commands triggering alarms and associated processes (that can be software based).","The source region is the range of pressures and temperatures that trigger a ‘code red’ alert and the destination region the command triggering the ‘code red’ alarm.","EXAMPLE 4 The source context and region is as in the example 3.The destination context is the list of the relevance status of the kuark and the region the actual relevance status of the kuark, based on the source region.","That is, the ‘code red’ alert is given a relevance status (perhaps expressed as a probability) based on the environment.","Kuark Properties The common properties of a kuark are: univeral id; title; explanation (multimedia); time and date created; author; time and date when last modified; person who last modified agent; version number; domain of discourse source context (what it knows about) 12; destination context 15; applicability source region or pattern 11 (can be identical to source context); relevance status to an enquiry (available, candidate, rejected, definite); mapping 13 (the process that produces the outcome based on the state of source context) outcome result of the mapping between source and destination context; the mapping determines the destination region or pattern; status (enabled/disabled/archived); state of source context at time of last firing (describes source context and region); state of destination context after last firing (describes destination region); links to other kuarks the hierarchical parent's universal id (more than one parents could be allowed); the id of the previous version kuark; the ids of the kuarks that are part of this kuark (see later for multi-kuark kuarks); the ids of the kuarks this kuark belongs to or is associated with (see later for multi-kuark kuarks).","The property ‘links to other kuarks’ is the mechanism used to implement relationships between kuarks.","The fourth link mentioned may be implemented as separate kuark(s) designed to contain the IDs of the kuarks the kuark in question relates (or belongs) to.","Kuark Methods The main methods of a kuark are: monitor applicability (detect only what its context allows); determines whether environment fits within source region, if yes triggers/fires mapping; execute mapping (modifies destination state) mapping determines the outcome which is a subset of the destination context; create daughter/parent kuark; adopt existing kuark as daughter or parent; display internal state (contexts, regions, mapping); make copy each time it fires.","It is possible to implement these methods outside the current kuark, in a separate class or subroutine for example.","Relevance Kuark Referring now to FIG.","8, a relevance kuark 20 is a kuark 10 (as described previously) that determines the relevance of the environment.","Depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping 23 that determines (calculates) the relevance of the environment.","The relevance can be expressed as a probability.","The destination context 15 is made of the different relevance values that the environment can take with respect to the source context and region.","For example, the values could be ‘definitely relevant’, ‘possibly relevant’, ‘relevant’, ‘likely not relevant’, ‘not relevant’.","Procedure Kuark Referring now to FIG.","9, a procedure kuark 30 is one that contains a (usually small) program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context.","The kuark executes its procedure mapping 33, when the environment is compatible with the source context and the region.","That is, the input to the procedure is the source region in the source context.","The destination context describes the range of outputs the procedure can produce when run, and the destination region or pattern is the actual result of the execution of the mapping, using an instance of the environment.","The procedure mapping can be a trivial mapping.","It can simply display a fixed pattern and an explanation in a fixed destination context.","The context item, see later, is an implementation of the trivial procedure kuark.","Relevance kuarks tend to have the same destination context, whereas procedure kuarks typically have context that are strongly application dependent and that can vary within an application.","Kuarks and Object Oriented Programming Kuarks can be implemented as classes and objects using an object oriented language such as Java.","A kuark is a class in OOP and some of its properties are static, final, etc.","A kuark that is given values for its source and destination contexts and regions are objects or instances of the corresponding kuark class.","Multi-Kuark Systems or Applications This section describes how kuarks can be used together to increase the power of applications.","Multi-Kuark Kitems A system made of several kuarks is still a kuark with all the allowed properties and methods.","However, the term kuark is reserved for simple (or elementary) kuarks, as described above.","A multi-kuark kuark is named a kitem.","Kitems are constructed as follows: the designer identifies the kuarks (or kitems) to be included in the new kitem; the designer clicks ‘construct kitem’.","The construction process simply puts a wrapper around the kuarks and kitems to include in the new kitem.","All the component kuark and kitems keep their identities.","The new kitem can have its own properties and methods in addition to those of its component kitems.","The new kitem has its own title and explanation, and can also have its own source and destination contexts and regions, and its own mapping.","One can also view the new kuark as having undergone a construction process that carried the following operations: concatenates the source contexts; concatenates the destination contexts; concatenates the source regions; concatenates the destination regions; concatenates the mappings; concatenates the explanations.","Here concatenates means ‘take the union’ of the entities in question; for mappings, it is the juxtaposition of the individual mappings.","The kitem construction process also builds the list of kuarks or kitems that are part of the new kitem.","Typically, when a kitem is built, all its components (kuarks or kitems) keep their individual contexts.","Alternatively the kitem construction process can also be set so that all components get the same source and destination contexts, that is the concatenation of the individual contexts of the kitem components.","Kitems have explicit domains of discourse, the source and destination contexts, and explicit regions; kitems are ‘aware’ of their domains of discourse and when their knowledge, expressed as their mappings and destination regions, can be applied.","Not all kuarks can be assembled in kitems as some concatenation may not be allowed; this issue is not treated further here.","Refer later for different architectures for multi-kitem systems.","Kitem Hierarchy When defining kitems for an application, it is often convenient to organise them as a hierarchy of objects, as in a file system for example.","In fact, all kitems are defined within a hierarchical structure similar to that of the domain names on the Internet.","Each kitem has a parent, except the root kitem which is the top of the tree.","The hierarchical organisation of the kitems imposes no restriction on how they can be grouped (in multi-kitem systems) to achieve some purposes in an application.","That is, composed kitems can be assembled out of any kitem from any location in the hierarchy.","Kitems and Object Oriented Programming A kitem as outlined above, together with its GKMS features, mean that kitems can be implemented using an object oriented language, as objects or instances of the kuark or kitem classes.","As indicated above, a kuark or kitem can be represented as an object using an object oriented language such as Java.","A multi-kuark kitem as outlined above, can be expressed as an object containing another object (or a class with sub-classes) which preserves all the features for knowledge items disclosed in the GKMS patent.","This means that kitem objects can be generated without programming.","The other features disclosed in the GKMS patent also apply to kitems implemented as classes.","Rationale for Multi-Kitem Systems The advantages of building applications using kitems are very significant: the methods and properties can be used across the hierarchy (for example, a method to display the components kitems of a kitem can be re-used at all levels in the hierarchy of kitems); kitems provide a unified way for thinking and implementing applications; kitems can be fashioned as simple building blocks that offer maximum re-use; kitems implement knowledge at every level (in every kitem) that make up the application; knowledge permeates the design and the use of the application; applications can be largely soft-coded, instead of hard-coded; applications can be developed rapidly (RAD: rapid application development).","Links Between Kitems Kitems can be linked.","A link has a source or origin kitem and a destination kitem.","The links can be stored inside the kitem or kuark, as a property of the kitem.","An alternative is to store inside the kitem the address of the ‘link kitem’, that is, the kitem that is designed to contain the links that relate to a source kitem.","In a link kitem, the source context contain the ID of the source kitem and the type of link (see above for list of possible links), and the destination regions contains the IDs of the corresponding links (the destination context is the domain of discourse of the source kitem).","It is also possible, indeed often preferable, to have the source and destination contexts and regions for a kitem expressed via links.","That is, one or several link kitems contain the source and destination context, and the regions (their kitems and values).","Multi-Kitem Architectures This section discusses the architectures available for building multi-kuark or multi-kitem systems.","In particular, these architectures can be used to build the display and executable kitems.","Serial Architecture In the serial architecture 40, the destination context of one kitem 41 is used as the source context (or part of the source context) of another 42.More than two kitems can be chained, as illustrated in FIG.","10.The state of the environment is used as input to kitem 41 (and perhaps to other kitems).","Similarly, the serial kitem output is the output from kitem n 45 (and perhaps from other kitems).","In the most simple case, the source context is the source context of kitem 41 and the destination context the destination context of kitem n. In general, the source context of the serial kitem is a subset of the source contexts of all its ‘internal kitems’.","Similarly for the destination context and for the regions in these contexts.","The source context of an internal kitem can comprise some elements from the environment in addition to the output from the previous kitem.","The serial kitem is a kitem and has all the properties of kitems discussed above.","Parallel Architecture Referring now to FIG.","11, a parallel kitem 50 is made of kitems that have independent (or disjointed), but can also have overlapping, source contexts.","The power of parallel kitems derives from the links that can be established between its internal kitems.","The objective is to ensure that the information and knowledge contained in two kitems becomes linked.","For example, a kitem could describe clients and another projects; and a link would connect a client with a project.","A link has an origin and a destination.","Inside a parallel kitem, the component kitems are treated as peers, and links are defined between peers.","When the class of a parallel kitem is defined, the operator has to specify which kitems are instantiated as objects when the parallel kitem object is created.","In linked kitems, it is convenient, but not mandatory, to instantiate the origin kitem but not the destination kitem.","Links can be unidirectional or bidirectional.","For example, from clients to projects, or from clients to projects and from projects to clients.","Hierarchical Kitems The kitems that are part of a parallel (or serial kitem) keep their properties, notably the one identifying their parents.","Therefore the parallel kitem can, in fact, contain a hierarchy of kitems even if they are organised in a flat hierarchical (that is, parallel) way in the parallel kitem.","A Knowledge Application as A Parallel Architecture Kitem An application or knowledge base is a set of knowledge items (kitems) defined with respect to a domain of discourse (source and destination contexts and mappings available).","These kitems are arranged in parallel and some of them can be linked.","For example, in an application all the knowledge items are typically linked (that is refer) to the same domain of discourse, as explained above.","Hybrid Architecture It is possible to define a hybrid architecture, with the hybrid kitem containing some kitems linked in a serial way and others in a parallel way.","Meta-Kitems and Meta-Knowledge In FIGS.","10 and 11, each kitem is a class that supports the creation of objects, or knowledge elements.","It is desirable to be able to define a class that can have attributes and methods that span more than one of the kitems contained in the parallel, serial or hybrid kitem.","This can be achieved by giving the outside kitem (eg: the serial, parallel or hybrid kitem) access to all the properties and methods of the contained kitems, in addition to the properties that belong only to the outside kitems.","This means that it is possible to define meta-knowledge about the kitems contained in the outside kitems.","The outside kitem, when instanced as an object, can observe the behaviour of the kitems in it and take actions when necessary.","The outside kitems are referred to as meta-kitems able to express meta-knowledge.","Display and Executable Kitems In this section we consider two different types of very useful kitems: the display kitem and executable kitem.","We show that each can be constructed using one relevance kuark and one procedure kuark.","Display Kitem The display kitem is a serial kitem comprising two kuarks.","The first kuark is a relevance kuark.","It uses its region and the state of the environment to determines the relevance of the display kitem.","The second kuark has the relevance (the output of the first kuark) as source context and region.","The material to be displayed comprises the destination region and the explanation (the destination region can be the explanation.","Context Kitem Contexts and regions are made of elements or objects that describe the contexts and regions.","Each such object can have values.","Here we show that kitems are suitable for describing and implementing these context objects.","A context object is a kitem as shown below (only some properties are mentioned): kitem tile → object title source context → object values source region → instantiated values destination context → same as source context destination region → same as source region mapping → maps source to destination (contexts and regions) explanation → optional.","Explanation Kitem An explanation object is a kitem as shown below (only some properties are mentioned): kitem title → explanation title (can be ‘explanation’) source context → not used source region → not used destination context → not used destination region → not used mapping → not used explanation → multimedia explanation.","Context Element A context element is a kitem made of, typically, a context kitem and an explanation kitem (some context items can have more than one explanation).","To create such a kitem, the designer defines a parallel kitem using the procedure described in the GKMS patent.","It is also possible to have a context item made of a single context kitem, by using its explanation instead of the explanation kitem.","This solution does not work if more than one explanation is required.","For example, an explanation relating to the meaning of the context item and another relating to its use in a knowledge item.","Context Space (Source Or Destination) The context space is a kitem made of context items (also kitems) using the parallel architecture.","Typically no links are used between context items.","Executable Kitem The executable kitem is a serial kitem comprising two kuarks.","The first kuark uses its region and the state of the environment to determines the relevance of the executable kitem.","The second kuark has the relevance (the output of the first kuark) as region and as part of its source context.","When the relevance is not ‘null’, the second kuark executes its mapping to produce the destination region.","The inputs to the executable mapping are elements of the second kuark's source context that can be additional to the relevance context.","Knowledge Item and Knowledge Item Definition A knowledge item is a kitem comprising a source context, a source region, a destination context, a destination region, a mapping and an explanation.","Defining a knowledge item, as described in the GKMS patent, involves defining a region in the source context (and other things).","This is done using the context space kitem, that is made of context objects (or elements).","The architecture of a knowledge item is normally that of a parallel kitem.","It is often desirable to explain why a certain context element is part of a knowledge item or how to use or interpret this context element when it is attached to the knowledge item.","This can be achieved simply by giving the user defining the knowledge item the facility to add an explantion kitem to the list of kitem that are part of the knowledge item, and linking this explanation kitem to the context element (kitem) in question.","Alternatively, the explanation can be part of the context element kitem.","That is, the explanation kitem is already linked to the context element (with reference to OOP, the context element class ‘contains’ the explanation).","A knowledge item is defined with respect to a domain of discourse, defined by the source and destination context and the mappings available at definition time.","It would not be efficient to include the contexts kitems in the knowledge item.","Instead, the knowledge item is linked to the context space kitems.","This is in agreement with the definition of links in the parallel architecture kitem.","Kitem Hardcoding We have discussed how kitems can be implemented using other kitems.","However it is always possible to implement a kitem as a single hardcoded entity.","For example, in the GKMS patent, the context elements and the knowledge items were implemented as single entities with their specific architectures.","When this is done, many of the modular and flexible features of the kuark-kitem model are lost.","When a similar effect is desired, it is preferable to implement the kitem as a permanently compiled multi-kitem kitem (see later).","Knowledge Processing in Multi-Kitem Systems A multi-kitem system is a system comprising a set of kitems representing the body of knowledge about a certain domain of discourse.","Typically the domain of discourse is identical for all the kitems in the system, although it is not essential.","Such a system is identified as a knowledge module or database.","Knowledge processing involves the system (or server) running a question-answer session.","The system asks questions, get answers and, on the basis of these answers, asks further questions until the correct answers are found or no answers exist.","The steps involved are: identify the most important questions (the importance is determined by the ability of the answers to the question to lead to the correct answers as quickly as possible); asks these questions; on the basis of the answers to the questions, determine a) whether any answers can be found, and b) the next best questions to ask.","The processing refers to: the identification of the correct answers available in the knowledge base, on the basis of the information available so far in the consultation; and the identification of the most promising provisional answers available in the knowledge base, on the basis of the information available so far in the consultation; and the identification of the most important questions to ask on the basis of the information available.","Searching for relevant or important knowledge Knowledge access in a multi-kitem system (a knowledge module) follows the same pattern as for a GKMS knowledge base; that is, it is a question-answer session or consultation.","The consultation process is managed with, and recorded in, a document.","This document can also be a kitem called the consultation kitem.","That is, the consultation kitem has all the properties and methods of the kitem class.","All the utilities for kuarks and kitems can be used with consultation kitems.","Searching involves identifying those kitems that have source regions compatible with the enquiry (see GKMS patent for meaning of compatible).","It means determining the ‘relevance status’ (available, candidate, rejected or definite) of a kitem with respect to the enquiry or consultation so far.","Searches can be synchronous or asynchronous.","An available status is the default status of a kitem before a search takes place; a candidate kitem is one that is not incompatible with the enquiry so far; a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.","Synchronous Searches Database Search The database search is a SQL search or equivalent.","This means that the kitems are stored as records in a database.","This is not discussed further in this document Full Text Search The full text search has to process each relevant field in each record where each record describes a knowledge element.","This process can be slow; it is not discussed further in this document.","Context Tree Search with Processing Delegated to A Search Agent The source context is organised as a tree structure.","A source region, which determines whether a kitem will fire (firing depends on the region's compatibility with an enquiry), is a subset of the source context.","Each kitem (eg: knowledge element) can be indexed against the context tree.","This means that each object in the context possesses a list of the kitems it is associated with.","The context tree is a formalisation of one of the kuarks'properties (see above).","During processing, all the indexed kitems to the questions (context objects) asked and answered so far in the consultation have their relevance status checked and modified if appropriate.","Kitems which become definite can be presented to the user, candidates remain contenders for definite status depending on future answers, and rejected items will not be considered any longer.","At the implementation level, the context tree is a modified context space kitem.","The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.","These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.","The search process updates the status of the kitems according to the enquiry.","Context Tree Search with Processing Delegated to Each Kitem The search process (also a kitem) flags these elements in the context tree that correspond to the questions answered so far.","Each context tree element is itself a kitem and its role is to determine whether all the indexed elements have taken into account the values of the context tree element.","If they do this, it passes the value(s) to the indexed kitems and waits until all the responses have been received.","The search process waits until all the tree elements corresponding to the enquiry so far have replied.","When all have replied, the enquiry has been processed (the knowledge elements have determined their status based on the enquiry so far).","Synchronisation is assured and the next step in the enquiry (presenting the results to the user and waiting for its input) can proceed.","At the implementation level, the context tree is a modified context space kitem.","The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.","These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.","Each kitem updates its status according to the enquiry and informs the searc process.","Asynchronous Searches Processing Delegated to Each Kitem In asynchronous searches, the processing is distributed and controlled by each item.","There is no central authority that decides what to do when.","Each kitem (or agent) monitors its environment and reacts appropriately.","In doing so, it may change the environment and trigger new actions by other kitems.","This is in contrast to synchronous searches in which it is the search process that determines whether a kitem is applicable or not.","In particular, in asynchronous searches it is each kitem that determines whether it is applicable to the environment as it is detected via the source context and the source region.","In asynchronous searches, the source region determines whether the outcome of the kitem becomes applicable.","That is, the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.","The state of the relevance region determines what happens to the application region.","In the case of non-executable mappings (or display mappings) the process is as illustrated in FIG.","8; the relevance mapping ‘releases’ the destination region to the kuark output.","State of Environment (or Context) Communicated to the Items As indicated above, each kitem has a unique ID or address that determines its location in a distributed memory space or network (such as the Internet for example).","It also possesses a set of applicability addresses that correspond to the kitems in its source region.","During processing, the enquiry process submits each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.","In this model, each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process.","The enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.","These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry (or enquiry step).","Each kitem can decide whether to answer more than once to an enquiry from the same origin or process.","It is up to the enquiry process to manage all the replies it gets from the network.","The solution outlined here is applicable to distributed processing.","The Kitems Seek to Know the Status of their Source Context Another model for asynchronous searches is to give even more responsibility to the distributed kitems than in the previous description.","In this model, each kitem has the address of each of the context objects in its context and in its region (a subset of the context).","The kitem interrogates the network at regular interval to determine whether the status of the region, and hence of the applicability of the kitem, has changed.","This model means that the kitem takes the intiative (this has some important applications not discussed here).","However during searching, the model imposes much traffic on the network that is of no benefit, unless the context object has changed since the last status check.","Another problem is that there may be an unacceptable delay between status checks, during which time, if the status has changed, the kitem doing the interrogating cannot determine its applicability.","Cascading Knowledge Cascading refers to a knowledge element having, as part of its destination region, an object that belongs to the source region of another knowledge element.","When the first knowledge element fires, it may change its source region in a way that impact on the applicability of the second knowledge element.","The second knowledge element may have to fire, not as a direct effect of the enquiry but as an indirect effect, that is, a consequence of another knowledge element having changed its state.","This process can extend to any arbitrary number of knowledge elements, called cascading knowledge elements.","In order to ensure that all the cascaded elements update their state if appropriate, it is necessary to a) link the cascaded elements, and b) have the search process update the relevance status of these cascaded knowledge elements (synchronous search) or c) to communicate the state of the environment (outputs of previously fired knowledge elements) to the linked cascaded knowledge elements (asynchronous search).","Validity of the Relevant Knowledge For simple knowledge bases on a single server it is a relatively simple matter to ensure that all kitems they contain have the same domain of discourse.","This, however, does not apply for large or distributed knowledge bases where the knowledge comes from different sources.","In general, therefore, there is no certainty that all the knowledge items (kitems) accessed during a search have the same source and destination contexts.","In fact it is unlikely.","This means that a user inspecting a definite kitem (with respect to an enquiry) may not know what the domain of discourse of that kitem might be.","This information is very important as it defines the context for the knowledge in the kitem in question.","A solution to this situation is to give the user the ability to inspect the source and destination context of every kitem that is provided by the search process either as a candidate or definite kitem.","Identifying the Most Promising Questions Synchronous Searches Context Tree Search with Processing Delegated to A Search Agent Context objects which index a large number of available or candidate kitems are potentially very important when used as questions.","This is because their answers have the potential to impact on the relevance status of a large number of kitems.","This indicates that the importance of these context objects as questions in an enquiry is determined by the number of kitems with status available or candidate they index.","As the context tree is updated each time a question is considered, the most important questions to ask next depend on the consultation so far; that is, on the state of the context tree after the previous enquiry has been processed.","Therefore the questions are selected dynamically.","Context Tree Search with Processing Delegated to Each Kitem As explained above, the search process waits until all the tree elements corresponding to the enquiry so far have replied.","It is the responsibility of the enquiry process to collect these replies and to count the number that come from each of the tree context element: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.","In addition, the enquiry process can perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relerance status), this process can also be asynchronous it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.","Asynchronous Searches Processing Delegated to Each Kitem The enquiry process (that sends the enquiry over the network for kitems to process it) performs several tasks: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.","In addition, the enquiry process can perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relevance status), this process can be asynchronous; it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.","Search for Hidden Knowledge Hidden knowledge refers to knowledge that may be relevant to an enquiry but that is not yet linked to any question answered in the enquiry process so far.","The question is how to identify this hidden knowledge and how to extract its ‘best questions’.","In situations where the domain of discourse of a body of knowledge is known, one can simply identify the kitems with the largest number of indexed kitems with relevance status available or candidate (see context tree above).","There is no simple solutions when the domain of discourse of a body of knowledge is not known.","Processing Speed Synchronous Searches Context Tree Search with Processing Delegated to A Search Agent Synchronous searches can be very quick as they are under the control of one process that can be optimised, based on known conditions in the server.","Context Tree Search with Processing Delegated to Each Kitem This processing can be distributed across a network, but it is infrequent.","This processing is slower than the previous one as it imposes communications overheads between kitems (those doing some processing and the enquiry process).","Nevertheless, the enquiry process runs on a single server and can be optimised.","When the process is distributed, several kitems can process simultaneously; this can partially offset the communication overheads.","Asynchronous Searches Processing Delegated to Each Kitem This processing is slowest unless the number of kitems doing processing is large enough to offset the communication overhead between kitems and the network delays.","Compilation Compilation is the process that takes a collection of kitems and organises them for the purpose of speeding up processing.","The result of compilation is a kitem.","In general, compilation is applied to any kitem.","In practice it is often applied to all the kitems that belong to a domain of discourse (also a kitem) on a single server.","That is, all the kitems involved have domains of discourse that are subset of the compiled domain of discourse.","Compilation is also applied to a group of kitems contained in a serial, parallel or hybrid kitem.","The process of producing a context tree is a compilation of the kitems in an application or domain of discourse.","Compilation keeps the identity of the kitems and all the data and information in these kitems that pertain to their execution.","Keeping the identity of these kitems enables the non-executable parts of the kitems to be retrieved at execution time on a ‘as needed’ basis.","The processing model for the multi-agent systems described in this document imposes, like the Java language, that kitems call other kitems that in turn may call other kitems, etc, until the processing can be completed.","Once this is done, the results of the processing need to travel up the hierarchy of kitems, back to the initiating process.","The compilation process is designed to speed up the ‘down-hierarchy’ and ‘up-hierarchy’ travelling described above.","The context tree compilation achieves this purpose by ‘flattening’ the hierarchy to a single branch tree, where each context element (or node) in the branch contains all the kitems that have that context element as part of their regions.","There are two types of compilation: Just-in-time compilation imposes the compiled kitem to take the latest version of each kitem in its process, in particular the latest version of the source and destination regions; or Permanent compilation accepts that the kitems do not change (between compilations) and that their attributes and methods can be used without checking them.","Therefore they are included in the compiled kitem.","This is faster than the partial compilation.","Compilation can be be carried out to different depths: 1.build context tree at runtime; 2.get regions of indexed kitems in the context tree at runtime; 3.include regions of indexed kitems in the context tree; Compilations of type 1 and 2 above are just-in-time compilations.","They updates the context tree each time the search process is triggered; permanent compilation produces the context tree and leaves it untouched until another compilation is carried out.","Agents as Kitem Systems An agent, as illustrated in FIG.","5, can be represented as a kitem 60 with the structure illustrated in FIG.","12.: Beliefs 61, Desires 62 and Intentions 63 are parallel kitems (their respective components are also kitems); the Beliefs 61, Desires 62 and Intentions 63 kitems are organised in a serial architecture.","That is, the agent kitem is a serial kitem; the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.","It is recommended but not essential that the kitems that comprise the agent operate in a synchronous mode.","Internal synchronous processing is possible, even if a multi-agent systems behaves asynchronously.","As an agent is a kitem, a multi-agent system is a multi-kitem system with a parallel architecture and continuous asynchronous processing.","Kitems Needed for A Workable Solutions The kitems needed to produce a workable implementation for knowledge management are shown below: Context Management Context Elements A context element is a display kitem (a serial kitem made of two kuarks).","Source and Destination Contexts A source or destination context kitem is a parallel display kitem.","Its members comprise the list of all the context elements defined in the domain of discourse.","Context Executable Kitems Edit a context element Edit a context space Display a context space as a tree A source or destination context tree is an executable kitem.","The display kitem can display the elements that belong to the source and destination kitems.","Its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context.","Knowledge Definition Knowledge Editing Kitem The knowledge editing kitem is an executable kitem that has: As source space: the source and destination contexts.","As destination space: the source and destination contexts.","As source region: a subset of the source context that defines the applicability of the knowledge kitem.","As mapping: a link to a subset of the destination context, or a procedure or executable (sub-)kitem.","As destination region: a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure.","An explanation.","Knowledge Access and Display Search Kitem The search kitem is an executable kitem with: as source context: the source and destination contexts; as source region: the enquiry as defined so far; as destination context: the kitems in the domain of discourse (if known); as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry; as destination region: the candidate and definite kitems ‘retrieved’ by the mapping.","Enquiry or Consolation Kitem The enquiry kitem is an executable kitem with: as source space: the enquiry (the consultation process, showing each step in the question-answer session); as destination space: the status of the enquiry (answered, not answered, etc.","), the ids of the definite kitems.","Display Kitem The display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.","Administration of Contexts and Other Kitems The hierarchical (tree) structure of the kitems gives a very convenient way of controlling user access to kitems: the administrator can decide which branch of the hierarchy users can have access to and the type of access for the branch and each kitem in the branch; access control can also be carried out at the level of the kitem.","Dynamic Kitem Management The objective of dynamic kitem management is to be able to manage the integrity of the knowledge base when a kitem is edited.","This issue is important as kitems (eg: a context kitem) can be used in many knowledge elements and a change in such a kitem can have impacts on the validity of these knowledge elements (from a knowledge viewpoint, not operational viewpoint).","It is essential that the users are not left in the dark about the potential ramification of their changes and that they are helped in checking the validity of some affected kitems.","As much as possible, the system should ensure the integrity of the knowledge itself.","The process for achieving the above is to: use the enquiry process (by the system or the user) to retrieve all kitems that could be affected (the system or the user can specify the profile of the kitems affected by the change); make the changes automatically if acceptable; or ask the user to review/recertify the kitems affected (the system needs to provide guidance in this process).","The profile mentioned above refers to the values given to kitems in the source and destination context.","The system treats these values as an enquiry and retrieves all the kitems that are compatible with the enquiry.","Change to properties All the properties of the kitems can be changed.","Potentially, all the kitems that are linked to the kitem being edited are affected.","Some properties and the way their editing can be handled is listed below.","Change to Source Context and Region If a new context object is added: the system asks the user to specify the profile of the kitems that may be affected, the system retrieves these kitems and the user is asked to recertify them.","If an existing context object is edited or deleted: the system retrieves all the kitems that are related to this kitem (that contain it as part of a link for example) and asks the user to recertify them and to edit them if necessary before doing so.","Change to Destination Context and Region As for a change to the source context and region.","Change to Mapping The system retrieves all the kitems that are related to this kitem (that contain it as part of a link or region for example) and asks the user to recertify them and to edit them if necessary.","Change to Status (Enabled/Disabled/Archived) The system retrieves all the kitems that are related to this kitem (that contain it as part of a link or region for example) and asks the user to recertify them and to edit them if necessary.","Change to the ‘Links to Other Kitems’ No checks required, the change is localised.","Consistency Issues Consistency issues may arise in a knowledge base when some kitems have the following features: kitems have overlapping (at least one common kitem with at least a common value) or identical destination regions with non-identical source regions; kitems have disjointed destination regions with overlapping or identical source regions.","The system can easily have an agent that checks all the kitems in a knowledge base and identifies these kitems that may be inconsistent.","The system presents the kitems to their authors for editing if necessary and recertification.","Missing Knowledge Missing knowledge refers to parts of a domain of discourse for which there is no knowledge or inadequate knowledge.","When the domain of discourse for a set of kitems is explicit, it is a simple matter to check that the union of the source regions in the kitems cover the entire source context.","If it is not the case, it means that the some additional knowledge is required.","The user can be informed and the location of this missing knowledge presented.","The location of the missing knowledge can be pointed out on each dimension (or context kitem) of the source context.","The user selects a context item and a value (or range of values) for which there is no region, and the system responds by showing the other context items and their values for which there is no region.","The user then selects a second context item and gives it some values (where there is no region), and so on for the other kitems if desired, until the user is confident of being able to add some missing knowledge.","New Kitems When a new knowledge element is being saved (the user has finished its definition), the system checks that it is consistent with the other knowledge elements in the knowledge base (see section 12.2).","The system also retrieves all kitems that overlap with the new kitem's source and/or destination regions.","The system informs the user that these kitems may be affected (their knowledge may be superseeded by the new kitem) and presents them to the user for checking.","The system can also ask the user to define the profile of the kitems that may be superseeded by the new kitem.","Terminology Term Explanation Applicability region The region in the source context that determines whether the kitem is applicable.","Application context The context for the application made of the source or space and destination contexts.","Available kitem The status of a kitem that is available for an enquiry (the other status are definite, candidate and rejected.","Candidate kitem A kitem that is compatible with the enquiry so far but for which the applicability conditions are not completely met.","Definite kitem A kitem that is compatible with the enquiry so far with its applicability conditions completely met.","Destination context The space of solutions considered by an application or a kitem.","Domain of discourse The space of problems and solutions (or situations and outcomes) considered by an application or a kitem; it is made of the source and destination contexts.","Enquriy process The agent or process that controls the question- answer session or consultation.","Executable mapping A mapping that takes an input from the source context and applies an algorithm to it to produce an outcome.","Kitem A object or element that comprises a source context and region, a destination context and region, and a mapping.","Knowledge item A kitem that is used to express knowledge.","Kuark An elementary kitem (the smallest building block) that can be used to build other kitems and/or complete applications.","Mapping The transformation that is applied to a source region to produce a destination region or outcome.","Outcome Another term for destionation region or solution.","Parallel kitem A kitem comprising other kitems organised in parallel.","Rejected kitem An kitem that is not compatible with an enquiry.","Relevance context The destination context that expresses the range of possible relevance for kitems in an application.","Relevance region A region in the relevance context.","Relevance status The relevance of a kitem with respect to an enquiry (see GKMS patent for details).","Serial kitem A kitem comprising other kitems organised in series.","Solution Another term for destination region or outcome.","Source context The space of outcomes considered by an application or a kitem."]],"string":"[\n [\n \"Generic knowledge agents\",\n \"This invention concerns a generic knowledge software agent, or kuark, existing within a knowledge environment.\",\n \"The kuark comprises: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark, and a destination context of information identifiable by the kuark.\",\n \"The kuark has a defined source region, or pattern, within the and problems source context and a mapping between that source region and a defined destination region, or pattern, within the destination context.\",\n \"The kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.\",\n \"It also concerns systems or kitems, made up of kuarks and a process for constructing them.\",\n \"It also concerns mult-kitem systems and searching processes for those systems.\",\n \"Finally it also concerns software agents and a knowledge management system comprising kitems.\"\n ],\n [\n \"1.A generic knowledge software agent, or kuark, existing within a knowledge environment, and comprising: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark and a destination context of information identifiable by the kuark; the kuark having a defined source region, or pattern, within the source context and a mapping between that source region and a defined destination region, or pattern, within the destination context; and the kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.\",\n \"2.A software agent according to claim 1, and further comprising: a univeral id; a title; an explanation, which may be multimedia; a time and date created; an author; a time and date when last modified; a person who last modified agent; a version number; a status to an enquiry, such as available, candidate, rejected, definite; a description of the source context at time of last firing; a description of destination context after last firing; or, links to other kuarks.\",\n \"3.A software agent according to claim 2, where the links are: the hierarchical parent's universal id, and more than one parents could be allowed; the id of the previous version kuark; the ids of the kuarks that are part of this kuark; or, the ids of the kuarks this kuark belongs to or is associated with.\",\n \"4.A software agent according to claim 1, operating to: create a daughter/parent kuark; adopt an existing kuark as daughter or parent; display an internal state (contexts, regions, mapping); or make a copy each time it fires.\",\n \"5.A software agent according to claim 1 where, depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping that determines the relevance of the environment.\",\n \"6.A software agent according to claim 1 where, the mapping is a program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context, and.\",\n \"the mapping executes when the environment is compatible with the source context and the region.\",\n \"7.A software agent according to claim 1, implemented as classes and objects using an object oriented language.\",\n \"8.A system, or kitem, comprising more than one software agent according to claim 1, where the kitem has its own properties and methods in addition to those of its components 9.A process for constructing a system, or kitem, according to claim 8 from its components, comprising the steps of: concatenating their source contexts; concatenating their destination contexts; concatenating their source regions; concatenating their destination regions; concatenating their mappings; concatenating their explanations.\",\n \"10.A process according to claim 9, comprising the additional step of building a list of the component kuarks or kitems.\",\n \"11.A system, or kitem, according to claim 8, where all its components (kuarks or kitems) keep their individual contexts.\",\n \"12.A system, or kitem, according to claim 8, where all its components have their individual contexts concatenated.\",\n \"13.A system, or kitem, according to claim 8, expressed as an object containing another object.\",\n \"14.A multi-kitem system comprising two or more systems, or kitems, according to claim 8, where the kitems are linked to one another.\",\n \"15.A multi-kitem system according to claim 14, comprising a serial arrangement of kitems in which the destination context of one kitem is used as at least part of the source context of another.\",\n \"16.A multi-kitem system according to claim 14, comprising a parallel arrangement of kitems in which the kitems that have independent (or disjointed), but can also have overlapping, source contexts.\",\n \"17.A kitem according to claim 8, including within itself a multi-kitem system and having access to all the properties and methods of kitems contained within it, in addition to the properties that belong only to that kitem.\",\n \"18.A kitem according to claim 15, comprising two kuarks, where the first kuark, depending on the the state of the environment with respect to the source context and source region, fires a relevance mapping that determines the relevance of the environment, and the second kuark uses the output of the first kuark as its source context and region, and a has a mapping which is a program or procedure that produces material to be displayed as output when the relevance mapping fires.\",\n \"19.A kitem according to claim 15, comprising two kuarks, where the first kuark, depending on the the state of the environment with respect to the source context and source region, fires a relevance mapping that determines the relevance of the environment, and the second kuark uses the output of the first kuark as part of its source context and its source region, and a has a mapping which is a program or procedure that produces a destination region as output when the relevance mapping fires.\",\n \"20.A searching process for identifying those kitems in a multi-kitem system according to claim 14, that have source regions compatible with an enquiry, the process comprising the step of: determining the ‘relevance status’ of a kitem with respect to the enquiry or consultation so far, where an available status is the default status of a kitem before a search takes place, a candidate kitem is one that is not incompatible with the enquiry so far, and a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.\",\n \"21.A searching process according to claim 20, where the source context is organised as a tree structure, and each kitem is indexed against the context tree so that each object in the context possesses a list of the kitems it is associated with, and during the search questions are context objects and all the indexed kitems to the questions asked and answered so far in the consultation have their relevance status checked and modified if appropriate.\",\n \"22.A searching process according to claim 21, where the tree structure is a modified context space kitem, and the enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.\",\n \"23.A searching process according to claim 20, where the source context is organised as a tree structure in which each element is a kitem and the search process is also a kitem which flags the kitems in the tree structurre that correspond to the questions answered so far.\",\n \"24.A searching process according to claim 23, where context tree element determines whether all the indexed elements have taken into account the values of the context tree element, and if so, it passes the value(s) to the indexed kitems and waits until all the responses have been received before the next step in the enquiry can proceed.\",\n \"25.A searching process according to claim 24, where the tree structure is a modified context space kitem, and the enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.\",\n \"26.A searching process according to claim 20, where the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.\",\n \"27.A searching process according to claim 20, where each kitem has a unique ID or address that determines its location in a distributed memory space or network, it also possess a set of applicability addresses that correspond to the kitems in its source region, and during processing, the enquiry process submits each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.\",\n \"28.A searching process according to claim 27, where each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process; and the enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.\",\n \"These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry, or enquiry step.\",\n \"29.An agent comprising a kitem according to claim 15, and having the following structure: Beliefs, Desires and Intentions are parallel kitems and their respective components are also kitems; and the Beliefs, Desires and Intentions kitems are organised in a serial architecture, that is the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.\",\n \"30.A knowledge management system, comprising: a context element is a display kitem according to claim 18; a source or destination context kitem is a parallel display kitem, and its members comprise the list of all the context elements defined in the domain of discourse; a context executable kitem to edit a context element, edit a context space or display a context space as a tree; a source or destination context tree as an executable kitem, where the display kitem display the elements that belong to the source and destination kitems, its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context; a knowledge editing kitem is an executable kitem that has: as source space: the source and destination contexts, as destination space: the source and destination contexts, as source region a subset of the source context that defines the applicability of the knowledge kitem, as mapping a link to a subset of the destination context, or a procedure or executable (sub-)kitem, as destination region a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure, and an explanation; a search kitem is an executable kitem with: as source context the source and destination contexts, as source region the enquiry as defined so far, as destination context the kitems in the domain of discourse (if known), as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry, as destination region the candidate and definite kitems ‘retrieved’ by the mapping; an enquiry kitem is an executable kitem with: as source space the enquiry (the consultation process, showing each step in the question-answer session), as destination space the status of the enquiry (answered, not answered, etc.\",\n \"), the ids of the definite kitems; a display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.\"\n ],\n [\n \"<SOH> BACKGROUND ART <EOH>The model for the agents uses the inventions described in International Patent Application No.\",\n \"PCT/AU99/00501 entitled ‘Generic Knowledge Management System’ (GKMS), and Australian provisional patent application no PR0852 entitled ‘Networked Knowledge Management And Learning’ (NKML) both of which are incorporated herein by reference.\",\n \"A summary of these knowledge models will now be given with reference to FIGS.\",\n \"1 to 4 .\",\n \"A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.\",\n \"Knowledge is mapped as regions (or patterns) in a source (or problem) space that are linked to regions (or patterns) in a destination (or solution) space, as illustrated in FIG.\",\n \"1 .\",\n \"The interactions for defining the problem and solution spaces, and for defining the mappings, and the interactions for accessing the knowledge take place in a network of client-servers, as illustrated in FIG.\",\n \"2 .\",\n \"FIGS.\",\n \"3 and 4 show the decision-making process and the knowledge capture process, and how knowledge can be improved on an on-going basis.\",\n \"FIG.\",\n \"4 is an extension of FIG.\",\n \"3 in which the consequences of the decisions taken by a GKMS are evaluated and used to qualify the knowledge in the GKMS.\",\n \"Once again it involves signal exchanges between servers and clients.\",\n \"The accepted architecture for agents is summarized in FIGS.\",\n \"5 and 6 .\",\n \"FIG.\",\n \"5 illustrates the BDI model, that is agents have Beliefs, Desires and Intentions.\",\n \"FIG.\",\n \"5 shows that in each part of the BDI model, agents need knowledge to be able to operate.\",\n \"They must have belief which is knowledge about what their role in existence is.\",\n \"Their desires or goals represent knowledge about what they are capable of and their intentions represent the knowledge about implementing the goals.\",\n \"Similarly, each of the DBI boxes in FIG.\",\n \"5 is made of components, each representing a kind of knowledge in its category.\",\n \"All these different types of knowledge can be easily and conveniently represented using the GKMS model for knowledge.\",\n \"In addition, the use of that knowledge can be supported by the knowledge processing described in the GKMS patent.\",\n \"This means that the advantages of the GKMS model for knowledge representation and processing can become available to agents.\",\n \"It is also important to note that the use of the GKMS knowledge model for representation and processing is not restricted to the categories of knowledge shown in FIG.\",\n \"5 , but to any knowledge that may be useful in agents.\",\n \"FIG.\",\n \"6 shows how an agent situated in an environment interacts with this environment.\",\n \"Th interaction is very similar to FIGS.\",\n \"3 and 4 which show how interaction with the environment and learning can take place in the GKMS model.\",\n \"Therefore the GKMS model is also suitable for dealing with agents interacting with their environments and learning in the process.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The invention is a generic knowledge software agent, or kuark, existing within a knowledge environment, and comprising: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark and a destination context of information identifiable by the kuark; the kuark having a defined source region, or pattern, within the source context and a mapping between that source region and a defined destination region, or pattern, within the destination context; and the kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.\",\n \"So the kuark makes predetermined information automatically available to a user when input criteria are met.\",\n \"The kuark is only able to detect parameters that fall within its defined source context and when the environment satisfies the source region the mapping is triggered.\",\n \"The source region may be identicle to the source context, or it may be a subset within it.\",\n \"A kuark may comprise many other elements, such as: a univeral id; a title; an explanation, which may be multimedia; a time and date created; an author; a time and date when last modified; a person who last modified agent; a version number; a status to an enquiry, such as available, candidate, rejected, definite; a description of the source context at time of last firing; a description of destination context after last firing; and, links to other kuarks.\",\n \"The links implement relationships to other kuarks.\",\n \"The are four links: The hierarchical parent's universal id, and more than one parents could be allowed; The id of the previous version kuark; The ids of the kuarks that are part of this kuark; The ids of the kuarks this kuark belongs to or is associated with.\",\n \"This last link may be implemented as separate kuark(s) designed to contain the IDs of the kuarks the kuark in question relates (or belongs) to.\",\n \"The kuark may also operate to do the following: Create a daughter/parent kuark; Adopt an existing kuark as daughter or parent; Display an internal state (contexts, regions, mapping); and Make a copy each time it fires.\",\n \"It is possible to implement these methods outside the current kuark, in a separate class or subroutine for example.\",\n \"A first special kuark, known as a ‘relevance kuark’, determines the relevance of the environment, and depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping that determines (calculates) the relevance of the environment.\",\n \"The relevance can be expressed as a probability.\",\n \"The destination context may be made of the different relevance values that the environment can take with respect to the source context and region.\",\n \"For example, the values could be ‘definitely relevant’, ‘possibly relevant’, ‘relevant’, ‘likely not relevant’, ‘not relevant’.\",\n \"Another special kuark is known as a ‘procedure kuark’, and contains as a mapping a (usually small) program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context.\",\n \"This kuark executes its mapping when the environment is compatible with the source context and the region.\",\n \"That is, the input to the procedure is the source region in the source context.\",\n \"The destination context describes the range of outputs the procedure can produce when run, and the destination region or pattern is the actual result of the execution of the mapping, using an instance of the environment.\",\n \"Kuarks may be implemented as classes and objects using an object oriented language such as Java.\",\n \"A kuark is a class in OOP and some of its properties are static, final, etc.\",\n \"A kuark that is given values for its source and destination contexts and regions are objects or instances of the corresponding kuark class.\",\n \"A system made of several kuarks is still a kuark with all the allowed properties and methods, but is known as a ‘kitem’.\",\n \"In a kitem all the component kuark and kitems may keep their identities.\",\n \"The new kitem may have its own properties and methods in addition to those of its component kitems.\",\n \"The new kitem may have its own title and explanation, and may also have its own source and destination contexts and regions, and its own mapping.\",\n \"A kitem may be constructed form kuarks and kitems by the following process: concatenating the source contexts; concatenating the destination contexts; concatenating the source regions; concatenating the destination regions; concatenating the mappings; concatenating the explanations.\",\n \"Here concatenates means ‘take the union’ of the entities in question; for mappings, it is the juxtaposition of the individual mappings.\",\n \"The kitem construction process may also build the list of kuarks or kitems that are part of the new kitem.\",\n \"Typically, when a kitem is built, all its components (kuarks or kitems) keep their individual contexts.\",\n \"Alternatively the kitem construction process may be set so that all components get the same source and destination contexts, that is the concatenation of the individual contexts of the kitem components.\",\n \"A kuark or kitem can be represented as an object using an object oriented language such as Java.\",\n \"A kitem can be expressed as an object containing another object (or a class with sub-classes) which preserves all the features for knowledge items disclosed in the GKMS patent.\",\n \"This means that kitem objects can be generated without programming.\",\n \"The other features disclosed in the GKMS patent also apply to kitems implemented as classes.\",\n \"Kitems can be linked.\",\n \"A link has a source or origin kitem and a destination kitem.\",\n \"The links can be stored inside the kitem or kuark, as a property of the kitem.\",\n \"An alternative is to store inside the kitem the address of the ‘link kitem’, that is, the kitem that is designed to contain the links that relate to a source kitem.\",\n \"A multi-kuark or multi-kitem system may comprise a serial arrangement, in which the destination context of one kitem is used as the source context (or part of the source context) of another.\",\n \"More than two kitems can be chained.\",\n \"Alternatively, a parallel kitem is made of kitems that have independent (or disjointed), but can also have overlapping, source contexts.\",\n \"Inside a parallel kitem, the component kitems are treated as peers, and links are defined between peers.\",\n \"An application or knowledge base may comprise a set of knowledge items, kitems, defined with respect to a domain of discourse, where the kitems are arranged in parallel.\",\n \"Some of the kitems may be linked in series or in parallel.\",\n \"A kitem may be a class that supports the creation of objects, or knowledge elements, where the kitem has access to all the properties and methods of other kitems contained within it, in addition to the properties that belong only to that kitem.\",\n \"Such a kitem is referred to as a ‘meta-kitem’ and is able to express meta-knowledge.\",\n \"A display kitem is a serial kitem comprising two kuarks.\",\n \"The first kuark is a relevance kuark.\",\n \"It uses its region and the state of the environment to determines the relevance of the display kitem.\",\n \"The second kuark is a procedure kuark and has the relevance (the output of the first kuark) as source context and region.\",\n \"The material to be displayed comprises the destination region and the explanation.\",\n \"An executable kitem is a serial kitem comprising two kuarks.\",\n \"The first kuark is a relevance kuark.\",\n \"It uses its region and the state of the environment to determines the relevance of the executable kitem.\",\n \"The second kuark is a procudure kuark.\",\n \"It has the relevance (the output of the first kuark) as region and as part of its source context.\",\n \"When the relevance is not ‘null’, the second kuark executes its mapping to produce the destination region.\",\n \"The inputs to the executable mapping are elements of the second kuark's source context that can be additional to the relevance context.\",\n \"A knowledge item is a kitem comprising a source context, a source region, a destination context, a destination region, a mapping and an explanation.\",\n \"Searching involves identifying those kitems that have source regions compatible with the enquiry.\",\n \"It means determining the ‘relevance status’(available, candidate, rejected or definite) of a kitem with respect to the enquiry or consultation so far.\",\n \"Searches may be synchronous or asynchronous.\",\n \"An available status is the default status of a kitem before a search takes place; a candidate kitem is one that is not incompatible with the enquiry so far; a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.\",\n \"The source context may be organised as a tree structure.\",\n \"Each kitem may be indexed against the context tree.\",\n \"This means that each object in the context possesses a list of the kitems it is associated with.\",\n \"During processing, all the indexed kitems to the questions (context objects) asked and answered so far in the consultation have their relevance status checked and modified if appropriate.\",\n \"Kitems which become definite can be presented to the user, candidates remain contenders for definite status depending on future answers, and rejected items will not be considered any longer.\",\n \"At the implementation level, the context tree is a modified context space kitem.\",\n \"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.\",\n \"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.\",\n \"The search process updates the status of the kitems according to the enquiry.\",\n \"Alternatively, the search process (also a kitem) may flag the elements in the context tree that correspond to the questions answered so far.\",\n \"Each context tree element is itself a kitem and its role is to determine whether all the indexed elements have taken into account the values of the context tree element.\",\n \"If they do this, it passes the value(s) to the indexed kitems and waits until all the responses have been received.\",\n \"The search process waits until all the tree elements corresponding to the enquiry so far have replied.\",\n \"When all have replied, the enquiry has been processed (the knowledge elements have determined their status based on the enquiry so far).\",\n \"Synchronisation is assured and the next step in the enquiry (presenting the results to the user and waiting for its input) can proceed.\",\n \"At the implementation level, the context tree is a modified context space kitem.\",\n \"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.\",\n \"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.\",\n \"Each kitem updates its status according to the enquiry and informs the searc process.\",\n \"Alternatively, in asynchronous searches, the source region may determine whether the outcome of the kitem becomes applicable.\",\n \"That is, the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.\",\n \"The state of the relevance region determines what happens to the application region.\",\n \"Where each kitem has a unique ID or address that determines its location in a distributed memory space or network, it may also possess a set of applicability addresses that correspond to the kitems in its source region.\",\n \"During processing, the enquiry process may submit each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.\",\n \"In this model, each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process.\",\n \"The enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.\",\n \"These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry (or enquiry step).\",\n \"Each kitem can decide whether to answer more than once to an enquiry from the same origin or process.\",\n \"It is up to the enquiry process to manage all the replies it gets from the network.\",\n \"The solution outlined here is applicable to distributed processing.\",\n \"Another model for asynchronous searches is to give even more responsibility to the distributed kitems, and each kitem has the address of each of the context objects in its context and in its region (a subset of the context).\",\n \"The kitem interrogates the network at regular interval to determine whether the status of the region, and hence of the applicability of the kitem, has changed.\",\n \"Cascading refers to a knowledge element having, as part of its destination region, an object that belongs to the source region of another knowledge element.\",\n \"When the first knowledge element fires, it may change its source region in a way that impact on the applicability of the second knowledge element.\",\n \"The second knowledge element may have to fire, not as a direct effect of the enquiry but as an indirect effect, that is, a consequence of another knowledge element having changed its state.\",\n \"This process can extend to any arbitrary number of knowledge elements, called cascading knowledge elements.\",\n \"In order to ensure that all the cascaded elements update their state if appropriate, it is necessary to a) link the cascaded elements, and b) have the search process update the relevance status of these cascaded knowledge elements (synchronous search) or c) to communicate the state of the environment (outputs of previously fired knowledge elements) to the linked cascaded knowledge elements (asynchronous search).\",\n \"In a multi-kitem system, knowledge processing involves running a question-answer session.\",\n \"The system asks questions, get answers and, on the basis of these answers, asks further questions until the correct answers are found or no answers exist.\",\n \"The steps involved are: identifying the most important questions (the importance is determined by the ability of the answers to the question to lead to the correct answers as quickly as possible); asking these questions; on the basis of the answers to the questions, determining whether any answers can be found, and the next best questions to ask.\",\n \"Identifying the most promising questions may involve a synchronous search through a context tree with processing delegated to a search agent.\",\n \"As the context tree is updated each time a question is considered, the most important questions to ask next depend on the consultation so far; that is, on the state of the context tree after the previous enquiry has been processed.\",\n \"Therefore the questions are selected dynamically.\",\n \"Alternatively the search may have processing delegated to each kitem.\",\n \"Context tree search with processing delegated to each kitem As explained above, the search process waits until all the tree elements corresponding to the enquiry so far have replied.\",\n \"It is the responsibility of the enquiry process to collect these replies and to count the number that come from each of the tree context element: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.\",\n \"In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relerance status), this process can also be asynchronous it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.\",\n \"In an asynchronous searches the enquiry process (that sends the enquiry over the network for kitems to process it) may perform several tasks: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.\",\n \"In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relevance status), this process can be asynchronous; it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.\",\n \"In situations where the domain of discourse of a body of knowledge is known, one can identify the kitems with the largest number of indexed kitems with relevance status available or candidate in order to find hidden knowledge.\",\n \"The process of producing a context tree is a compilation of the kitems in an application or domain of discourse.\",\n \"Compilation keeps the identity of the kitems and all the data and information in these kitems that pertain to their execution.\",\n \"Keeping the identity of these kitems enables the non-executable parts of the kitems to be retrieved at execution time on a ‘as needed’ basis.\",\n \"There are two types of compilation: Just-in-time compilation imposes the compiled kitem to take the latest version of each kitem in its process, in particular the latest version of the source and destination regions; or Permanent compilation accepts that the kitems do not change (between compilations) and that their attributes and methods can be used without checking them.\",\n \"Therefore they are included in the compiled kitem.\",\n \"This is faster than the partial compilation.\",\n \"An agent may be represented as a kitem with the following structure: Beliefs 61 , Desires 62 and Intentions 63 are parallel kitems (their respective components are also kitems); the Beliefs 61 , Desires 62 and Intentions 63 kitems are organised in a serial architecture.\",\n \"That is, the agent kitem is a serial kitem; the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.\",\n \"The kitems needed to produce a workable implementation for knowledge management are shown below: A context element is a display kitem (a serial kitem made of two kuarks).\",\n \"A source or destination context kitem is a parallel display kitem.\",\n \"Its members comprise the list of all the context elements defined in the domain of discourse.\",\n \"Context executable kitems may edit a context element, edit a context space or display a context space as a tree.\",\n \"A source or destination context tree is an executable kitem.\",\n \"The display kitem may display the elements that belong to the source and destination kitems.\",\n \"Its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context.\",\n \"A knowledge editing kitem is an executable kitem that has: As source space: the source and destination contexts.\",\n \"As destination space: the source and destination contexts.\",\n \"As source region: a subset of the source context that defines the applicability of the knowledge kitem.\",\n \"As mapping: a link to a subset of the destination context, or a procedure or executable (sub-)kitem.\",\n \"As destination region: a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure.\",\n \"An explanation.\",\n \"A search kitem is an executable kitem with: as source context: the source and destination contexts; as source region: the enquiry as defined so far; as destination context: the kitems in the domain of discourse (if known); as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry; as destination region: the candidate and definite kitems ‘retrieved’ by the mapping.\",\n \"An enquiry kitem is an executable kitem with: as source space: the enquiry (the consultation process, showing each step in the question-answer session); as destination space: the status of the enquiry (answered, not answered, etc.\",\n \"), the ids of the definite kitems.\",\n \"A display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.\",\n \"This technology supports rapid application development (RAD).\",\n \"RAD is a very important commercial feature for software.\",\n \"It holds the promise of speeding up software development, implementation and customization.\",\n \"RAD, or any significant progress in this area, has the potential to save, worldwide, enormous sums (billion of dollars) in development, and to reduce the total cost of ownership of software solutions over their lifetimes.\",\n \"RAD is also a very serious technical challenge for designers.\",\n \"Agents hold the promise of autonomous and intelligent software that is capable of co-operating with other agents (and human beings) towards achieving a goal.\",\n \"In addition agents, with proper architecture, should scale up.\",\n \"That is, they could provide a promising platform for building complex, adaptable systems that could have knoweldge and perhaps exhibit intelligent behaviour.\",\n \"Currently agents are difficult to design and buid.\",\n \"This is because agents draw on several disciplines, each one bringing its own theoretical and practical difficulties to design and development.\",\n \"They are: control theory, cognitive psychology and artificial intelligence.\"\n ],\n [\n \"TECHNICAL FIELD This invention concerns generic knowledge software agents, or kuarks, and their use as the building blocks for agent based applications.\",\n \"It also concerns systems or kitems, made up of kuarks and a process for constructing them.\",\n \"It also concerns mult-kitem systems and searching processes for those systems.\",\n \"Finally it also cocerns software agents and a knowledge management system comprising kitems.\",\n \"BACKGROUND ART The model for the agents uses the inventions described in International Patent Application No.\",\n \"PCT/AU99/00501 entitled ‘Generic Knowledge Management System’ (GKMS), and Australian provisional patent application no PR0852 entitled ‘Networked Knowledge Management And Learning’ (NKML) both of which are incorporated herein by reference.\",\n \"A summary of these knowledge models will now be given with reference to FIGS.\",\n \"1 to 4.A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.\",\n \"Knowledge is mapped as regions (or patterns) in a source (or problem) space that are linked to regions (or patterns) in a destination (or solution) space, as illustrated in FIG.\",\n \"1.The interactions for defining the problem and solution spaces, and for defining the mappings, and the interactions for accessing the knowledge take place in a network of client-servers, as illustrated in FIG.\",\n \"2.FIGS.\",\n \"3 and 4 show the decision-making process and the knowledge capture process, and how knowledge can be improved on an on-going basis.\",\n \"FIG.\",\n \"4 is an extension of FIG.\",\n \"3 in which the consequences of the decisions taken by a GKMS are evaluated and used to qualify the knowledge in the GKMS.\",\n \"Once again it involves signal exchanges between servers and clients.\",\n \"The accepted architecture for agents is summarized in FIGS.\",\n \"5 and 6.FIG.\",\n \"5 illustrates the BDI model, that is agents have Beliefs, Desires and Intentions.\",\n \"FIG.\",\n \"5 shows that in each part of the BDI model, agents need knowledge to be able to operate.\",\n \"They must have belief which is knowledge about what their role in existence is.\",\n \"Their desires or goals represent knowledge about what they are capable of and their intentions represent the knowledge about implementing the goals.\",\n \"Similarly, each of the DBI boxes in FIG.\",\n \"5 is made of components, each representing a kind of knowledge in its category.\",\n \"All these different types of knowledge can be easily and conveniently represented using the GKMS model for knowledge.\",\n \"In addition, the use of that knowledge can be supported by the knowledge processing described in the GKMS patent.\",\n \"This means that the advantages of the GKMS model for knowledge representation and processing can become available to agents.\",\n \"It is also important to note that the use of the GKMS knowledge model for representation and processing is not restricted to the categories of knowledge shown in FIG.\",\n \"5, but to any knowledge that may be useful in agents.\",\n \"FIG.\",\n \"6 shows how an agent situated in an environment interacts with this environment.\",\n \"Th interaction is very similar to FIGS.\",\n \"3 and 4 which show how interaction with the environment and learning can take place in the GKMS model.\",\n \"Therefore the GKMS model is also suitable for dealing with agents interacting with their environments and learning in the process.\",\n \"SUMMARY OF THE INVENTION The invention is a generic knowledge software agent, or kuark, existing within a knowledge environment, and comprising: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark and a destination context of information identifiable by the kuark; the kuark having a defined source region, or pattern, within the source context and a mapping between that source region and a defined destination region, or pattern, within the destination context; and the kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.\",\n \"So the kuark makes predetermined information automatically available to a user when input criteria are met.\",\n \"The kuark is only able to detect parameters that fall within its defined source context and when the environment satisfies the source region the mapping is triggered.\",\n \"The source region may be identicle to the source context, or it may be a subset within it.\",\n \"A kuark may comprise many other elements, such as: a univeral id; a title; an explanation, which may be multimedia; a time and date created; an author; a time and date when last modified; a person who last modified agent; a version number; a status to an enquiry, such as available, candidate, rejected, definite; a description of the source context at time of last firing; a description of destination context after last firing; and, links to other kuarks.\",\n \"The links implement relationships to other kuarks.\",\n \"The are four links: The hierarchical parent's universal id, and more than one parents could be allowed; The id of the previous version kuark; The ids of the kuarks that are part of this kuark; The ids of the kuarks this kuark belongs to or is associated with.\",\n \"This last link may be implemented as separate kuark(s) designed to contain the IDs of the kuarks the kuark in question relates (or belongs) to.\",\n \"The kuark may also operate to do the following: Create a daughter/parent kuark; Adopt an existing kuark as daughter or parent; Display an internal state (contexts, regions, mapping); and Make a copy each time it fires.\",\n \"It is possible to implement these methods outside the current kuark, in a separate class or subroutine for example.\",\n \"A first special kuark, known as a ‘relevance kuark’, determines the relevance of the environment, and depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping that determines (calculates) the relevance of the environment.\",\n \"The relevance can be expressed as a probability.\",\n \"The destination context may be made of the different relevance values that the environment can take with respect to the source context and region.\",\n \"For example, the values could be ‘definitely relevant’, ‘possibly relevant’, ‘relevant’, ‘likely not relevant’, ‘not relevant’.\",\n \"Another special kuark is known as a ‘procedure kuark’, and contains as a mapping a (usually small) program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context.\",\n \"This kuark executes its mapping when the environment is compatible with the source context and the region.\",\n \"That is, the input to the procedure is the source region in the source context.\",\n \"The destination context describes the range of outputs the procedure can produce when run, and the destination region or pattern is the actual result of the execution of the mapping, using an instance of the environment.\",\n \"Kuarks may be implemented as classes and objects using an object oriented language such as Java.\",\n \"A kuark is a class in OOP and some of its properties are static, final, etc.\",\n \"A kuark that is given values for its source and destination contexts and regions are objects or instances of the corresponding kuark class.\",\n \"A system made of several kuarks is still a kuark with all the allowed properties and methods, but is known as a ‘kitem’.\",\n \"In a kitem all the component kuark and kitems may keep their identities.\",\n \"The new kitem may have its own properties and methods in addition to those of its component kitems.\",\n \"The new kitem may have its own title and explanation, and may also have its own source and destination contexts and regions, and its own mapping.\",\n \"A kitem may be constructed form kuarks and kitems by the following process: concatenating the source contexts; concatenating the destination contexts; concatenating the source regions; concatenating the destination regions; concatenating the mappings; concatenating the explanations.\",\n \"Here concatenates means ‘take the union’ of the entities in question; for mappings, it is the juxtaposition of the individual mappings.\",\n \"The kitem construction process may also build the list of kuarks or kitems that are part of the new kitem.\",\n \"Typically, when a kitem is built, all its components (kuarks or kitems) keep their individual contexts.\",\n \"Alternatively the kitem construction process may be set so that all components get the same source and destination contexts, that is the concatenation of the individual contexts of the kitem components.\",\n \"A kuark or kitem can be represented as an object using an object oriented language such as Java.\",\n \"A kitem can be expressed as an object containing another object (or a class with sub-classes) which preserves all the features for knowledge items disclosed in the GKMS patent.\",\n \"This means that kitem objects can be generated without programming.\",\n \"The other features disclosed in the GKMS patent also apply to kitems implemented as classes.\",\n \"Kitems can be linked.\",\n \"A link has a source or origin kitem and a destination kitem.\",\n \"The links can be stored inside the kitem or kuark, as a property of the kitem.\",\n \"An alternative is to store inside the kitem the address of the ‘link kitem’, that is, the kitem that is designed to contain the links that relate to a source kitem.\",\n \"A multi-kuark or multi-kitem system may comprise a serial arrangement, in which the destination context of one kitem is used as the source context (or part of the source context) of another.\",\n \"More than two kitems can be chained.\",\n \"Alternatively, a parallel kitem is made of kitems that have independent (or disjointed), but can also have overlapping, source contexts.\",\n \"Inside a parallel kitem, the component kitems are treated as peers, and links are defined between peers.\",\n \"An application or knowledge base may comprise a set of knowledge items, kitems, defined with respect to a domain of discourse, where the kitems are arranged in parallel.\",\n \"Some of the kitems may be linked in series or in parallel.\",\n \"A kitem may be a class that supports the creation of objects, or knowledge elements, where the kitem has access to all the properties and methods of other kitems contained within it, in addition to the properties that belong only to that kitem.\",\n \"Such a kitem is referred to as a ‘meta-kitem’ and is able to express meta-knowledge.\",\n \"A display kitem is a serial kitem comprising two kuarks.\",\n \"The first kuark is a relevance kuark.\",\n \"It uses its region and the state of the environment to determines the relevance of the display kitem.\",\n \"The second kuark is a procedure kuark and has the relevance (the output of the first kuark) as source context and region.\",\n \"The material to be displayed comprises the destination region and the explanation.\",\n \"An executable kitem is a serial kitem comprising two kuarks.\",\n \"The first kuark is a relevance kuark.\",\n \"It uses its region and the state of the environment to determines the relevance of the executable kitem.\",\n \"The second kuark is a procudure kuark.\",\n \"It has the relevance (the output of the first kuark) as region and as part of its source context.\",\n \"When the relevance is not ‘null’, the second kuark executes its mapping to produce the destination region.\",\n \"The inputs to the executable mapping are elements of the second kuark's source context that can be additional to the relevance context.\",\n \"A knowledge item is a kitem comprising a source context, a source region, a destination context, a destination region, a mapping and an explanation.\",\n \"Searching involves identifying those kitems that have source regions compatible with the enquiry.\",\n \"It means determining the ‘relevance status’(available, candidate, rejected or definite) of a kitem with respect to the enquiry or consultation so far.\",\n \"Searches may be synchronous or asynchronous.\",\n \"An available status is the default status of a kitem before a search takes place; a candidate kitem is one that is not incompatible with the enquiry so far; a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.\",\n \"The source context may be organised as a tree structure.\",\n \"Each kitem may be indexed against the context tree.\",\n \"This means that each object in the context possesses a list of the kitems it is associated with.\",\n \"During processing, all the indexed kitems to the questions (context objects) asked and answered so far in the consultation have their relevance status checked and modified if appropriate.\",\n \"Kitems which become definite can be presented to the user, candidates remain contenders for definite status depending on future answers, and rejected items will not be considered any longer.\",\n \"At the implementation level, the context tree is a modified context space kitem.\",\n \"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.\",\n \"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.\",\n \"The search process updates the status of the kitems according to the enquiry.\",\n \"Alternatively, the search process (also a kitem) may flag the elements in the context tree that correspond to the questions answered so far.\",\n \"Each context tree element is itself a kitem and its role is to determine whether all the indexed elements have taken into account the values of the context tree element.\",\n \"If they do this, it passes the value(s) to the indexed kitems and waits until all the responses have been received.\",\n \"The search process waits until all the tree elements corresponding to the enquiry so far have replied.\",\n \"When all have replied, the enquiry has been processed (the knowledge elements have determined their status based on the enquiry so far).\",\n \"Synchronisation is assured and the next step in the enquiry (presenting the results to the user and waiting for its input) can proceed.\",\n \"At the implementation level, the context tree is a modified context space kitem.\",\n \"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.\",\n \"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.\",\n \"Each kitem updates its status according to the enquiry and informs the searc process.\",\n \"Alternatively, in asynchronous searches, the source region may determine whether the outcome of the kitem becomes applicable.\",\n \"That is, the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.\",\n \"The state of the relevance region determines what happens to the application region.\",\n \"Where each kitem has a unique ID or address that determines its location in a distributed memory space or network, it may also possess a set of applicability addresses that correspond to the kitems in its source region.\",\n \"During processing, the enquiry process may submit each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.\",\n \"In this model, each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process.\",\n \"The enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.\",\n \"These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry (or enquiry step).\",\n \"Each kitem can decide whether to answer more than once to an enquiry from the same origin or process.\",\n \"It is up to the enquiry process to manage all the replies it gets from the network.\",\n \"The solution outlined here is applicable to distributed processing.\",\n \"Another model for asynchronous searches is to give even more responsibility to the distributed kitems, and each kitem has the address of each of the context objects in its context and in its region (a subset of the context).\",\n \"The kitem interrogates the network at regular interval to determine whether the status of the region, and hence of the applicability of the kitem, has changed.\",\n \"Cascading refers to a knowledge element having, as part of its destination region, an object that belongs to the source region of another knowledge element.\",\n \"When the first knowledge element fires, it may change its source region in a way that impact on the applicability of the second knowledge element.\",\n \"The second knowledge element may have to fire, not as a direct effect of the enquiry but as an indirect effect, that is, a consequence of another knowledge element having changed its state.\",\n \"This process can extend to any arbitrary number of knowledge elements, called cascading knowledge elements.\",\n \"In order to ensure that all the cascaded elements update their state if appropriate, it is necessary to a) link the cascaded elements, and b) have the search process update the relevance status of these cascaded knowledge elements (synchronous search) or c) to communicate the state of the environment (outputs of previously fired knowledge elements) to the linked cascaded knowledge elements (asynchronous search).\",\n \"In a multi-kitem system, knowledge processing involves running a question-answer session.\",\n \"The system asks questions, get answers and, on the basis of these answers, asks further questions until the correct answers are found or no answers exist.\",\n \"The steps involved are: identifying the most important questions (the importance is determined by the ability of the answers to the question to lead to the correct answers as quickly as possible); asking these questions; on the basis of the answers to the questions, determining whether any answers can be found, and the next best questions to ask.\",\n \"Identifying the most promising questions may involve a synchronous search through a context tree with processing delegated to a search agent.\",\n \"As the context tree is updated each time a question is considered, the most important questions to ask next depend on the consultation so far; that is, on the state of the context tree after the previous enquiry has been processed.\",\n \"Therefore the questions are selected dynamically.\",\n \"Alternatively the search may have processing delegated to each kitem.\",\n \"Context tree search with processing delegated to each kitem As explained above, the search process waits until all the tree elements corresponding to the enquiry so far have replied.\",\n \"It is the responsibility of the enquiry process to collect these replies and to count the number that come from each of the tree context element: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.\",\n \"In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relerance status), this process can also be asynchronous it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.\",\n \"In an asynchronous searches the enquiry process (that sends the enquiry over the network for kitems to process it) may perform several tasks: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.\",\n \"In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relevance status), this process can be asynchronous; it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.\",\n \"In situations where the domain of discourse of a body of knowledge is known, one can identify the kitems with the largest number of indexed kitems with relevance status available or candidate in order to find hidden knowledge.\",\n \"The process of producing a context tree is a compilation of the kitems in an application or domain of discourse.\",\n \"Compilation keeps the identity of the kitems and all the data and information in these kitems that pertain to their execution.\",\n \"Keeping the identity of these kitems enables the non-executable parts of the kitems to be retrieved at execution time on a ‘as needed’ basis.\",\n \"There are two types of compilation: Just-in-time compilation imposes the compiled kitem to take the latest version of each kitem in its process, in particular the latest version of the source and destination regions; or Permanent compilation accepts that the kitems do not change (between compilations) and that their attributes and methods can be used without checking them.\",\n \"Therefore they are included in the compiled kitem.\",\n \"This is faster than the partial compilation.\",\n \"An agent may be represented as a kitem with the following structure: Beliefs 61, Desires 62 and Intentions 63 are parallel kitems (their respective components are also kitems); the Beliefs 61, Desires 62 and Intentions 63 kitems are organised in a serial architecture.\",\n \"That is, the agent kitem is a serial kitem; the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.\",\n \"The kitems needed to produce a workable implementation for knowledge management are shown below: A context element is a display kitem (a serial kitem made of two kuarks).\",\n \"A source or destination context kitem is a parallel display kitem.\",\n \"Its members comprise the list of all the context elements defined in the domain of discourse.\",\n \"Context executable kitems may edit a context element, edit a context space or display a context space as a tree.\",\n \"A source or destination context tree is an executable kitem.\",\n \"The display kitem may display the elements that belong to the source and destination kitems.\",\n \"Its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context.\",\n \"A knowledge editing kitem is an executable kitem that has: As source space: the source and destination contexts.\",\n \"As destination space: the source and destination contexts.\",\n \"As source region: a subset of the source context that defines the applicability of the knowledge kitem.\",\n \"As mapping: a link to a subset of the destination context, or a procedure or executable (sub-)kitem.\",\n \"As destination region: a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure.\",\n \"An explanation.\",\n \"A search kitem is an executable kitem with: as source context: the source and destination contexts; as source region: the enquiry as defined so far; as destination context: the kitems in the domain of discourse (if known); as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry; as destination region: the candidate and definite kitems ‘retrieved’ by the mapping.\",\n \"An enquiry kitem is an executable kitem with: as source space: the enquiry (the consultation process, showing each step in the question-answer session); as destination space: the status of the enquiry (answered, not answered, etc.\",\n \"), the ids of the definite kitems.\",\n \"A display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.\",\n \"This technology supports rapid application development (RAD).\",\n \"RAD is a very important commercial feature for software.\",\n \"It holds the promise of speeding up software development, implementation and customization.\",\n \"RAD, or any significant progress in this area, has the potential to save, worldwide, enormous sums (billion of dollars) in development, and to reduce the total cost of ownership of software solutions over their lifetimes.\",\n \"RAD is also a very serious technical challenge for designers.\",\n \"Agents hold the promise of autonomous and intelligent software that is capable of co-operating with other agents (and human beings) towards achieving a goal.\",\n \"In addition agents, with proper architecture, should scale up.\",\n \"That is, they could provide a promising platform for building complex, adaptable systems that could have knoweldge and perhaps exhibit intelligent behaviour.\",\n \"Currently agents are difficult to design and buid.\",\n \"This is because agents draw on several disciplines, each one bringing its own theoretical and practical difficulties to design and development.\",\n \"They are: control theory, cognitive psychology and artificial intelligence.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS The background to the invention has been described above with reference to the following drawings: FIG.\",\n \"1 is a diagram illustrating knowledge as mappings between a problem space and a solution space.\",\n \"FIG.\",\n \"2 is a diagram illustrating a network of client-servers, each able to run agents.\",\n \"FIG.\",\n \"3 is a diagram illustrating a knowledge capture loop in the GKMS.\",\n \"FIG.\",\n \"4 is a diagram illustrating an expanded knowledge capture loop in an on-the-job problem-solving system.\",\n \"FIG.\",\n \"5 is a diagram illustrating the agent DBI model.\",\n \"FIG.\",\n \"6 is a diagram illustrating an agent situated in and interacting with its environment.\",\n \"The invention will now be described with reference to the following drawings: FIG.\",\n \"7 is a diagram illustrating a generic elementary cognitive element (kuark).\",\n \"FIG.\",\n \"8 is a diagram illustrating a relevance kuark.\",\n \"FIG.\",\n \"9 is a diagram illustrating a procedure kuark.\",\n \"FIG.\",\n \"10 is a diagram illustrating a serial architecture.\",\n \"FIG.\",\n \"11 is a diagram illustrating a parallel architecture.\",\n \"FIG.\",\n \"12 is a diagram illustrating the agent implementing the DBI model as a serial kitem.\",\n \"BEST MODES OF THE INVENTION Generic Elementary Cognitive Agent (Kuark) Referring to FIG.\",\n \"7, the generic elementary cognitivey agent (kuark) 10 is the building block of agent-based applications.\",\n \"The kuark knowledge model implements and extends the GKMS model described in the GKMS patent.\",\n \"The kuark 10 monitors the environment by detecting all environment parameters compatible with the source region 11 and source context 12.When the source region 11, which determines the applicability of the mapping 13, is satisfied, the mapping 13 fires.\",\n \"The mapping determines the output region 14 in the destination context 15 and makes the region available to the output, and hence to the environment.\",\n \"Regions are sometimes referred to as patterns.\",\n \"EXAMPLE 1 The source context comprises two sets of integers and the destination context one set of integers.\",\n \"The source region is the positive integers for the two sets, the mapping a multiplication of the two inputs accepted in the source region.\",\n \"The destination region is the result of the multiplication of the two positive numbers.\",\n \"EXAMPLE 2 The source context is the state of a logical variable and the region the value ‘true’ of that variable.\",\n \"The destination context is the state of the same variable and the mapping the ‘negation’ of the original value, that is ‘false’.\",\n \"EXAMPLE 3 The source context is the temperature and pressure inside a boiler and the destination context a list of commands triggering alarms and associated processes (that can be software based).\",\n \"The source region is the range of pressures and temperatures that trigger a ‘code red’ alert and the destination region the command triggering the ‘code red’ alarm.\",\n \"EXAMPLE 4 The source context and region is as in the example 3.The destination context is the list of the relevance status of the kuark and the region the actual relevance status of the kuark, based on the source region.\",\n \"That is, the ‘code red’ alert is given a relevance status (perhaps expressed as a probability) based on the environment.\",\n \"Kuark Properties The common properties of a kuark are: univeral id; title; explanation (multimedia); time and date created; author; time and date when last modified; person who last modified agent; version number; domain of discourse source context (what it knows about) 12; destination context 15; applicability source region or pattern 11 (can be identical to source context); relevance status to an enquiry (available, candidate, rejected, definite); mapping 13 (the process that produces the outcome based on the state of source context) outcome result of the mapping between source and destination context; the mapping determines the destination region or pattern; status (enabled/disabled/archived); state of source context at time of last firing (describes source context and region); state of destination context after last firing (describes destination region); links to other kuarks the hierarchical parent's universal id (more than one parents could be allowed); the id of the previous version kuark; the ids of the kuarks that are part of this kuark (see later for multi-kuark kuarks); the ids of the kuarks this kuark belongs to or is associated with (see later for multi-kuark kuarks).\",\n \"The property ‘links to other kuarks’ is the mechanism used to implement relationships between kuarks.\",\n \"The fourth link mentioned may be implemented as separate kuark(s) designed to contain the IDs of the kuarks the kuark in question relates (or belongs) to.\",\n \"Kuark Methods The main methods of a kuark are: monitor applicability (detect only what its context allows); determines whether environment fits within source region, if yes triggers/fires mapping; execute mapping (modifies destination state) mapping determines the outcome which is a subset of the destination context; create daughter/parent kuark; adopt existing kuark as daughter or parent; display internal state (contexts, regions, mapping); make copy each time it fires.\",\n \"It is possible to implement these methods outside the current kuark, in a separate class or subroutine for example.\",\n \"Relevance Kuark Referring now to FIG.\",\n \"8, a relevance kuark 20 is a kuark 10 (as described previously) that determines the relevance of the environment.\",\n \"Depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping 23 that determines (calculates) the relevance of the environment.\",\n \"The relevance can be expressed as a probability.\",\n \"The destination context 15 is made of the different relevance values that the environment can take with respect to the source context and region.\",\n \"For example, the values could be ‘definitely relevant’, ‘possibly relevant’, ‘relevant’, ‘likely not relevant’, ‘not relevant’.\",\n \"Procedure Kuark Referring now to FIG.\",\n \"9, a procedure kuark 30 is one that contains a (usually small) program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context.\",\n \"The kuark executes its procedure mapping 33, when the environment is compatible with the source context and the region.\",\n \"That is, the input to the procedure is the source region in the source context.\",\n \"The destination context describes the range of outputs the procedure can produce when run, and the destination region or pattern is the actual result of the execution of the mapping, using an instance of the environment.\",\n \"The procedure mapping can be a trivial mapping.\",\n \"It can simply display a fixed pattern and an explanation in a fixed destination context.\",\n \"The context item, see later, is an implementation of the trivial procedure kuark.\",\n \"Relevance kuarks tend to have the same destination context, whereas procedure kuarks typically have context that are strongly application dependent and that can vary within an application.\",\n \"Kuarks and Object Oriented Programming Kuarks can be implemented as classes and objects using an object oriented language such as Java.\",\n \"A kuark is a class in OOP and some of its properties are static, final, etc.\",\n \"A kuark that is given values for its source and destination contexts and regions are objects or instances of the corresponding kuark class.\",\n \"Multi-Kuark Systems or Applications This section describes how kuarks can be used together to increase the power of applications.\",\n \"Multi-Kuark Kitems A system made of several kuarks is still a kuark with all the allowed properties and methods.\",\n \"However, the term kuark is reserved for simple (or elementary) kuarks, as described above.\",\n \"A multi-kuark kuark is named a kitem.\",\n \"Kitems are constructed as follows: the designer identifies the kuarks (or kitems) to be included in the new kitem; the designer clicks ‘construct kitem’.\",\n \"The construction process simply puts a wrapper around the kuarks and kitems to include in the new kitem.\",\n \"All the component kuark and kitems keep their identities.\",\n \"The new kitem can have its own properties and methods in addition to those of its component kitems.\",\n \"The new kitem has its own title and explanation, and can also have its own source and destination contexts and regions, and its own mapping.\",\n \"One can also view the new kuark as having undergone a construction process that carried the following operations: concatenates the source contexts; concatenates the destination contexts; concatenates the source regions; concatenates the destination regions; concatenates the mappings; concatenates the explanations.\",\n \"Here concatenates means ‘take the union’ of the entities in question; for mappings, it is the juxtaposition of the individual mappings.\",\n \"The kitem construction process also builds the list of kuarks or kitems that are part of the new kitem.\",\n \"Typically, when a kitem is built, all its components (kuarks or kitems) keep their individual contexts.\",\n \"Alternatively the kitem construction process can also be set so that all components get the same source and destination contexts, that is the concatenation of the individual contexts of the kitem components.\",\n \"Kitems have explicit domains of discourse, the source and destination contexts, and explicit regions; kitems are ‘aware’ of their domains of discourse and when their knowledge, expressed as their mappings and destination regions, can be applied.\",\n \"Not all kuarks can be assembled in kitems as some concatenation may not be allowed; this issue is not treated further here.\",\n \"Refer later for different architectures for multi-kitem systems.\",\n \"Kitem Hierarchy When defining kitems for an application, it is often convenient to organise them as a hierarchy of objects, as in a file system for example.\",\n \"In fact, all kitems are defined within a hierarchical structure similar to that of the domain names on the Internet.\",\n \"Each kitem has a parent, except the root kitem which is the top of the tree.\",\n \"The hierarchical organisation of the kitems imposes no restriction on how they can be grouped (in multi-kitem systems) to achieve some purposes in an application.\",\n \"That is, composed kitems can be assembled out of any kitem from any location in the hierarchy.\",\n \"Kitems and Object Oriented Programming A kitem as outlined above, together with its GKMS features, mean that kitems can be implemented using an object oriented language, as objects or instances of the kuark or kitem classes.\",\n \"As indicated above, a kuark or kitem can be represented as an object using an object oriented language such as Java.\",\n \"A multi-kuark kitem as outlined above, can be expressed as an object containing another object (or a class with sub-classes) which preserves all the features for knowledge items disclosed in the GKMS patent.\",\n \"This means that kitem objects can be generated without programming.\",\n \"The other features disclosed in the GKMS patent also apply to kitems implemented as classes.\",\n \"Rationale for Multi-Kitem Systems The advantages of building applications using kitems are very significant: the methods and properties can be used across the hierarchy (for example, a method to display the components kitems of a kitem can be re-used at all levels in the hierarchy of kitems); kitems provide a unified way for thinking and implementing applications; kitems can be fashioned as simple building blocks that offer maximum re-use; kitems implement knowledge at every level (in every kitem) that make up the application; knowledge permeates the design and the use of the application; applications can be largely soft-coded, instead of hard-coded; applications can be developed rapidly (RAD: rapid application development).\",\n \"Links Between Kitems Kitems can be linked.\",\n \"A link has a source or origin kitem and a destination kitem.\",\n \"The links can be stored inside the kitem or kuark, as a property of the kitem.\",\n \"An alternative is to store inside the kitem the address of the ‘link kitem’, that is, the kitem that is designed to contain the links that relate to a source kitem.\",\n \"In a link kitem, the source context contain the ID of the source kitem and the type of link (see above for list of possible links), and the destination regions contains the IDs of the corresponding links (the destination context is the domain of discourse of the source kitem).\",\n \"It is also possible, indeed often preferable, to have the source and destination contexts and regions for a kitem expressed via links.\",\n \"That is, one or several link kitems contain the source and destination context, and the regions (their kitems and values).\",\n \"Multi-Kitem Architectures This section discusses the architectures available for building multi-kuark or multi-kitem systems.\",\n \"In particular, these architectures can be used to build the display and executable kitems.\",\n \"Serial Architecture In the serial architecture 40, the destination context of one kitem 41 is used as the source context (or part of the source context) of another 42.More than two kitems can be chained, as illustrated in FIG.\",\n \"10.The state of the environment is used as input to kitem 41 (and perhaps to other kitems).\",\n \"Similarly, the serial kitem output is the output from kitem n 45 (and perhaps from other kitems).\",\n \"In the most simple case, the source context is the source context of kitem 41 and the destination context the destination context of kitem n. In general, the source context of the serial kitem is a subset of the source contexts of all its ‘internal kitems’.\",\n \"Similarly for the destination context and for the regions in these contexts.\",\n \"The source context of an internal kitem can comprise some elements from the environment in addition to the output from the previous kitem.\",\n \"The serial kitem is a kitem and has all the properties of kitems discussed above.\",\n \"Parallel Architecture Referring now to FIG.\",\n \"11, a parallel kitem 50 is made of kitems that have independent (or disjointed), but can also have overlapping, source contexts.\",\n \"The power of parallel kitems derives from the links that can be established between its internal kitems.\",\n \"The objective is to ensure that the information and knowledge contained in two kitems becomes linked.\",\n \"For example, a kitem could describe clients and another projects; and a link would connect a client with a project.\",\n \"A link has an origin and a destination.\",\n \"Inside a parallel kitem, the component kitems are treated as peers, and links are defined between peers.\",\n \"When the class of a parallel kitem is defined, the operator has to specify which kitems are instantiated as objects when the parallel kitem object is created.\",\n \"In linked kitems, it is convenient, but not mandatory, to instantiate the origin kitem but not the destination kitem.\",\n \"Links can be unidirectional or bidirectional.\",\n \"For example, from clients to projects, or from clients to projects and from projects to clients.\",\n \"Hierarchical Kitems The kitems that are part of a parallel (or serial kitem) keep their properties, notably the one identifying their parents.\",\n \"Therefore the parallel kitem can, in fact, contain a hierarchy of kitems even if they are organised in a flat hierarchical (that is, parallel) way in the parallel kitem.\",\n \"A Knowledge Application as A Parallel Architecture Kitem An application or knowledge base is a set of knowledge items (kitems) defined with respect to a domain of discourse (source and destination contexts and mappings available).\",\n \"These kitems are arranged in parallel and some of them can be linked.\",\n \"For example, in an application all the knowledge items are typically linked (that is refer) to the same domain of discourse, as explained above.\",\n \"Hybrid Architecture It is possible to define a hybrid architecture, with the hybrid kitem containing some kitems linked in a serial way and others in a parallel way.\",\n \"Meta-Kitems and Meta-Knowledge In FIGS.\",\n \"10 and 11, each kitem is a class that supports the creation of objects, or knowledge elements.\",\n \"It is desirable to be able to define a class that can have attributes and methods that span more than one of the kitems contained in the parallel, serial or hybrid kitem.\",\n \"This can be achieved by giving the outside kitem (eg: the serial, parallel or hybrid kitem) access to all the properties and methods of the contained kitems, in addition to the properties that belong only to the outside kitems.\",\n \"This means that it is possible to define meta-knowledge about the kitems contained in the outside kitems.\",\n \"The outside kitem, when instanced as an object, can observe the behaviour of the kitems in it and take actions when necessary.\",\n \"The outside kitems are referred to as meta-kitems able to express meta-knowledge.\",\n \"Display and Executable Kitems In this section we consider two different types of very useful kitems: the display kitem and executable kitem.\",\n \"We show that each can be constructed using one relevance kuark and one procedure kuark.\",\n \"Display Kitem The display kitem is a serial kitem comprising two kuarks.\",\n \"The first kuark is a relevance kuark.\",\n \"It uses its region and the state of the environment to determines the relevance of the display kitem.\",\n \"The second kuark has the relevance (the output of the first kuark) as source context and region.\",\n \"The material to be displayed comprises the destination region and the explanation (the destination region can be the explanation.\",\n \"Context Kitem Contexts and regions are made of elements or objects that describe the contexts and regions.\",\n \"Each such object can have values.\",\n \"Here we show that kitems are suitable for describing and implementing these context objects.\",\n \"A context object is a kitem as shown below (only some properties are mentioned): kitem tile → object title source context → object values source region → instantiated values destination context → same as source context destination region → same as source region mapping → maps source to destination (contexts and regions) explanation → optional.\",\n \"Explanation Kitem An explanation object is a kitem as shown below (only some properties are mentioned): kitem title → explanation title (can be ‘explanation’) source context → not used source region → not used destination context → not used destination region → not used mapping → not used explanation → multimedia explanation.\",\n \"Context Element A context element is a kitem made of, typically, a context kitem and an explanation kitem (some context items can have more than one explanation).\",\n \"To create such a kitem, the designer defines a parallel kitem using the procedure described in the GKMS patent.\",\n \"It is also possible to have a context item made of a single context kitem, by using its explanation instead of the explanation kitem.\",\n \"This solution does not work if more than one explanation is required.\",\n \"For example, an explanation relating to the meaning of the context item and another relating to its use in a knowledge item.\",\n \"Context Space (Source Or Destination) The context space is a kitem made of context items (also kitems) using the parallel architecture.\",\n \"Typically no links are used between context items.\",\n \"Executable Kitem The executable kitem is a serial kitem comprising two kuarks.\",\n \"The first kuark uses its region and the state of the environment to determines the relevance of the executable kitem.\",\n \"The second kuark has the relevance (the output of the first kuark) as region and as part of its source context.\",\n \"When the relevance is not ‘null’, the second kuark executes its mapping to produce the destination region.\",\n \"The inputs to the executable mapping are elements of the second kuark's source context that can be additional to the relevance context.\",\n \"Knowledge Item and Knowledge Item Definition A knowledge item is a kitem comprising a source context, a source region, a destination context, a destination region, a mapping and an explanation.\",\n \"Defining a knowledge item, as described in the GKMS patent, involves defining a region in the source context (and other things).\",\n \"This is done using the context space kitem, that is made of context objects (or elements).\",\n \"The architecture of a knowledge item is normally that of a parallel kitem.\",\n \"It is often desirable to explain why a certain context element is part of a knowledge item or how to use or interpret this context element when it is attached to the knowledge item.\",\n \"This can be achieved simply by giving the user defining the knowledge item the facility to add an explantion kitem to the list of kitem that are part of the knowledge item, and linking this explanation kitem to the context element (kitem) in question.\",\n \"Alternatively, the explanation can be part of the context element kitem.\",\n \"That is, the explanation kitem is already linked to the context element (with reference to OOP, the context element class ‘contains’ the explanation).\",\n \"A knowledge item is defined with respect to a domain of discourse, defined by the source and destination context and the mappings available at definition time.\",\n \"It would not be efficient to include the contexts kitems in the knowledge item.\",\n \"Instead, the knowledge item is linked to the context space kitems.\",\n \"This is in agreement with the definition of links in the parallel architecture kitem.\",\n \"Kitem Hardcoding We have discussed how kitems can be implemented using other kitems.\",\n \"However it is always possible to implement a kitem as a single hardcoded entity.\",\n \"For example, in the GKMS patent, the context elements and the knowledge items were implemented as single entities with their specific architectures.\",\n \"When this is done, many of the modular and flexible features of the kuark-kitem model are lost.\",\n \"When a similar effect is desired, it is preferable to implement the kitem as a permanently compiled multi-kitem kitem (see later).\",\n \"Knowledge Processing in Multi-Kitem Systems A multi-kitem system is a system comprising a set of kitems representing the body of knowledge about a certain domain of discourse.\",\n \"Typically the domain of discourse is identical for all the kitems in the system, although it is not essential.\",\n \"Such a system is identified as a knowledge module or database.\",\n \"Knowledge processing involves the system (or server) running a question-answer session.\",\n \"The system asks questions, get answers and, on the basis of these answers, asks further questions until the correct answers are found or no answers exist.\",\n \"The steps involved are: identify the most important questions (the importance is determined by the ability of the answers to the question to lead to the correct answers as quickly as possible); asks these questions; on the basis of the answers to the questions, determine a) whether any answers can be found, and b) the next best questions to ask.\",\n \"The processing refers to: the identification of the correct answers available in the knowledge base, on the basis of the information available so far in the consultation; and the identification of the most promising provisional answers available in the knowledge base, on the basis of the information available so far in the consultation; and the identification of the most important questions to ask on the basis of the information available.\",\n \"Searching for relevant or important knowledge Knowledge access in a multi-kitem system (a knowledge module) follows the same pattern as for a GKMS knowledge base; that is, it is a question-answer session or consultation.\",\n \"The consultation process is managed with, and recorded in, a document.\",\n \"This document can also be a kitem called the consultation kitem.\",\n \"That is, the consultation kitem has all the properties and methods of the kitem class.\",\n \"All the utilities for kuarks and kitems can be used with consultation kitems.\",\n \"Searching involves identifying those kitems that have source regions compatible with the enquiry (see GKMS patent for meaning of compatible).\",\n \"It means determining the ‘relevance status’ (available, candidate, rejected or definite) of a kitem with respect to the enquiry or consultation so far.\",\n \"Searches can be synchronous or asynchronous.\",\n \"An available status is the default status of a kitem before a search takes place; a candidate kitem is one that is not incompatible with the enquiry so far; a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.\",\n \"Synchronous Searches Database Search The database search is a SQL search or equivalent.\",\n \"This means that the kitems are stored as records in a database.\",\n \"This is not discussed further in this document Full Text Search The full text search has to process each relevant field in each record where each record describes a knowledge element.\",\n \"This process can be slow; it is not discussed further in this document.\",\n \"Context Tree Search with Processing Delegated to A Search Agent The source context is organised as a tree structure.\",\n \"A source region, which determines whether a kitem will fire (firing depends on the region's compatibility with an enquiry), is a subset of the source context.\",\n \"Each kitem (eg: knowledge element) can be indexed against the context tree.\",\n \"This means that each object in the context possesses a list of the kitems it is associated with.\",\n \"The context tree is a formalisation of one of the kuarks'properties (see above).\",\n \"During processing, all the indexed kitems to the questions (context objects) asked and answered so far in the consultation have their relevance status checked and modified if appropriate.\",\n \"Kitems which become definite can be presented to the user, candidates remain contenders for definite status depending on future answers, and rejected items will not be considered any longer.\",\n \"At the implementation level, the context tree is a modified context space kitem.\",\n \"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.\",\n \"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.\",\n \"The search process updates the status of the kitems according to the enquiry.\",\n \"Context Tree Search with Processing Delegated to Each Kitem The search process (also a kitem) flags these elements in the context tree that correspond to the questions answered so far.\",\n \"Each context tree element is itself a kitem and its role is to determine whether all the indexed elements have taken into account the values of the context tree element.\",\n \"If they do this, it passes the value(s) to the indexed kitems and waits until all the responses have been received.\",\n \"The search process waits until all the tree elements corresponding to the enquiry so far have replied.\",\n \"When all have replied, the enquiry has been processed (the knowledge elements have determined their status based on the enquiry so far).\",\n \"Synchronisation is assured and the next step in the enquiry (presenting the results to the user and waiting for its input) can proceed.\",\n \"At the implementation level, the context tree is a modified context space kitem.\",\n \"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.\",\n \"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.\",\n \"Each kitem updates its status according to the enquiry and informs the searc process.\",\n \"Asynchronous Searches Processing Delegated to Each Kitem In asynchronous searches, the processing is distributed and controlled by each item.\",\n \"There is no central authority that decides what to do when.\",\n \"Each kitem (or agent) monitors its environment and reacts appropriately.\",\n \"In doing so, it may change the environment and trigger new actions by other kitems.\",\n \"This is in contrast to synchronous searches in which it is the search process that determines whether a kitem is applicable or not.\",\n \"In particular, in asynchronous searches it is each kitem that determines whether it is applicable to the environment as it is detected via the source context and the source region.\",\n \"In asynchronous searches, the source region determines whether the outcome of the kitem becomes applicable.\",\n \"That is, the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.\",\n \"The state of the relevance region determines what happens to the application region.\",\n \"In the case of non-executable mappings (or display mappings) the process is as illustrated in FIG.\",\n \"8; the relevance mapping ‘releases’ the destination region to the kuark output.\",\n \"State of Environment (or Context) Communicated to the Items As indicated above, each kitem has a unique ID or address that determines its location in a distributed memory space or network (such as the Internet for example).\",\n \"It also possesses a set of applicability addresses that correspond to the kitems in its source region.\",\n \"During processing, the enquiry process submits each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.\",\n \"In this model, each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process.\",\n \"The enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.\",\n \"These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry (or enquiry step).\",\n \"Each kitem can decide whether to answer more than once to an enquiry from the same origin or process.\",\n \"It is up to the enquiry process to manage all the replies it gets from the network.\",\n \"The solution outlined here is applicable to distributed processing.\",\n \"The Kitems Seek to Know the Status of their Source Context Another model for asynchronous searches is to give even more responsibility to the distributed kitems than in the previous description.\",\n \"In this model, each kitem has the address of each of the context objects in its context and in its region (a subset of the context).\",\n \"The kitem interrogates the network at regular interval to determine whether the status of the region, and hence of the applicability of the kitem, has changed.\",\n \"This model means that the kitem takes the intiative (this has some important applications not discussed here).\",\n \"However during searching, the model imposes much traffic on the network that is of no benefit, unless the context object has changed since the last status check.\",\n \"Another problem is that there may be an unacceptable delay between status checks, during which time, if the status has changed, the kitem doing the interrogating cannot determine its applicability.\",\n \"Cascading Knowledge Cascading refers to a knowledge element having, as part of its destination region, an object that belongs to the source region of another knowledge element.\",\n \"When the first knowledge element fires, it may change its source region in a way that impact on the applicability of the second knowledge element.\",\n \"The second knowledge element may have to fire, not as a direct effect of the enquiry but as an indirect effect, that is, a consequence of another knowledge element having changed its state.\",\n \"This process can extend to any arbitrary number of knowledge elements, called cascading knowledge elements.\",\n \"In order to ensure that all the cascaded elements update their state if appropriate, it is necessary to a) link the cascaded elements, and b) have the search process update the relevance status of these cascaded knowledge elements (synchronous search) or c) to communicate the state of the environment (outputs of previously fired knowledge elements) to the linked cascaded knowledge elements (asynchronous search).\",\n \"Validity of the Relevant Knowledge For simple knowledge bases on a single server it is a relatively simple matter to ensure that all kitems they contain have the same domain of discourse.\",\n \"This, however, does not apply for large or distributed knowledge bases where the knowledge comes from different sources.\",\n \"In general, therefore, there is no certainty that all the knowledge items (kitems) accessed during a search have the same source and destination contexts.\",\n \"In fact it is unlikely.\",\n \"This means that a user inspecting a definite kitem (with respect to an enquiry) may not know what the domain of discourse of that kitem might be.\",\n \"This information is very important as it defines the context for the knowledge in the kitem in question.\",\n \"A solution to this situation is to give the user the ability to inspect the source and destination context of every kitem that is provided by the search process either as a candidate or definite kitem.\",\n \"Identifying the Most Promising Questions Synchronous Searches Context Tree Search with Processing Delegated to A Search Agent Context objects which index a large number of available or candidate kitems are potentially very important when used as questions.\",\n \"This is because their answers have the potential to impact on the relevance status of a large number of kitems.\",\n \"This indicates that the importance of these context objects as questions in an enquiry is determined by the number of kitems with status available or candidate they index.\",\n \"As the context tree is updated each time a question is considered, the most important questions to ask next depend on the consultation so far; that is, on the state of the context tree after the previous enquiry has been processed.\",\n \"Therefore the questions are selected dynamically.\",\n \"Context Tree Search with Processing Delegated to Each Kitem As explained above, the search process waits until all the tree elements corresponding to the enquiry so far have replied.\",\n \"It is the responsibility of the enquiry process to collect these replies and to count the number that come from each of the tree context element: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.\",\n \"In addition, the enquiry process can perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relerance status), this process can also be asynchronous it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.\",\n \"Asynchronous Searches Processing Delegated to Each Kitem The enquiry process (that sends the enquiry over the network for kitems to process it) performs several tasks: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.\",\n \"In addition, the enquiry process can perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relevance status), this process can be asynchronous; it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.\",\n \"Search for Hidden Knowledge Hidden knowledge refers to knowledge that may be relevant to an enquiry but that is not yet linked to any question answered in the enquiry process so far.\",\n \"The question is how to identify this hidden knowledge and how to extract its ‘best questions’.\",\n \"In situations where the domain of discourse of a body of knowledge is known, one can simply identify the kitems with the largest number of indexed kitems with relevance status available or candidate (see context tree above).\",\n \"There is no simple solutions when the domain of discourse of a body of knowledge is not known.\",\n \"Processing Speed Synchronous Searches Context Tree Search with Processing Delegated to A Search Agent Synchronous searches can be very quick as they are under the control of one process that can be optimised, based on known conditions in the server.\",\n \"Context Tree Search with Processing Delegated to Each Kitem This processing can be distributed across a network, but it is infrequent.\",\n \"This processing is slower than the previous one as it imposes communications overheads between kitems (those doing some processing and the enquiry process).\",\n \"Nevertheless, the enquiry process runs on a single server and can be optimised.\",\n \"When the process is distributed, several kitems can process simultaneously; this can partially offset the communication overheads.\",\n \"Asynchronous Searches Processing Delegated to Each Kitem This processing is slowest unless the number of kitems doing processing is large enough to offset the communication overhead between kitems and the network delays.\",\n \"Compilation Compilation is the process that takes a collection of kitems and organises them for the purpose of speeding up processing.\",\n \"The result of compilation is a kitem.\",\n \"In general, compilation is applied to any kitem.\",\n \"In practice it is often applied to all the kitems that belong to a domain of discourse (also a kitem) on a single server.\",\n \"That is, all the kitems involved have domains of discourse that are subset of the compiled domain of discourse.\",\n \"Compilation is also applied to a group of kitems contained in a serial, parallel or hybrid kitem.\",\n \"The process of producing a context tree is a compilation of the kitems in an application or domain of discourse.\",\n \"Compilation keeps the identity of the kitems and all the data and information in these kitems that pertain to their execution.\",\n \"Keeping the identity of these kitems enables the non-executable parts of the kitems to be retrieved at execution time on a ‘as needed’ basis.\",\n \"The processing model for the multi-agent systems described in this document imposes, like the Java language, that kitems call other kitems that in turn may call other kitems, etc, until the processing can be completed.\",\n \"Once this is done, the results of the processing need to travel up the hierarchy of kitems, back to the initiating process.\",\n \"The compilation process is designed to speed up the ‘down-hierarchy’ and ‘up-hierarchy’ travelling described above.\",\n \"The context tree compilation achieves this purpose by ‘flattening’ the hierarchy to a single branch tree, where each context element (or node) in the branch contains all the kitems that have that context element as part of their regions.\",\n \"There are two types of compilation: Just-in-time compilation imposes the compiled kitem to take the latest version of each kitem in its process, in particular the latest version of the source and destination regions; or Permanent compilation accepts that the kitems do not change (between compilations) and that their attributes and methods can be used without checking them.\",\n \"Therefore they are included in the compiled kitem.\",\n \"This is faster than the partial compilation.\",\n \"Compilation can be be carried out to different depths: 1.build context tree at runtime; 2.get regions of indexed kitems in the context tree at runtime; 3.include regions of indexed kitems in the context tree; Compilations of type 1 and 2 above are just-in-time compilations.\",\n \"They updates the context tree each time the search process is triggered; permanent compilation produces the context tree and leaves it untouched until another compilation is carried out.\",\n \"Agents as Kitem Systems An agent, as illustrated in FIG.\",\n \"5, can be represented as a kitem 60 with the structure illustrated in FIG.\",\n \"12.: Beliefs 61, Desires 62 and Intentions 63 are parallel kitems (their respective components are also kitems); the Beliefs 61, Desires 62 and Intentions 63 kitems are organised in a serial architecture.\",\n \"That is, the agent kitem is a serial kitem; the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.\",\n \"It is recommended but not essential that the kitems that comprise the agent operate in a synchronous mode.\",\n \"Internal synchronous processing is possible, even if a multi-agent systems behaves asynchronously.\",\n \"As an agent is a kitem, a multi-agent system is a multi-kitem system with a parallel architecture and continuous asynchronous processing.\",\n \"Kitems Needed for A Workable Solutions The kitems needed to produce a workable implementation for knowledge management are shown below: Context Management Context Elements A context element is a display kitem (a serial kitem made of two kuarks).\",\n \"Source and Destination Contexts A source or destination context kitem is a parallel display kitem.\",\n \"Its members comprise the list of all the context elements defined in the domain of discourse.\",\n \"Context Executable Kitems Edit a context element Edit a context space Display a context space as a tree A source or destination context tree is an executable kitem.\",\n \"The display kitem can display the elements that belong to the source and destination kitems.\",\n \"Its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context.\",\n \"Knowledge Definition Knowledge Editing Kitem The knowledge editing kitem is an executable kitem that has: As source space: the source and destination contexts.\",\n \"As destination space: the source and destination contexts.\",\n \"As source region: a subset of the source context that defines the applicability of the knowledge kitem.\",\n \"As mapping: a link to a subset of the destination context, or a procedure or executable (sub-)kitem.\",\n \"As destination region: a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure.\",\n \"An explanation.\",\n \"Knowledge Access and Display Search Kitem The search kitem is an executable kitem with: as source context: the source and destination contexts; as source region: the enquiry as defined so far; as destination context: the kitems in the domain of discourse (if known); as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry; as destination region: the candidate and definite kitems ‘retrieved’ by the mapping.\",\n \"Enquiry or Consolation Kitem The enquiry kitem is an executable kitem with: as source space: the enquiry (the consultation process, showing each step in the question-answer session); as destination space: the status of the enquiry (answered, not answered, etc.\",\n \"), the ids of the definite kitems.\",\n \"Display Kitem The display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.\",\n \"Administration of Contexts and Other Kitems The hierarchical (tree) structure of the kitems gives a very convenient way of controlling user access to kitems: the administrator can decide which branch of the hierarchy users can have access to and the type of access for the branch and each kitem in the branch; access control can also be carried out at the level of the kitem.\",\n \"Dynamic Kitem Management The objective of dynamic kitem management is to be able to manage the integrity of the knowledge base when a kitem is edited.\",\n \"This issue is important as kitems (eg: a context kitem) can be used in many knowledge elements and a change in such a kitem can have impacts on the validity of these knowledge elements (from a knowledge viewpoint, not operational viewpoint).\",\n \"It is essential that the users are not left in the dark about the potential ramification of their changes and that they are helped in checking the validity of some affected kitems.\",\n \"As much as possible, the system should ensure the integrity of the knowledge itself.\",\n \"The process for achieving the above is to: use the enquiry process (by the system or the user) to retrieve all kitems that could be affected (the system or the user can specify the profile of the kitems affected by the change); make the changes automatically if acceptable; or ask the user to review/recertify the kitems affected (the system needs to provide guidance in this process).\",\n \"The profile mentioned above refers to the values given to kitems in the source and destination context.\",\n \"The system treats these values as an enquiry and retrieves all the kitems that are compatible with the enquiry.\",\n \"Change to properties All the properties of the kitems can be changed.\",\n \"Potentially, all the kitems that are linked to the kitem being edited are affected.\",\n \"Some properties and the way their editing can be handled is listed below.\",\n \"Change to Source Context and Region If a new context object is added: the system asks the user to specify the profile of the kitems that may be affected, the system retrieves these kitems and the user is asked to recertify them.\",\n \"If an existing context object is edited or deleted: the system retrieves all the kitems that are related to this kitem (that contain it as part of a link for example) and asks the user to recertify them and to edit them if necessary before doing so.\",\n \"Change to Destination Context and Region As for a change to the source context and region.\",\n \"Change to Mapping The system retrieves all the kitems that are related to this kitem (that contain it as part of a link or region for example) and asks the user to recertify them and to edit them if necessary.\",\n \"Change to Status (Enabled/Disabled/Archived) The system retrieves all the kitems that are related to this kitem (that contain it as part of a link or region for example) and asks the user to recertify them and to edit them if necessary.\",\n \"Change to the ‘Links to Other Kitems’ No checks required, the change is localised.\",\n \"Consistency Issues Consistency issues may arise in a knowledge base when some kitems have the following features: kitems have overlapping (at least one common kitem with at least a common value) or identical destination regions with non-identical source regions; kitems have disjointed destination regions with overlapping or identical source regions.\",\n \"The system can easily have an agent that checks all the kitems in a knowledge base and identifies these kitems that may be inconsistent.\",\n \"The system presents the kitems to their authors for editing if necessary and recertification.\",\n \"Missing Knowledge Missing knowledge refers to parts of a domain of discourse for which there is no knowledge or inadequate knowledge.\",\n \"When the domain of discourse for a set of kitems is explicit, it is a simple matter to check that the union of the source regions in the kitems cover the entire source context.\",\n \"If it is not the case, it means that the some additional knowledge is required.\",\n \"The user can be informed and the location of this missing knowledge presented.\",\n \"The location of the missing knowledge can be pointed out on each dimension (or context kitem) of the source context.\",\n \"The user selects a context item and a value (or range of values) for which there is no region, and the system responds by showing the other context items and their values for which there is no region.\",\n \"The user then selects a second context item and gives it some values (where there is no region), and so on for the other kitems if desired, until the user is confident of being able to add some missing knowledge.\",\n \"New Kitems When a new knowledge element is being saved (the user has finished its definition), the system checks that it is consistent with the other knowledge elements in the knowledge base (see section 12.2).\",\n \"The system also retrieves all kitems that overlap with the new kitem's source and/or destination regions.\",\n \"The system informs the user that these kitems may be affected (their knowledge may be superseeded by the new kitem) and presents them to the user for checking.\",\n \"The system can also ask the user to define the profile of the kitems that may be superseeded by the new kitem.\",\n \"Terminology Term Explanation Applicability region The region in the source context that determines whether the kitem is applicable.\",\n \"Application context The context for the application made of the source or space and destination contexts.\",\n \"Available kitem The status of a kitem that is available for an enquiry (the other status are definite, candidate and rejected.\",\n \"Candidate kitem A kitem that is compatible with the enquiry so far but for which the applicability conditions are not completely met.\",\n \"Definite kitem A kitem that is compatible with the enquiry so far with its applicability conditions completely met.\",\n \"Destination context The space of solutions considered by an application or a kitem.\",\n \"Domain of discourse The space of problems and solutions (or situations and outcomes) considered by an application or a kitem; it is made of the source and destination contexts.\",\n \"Enquriy process The agent or process that controls the question- answer session or consultation.\",\n \"Executable mapping A mapping that takes an input from the source context and applies an algorithm to it to produce an outcome.\",\n \"Kitem A object or element that comprises a source context and region, a destination context and region, and a mapping.\",\n \"Knowledge item A kitem that is used to express knowledge.\",\n \"Kuark An elementary kitem (the smallest building block) that can be used to build other kitems and/or complete applications.\",\n \"Mapping The transformation that is applied to a source region to produce a destination region or outcome.\",\n \"Outcome Another term for destionation region or solution.\",\n \"Parallel kitem A kitem comprising other kitems organised in parallel.\",\n \"Rejected kitem An kitem that is not compatible with an enquiry.\",\n \"Relevance context The destination context that expresses the range of possible relevance for kitems in an application.\",\n \"Relevance region A region in the relevance context.\",\n \"Relevance status The relevance of a kitem with respect to an enquiry (see GKMS patent for details).\",\n \"Serial kitem A kitem comprising other kitems organised in series.\",\n \"Solution Another term for destination region or outcome.\",\n \"Source context The space of outcomes considered by an application or a kitem.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10450975"}}},{"rowIdx":292,"cells":{"text":{"kind":"list like","value":[["Support and guiding structure for friction element in disc brakes","Method and apparatus for friction element control in spot-type automotive disc brakes provides for use in a disc brake (10) of the kind comprising a fixed caliper (32) and at least one axially slidable disc (12, 14), a friction element mounting in which the friction clement (20) is supported on lengthwise guides (52) in the region of its circumferentially spaced ends (62, 64) and is further supported by an axially-extending abutment element (66) fixed to its upper edge and received in a channel (68) in the brake caliper (32).","The abutment element an its channel serve to provide improved support for the friction element in resisting torque applied to the friction element during braking."],["1-9.","(canceled) 10.A method of mounting a friction element in a spot-type automotive disc brake, the disc brake comprising: a) at least one rotatable brake disc; b) a rotatable mounting for said brake disc to permit such rotation and which is adapted to drive said brake disc and to have exerted thereon a braking effect by said brake disc when the disc brake is actuated; c) at least one pair of friction elements adapted to frictionally engage braking surfaces on opposite sides of said brake disc to effect braking on actuation of actuation means therefor and having a line of action intersecting said friction elements; d) said brake disc being axially slidable in use with respect to said mounting therefor under the action of said friction elements and said actuation device therefor; e) a non-rotatable axially fixed mounting for said friction elements adapted to permit axial sliding movement of at least one of said friction elements into and out of frictional engagement with said disc while locating same and resisting movement of same under the action of friction forces generated by engagement of same with said at least one disc; f) axially-extending guide structure being provided in relation to said non-rotatable mounting and adapted to act on said at least one friction element at two spaced locations thereof; and said method comprising; g) providing abutment structure adapted to resist torque applied to said at least one friction element and comprising an abutment element located intermediate said spaced locations of said friction element, and the method comprising causing same to resist torque applied to said friction element during use of said brake while permitting said friction element to move towards and away from said disc.","11.A method of mounting a friction element in a disc brake comprising an axially slidable disc, the method comprising providing guide structure adapted to act at two spaced locations on said friction element between said friction element and a non-rotatable mounting therefor, and said method comprising providing abutment structure located between said spaced locations on said friction element, and said method comprising causing said abutment structure to resist torque applied to said friction element during use of said brake.","12.A brake friction element comprising abutment structure located between guide structure at spaced locations of said friction element and adapted to resist torque applied to said friction element during use in a brake system.","13.A spot-type automotive disc brake comprising: a) at least one rotatable brake disc; b) a rotatable mounting for said brake disc to permit such rotation and which is adapted to drive said brake disc and to have exerted thereon a braking effect by said brake disc when the disc brake is actuated; c) at least one pair of friction elements adapted to frictionally engage braking surfaces on opposite sides of said brake disc to effect braking on actuation of actuation device therefor and having a line of action intersecting said friction elements; d) said brake disc being axially slidable in use with respect to said mounting therefor under the action of said friction elements and said actuation device therefor; e) a non-rotatable axially fixed mounting for said friction elements adapted to permit axial sliding movement of at least one of said friction elements into and out of frictional engagement with said disc while locating same and resisting movement of same under the action of friction forces generated by engagement of same with said at lest one disc; f) axially-extending guide structure being provided in relation to said non-rotatable mounting and adapted to act on said at least one friction element at two spaced locations thereof; and g) abutment structure adapted to resist torque applied to said at least on friction element and comprising an abutment element located intermediate said spaced locations of said friction element, and adapted to resist torque applied to said friction element during use of said brake while permitting said friction element to move towards and away from said disc.","14.A disc brake comprising an axially slidable disc, and guide structure adapted to act at two spaced locations on a friction element and between said friction element and a non-rotatable mounting therefor, and said brake comprising abutment structure located intermediate said spaced locations and adapted to resist torque applied to said friction element during use of said brake.","15.A brake friction element comprising abutment structure located between circumferentially spaced ends of said friction element and adapted to resist torque applied to said friction element during use in a brake system.","16.A spot-type automotive disc brake according to claim 13, wherein said abutment structure is located generally radially in-line with the line of action of said actuation device.","17.A spot-type automotive disc brake according to claim 13, wherein said abutment structure comprises an abutment element fixed at the upper periphery of said at least one friction element and extending lengthwise in the direction of sliding brake-actuating motion of said friction element and slidingly received in a complementary channel formed in said non-rotatable mounting or caliper.","18.A brake according to claim 17, wherein said abutment element having a length-to-width ratio of at least in the range of 1 to 1 to 3 to 3."],["This invention relates to friction element control in disc brakes.","An example of the application of the invention is to the control of friction elements in spot-type automotive disc brakes, particularly such brakes of the kind in which a fixed caliper and sliding friction elements are employed.","The invention may find an application outside the strict limits of the technical field of the disc brakes disclosed in the specific embodiments described below, but principally the invention is concerned with controlling the movement of friction elements in relation to the associated structures of a disc brake, particularly the disc or discs themselves (which rotate during use), and the relatively fixed or non-rotatable structures such as that of the caliper on which the friction elements are mounted.","Generally, the friction elements with which the invention is concerned are those which are not actually fixed to a position-defined structure such as the end plate of a fixed caliper and thus, the invention is particularly applicable to friction elements of the kind employed as the middle one of three friction elements interleaved with twin discs in a fixed caliper/sliding disc assembly as disclosed below.","In such a disc brake, a need arises for excellent dynamic control of the movement/position/attitude of the friction elements during brake application in order to maximise the effectiveness of the brake and minimise the extent of uneven wear during use.","In this connection, we have already disclosed in co-pending applications arrangements whereby the friction elements are provided with anti-tilt systems to maintain as far as possible the parallel and co-planar relationship of the friction-producing faces of the friction elements and of the brake discs themselves, and this has largely been achieved by means of spring arrangements exerting substantial resilient forces on the sliding disc assemblies and likewise on the sliding friction elements assemblies to achieve the necessary degree of control under the dynamic conditions of use.","Developments thus far have enabled us to provide a satisfactory degree of control in respect of those functions which may be conveniently grouped under the heading “anti-tilt control”, but we have discovered that there is a further area in relation to friction element control which requires attention in order to provide further advances in relation to the dynamic aspects of the performance of the disc brake itself.","These further factors which require attention concern principally the question of the response of the friction elements to the effect of the torque exerted thereon by the frictional forces during brake application.","Thus, it will be readily understood that upon brake application and frictional engagement of the friction pads with the rotating disc, the applied forces acting on the friction elements produce a torque due to the rotary motion of the brake discs and such torque or turning effect on the pad and thus on its backing plate and thus on the friction element as a whole can cause destabilisation of the friction elements leading to a reduction in brake efficiency, or possibly uneven wear of the friction pads, and an object of the present invention is to provide improvements in this regard in relation to friction elements for disc brakes, or indeed improvements generally.","According to the invention there is provided a method and apparatus as defined in the accompanying claims.","In embodiments of the invention described below the axially slidable friction elements are provided with the basic guide means and supporting means whereby they are able to execute supported axial movement for frictional engagement with the brake discs.","However, in addition, the slidable friction elements are provided with abutment or torque-transmitting means in the form of an abutment or torque-transmitting element and an associated abutment or torque-transmitting channel in which the abutment or torque-transmitting element is received.","These structures (provided respectively at the upper edge of the friction element and at the underside of the brake caliper) serve to provide an important abutment or torque-transmitting function for the friction elements whereby the potentially twisting effect of the braking torque applied by the rotating brake discs is effectively neutralised by the abutting engagement of the abutment or torque-transmitting element with its corresponding abutment or torque-transmitting channel.","Moreover, although it would be possible to provide the abutment or torque-transmitting element as merely an upstanding peg or the like structure having the same thickness as the remainder of the friction element backing plate (and some benefit in terms of torque neutralisation would undoubtably be available), in the embodiments the abutment or torque-transmitting element is constructed so as to have a length dimension (extending lengthwise in the direction of the thickness of the backing plate material) whereby its torque neutralisation effects are significantly enhanced.","It also potentially assists in guiding its friction element.","A particular feature of the embodiments is the location of the abutment or torque-transmitting element in relation to the line of action of the actuation means for the brake, such as the centre line of the actuating piston.","In the embodiments, the abutment or torque-transmitting element is positioned directly above the line of action of the piston, and this is the preferred position.","In the case where twin actuating pistons are provided, then a central position between the pistons (and thus effectively at the centre of pressure produced by the pistons) is preferred.","Embodiments of the invention will now be described by way of example with reference to the accompanying drawings in which: FIG.","1 shows, in block diagram format a spot-type automotive disc brake comprising a pair of axially slidable discs and associated friction elements, an actuating mechanism therefor and a fixed caliper or bridge structure overlying same; FIG.","2 shows on a somewhat larger scale an exploded perspective view of the caliper and an associated friction element with an abutment or torque-transmitting element shown at an upper edge of the friction element together with an associated abutment or torque-transmitting channel to receive same; FIG.","3 shows a side elevation view of the assembly of FIG.","2 in its assembled condition; and FIG.","4 shows a plan view, as seen in the direction of arrow IV in FIG.","3, of the assembly of FIGS.","2 and 3.As shown in FIG.","1 a spot-type automotive disc brake 10 comprises a pair of rotatable brake discs 12, 14, a rotatable mounting 16 for the brake discs to permit rotation of the discs and which is adapted to drive the brake discs and have exerted thereon the braking effect by the discs when the disc brake 10 is actuated.","Two pairs of friction elements 18, 20 and 22 are provided and are adapted to frictionally engage braking surfaces 24, 26 provided at opposite sides of brake discs 12, 14 to effect braking on actuating actuation means for the brake.","Central friction element 20 is double-sided for frictional engagement with the mutually-inwardly facing braking surfaces 24, 26 of brake discs 12, 14 and is provided with appropriately facing friction pad material accordingly.","Friction elements 18, 20, 22 comprise (as shown in FIGS.","3 and 4) in each case a generally flat metal backing plate 28 and secured thereto and standing proud thereof a body of friction material of known construction for high durability frictional engagement with the relevant braking surface of the relevant brake disc.","In the case of central friction element 20, the friction material is provided at both faces of the backing plate 28.Brake discs 12, 14 are axially slidable in use with respect to their rotatable mounting 16 under the action of friction elements 18, 20, 22 and the actuation means (to be described below) therefor during braking.","For example the brake discs may be keyed to the rotatable mounting or hub 16 at three or more locations and resilient means may act there between.","A non-rotatable mounting 32 for friction elements 18, 20, 22 is provided comprising a caliper or bridge structure 34 which is mounted on a fixed structure of the vehicle to be braked, for example on the wheel mounting and which straddles the brake discs 12, 14 and also provides a mounting for actuation means 36,38 (indicated diagrammatically) which applies inwardly directed braking forces to the outer friction elements 18, 22, thereby causing frictional engagement with the brake discs 12, 14, and slight sliding movement of those discs with respect to their rotatable mounting 16.In FIG.","1 of course it can be seen that the clearances between the structures have been greatly exaggerated for simplicity of diagrammatic illustration.","The actuation means 36, 38 could comprise a pair of piston and cylinder assemblies.","However only one such is strictly needed since the actuation means can be one-sided with a fixed structure at one side or the other of the assembly of discs and friction elements (which fixed structure could simply be a stop extending from caliper 34), and against which fixed structure the assembly is pushed by the single actuation means.","Further details in this regard may be found in our co-pending applications WO 98/26192 (docket 2558).","Non-rotatable mounting 32 for the friction elements 18 to 22 is adapted to permit sliding movement of the friction elements into and out of frictional engagement with the brake discs while resisting movement of the friction elements under the action of frictional forces generated by engagement of the friction elements with the discs 12, 14.Turning now to the details of the structures shown in FIGS.","2, 3 and 4, we mention first that the friction element illustrated is taken to be the central friction element 20 which (though not shown in FIG.","2) is provided as shown in FIGS.","3 and 4 with pads 40, 42 of friction material facing axially at its opposite sides.","Other structures seen in the drawings include the casting 44 to which caliper 34 is secured and from which the actuating piston 46 can extend under the action of the usual hydraulic and driver-controlled brake actuation system having a line of action 48 which of course intersects the friction elements, this location being shown in FIG.","2 at 50 which identifies the line of action of the centre of piston 46.As also shown in FIGS.","2 to 4 axially-extending primary abutment or torque-transmitting means 52 is provided in relation to caliper 34 and is adapted to act on the slidable friction elements 20, 22 (end friction element 18 being secured or fixed to an end plate (not shown) extending downwards from the outboard end of caliper 34).","Primary guide means 52 comprises axially-extending guide rails 54, 56, one at each side of caliper 34 and adapted to receive corresponding hook elements 58, 60 provided at spaced locations namely the opposite circumferentially-spaced ends 62, 64 of friction element 20.Thus, guide rails 54, 56 and hook elements 58, 60 serve to ensure that friction element 20 (and likewise sliding friction element 22 in a similar way) is able to hang from the caliper for sliding axial movement lengthwise thereof towards and away from the brake discs 12, 14.In addition to the guide rails 54, 56 and hook elements 58, 60 there is provided further abutment or torque-transmitting means comprising an abutment or torque-transmitting element 66 located intermediate spaced locations namely the opposite ends 62, 64 of the friction element and adapted to enable the friction element to resist torque from frictional forces applied to it during use of the brake, while permitting the friction element to move towards and away from the brake discs.","Abutment or torque-transmitting element 66 is received in an abutment or torque-transmitting channel 68 formed in the underside 70 of caliper 34.It will be noted from FIG.","2 that abutment or torqur-transmitting element 66 is generally radially in-line with the line of action 48 of actuating piston 46 and indeed is directly above the piston centre line point 50.FIG.","2 of the drawings shows the proportions of abutment or torque-transmitting element 66 in a clear manner with respect to the remainder of the friction element structure.","Thus, the abutment or torque-transmitting element has an axial length L and a traverse width W such that L/W lies in the range of 2 to 3.In general, the ratio L/W should preferably be at least 1 and more preferably still in the range of 1 to 3.Such a ratio has the effect of maximising the abutment or torque-transmitting influence of the abutment or torque-transmitting element in relation to frictionally-developed torque forces applied to the friction element during use, thereby minimising the tendency for taper wear to develop in the pads 40, 42 of friction material.","The significance of the aspect ratio L/W in terms of maximising the torque-transmitting effect of the abutment or torque-transmitting element is discussed above.","So far as the length aspects of the dimensions of the abutment element 66 are concerned, the following applies.","As shown in FIG.","3, there is shown at 72 an indication of the possibility of friction element tilt in an axial plane which can occur when the brake is in its off condition.","Such tilting is limited by engagement of abutment element 66 with the top of groove 68.The possibility of tilt in the mode indicated at 72 is decreased with increasing length of the abutment element 66.Likewise, a similar tilt-inhibiting effect is exerted by minimising clearance between the top of the abutment element 66 and the top of groove 68.In the on condition of the brake, as indicated in FIG.","3, the brake-applying force Fp is indicated at 74 and acts along line 48 on the friction element 20 and has a turning (or taper wear) moment Fp×xp at the element 66 where xp is the radial distance (identified at 76) of off-set of line 48 from the top of channel 68.This force is resisted by a force at the end of the abutment element 66 acting on the top of groove 68 accordingly.","This function likewise is enhanced by increasing the length of abutment or torque-transmitting element 66.In use, disc brake 10 operates in substantially the usual manner, which is implicit from the above description, so far as concerns the general mode of frictional engagement of the relevant surfaces.","So far as guidance of the friction elements during use is concerned, the friction elements are supported on guide rails 54, 56 through hook elements 58, 60 and frictionally-developed torque forces applied to the pads 40, 42 of friction material which tend to produce twisting forces arising from the rotation of the brake discs are largely taken out of the friction element structure by the abutment or torque-transmitting element 66 and the engagement of same with the side walls of abutment or torque-transmitting channel 68 in which the abutment or torque-transmitting element is a close sliding fit.","Indeed, in principle, the tolerances in relation to the sliding fit of abutment or torque-transmitting element 66 within abutment or torque-transmitting channel 68 are tighter (meaning a closer fit) than exists between hook elements 58, 60 and guide rails 54, 56.As a result, wear on the pads 40, 42 or friction material is greatly improved in terms of its uniformity throughout the total working area of the friction material.","Amongst other modifications which could be made in the above embodiment while remaining within the scope of the appended claims are changes to the shape and dimensions and exact location of the abutment or torque-transmitting element 66 in relation to caliper 34."]],"string":"[\n [\n \"Support and guiding structure for friction element in disc brakes\",\n \"Method and apparatus for friction element control in spot-type automotive disc brakes provides for use in a disc brake (10) of the kind comprising a fixed caliper (32) and at least one axially slidable disc (12, 14), a friction element mounting in which the friction clement (20) is supported on lengthwise guides (52) in the region of its circumferentially spaced ends (62, 64) and is further supported by an axially-extending abutment element (66) fixed to its upper edge and received in a channel (68) in the brake caliper (32).\",\n \"The abutment element an its channel serve to provide improved support for the friction element in resisting torque applied to the friction element during braking.\"\n ],\n [\n \"1-9.\",\n \"(canceled) 10.A method of mounting a friction element in a spot-type automotive disc brake, the disc brake comprising: a) at least one rotatable brake disc; b) a rotatable mounting for said brake disc to permit such rotation and which is adapted to drive said brake disc and to have exerted thereon a braking effect by said brake disc when the disc brake is actuated; c) at least one pair of friction elements adapted to frictionally engage braking surfaces on opposite sides of said brake disc to effect braking on actuation of actuation means therefor and having a line of action intersecting said friction elements; d) said brake disc being axially slidable in use with respect to said mounting therefor under the action of said friction elements and said actuation device therefor; e) a non-rotatable axially fixed mounting for said friction elements adapted to permit axial sliding movement of at least one of said friction elements into and out of frictional engagement with said disc while locating same and resisting movement of same under the action of friction forces generated by engagement of same with said at least one disc; f) axially-extending guide structure being provided in relation to said non-rotatable mounting and adapted to act on said at least one friction element at two spaced locations thereof; and said method comprising; g) providing abutment structure adapted to resist torque applied to said at least one friction element and comprising an abutment element located intermediate said spaced locations of said friction element, and the method comprising causing same to resist torque applied to said friction element during use of said brake while permitting said friction element to move towards and away from said disc.\",\n \"11.A method of mounting a friction element in a disc brake comprising an axially slidable disc, the method comprising providing guide structure adapted to act at two spaced locations on said friction element between said friction element and a non-rotatable mounting therefor, and said method comprising providing abutment structure located between said spaced locations on said friction element, and said method comprising causing said abutment structure to resist torque applied to said friction element during use of said brake.\",\n \"12.A brake friction element comprising abutment structure located between guide structure at spaced locations of said friction element and adapted to resist torque applied to said friction element during use in a brake system.\",\n \"13.A spot-type automotive disc brake comprising: a) at least one rotatable brake disc; b) a rotatable mounting for said brake disc to permit such rotation and which is adapted to drive said brake disc and to have exerted thereon a braking effect by said brake disc when the disc brake is actuated; c) at least one pair of friction elements adapted to frictionally engage braking surfaces on opposite sides of said brake disc to effect braking on actuation of actuation device therefor and having a line of action intersecting said friction elements; d) said brake disc being axially slidable in use with respect to said mounting therefor under the action of said friction elements and said actuation device therefor; e) a non-rotatable axially fixed mounting for said friction elements adapted to permit axial sliding movement of at least one of said friction elements into and out of frictional engagement with said disc while locating same and resisting movement of same under the action of friction forces generated by engagement of same with said at lest one disc; f) axially-extending guide structure being provided in relation to said non-rotatable mounting and adapted to act on said at least one friction element at two spaced locations thereof; and g) abutment structure adapted to resist torque applied to said at least on friction element and comprising an abutment element located intermediate said spaced locations of said friction element, and adapted to resist torque applied to said friction element during use of said brake while permitting said friction element to move towards and away from said disc.\",\n \"14.A disc brake comprising an axially slidable disc, and guide structure adapted to act at two spaced locations on a friction element and between said friction element and a non-rotatable mounting therefor, and said brake comprising abutment structure located intermediate said spaced locations and adapted to resist torque applied to said friction element during use of said brake.\",\n \"15.A brake friction element comprising abutment structure located between circumferentially spaced ends of said friction element and adapted to resist torque applied to said friction element during use in a brake system.\",\n \"16.A spot-type automotive disc brake according to claim 13, wherein said abutment structure is located generally radially in-line with the line of action of said actuation device.\",\n \"17.A spot-type automotive disc brake according to claim 13, wherein said abutment structure comprises an abutment element fixed at the upper periphery of said at least one friction element and extending lengthwise in the direction of sliding brake-actuating motion of said friction element and slidingly received in a complementary channel formed in said non-rotatable mounting or caliper.\",\n \"18.A brake according to claim 17, wherein said abutment element having a length-to-width ratio of at least in the range of 1 to 1 to 3 to 3.\"\n ],\n [\n \"This invention relates to friction element control in disc brakes.\",\n \"An example of the application of the invention is to the control of friction elements in spot-type automotive disc brakes, particularly such brakes of the kind in which a fixed caliper and sliding friction elements are employed.\",\n \"The invention may find an application outside the strict limits of the technical field of the disc brakes disclosed in the specific embodiments described below, but principally the invention is concerned with controlling the movement of friction elements in relation to the associated structures of a disc brake, particularly the disc or discs themselves (which rotate during use), and the relatively fixed or non-rotatable structures such as that of the caliper on which the friction elements are mounted.\",\n \"Generally, the friction elements with which the invention is concerned are those which are not actually fixed to a position-defined structure such as the end plate of a fixed caliper and thus, the invention is particularly applicable to friction elements of the kind employed as the middle one of three friction elements interleaved with twin discs in a fixed caliper/sliding disc assembly as disclosed below.\",\n \"In such a disc brake, a need arises for excellent dynamic control of the movement/position/attitude of the friction elements during brake application in order to maximise the effectiveness of the brake and minimise the extent of uneven wear during use.\",\n \"In this connection, we have already disclosed in co-pending applications arrangements whereby the friction elements are provided with anti-tilt systems to maintain as far as possible the parallel and co-planar relationship of the friction-producing faces of the friction elements and of the brake discs themselves, and this has largely been achieved by means of spring arrangements exerting substantial resilient forces on the sliding disc assemblies and likewise on the sliding friction elements assemblies to achieve the necessary degree of control under the dynamic conditions of use.\",\n \"Developments thus far have enabled us to provide a satisfactory degree of control in respect of those functions which may be conveniently grouped under the heading “anti-tilt control”, but we have discovered that there is a further area in relation to friction element control which requires attention in order to provide further advances in relation to the dynamic aspects of the performance of the disc brake itself.\",\n \"These further factors which require attention concern principally the question of the response of the friction elements to the effect of the torque exerted thereon by the frictional forces during brake application.\",\n \"Thus, it will be readily understood that upon brake application and frictional engagement of the friction pads with the rotating disc, the applied forces acting on the friction elements produce a torque due to the rotary motion of the brake discs and such torque or turning effect on the pad and thus on its backing plate and thus on the friction element as a whole can cause destabilisation of the friction elements leading to a reduction in brake efficiency, or possibly uneven wear of the friction pads, and an object of the present invention is to provide improvements in this regard in relation to friction elements for disc brakes, or indeed improvements generally.\",\n \"According to the invention there is provided a method and apparatus as defined in the accompanying claims.\",\n \"In embodiments of the invention described below the axially slidable friction elements are provided with the basic guide means and supporting means whereby they are able to execute supported axial movement for frictional engagement with the brake discs.\",\n \"However, in addition, the slidable friction elements are provided with abutment or torque-transmitting means in the form of an abutment or torque-transmitting element and an associated abutment or torque-transmitting channel in which the abutment or torque-transmitting element is received.\",\n \"These structures (provided respectively at the upper edge of the friction element and at the underside of the brake caliper) serve to provide an important abutment or torque-transmitting function for the friction elements whereby the potentially twisting effect of the braking torque applied by the rotating brake discs is effectively neutralised by the abutting engagement of the abutment or torque-transmitting element with its corresponding abutment or torque-transmitting channel.\",\n \"Moreover, although it would be possible to provide the abutment or torque-transmitting element as merely an upstanding peg or the like structure having the same thickness as the remainder of the friction element backing plate (and some benefit in terms of torque neutralisation would undoubtably be available), in the embodiments the abutment or torque-transmitting element is constructed so as to have a length dimension (extending lengthwise in the direction of the thickness of the backing plate material) whereby its torque neutralisation effects are significantly enhanced.\",\n \"It also potentially assists in guiding its friction element.\",\n \"A particular feature of the embodiments is the location of the abutment or torque-transmitting element in relation to the line of action of the actuation means for the brake, such as the centre line of the actuating piston.\",\n \"In the embodiments, the abutment or torque-transmitting element is positioned directly above the line of action of the piston, and this is the preferred position.\",\n \"In the case where twin actuating pistons are provided, then a central position between the pistons (and thus effectively at the centre of pressure produced by the pistons) is preferred.\",\n \"Embodiments of the invention will now be described by way of example with reference to the accompanying drawings in which: FIG.\",\n \"1 shows, in block diagram format a spot-type automotive disc brake comprising a pair of axially slidable discs and associated friction elements, an actuating mechanism therefor and a fixed caliper or bridge structure overlying same; FIG.\",\n \"2 shows on a somewhat larger scale an exploded perspective view of the caliper and an associated friction element with an abutment or torque-transmitting element shown at an upper edge of the friction element together with an associated abutment or torque-transmitting channel to receive same; FIG.\",\n \"3 shows a side elevation view of the assembly of FIG.\",\n \"2 in its assembled condition; and FIG.\",\n \"4 shows a plan view, as seen in the direction of arrow IV in FIG.\",\n \"3, of the assembly of FIGS.\",\n \"2 and 3.As shown in FIG.\",\n \"1 a spot-type automotive disc brake 10 comprises a pair of rotatable brake discs 12, 14, a rotatable mounting 16 for the brake discs to permit rotation of the discs and which is adapted to drive the brake discs and have exerted thereon the braking effect by the discs when the disc brake 10 is actuated.\",\n \"Two pairs of friction elements 18, 20 and 22 are provided and are adapted to frictionally engage braking surfaces 24, 26 provided at opposite sides of brake discs 12, 14 to effect braking on actuating actuation means for the brake.\",\n \"Central friction element 20 is double-sided for frictional engagement with the mutually-inwardly facing braking surfaces 24, 26 of brake discs 12, 14 and is provided with appropriately facing friction pad material accordingly.\",\n \"Friction elements 18, 20, 22 comprise (as shown in FIGS.\",\n \"3 and 4) in each case a generally flat metal backing plate 28 and secured thereto and standing proud thereof a body of friction material of known construction for high durability frictional engagement with the relevant braking surface of the relevant brake disc.\",\n \"In the case of central friction element 20, the friction material is provided at both faces of the backing plate 28.Brake discs 12, 14 are axially slidable in use with respect to their rotatable mounting 16 under the action of friction elements 18, 20, 22 and the actuation means (to be described below) therefor during braking.\",\n \"For example the brake discs may be keyed to the rotatable mounting or hub 16 at three or more locations and resilient means may act there between.\",\n \"A non-rotatable mounting 32 for friction elements 18, 20, 22 is provided comprising a caliper or bridge structure 34 which is mounted on a fixed structure of the vehicle to be braked, for example on the wheel mounting and which straddles the brake discs 12, 14 and also provides a mounting for actuation means 36,38 (indicated diagrammatically) which applies inwardly directed braking forces to the outer friction elements 18, 22, thereby causing frictional engagement with the brake discs 12, 14, and slight sliding movement of those discs with respect to their rotatable mounting 16.In FIG.\",\n \"1 of course it can be seen that the clearances between the structures have been greatly exaggerated for simplicity of diagrammatic illustration.\",\n \"The actuation means 36, 38 could comprise a pair of piston and cylinder assemblies.\",\n \"However only one such is strictly needed since the actuation means can be one-sided with a fixed structure at one side or the other of the assembly of discs and friction elements (which fixed structure could simply be a stop extending from caliper 34), and against which fixed structure the assembly is pushed by the single actuation means.\",\n \"Further details in this regard may be found in our co-pending applications WO 98/26192 (docket 2558).\",\n \"Non-rotatable mounting 32 for the friction elements 18 to 22 is adapted to permit sliding movement of the friction elements into and out of frictional engagement with the brake discs while resisting movement of the friction elements under the action of frictional forces generated by engagement of the friction elements with the discs 12, 14.Turning now to the details of the structures shown in FIGS.\",\n \"2, 3 and 4, we mention first that the friction element illustrated is taken to be the central friction element 20 which (though not shown in FIG.\",\n \"2) is provided as shown in FIGS.\",\n \"3 and 4 with pads 40, 42 of friction material facing axially at its opposite sides.\",\n \"Other structures seen in the drawings include the casting 44 to which caliper 34 is secured and from which the actuating piston 46 can extend under the action of the usual hydraulic and driver-controlled brake actuation system having a line of action 48 which of course intersects the friction elements, this location being shown in FIG.\",\n \"2 at 50 which identifies the line of action of the centre of piston 46.As also shown in FIGS.\",\n \"2 to 4 axially-extending primary abutment or torque-transmitting means 52 is provided in relation to caliper 34 and is adapted to act on the slidable friction elements 20, 22 (end friction element 18 being secured or fixed to an end plate (not shown) extending downwards from the outboard end of caliper 34).\",\n \"Primary guide means 52 comprises axially-extending guide rails 54, 56, one at each side of caliper 34 and adapted to receive corresponding hook elements 58, 60 provided at spaced locations namely the opposite circumferentially-spaced ends 62, 64 of friction element 20.Thus, guide rails 54, 56 and hook elements 58, 60 serve to ensure that friction element 20 (and likewise sliding friction element 22 in a similar way) is able to hang from the caliper for sliding axial movement lengthwise thereof towards and away from the brake discs 12, 14.In addition to the guide rails 54, 56 and hook elements 58, 60 there is provided further abutment or torque-transmitting means comprising an abutment or torque-transmitting element 66 located intermediate spaced locations namely the opposite ends 62, 64 of the friction element and adapted to enable the friction element to resist torque from frictional forces applied to it during use of the brake, while permitting the friction element to move towards and away from the brake discs.\",\n \"Abutment or torque-transmitting element 66 is received in an abutment or torque-transmitting channel 68 formed in the underside 70 of caliper 34.It will be noted from FIG.\",\n \"2 that abutment or torqur-transmitting element 66 is generally radially in-line with the line of action 48 of actuating piston 46 and indeed is directly above the piston centre line point 50.FIG.\",\n \"2 of the drawings shows the proportions of abutment or torque-transmitting element 66 in a clear manner with respect to the remainder of the friction element structure.\",\n \"Thus, the abutment or torque-transmitting element has an axial length L and a traverse width W such that L/W lies in the range of 2 to 3.In general, the ratio L/W should preferably be at least 1 and more preferably still in the range of 1 to 3.Such a ratio has the effect of maximising the abutment or torque-transmitting influence of the abutment or torque-transmitting element in relation to frictionally-developed torque forces applied to the friction element during use, thereby minimising the tendency for taper wear to develop in the pads 40, 42 of friction material.\",\n \"The significance of the aspect ratio L/W in terms of maximising the torque-transmitting effect of the abutment or torque-transmitting element is discussed above.\",\n \"So far as the length aspects of the dimensions of the abutment element 66 are concerned, the following applies.\",\n \"As shown in FIG.\",\n \"3, there is shown at 72 an indication of the possibility of friction element tilt in an axial plane which can occur when the brake is in its off condition.\",\n \"Such tilting is limited by engagement of abutment element 66 with the top of groove 68.The possibility of tilt in the mode indicated at 72 is decreased with increasing length of the abutment element 66.Likewise, a similar tilt-inhibiting effect is exerted by minimising clearance between the top of the abutment element 66 and the top of groove 68.In the on condition of the brake, as indicated in FIG.\",\n \"3, the brake-applying force Fp is indicated at 74 and acts along line 48 on the friction element 20 and has a turning (or taper wear) moment Fp×xp at the element 66 where xp is the radial distance (identified at 76) of off-set of line 48 from the top of channel 68.This force is resisted by a force at the end of the abutment element 66 acting on the top of groove 68 accordingly.\",\n \"This function likewise is enhanced by increasing the length of abutment or torque-transmitting element 66.In use, disc brake 10 operates in substantially the usual manner, which is implicit from the above description, so far as concerns the general mode of frictional engagement of the relevant surfaces.\",\n \"So far as guidance of the friction elements during use is concerned, the friction elements are supported on guide rails 54, 56 through hook elements 58, 60 and frictionally-developed torque forces applied to the pads 40, 42 of friction material which tend to produce twisting forces arising from the rotation of the brake discs are largely taken out of the friction element structure by the abutment or torque-transmitting element 66 and the engagement of same with the side walls of abutment or torque-transmitting channel 68 in which the abutment or torque-transmitting element is a close sliding fit.\",\n \"Indeed, in principle, the tolerances in relation to the sliding fit of abutment or torque-transmitting element 66 within abutment or torque-transmitting channel 68 are tighter (meaning a closer fit) than exists between hook elements 58, 60 and guide rails 54, 56.As a result, wear on the pads 40, 42 or friction material is greatly improved in terms of its uniformity throughout the total working area of the friction material.\",\n \"Amongst other modifications which could be made in the above embodiment while remaining within the scope of the appended claims are changes to the shape and dimensions and exact location of the abutment or torque-transmitting element 66 in relation to caliper 34.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10451031"}}},{"rowIdx":293,"cells":{"text":{"kind":"list like","value":[["Lugged cap forming system","Transfer apparatus and method are provided from first (15A, 16A) to second (15B, 16B) stations of tooling in a cap (11) making press.","This cap is biased against the first upper tools by a first airstream (50) introduced under the cap and moves upward with the first station punch (45).","As the punch approaches its top location, a transfer airstream (52) begins while the first airstream is still on, and moves the cap out through a transfer chute (18) to the second station.","The cap departs the chute and passes detents (67) on a pair of closed retention fingers (60) which define an extension of the transfer path from the chute into the open second station tools.","A vacuum (85) applied to a port in the second station punch then holds the cup against the rising upper tools.","When the punch clears the closing fingers and approaches its top location, an ejection airstream (87) commences to propel the finished cup via a discharge chute (19)."],["1.A system for forming container caps, comprising: a first station including first station tools comprising a blank punch tool and a cooperating compound die tool adapted for mounting in a reciprocating press for cyclic movement between an open position in which said tools are separated and a closed position in which said tools are closely spaced to form a cap from a metal blank separated from a supply of thin sheet metal during the closing motion of said tools and then to draw the blank into a cup shaped part with a top panel and a side wall extending from said top panel; a second station aligned with and spaced from said first station, said second station including second station tools for receiving parts from said first station and performing further operations thereon; a chute extending between said first and second stations at the open position of said first station tools to guide parts in transfer from the first station into the second station; means projecting a first airstream into the part to hold the top panel against said blank punch tool during separating motion of said first station tools upon forming of a part; means projecting a second airstream across said blank punch tool at its open position and into said chute so as to propel cap parts into said second station tools; and control means coordinated with the operation of the press for switching on said first and second airstream projecting means to (a) initiate the first airstream at the beginning of separation of said first station tools and maintaining the first airstream at least until said first station tools are fully separated, and (b) initiate the second airstream before said first station tools are fully separated and maintaining the second airstream sufficiently to propel the part through said chute.","2.A system as defined in claim 1, wherein said chute terminates adjacent the interface of the second station tools; a set of spaced apart guide fingers for forming a continuation of said chute, said fingers being movably supported on opposite sides of the second station tools for movement perpendicular to said second station tools such as to provide for closing of the second station tools with said fingers moved apart and for closing motion of said fingers into an extension of said chute when the second station tools are open.","3.A system as defined in claim 2, wherein said fingers have complementary tracks formed thereon to receive and guide a part exiting said chute, said tracks having inwardly curved end sections which align with the center of the second station tools to provide a termination of the path of a cap part entering the second station tools, and cam means operated by the press in coordination with opening and closing of the second section tools to move the fingers apart when the tools are closing and to close the fingers into a chute extension when the tools are opening.","4.A system as defined in claim 3, wherein said finger tracks include detents spaced from said curved end sections to define a centered position of the incoming parts.","5.A system as defined in claim 1 wherein said second station tools are constructed to form lugs on a rolled rim of the cap parts.","6.A system for forming container caps, comprising: a first station including first station tools comprising a blank punch tool and a cooperating compound die tool adapted for mounting in a reciprocating press for cyclic movement between an open position in which said tools are separated and a closed interface position in which said tools are closely spaced (a) to form a cap from a metal blank separated from a supply of thin sheet metal during the closing motion of said tools and (b) then to draw the blank into a cup shaped part with a top panel and a side wall extending from said top panel, said tools also form an outward rolled rim on the side wall; a second station aligned with and spaced from said first station, said second station including second station tools for receiving parts from said first station and performing a lug shaping operation on the rolled rim; a chute extending between said first and second stations at the open position of said first station tools to guide parts in transfer from the first station into the second station, said chute terminating adjacent the interface of the second station tools; a set of spaced apart guide fingers for forming a continuation of said chute, said fingers being pivotally supported on opposite sides of the second station tools for opening and closing motion perpendicular to said second station tools such as to provide for closing of the second station tools with said fingers moved apart and for closing motion of said fingers into an extension of said chute when the second station tools are open; said fingers have complementary tracks formed thereon to receive and guide a part exiting said cute, said tracks having inwardly curved end sections which align with the center of the second station tools, said finger tracks include detents spaced from said curved end sections to define a centered position of the incoming parts and to provide a termination of the path of a cap part entering the second station tools; cam means operated by the press in coordination with opening and closing of the second section tools to move the fingers apart when the tools are closing and to close the fingers into a chute extension when the tools are opening; means projecting a first airstream into the part to hold the top panel against said blank punch tool during separating motion of said first station tools upon forming of a part; means projecting a second airstream across said blank punch tool at its open position and into said chute so as to propel cap parts into said second station; and control means coordinated with the operation of the press for switching on said first and second airstream projecting means to (a) initiate the first airstream at the beginning of separation of said first station tools and maintaining the first airstream at least until said first station tools are fully separated, and (b) initiate the second airstream before said first station tools are fully separated and maintaining the second airstream sufficiently to propel the part through said chute, said control means including an input pulse generator driven in synchronism with the cyclic operation of the press to generate a train of control pulses related to the angular position of the press crankshaft as it rotates to open and close the tools, whereby the initiation and termination of said airstream projecting means is synchronized with cyclic operation of the tools.","7.A method for discharging and transferring a cup shaped object from a work station, having upper and lower tooling which is opened and closed to form the object, along a transfer path, the open position of the upper tooling defining a ready position, comprising the steps of: (a) locating the object within the work station in the ready position by directing a first stream of pressurized gas against the object to cause the object to remain with the upper tooling upon opening of the tooling whereby the object is supported by the first stream against the upper tooling; (b) prior to locating the object in said ready position, initiating a second flow of pressurized gas through orifice means located adjacent to and directed across said ready position, thereby causing the transfer of said object from the work station when the object is released at the ready position; and (c) discontinuing the second flow of pressurized gas through said orifice means after the object has exited the open tooling.","8.The method of claim 7 including the additional steps of: (d) receiving the object in a chute extending to a second work station which defines the transfer path to adjacent second upper and lower tools of the second work station, (e) directing the object into an extension of the transfer path defined by pivoting fingers located at opposite sides of the second tools, and (f) opening the fingers when the second tools are closing, and closing the fingers when the second tools are opening such as to guide the object into a centered position between the second station tools."],["<SOH> BACKGROUND OF THE INVENTION <EOH>U.S.","Pat.","Nos.","6,082,944 and 6,015,062, assigned to the assignee of this application, disclose closure constructions for reclosable containers (e.g a can body) wherein a domed container end with a neck portion having a pour opening is provided with a reclosable lugged type of cap.","That invention provides a unique and versatile container for fluids, particularly for beverages, wherein various standard can bodies are provided with a two part end including a neck with a pour opening, a lug formation on the neck below the pour opening, a reclosable cap having a lug formation which can interlock with the lug formation on the neck and including a seal surrounding the pour opening, and thus capable of maintaining product under pressure.","The two part end is affixed to a can body by conventional double rolled seam attachment between the bottom of the neck and the rim of the can body.","However, it is possible to affix the domed end to a can body without a cap, fill the can though the pour opening, and then apply the cap.","With the possibility of expanded markets for these lugged caps, which are also useful on various jars and bottles, there is a need for a system (method and tooling) for producing lugged cap members in a single machine, e.g.","a reciprocating press fitted with appropriate tooling, which is capable of precise high speed (e.g.","in the range of 135 to 150 strokes/minute) to achieve acceptable commercial production of the cap.","To develop such production speeds it is desirable to divide the progressive tooling operations into more than one step, and this in turn requires a rapid and precise transfer system to move the partially completed caps from a first station to a second station, and precisely register the caps in the second station.","Prior art transfer systems are known for moving and registering can end shells, such as in U.S. Pat.","Nos.","4,770,022 and 4,895,012, however, the end shells are relatively flat disc-like objects with a quite small height to diameter ratio, whereas the caps made by the present invention have a substantially greater height with respect to their diameter.","Thus, the physical dimensions of the caps involved in this invention are quite different from can shells or easy self-opening can ends.","This in turn introduces needs not required or anticipated in shell transfer systems, for example with regard to tipping of the caps during high speed transfer operations.","Also, because of the relatively high press cycling, and need for precision in cap positioning and deceleration immediately after each transfer to another press station, there is a requirement for precise transfer of each cap from a first press station through high acceleration, very rapid transfer to the next press station, and high deceleration to a precisely defined stationary location at that next station."],["<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a transfer apparatus and method for a first to a second station of progressive tooling in a cap making press.","The caps are of a type having substantial height with respect to their diameter.","Thus a first station punch and die form a cap of generally inverted cup shape, with an outward curled rim, and a second station punch and die form lugs into that rim, requiring just two strokes of the press to sever a disc from a supply sheet, form it, and discharge a completed cap.","Thus, in the first station, a cap is formed except for lugs which are added to the cap in the second station.","The formed cap in the first station, which is in the nature of an inverted cup, has an outward curl formed on its lower edge during the initial up stoke of the press.","This cup is then biased against the upper forming punch, by a first airstream introduced into the cavity within the underside of the cap, and moves upward in contact with the first station punch.","As the punch approaches its top dead center location (tooling fully open), a second airstream begins, before the first station punch is fully raised and while the first airstream is still on.","This second airstream moves the cap off the raised first station punch, and into and through a transfer chute directed toward the second station.","At the second station, the cap departs the chute and passes detents on a pair of closed retention fingers which, with the tooling open, define an extension of the transfer path from the chute into the open second station tools.","As the second station tools begin to close and the partially finished cap is located, the fingers are opened and the tooling operates to form lugs into the cap rim.","As the second station tooling opens, a vacuum applied to a port in the second station punch begins to hold the finished cup against that punch tool, and when that punch clears the closing fingers and approaches its top dead center location, an ejection airstream commences to propel the finished cup from the tooling via a discharge chute."],["BACKGROUND OF THE INVENTION U.S. Pat.","Nos.","6,082,944 and 6,015,062, assigned to the assignee of this application, disclose closure constructions for reclosable containers (e.g a can body) wherein a domed container end with a neck portion having a pour opening is provided with a reclosable lugged type of cap.","That invention provides a unique and versatile container for fluids, particularly for beverages, wherein various standard can bodies are provided with a two part end including a neck with a pour opening, a lug formation on the neck below the pour opening, a reclosable cap having a lug formation which can interlock with the lug formation on the neck and including a seal surrounding the pour opening, and thus capable of maintaining product under pressure.","The two part end is affixed to a can body by conventional double rolled seam attachment between the bottom of the neck and the rim of the can body.","However, it is possible to affix the domed end to a can body without a cap, fill the can though the pour opening, and then apply the cap.","With the possibility of expanded markets for these lugged caps, which are also useful on various jars and bottles, there is a need for a system (method and tooling) for producing lugged cap members in a single machine, e.g.","a reciprocating press fitted with appropriate tooling, which is capable of precise high speed (e.g.","in the range of 135 to 150 strokes/minute) to achieve acceptable commercial production of the cap.","To develop such production speeds it is desirable to divide the progressive tooling operations into more than one step, and this in turn requires a rapid and precise transfer system to move the partially completed caps from a first station to a second station, and precisely register the caps in the second station.","Prior art transfer systems are known for moving and registering can end shells, such as in U.S. Pat.","Nos.","4,770,022 and 4,895,012, however, the end shells are relatively flat disc-like objects with a quite small height to diameter ratio, whereas the caps made by the present invention have a substantially greater height with respect to their diameter.","Thus, the physical dimensions of the caps involved in this invention are quite different from can shells or easy self-opening can ends.","This in turn introduces needs not required or anticipated in shell transfer systems, for example with regard to tipping of the caps during high speed transfer operations.","Also, because of the relatively high press cycling, and need for precision in cap positioning and deceleration immediately after each transfer to another press station, there is a requirement for precise transfer of each cap from a first press station through high acceleration, very rapid transfer to the next press station, and high deceleration to a precisely defined stationary location at that next station.","SUMMARY OF THE INVENTION The present invention provides a transfer apparatus and method for a first to a second station of progressive tooling in a cap making press.","The caps are of a type having substantial height with respect to their diameter.","Thus a first station punch and die form a cap of generally inverted cup shape, with an outward curled rim, and a second station punch and die form lugs into that rim, requiring just two strokes of the press to sever a disc from a supply sheet, form it, and discharge a completed cap.","Thus, in the first station, a cap is formed except for lugs which are added to the cap in the second station.","The formed cap in the first station, which is in the nature of an inverted cup, has an outward curl formed on its lower edge during the initial up stoke of the press.","This cup is then biased against the upper forming punch, by a first airstream introduced into the cavity within the underside of the cap, and moves upward in contact with the first station punch.","As the punch approaches its top dead center location (tooling fully open), a second airstream begins, before the first station punch is fully raised and while the first airstream is still on.","This second airstream moves the cap off the raised first station punch, and into and through a transfer chute directed toward the second station.","At the second station, the cap departs the chute and passes detents on a pair of closed retention fingers which, with the tooling open, define an extension of the transfer path from the chute into the open second station tools.","As the second station tools begin to close and the partially finished cap is located, the fingers are opened and the tooling operates to form lugs into the cap rim.","As the second station tooling opens, a vacuum applied to a port in the second station punch begins to hold the finished cup against that punch tool, and when that punch clears the closing fingers and approaches its top dead center location, an ejection airstream commences to propel the finished cup from the tooling via a discharge chute.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 is a bottom view of a container cap as provided by the invention.","FIG.","2 is a transverse cross-sectional view of a typical completed cap, as shown in FIG.","1.FIG.","3 is an over all perspective view of the tooling for a four-out cap making system.","FIG.","4 is an enlarged perspective view, with the die shoe removed, showing details of the tooling, including four first station tools in the center and four related second station tools outwardly in opposite directions of the first stations.","FIG.","5 is a further enlarged perspective view showing details of one pair of first and second stations of the tooling.","FIG.","6 is a cross-section view of a first station punch or upper tool.","FIG.","7 is a cross-section view of a first station die or lower tool.","FIG.","8 is a cross-section view illustrating the first station punch and die in closed position, and showing a cap formed except for an outward curl at the lower rim of the cap.","FIG.","9 is a cross-section view showing first station and corresponding second station tools, a transfer chute between them, the fingers which provide an extension of the chute into the second stage tool, and cams which control movement of the fingers.","FIGS.","10 and 11 are detail views of the top of the second station die, showing the open and closed positions of the fingers.","FIG.","12 is an enlarged cross-section view of the second station tools, and FIG.","12A is a further enlargement of the circled area in the center of FIG.","12.FIGS.","13A and 13B1 & 13B2 (three sheets) together are a schematic diagram of the pneumatic supply & control system of the press and tooling.","FIG.","14 is a flow diagram of the electronic control system for the tooling package.","DESCRIPTION OF PREFERRED EMBODIMENT The present invention provides a transfer apparatus and method for two station progressive tooling in a cap making press and system.","The caps are of a type having substantial height with respect to their diameter.","A typical such cap is shown in FIGS.","1 and 2, which is also shown in FIGS.","2, 4A and 4B of U.S. Pat.","No.","6,015,062.The preferably integral cap 11, in the general form of an inverted cup, includes a top panel 12, a peripheral sidewall 13, and a curled rim 14.In the present drawings, FIGS.","3-12A depict multi-lane progressive tooling comprising four lanes for simultaneously forming four caps 11, each lane comprising pairs of first and second tooling stations 15A, 15B, 16A, 16B, 17A,17B, and 18A, 18B, with the first stations 15A-18A arranged centrally of the tooling (FIGS.","3, 4 & 5) and corresponding second stations 16A-16B arranged outward of the first stations toward opposite sides of the upper and lower die plates 20A, 20B which support the upper (punch) and lower (die) tooling, and mount between the bed and slide of a reciprocating press (not shown).","Except for their orientation in the overall tooling package, the respective first station and second station tools are alike, and the following detailed description applies to all.","Taking stations 15A and 15B as examples, each pair of corresponding first and second stations has an associated transfer chute 19 (FIGS.","4, 5 & 9) between them, and each second station has a discharge chute 19A, the four of which are directed out opposite sides of the tooling (FIGS.","3 & 4).","Sheets of metal, with an appropriate pattern of lithographed materials for each cap, are fed centrally into the first stations by sheet feeding mechanism of known construction (not shown) which moves the sheets one at a time in step wise fashion, synchronized to the press strokes, along the feed path indicated by arrow IN in FIGS.","3 & 4.First Station The first station tools comprise an upper or blank punch tool 45 and a compound lower die.","During the initial operation of the first station tools, with the lithographed patterns aligned with respect to the first station tools, a blank is cut from the material (typically aluminum or thin cold rolled steel) on the down stroke of the press by blank punch 45.On the continuation of the down stroke, the blank punch and lower die tool cooperate such that the blank is drawn into a cup shaped cap part 11P (FIG.","8).","At the bottom of the stroke the panel shape 12 is formed into the top of cap part lip by the punch 45 and cooperating die 46 (FIGS.","6, 7 & 8).","On the up stroke, the lower curl ring 48, which is under spring pressure, raises with the blank punch.","The bottom edge of the cap part 11P is curled outward into the cavity 49 formed by curl ring 48 and blank punch 45, completing a formed cap 11 with an outward curled rim 15.The formed cap in the first station, which is in the nature of an inverted cup, is biased against the upper forming die by a first airstream introduced through passage 50 (see FIGS.","8 & 9) into the cap as the first station tooling opens, and causes the cap to follow upward against the bottom of the punch 45.During the upward travel of the cap, a second airstream is initiated through a nozzle 52 directed across the upper fist station tooling toward chute 18, and is at its full power when punch 45 (with a cap 11 held thereto by the upward directed first airstream) traverses the space between nozzle 52 and the entry 18E to chute 18 (see FIG.","9).","By the time the first station tooling reaches full open at the top-dead-center of a press stroke, the cap has actually been transferred into the second station 15B; see the Press Rotation timing chart below (page 8).","Thus, the first and second airstreams, appropriately switched on and off, together with the associated chute, constitute a first part of an essentially passive press transfer system.","Second Station At the second station, a separated pair of fingers 60 reach around the sides of the second station tooling (FIGS.","9, 10 & 11), defining partial sides of a receiving space 62 when the second station tooling is open, and an extension of the transfer chute when closed.","The fingers are supported by pivots 63 inward of their rear ends 61, and are biased into their closed position (FIG.","10, forward ends parallel) by spring mechanism 64.Each finger has a narrow ledge-like track 65 extending part way along its forward upper edge (FIGS.","10-12), facing each other and ending in a curved section 66.Extending horizontally inward over tracks 65 are spring-loaded ball detents 67 which, together with the curved track sections 66, define the termination of the transfer path for the incoming caps.","A cap propelled from the first station traverses the adjacent transfer chute 19 and enters receiving space 62, passing across ball detents 67 and resting against the curved track sections 66 (see FIG.","11).","The fingers and their operating mechanism form the remainder of the unique transfer system.","As the tooling proceeds to close during the beginning of a next stroke, fingers 60 are swung outward by descending cams 48 pressing against followers 49 on the rear ends 61 of the fingers to move the followers 49 inward and the forward section of the fingers outward (FIGS.","10 & 12) before the second station tools close.","The elongated cams 48 are mounted on the upper (die) tooling base (FIG.","12), and this action centers a cap (FIG.","11) and then opens the fingers associated with the second station tooling, before that tooling closes to form lugs on rim 15 of a cap 11.This closing action of fingers 60 is momentary, and they are then opened substantially before the second station tooling closes; see Press Rotation timing chart below.","In the second station 15B in the press, the tooling includes an upper punch 70 and a lower die 72.The punch tool 70 includes an annular knock out ring 71 with an inner shape conforming to a cap exterior, and a headed knock out pin 73 which is spring loaded toward a position flush with the lower edge of ring 71 (FIGS.","12 & 12A) when the tooling is opened.","In the lower die 72 there is a vertically extending tapered cam 74 mounted onto a base plate 75 and surrounded by a plurality of die pins 76 also mounted onto plate 75.The cam 74 and pins 76 project through a vertically movable, upwardly biased ring 78 which contains a plurality of lug forming dies 80, equal in number to pins 76 and movable laterally outward through forces from cam 74 as it is made to enter ring 78.The top of ring 78 has an upper surface 82 contoured to fit within a cap 11.Thus, when the second station tooling closes a cap is positioned with the curled rim 15 between the upper ends of pins 76 and the radially outward moving forming dies 80, to for the predetermined number of lugs in the rim (FIG.","1).","Then, the completed cap is held to the knockout pin by means of a vacuum created in passage 85 though the head of that pin 73, such that the cap is carried upward past an air nozzle 87 directed across the path of the rising upper tool toward an associated discharge chute 19A.","The vacuum is created by air flow through a small venturi (not shown) so the vacuum is relieved immediately upon cessation of air flow through that venturi.","A discharge air stream is started from nozzle 87 and moves the cap from the upper knock out ring and pin, into and through the associated discharge chute 19.Press Rotation Relation to Tooling Function 1st Station 0° Top of the Stroke; Top Dead Center 140° Material is Blanked 180° Form & draw complete; Bottom Dead Center; Cap Overall Height ˜0.810 inch 190° Cap Curl complete; Overall Height is ˜0.625 inch 190° 1st Air turned on; Blows cap against punch tool 220° Blank punch exits Die tool, cap against its face 230° 2nd Transfer Air on; Blows cap into transfer chute 330° 1st Cap arrives into catch fingers at 2nd Station.","2nd Station 0° Top of a stroke; Top Dead Center 60° Upper vacuum is turned on 136° Catch fingers start open, upper vacuum holds 1st cap against upper knockout and tools close 180° Lugs formed in 1st cap; Bottom Dead Center 181° 1st cap (completed) and tools move up together 224° Catch fingers close completely after tooling passes 270° Upper vacuum to knockout turned off 280° Discharge air valve to nozzle is turned on 290° Knock out air to nozzle 87 is turned on 330° 2nd Cap arrives into catch fingers 336° 1st Cap actually moves to discharge position 335° Vacuum actually turns off on Cap #1 350° 1st Cap actually leaves press via chute 19 FIGS.","13A and 13B comprise a pneumatic diagram for the pneumatic portion of the press control.","A “shop air” source of compressed air is supplied to the various electrically controlled valves (which are all labeled) to direct air under pressure to the above mentioned parts of the tooling, under management control of the electronics control (FIG.","14) of the system.","The details of such controls are apparent to persons skilled in this art from these diagrams FIG.","14 is a block diagram of electrical/electronics controls for the system, which selectively turn on and off the compressed air to the nozzles providing lift or “blow up” air to hold the cap part against the rising upper tools in the first station, and providing a transfer air stream to quickly move a cap part into a transfer chute 19.The third (upper) control provides timed discharge airstreams through nozzles 87 to move the finished caps into the discharge chutes 19A.","A pulse generator PG is driven by the press crankshaft (not shown) in typical fashion to generate a train of pulses related to the angular position of the crankshaft as it rotates, and these pulses are directed to the system PLC (Programmable Logic Controller).","Since the diagram is divided into three functions which occur during a press cycle, the controller PLC is shown in each of the three diagram parts, but in fact one PLC is employed in the control system.","While the method herein described, and the form of apparatus for carrying this method into effect, constitute preferred embodiments of this invention, it is to be understood that the invention is not limited to this precise method and form of apparatus, and that changes may be made in either without departing from the scope of the invention, which is defined in the appended claims."]],"string":"[\n [\n \"Lugged cap forming system\",\n \"Transfer apparatus and method are provided from first (15A, 16A) to second (15B, 16B) stations of tooling in a cap (11) making press.\",\n \"This cap is biased against the first upper tools by a first airstream (50) introduced under the cap and moves upward with the first station punch (45).\",\n \"As the punch approaches its top location, a transfer airstream (52) begins while the first airstream is still on, and moves the cap out through a transfer chute (18) to the second station.\",\n \"The cap departs the chute and passes detents (67) on a pair of closed retention fingers (60) which define an extension of the transfer path from the chute into the open second station tools.\",\n \"A vacuum (85) applied to a port in the second station punch then holds the cup against the rising upper tools.\",\n \"When the punch clears the closing fingers and approaches its top location, an ejection airstream (87) commences to propel the finished cup via a discharge chute (19).\"\n ],\n [\n \"1.A system for forming container caps, comprising: a first station including first station tools comprising a blank punch tool and a cooperating compound die tool adapted for mounting in a reciprocating press for cyclic movement between an open position in which said tools are separated and a closed position in which said tools are closely spaced to form a cap from a metal blank separated from a supply of thin sheet metal during the closing motion of said tools and then to draw the blank into a cup shaped part with a top panel and a side wall extending from said top panel; a second station aligned with and spaced from said first station, said second station including second station tools for receiving parts from said first station and performing further operations thereon; a chute extending between said first and second stations at the open position of said first station tools to guide parts in transfer from the first station into the second station; means projecting a first airstream into the part to hold the top panel against said blank punch tool during separating motion of said first station tools upon forming of a part; means projecting a second airstream across said blank punch tool at its open position and into said chute so as to propel cap parts into said second station tools; and control means coordinated with the operation of the press for switching on said first and second airstream projecting means to (a) initiate the first airstream at the beginning of separation of said first station tools and maintaining the first airstream at least until said first station tools are fully separated, and (b) initiate the second airstream before said first station tools are fully separated and maintaining the second airstream sufficiently to propel the part through said chute.\",\n \"2.A system as defined in claim 1, wherein said chute terminates adjacent the interface of the second station tools; a set of spaced apart guide fingers for forming a continuation of said chute, said fingers being movably supported on opposite sides of the second station tools for movement perpendicular to said second station tools such as to provide for closing of the second station tools with said fingers moved apart and for closing motion of said fingers into an extension of said chute when the second station tools are open.\",\n \"3.A system as defined in claim 2, wherein said fingers have complementary tracks formed thereon to receive and guide a part exiting said chute, said tracks having inwardly curved end sections which align with the center of the second station tools to provide a termination of the path of a cap part entering the second station tools, and cam means operated by the press in coordination with opening and closing of the second section tools to move the fingers apart when the tools are closing and to close the fingers into a chute extension when the tools are opening.\",\n \"4.A system as defined in claim 3, wherein said finger tracks include detents spaced from said curved end sections to define a centered position of the incoming parts.\",\n \"5.A system as defined in claim 1 wherein said second station tools are constructed to form lugs on a rolled rim of the cap parts.\",\n \"6.A system for forming container caps, comprising: a first station including first station tools comprising a blank punch tool and a cooperating compound die tool adapted for mounting in a reciprocating press for cyclic movement between an open position in which said tools are separated and a closed interface position in which said tools are closely spaced (a) to form a cap from a metal blank separated from a supply of thin sheet metal during the closing motion of said tools and (b) then to draw the blank into a cup shaped part with a top panel and a side wall extending from said top panel, said tools also form an outward rolled rim on the side wall; a second station aligned with and spaced from said first station, said second station including second station tools for receiving parts from said first station and performing a lug shaping operation on the rolled rim; a chute extending between said first and second stations at the open position of said first station tools to guide parts in transfer from the first station into the second station, said chute terminating adjacent the interface of the second station tools; a set of spaced apart guide fingers for forming a continuation of said chute, said fingers being pivotally supported on opposite sides of the second station tools for opening and closing motion perpendicular to said second station tools such as to provide for closing of the second station tools with said fingers moved apart and for closing motion of said fingers into an extension of said chute when the second station tools are open; said fingers have complementary tracks formed thereon to receive and guide a part exiting said cute, said tracks having inwardly curved end sections which align with the center of the second station tools, said finger tracks include detents spaced from said curved end sections to define a centered position of the incoming parts and to provide a termination of the path of a cap part entering the second station tools; cam means operated by the press in coordination with opening and closing of the second section tools to move the fingers apart when the tools are closing and to close the fingers into a chute extension when the tools are opening; means projecting a first airstream into the part to hold the top panel against said blank punch tool during separating motion of said first station tools upon forming of a part; means projecting a second airstream across said blank punch tool at its open position and into said chute so as to propel cap parts into said second station; and control means coordinated with the operation of the press for switching on said first and second airstream projecting means to (a) initiate the first airstream at the beginning of separation of said first station tools and maintaining the first airstream at least until said first station tools are fully separated, and (b) initiate the second airstream before said first station tools are fully separated and maintaining the second airstream sufficiently to propel the part through said chute, said control means including an input pulse generator driven in synchronism with the cyclic operation of the press to generate a train of control pulses related to the angular position of the press crankshaft as it rotates to open and close the tools, whereby the initiation and termination of said airstream projecting means is synchronized with cyclic operation of the tools.\",\n \"7.A method for discharging and transferring a cup shaped object from a work station, having upper and lower tooling which is opened and closed to form the object, along a transfer path, the open position of the upper tooling defining a ready position, comprising the steps of: (a) locating the object within the work station in the ready position by directing a first stream of pressurized gas against the object to cause the object to remain with the upper tooling upon opening of the tooling whereby the object is supported by the first stream against the upper tooling; (b) prior to locating the object in said ready position, initiating a second flow of pressurized gas through orifice means located adjacent to and directed across said ready position, thereby causing the transfer of said object from the work station when the object is released at the ready position; and (c) discontinuing the second flow of pressurized gas through said orifice means after the object has exited the open tooling.\",\n \"8.The method of claim 7 including the additional steps of: (d) receiving the object in a chute extending to a second work station which defines the transfer path to adjacent second upper and lower tools of the second work station, (e) directing the object into an extension of the transfer path defined by pivoting fingers located at opposite sides of the second tools, and (f) opening the fingers when the second tools are closing, and closing the fingers when the second tools are opening such as to guide the object into a centered position between the second station tools.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>U.S.\",\n \"Pat.\",\n \"Nos.\",\n \"6,082,944 and 6,015,062, assigned to the assignee of this application, disclose closure constructions for reclosable containers (e.g a can body) wherein a domed container end with a neck portion having a pour opening is provided with a reclosable lugged type of cap.\",\n \"That invention provides a unique and versatile container for fluids, particularly for beverages, wherein various standard can bodies are provided with a two part end including a neck with a pour opening, a lug formation on the neck below the pour opening, a reclosable cap having a lug formation which can interlock with the lug formation on the neck and including a seal surrounding the pour opening, and thus capable of maintaining product under pressure.\",\n \"The two part end is affixed to a can body by conventional double rolled seam attachment between the bottom of the neck and the rim of the can body.\",\n \"However, it is possible to affix the domed end to a can body without a cap, fill the can though the pour opening, and then apply the cap.\",\n \"With the possibility of expanded markets for these lugged caps, which are also useful on various jars and bottles, there is a need for a system (method and tooling) for producing lugged cap members in a single machine, e.g.\",\n \"a reciprocating press fitted with appropriate tooling, which is capable of precise high speed (e.g.\",\n \"in the range of 135 to 150 strokes/minute) to achieve acceptable commercial production of the cap.\",\n \"To develop such production speeds it is desirable to divide the progressive tooling operations into more than one step, and this in turn requires a rapid and precise transfer system to move the partially completed caps from a first station to a second station, and precisely register the caps in the second station.\",\n \"Prior art transfer systems are known for moving and registering can end shells, such as in U.S. Pat.\",\n \"Nos.\",\n \"4,770,022 and 4,895,012, however, the end shells are relatively flat disc-like objects with a quite small height to diameter ratio, whereas the caps made by the present invention have a substantially greater height with respect to their diameter.\",\n \"Thus, the physical dimensions of the caps involved in this invention are quite different from can shells or easy self-opening can ends.\",\n \"This in turn introduces needs not required or anticipated in shell transfer systems, for example with regard to tipping of the caps during high speed transfer operations.\",\n \"Also, because of the relatively high press cycling, and need for precision in cap positioning and deceleration immediately after each transfer to another press station, there is a requirement for precise transfer of each cap from a first press station through high acceleration, very rapid transfer to the next press station, and high deceleration to a precisely defined stationary location at that next station.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a transfer apparatus and method for a first to a second station of progressive tooling in a cap making press.\",\n \"The caps are of a type having substantial height with respect to their diameter.\",\n \"Thus a first station punch and die form a cap of generally inverted cup shape, with an outward curled rim, and a second station punch and die form lugs into that rim, requiring just two strokes of the press to sever a disc from a supply sheet, form it, and discharge a completed cap.\",\n \"Thus, in the first station, a cap is formed except for lugs which are added to the cap in the second station.\",\n \"The formed cap in the first station, which is in the nature of an inverted cup, has an outward curl formed on its lower edge during the initial up stoke of the press.\",\n \"This cup is then biased against the upper forming punch, by a first airstream introduced into the cavity within the underside of the cap, and moves upward in contact with the first station punch.\",\n \"As the punch approaches its top dead center location (tooling fully open), a second airstream begins, before the first station punch is fully raised and while the first airstream is still on.\",\n \"This second airstream moves the cap off the raised first station punch, and into and through a transfer chute directed toward the second station.\",\n \"At the second station, the cap departs the chute and passes detents on a pair of closed retention fingers which, with the tooling open, define an extension of the transfer path from the chute into the open second station tools.\",\n \"As the second station tools begin to close and the partially finished cap is located, the fingers are opened and the tooling operates to form lugs into the cap rim.\",\n \"As the second station tooling opens, a vacuum applied to a port in the second station punch begins to hold the finished cup against that punch tool, and when that punch clears the closing fingers and approaches its top dead center location, an ejection airstream commences to propel the finished cup from the tooling via a discharge chute.\"\n ],\n [\n \"BACKGROUND OF THE INVENTION U.S. Pat.\",\n \"Nos.\",\n \"6,082,944 and 6,015,062, assigned to the assignee of this application, disclose closure constructions for reclosable containers (e.g a can body) wherein a domed container end with a neck portion having a pour opening is provided with a reclosable lugged type of cap.\",\n \"That invention provides a unique and versatile container for fluids, particularly for beverages, wherein various standard can bodies are provided with a two part end including a neck with a pour opening, a lug formation on the neck below the pour opening, a reclosable cap having a lug formation which can interlock with the lug formation on the neck and including a seal surrounding the pour opening, and thus capable of maintaining product under pressure.\",\n \"The two part end is affixed to a can body by conventional double rolled seam attachment between the bottom of the neck and the rim of the can body.\",\n \"However, it is possible to affix the domed end to a can body without a cap, fill the can though the pour opening, and then apply the cap.\",\n \"With the possibility of expanded markets for these lugged caps, which are also useful on various jars and bottles, there is a need for a system (method and tooling) for producing lugged cap members in a single machine, e.g.\",\n \"a reciprocating press fitted with appropriate tooling, which is capable of precise high speed (e.g.\",\n \"in the range of 135 to 150 strokes/minute) to achieve acceptable commercial production of the cap.\",\n \"To develop such production speeds it is desirable to divide the progressive tooling operations into more than one step, and this in turn requires a rapid and precise transfer system to move the partially completed caps from a first station to a second station, and precisely register the caps in the second station.\",\n \"Prior art transfer systems are known for moving and registering can end shells, such as in U.S. Pat.\",\n \"Nos.\",\n \"4,770,022 and 4,895,012, however, the end shells are relatively flat disc-like objects with a quite small height to diameter ratio, whereas the caps made by the present invention have a substantially greater height with respect to their diameter.\",\n \"Thus, the physical dimensions of the caps involved in this invention are quite different from can shells or easy self-opening can ends.\",\n \"This in turn introduces needs not required or anticipated in shell transfer systems, for example with regard to tipping of the caps during high speed transfer operations.\",\n \"Also, because of the relatively high press cycling, and need for precision in cap positioning and deceleration immediately after each transfer to another press station, there is a requirement for precise transfer of each cap from a first press station through high acceleration, very rapid transfer to the next press station, and high deceleration to a precisely defined stationary location at that next station.\",\n \"SUMMARY OF THE INVENTION The present invention provides a transfer apparatus and method for a first to a second station of progressive tooling in a cap making press.\",\n \"The caps are of a type having substantial height with respect to their diameter.\",\n \"Thus a first station punch and die form a cap of generally inverted cup shape, with an outward curled rim, and a second station punch and die form lugs into that rim, requiring just two strokes of the press to sever a disc from a supply sheet, form it, and discharge a completed cap.\",\n \"Thus, in the first station, a cap is formed except for lugs which are added to the cap in the second station.\",\n \"The formed cap in the first station, which is in the nature of an inverted cup, has an outward curl formed on its lower edge during the initial up stoke of the press.\",\n \"This cup is then biased against the upper forming punch, by a first airstream introduced into the cavity within the underside of the cap, and moves upward in contact with the first station punch.\",\n \"As the punch approaches its top dead center location (tooling fully open), a second airstream begins, before the first station punch is fully raised and while the first airstream is still on.\",\n \"This second airstream moves the cap off the raised first station punch, and into and through a transfer chute directed toward the second station.\",\n \"At the second station, the cap departs the chute and passes detents on a pair of closed retention fingers which, with the tooling open, define an extension of the transfer path from the chute into the open second station tools.\",\n \"As the second station tools begin to close and the partially finished cap is located, the fingers are opened and the tooling operates to form lugs into the cap rim.\",\n \"As the second station tooling opens, a vacuum applied to a port in the second station punch begins to hold the finished cup against that punch tool, and when that punch clears the closing fingers and approaches its top dead center location, an ejection airstream commences to propel the finished cup from the tooling via a discharge chute.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 is a bottom view of a container cap as provided by the invention.\",\n \"FIG.\",\n \"2 is a transverse cross-sectional view of a typical completed cap, as shown in FIG.\",\n \"1.FIG.\",\n \"3 is an over all perspective view of the tooling for a four-out cap making system.\",\n \"FIG.\",\n \"4 is an enlarged perspective view, with the die shoe removed, showing details of the tooling, including four first station tools in the center and four related second station tools outwardly in opposite directions of the first stations.\",\n \"FIG.\",\n \"5 is a further enlarged perspective view showing details of one pair of first and second stations of the tooling.\",\n \"FIG.\",\n \"6 is a cross-section view of a first station punch or upper tool.\",\n \"FIG.\",\n \"7 is a cross-section view of a first station die or lower tool.\",\n \"FIG.\",\n \"8 is a cross-section view illustrating the first station punch and die in closed position, and showing a cap formed except for an outward curl at the lower rim of the cap.\",\n \"FIG.\",\n \"9 is a cross-section view showing first station and corresponding second station tools, a transfer chute between them, the fingers which provide an extension of the chute into the second stage tool, and cams which control movement of the fingers.\",\n \"FIGS.\",\n \"10 and 11 are detail views of the top of the second station die, showing the open and closed positions of the fingers.\",\n \"FIG.\",\n \"12 is an enlarged cross-section view of the second station tools, and FIG.\",\n \"12A is a further enlargement of the circled area in the center of FIG.\",\n \"12.FIGS.\",\n \"13A and 13B1 & 13B2 (three sheets) together are a schematic diagram of the pneumatic supply & control system of the press and tooling.\",\n \"FIG.\",\n \"14 is a flow diagram of the electronic control system for the tooling package.\",\n \"DESCRIPTION OF PREFERRED EMBODIMENT The present invention provides a transfer apparatus and method for two station progressive tooling in a cap making press and system.\",\n \"The caps are of a type having substantial height with respect to their diameter.\",\n \"A typical such cap is shown in FIGS.\",\n \"1 and 2, which is also shown in FIGS.\",\n \"2, 4A and 4B of U.S. Pat.\",\n \"No.\",\n \"6,015,062.The preferably integral cap 11, in the general form of an inverted cup, includes a top panel 12, a peripheral sidewall 13, and a curled rim 14.In the present drawings, FIGS.\",\n \"3-12A depict multi-lane progressive tooling comprising four lanes for simultaneously forming four caps 11, each lane comprising pairs of first and second tooling stations 15A, 15B, 16A, 16B, 17A,17B, and 18A, 18B, with the first stations 15A-18A arranged centrally of the tooling (FIGS.\",\n \"3, 4 & 5) and corresponding second stations 16A-16B arranged outward of the first stations toward opposite sides of the upper and lower die plates 20A, 20B which support the upper (punch) and lower (die) tooling, and mount between the bed and slide of a reciprocating press (not shown).\",\n \"Except for their orientation in the overall tooling package, the respective first station and second station tools are alike, and the following detailed description applies to all.\",\n \"Taking stations 15A and 15B as examples, each pair of corresponding first and second stations has an associated transfer chute 19 (FIGS.\",\n \"4, 5 & 9) between them, and each second station has a discharge chute 19A, the four of which are directed out opposite sides of the tooling (FIGS.\",\n \"3 & 4).\",\n \"Sheets of metal, with an appropriate pattern of lithographed materials for each cap, are fed centrally into the first stations by sheet feeding mechanism of known construction (not shown) which moves the sheets one at a time in step wise fashion, synchronized to the press strokes, along the feed path indicated by arrow IN in FIGS.\",\n \"3 & 4.First Station The first station tools comprise an upper or blank punch tool 45 and a compound lower die.\",\n \"During the initial operation of the first station tools, with the lithographed patterns aligned with respect to the first station tools, a blank is cut from the material (typically aluminum or thin cold rolled steel) on the down stroke of the press by blank punch 45.On the continuation of the down stroke, the blank punch and lower die tool cooperate such that the blank is drawn into a cup shaped cap part 11P (FIG.\",\n \"8).\",\n \"At the bottom of the stroke the panel shape 12 is formed into the top of cap part lip by the punch 45 and cooperating die 46 (FIGS.\",\n \"6, 7 & 8).\",\n \"On the up stroke, the lower curl ring 48, which is under spring pressure, raises with the blank punch.\",\n \"The bottom edge of the cap part 11P is curled outward into the cavity 49 formed by curl ring 48 and blank punch 45, completing a formed cap 11 with an outward curled rim 15.The formed cap in the first station, which is in the nature of an inverted cup, is biased against the upper forming die by a first airstream introduced through passage 50 (see FIGS.\",\n \"8 & 9) into the cap as the first station tooling opens, and causes the cap to follow upward against the bottom of the punch 45.During the upward travel of the cap, a second airstream is initiated through a nozzle 52 directed across the upper fist station tooling toward chute 18, and is at its full power when punch 45 (with a cap 11 held thereto by the upward directed first airstream) traverses the space between nozzle 52 and the entry 18E to chute 18 (see FIG.\",\n \"9).\",\n \"By the time the first station tooling reaches full open at the top-dead-center of a press stroke, the cap has actually been transferred into the second station 15B; see the Press Rotation timing chart below (page 8).\",\n \"Thus, the first and second airstreams, appropriately switched on and off, together with the associated chute, constitute a first part of an essentially passive press transfer system.\",\n \"Second Station At the second station, a separated pair of fingers 60 reach around the sides of the second station tooling (FIGS.\",\n \"9, 10 & 11), defining partial sides of a receiving space 62 when the second station tooling is open, and an extension of the transfer chute when closed.\",\n \"The fingers are supported by pivots 63 inward of their rear ends 61, and are biased into their closed position (FIG.\",\n \"10, forward ends parallel) by spring mechanism 64.Each finger has a narrow ledge-like track 65 extending part way along its forward upper edge (FIGS.\",\n \"10-12), facing each other and ending in a curved section 66.Extending horizontally inward over tracks 65 are spring-loaded ball detents 67 which, together with the curved track sections 66, define the termination of the transfer path for the incoming caps.\",\n \"A cap propelled from the first station traverses the adjacent transfer chute 19 and enters receiving space 62, passing across ball detents 67 and resting against the curved track sections 66 (see FIG.\",\n \"11).\",\n \"The fingers and their operating mechanism form the remainder of the unique transfer system.\",\n \"As the tooling proceeds to close during the beginning of a next stroke, fingers 60 are swung outward by descending cams 48 pressing against followers 49 on the rear ends 61 of the fingers to move the followers 49 inward and the forward section of the fingers outward (FIGS.\",\n \"10 & 12) before the second station tools close.\",\n \"The elongated cams 48 are mounted on the upper (die) tooling base (FIG.\",\n \"12), and this action centers a cap (FIG.\",\n \"11) and then opens the fingers associated with the second station tooling, before that tooling closes to form lugs on rim 15 of a cap 11.This closing action of fingers 60 is momentary, and they are then opened substantially before the second station tooling closes; see Press Rotation timing chart below.\",\n \"In the second station 15B in the press, the tooling includes an upper punch 70 and a lower die 72.The punch tool 70 includes an annular knock out ring 71 with an inner shape conforming to a cap exterior, and a headed knock out pin 73 which is spring loaded toward a position flush with the lower edge of ring 71 (FIGS.\",\n \"12 & 12A) when the tooling is opened.\",\n \"In the lower die 72 there is a vertically extending tapered cam 74 mounted onto a base plate 75 and surrounded by a plurality of die pins 76 also mounted onto plate 75.The cam 74 and pins 76 project through a vertically movable, upwardly biased ring 78 which contains a plurality of lug forming dies 80, equal in number to pins 76 and movable laterally outward through forces from cam 74 as it is made to enter ring 78.The top of ring 78 has an upper surface 82 contoured to fit within a cap 11.Thus, when the second station tooling closes a cap is positioned with the curled rim 15 between the upper ends of pins 76 and the radially outward moving forming dies 80, to for the predetermined number of lugs in the rim (FIG.\",\n \"1).\",\n \"Then, the completed cap is held to the knockout pin by means of a vacuum created in passage 85 though the head of that pin 73, such that the cap is carried upward past an air nozzle 87 directed across the path of the rising upper tool toward an associated discharge chute 19A.\",\n \"The vacuum is created by air flow through a small venturi (not shown) so the vacuum is relieved immediately upon cessation of air flow through that venturi.\",\n \"A discharge air stream is started from nozzle 87 and moves the cap from the upper knock out ring and pin, into and through the associated discharge chute 19.Press Rotation Relation to Tooling Function 1st Station 0° Top of the Stroke; Top Dead Center 140° Material is Blanked 180° Form & draw complete; Bottom Dead Center; Cap Overall Height ˜0.810 inch 190° Cap Curl complete; Overall Height is ˜0.625 inch 190° 1st Air turned on; Blows cap against punch tool 220° Blank punch exits Die tool, cap against its face 230° 2nd Transfer Air on; Blows cap into transfer chute 330° 1st Cap arrives into catch fingers at 2nd Station.\",\n \"2nd Station 0° Top of a stroke; Top Dead Center 60° Upper vacuum is turned on 136° Catch fingers start open, upper vacuum holds 1st cap against upper knockout and tools close 180° Lugs formed in 1st cap; Bottom Dead Center 181° 1st cap (completed) and tools move up together 224° Catch fingers close completely after tooling passes 270° Upper vacuum to knockout turned off 280° Discharge air valve to nozzle is turned on 290° Knock out air to nozzle 87 is turned on 330° 2nd Cap arrives into catch fingers 336° 1st Cap actually moves to discharge position 335° Vacuum actually turns off on Cap #1 350° 1st Cap actually leaves press via chute 19 FIGS.\",\n \"13A and 13B comprise a pneumatic diagram for the pneumatic portion of the press control.\",\n \"A “shop air” source of compressed air is supplied to the various electrically controlled valves (which are all labeled) to direct air under pressure to the above mentioned parts of the tooling, under management control of the electronics control (FIG.\",\n \"14) of the system.\",\n \"The details of such controls are apparent to persons skilled in this art from these diagrams FIG.\",\n \"14 is a block diagram of electrical/electronics controls for the system, which selectively turn on and off the compressed air to the nozzles providing lift or “blow up” air to hold the cap part against the rising upper tools in the first station, and providing a transfer air stream to quickly move a cap part into a transfer chute 19.The third (upper) control provides timed discharge airstreams through nozzles 87 to move the finished caps into the discharge chutes 19A.\",\n \"A pulse generator PG is driven by the press crankshaft (not shown) in typical fashion to generate a train of pulses related to the angular position of the crankshaft as it rotates, and these pulses are directed to the system PLC (Programmable Logic Controller).\",\n \"Since the diagram is divided into three functions which occur during a press cycle, the controller PLC is shown in each of the three diagram parts, but in fact one PLC is employed in the control system.\",\n \"While the method herein described, and the form of apparatus for carrying this method into effect, constitute preferred embodiments of this invention, it is to be understood that the invention is not limited to this precise method and form of apparatus, and that changes may be made in either without departing from the scope of the invention, which is defined in the appended claims.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10451057"}}},{"rowIdx":294,"cells":{"text":{"kind":"list like","value":[["Diving aid","Diving aid for divers, at least comprising a power supply, alarm sound-generating means (3) and activating means (2) for activating the sound-generating means (3), which activating means (2) are designed in such a way that when the alarm device is in use said activating means can be activated by an uncontrolled shock load, and that the device comprises means (6) for attaching to the divers's body or the divers's equipment in an unmistakable orientation.","The diving aid according to the invention is preferably designed in the form to a wristwatch."],["1.Diving aid for reducing the dive stress of divers, in particular pre-dive stress, at least comprising a power supply, alarm sound-generating means and activating means for activating the alarm sound-generating means, which activating means are designed in such a way that when the alarm device is in use said activating means can be activated by an uncontrolled shock load, and which device also comprises means for attaching to the diver's body or the diver's equipment in an unmistakable orientation.","2.Diving aid according to claim 1, wherein the diving aid also comprises light-signal-generating means that are connected to the activating means.","3.Diving aid according to claim 1, wherein the activating means comprise a push-button.","4.Diving aid according to claim 1, wherein the activating means comprise an acceleration detector.","5.Diving aid according claim 1, wherein the diving aid comprises water detection means by means of which the device can be activated.","6.Diving aid according to claim 1, wherein the diving aid is designed in the form of a wristwatch.","7.Diving aid according to claim 1, wherein the means for attaching the diving aid to the diver's body or the diver's equipment comprise a clip.","8.Diving aid according to claim 1, wherein the diving aid forms part of an ordinary diving computer."],["The present invention relates to a diving aid.","A major problem encountered by both experienced and inexperienced divers during diving is so-called dive stress, in particular pre-dive stress.","This dive stress increases the likelihood of mistakes being made prior to and during diving, mistakes which can have fatal consequences.","The chance of a panic situation under water is also increased considerably by this dive stress.","In practice, it is found that most divers dive only once or twice a year.","These are the divers at greatest risk.","It has been found that when such an inexperienced diver is in distress or in danger he has only three seconds in which it is possible to take action.","In the first second the distress or danger is perceived, in the second second the diver becomes aware of the problem and his position, and in the third second the diver is in a panic.","The most common reaction of such divers is to swim as fast as possible towards the surface, with all the risks this involves.","In a panic situation inexperienced divers are found to be incapable of any coordinated movement, for example in order to activate an alarm device.","The known alarm devices for use as diving aids are therefore found to be inadequate.","In this connection reference is made to FR-A-2,695,747.This French patent application discloses an alarm device for attaching to the wrist.","This device comprises two parts that are rotatable relative to each other and can be locked relative to each other by means of a locking pin.","This locking pin has to be removed in order to activate the alarm device.","FR-A-2,755,023 also discloses an alarm device, which comprises a housing with a battery and sound-generating means.","The housing comprises a push-button for activating the alarm device.","This alarm device is attached to an eye present on the housing.","It has been found that a diver who is in a panic is incapable of removing a locking pin in a controlled manner or locating and pressing a push-button on a trailing device.","Divers also very often wear gloves during diving, which makes such actions even more difficult.","Furthermore, a fully equipped diver has very limited freedom of movement.","There are also alarm devices that are operated by compressed air and are therefore connected to the compressed air system of the diver.","A common problem is that the diver is not getting any air and there is a fault in the compressed air system.","Such devices are therefore not sufficiently reliable, even if the diver were capable of activating the activating means.","In other words, all devices known in the prior art require coordinated movements from the diver.","These devices are therefore found to be inadequate in practice.","Knowledge of this fact increases both pre-dive stress and dive stress among divers.","There is consequently a great demand for a diving aid that can give the diver a relaxed and safe feeling.","The object of the present invention is to meet this demand, and to that end the invention provides a diving aid at least comprising a power supply, alarm sound-generating means and activating means for activating the alarm sound-generating means, which activating means are designed in such a way that when the alarm device is in use said activating means can be activated by an uncontrolled shock load, and that the device comprises means for attaching to the diver's body or the diver's equipment in an unmistakable orientation.","The diving aid according to the invention is found to be extremely satisfactory, even in a situation where an inexperienced diver panics.","It appears that an inexperienced diver in a panic is in fact only capable of making an uncoordinated striking movement with one hand in the direction of the alarm device.","For the device according to the invention, this is sufficient to activate the latter and make co-divers aware of the distress or danger.","It has been proved in practice that knowledge of the substantially increased safety under water through use of the diving aid according to the invention substantially reduces dive stress even in the case of very experienced divers.","It is pointed out that alarm devices that can be activated by an uncontrolled shock load are known for use on dry land.","These devices are not, however, suitable for use under water.","In this connection; reference is made to GB-A-2,267,373, GB-A-2,295,910 and GB-A-2,316,784.Apart from alarm sound-generating means, other means can, of course, also be used for alerting co-divers.","The diving aid also preferably comprises light-signal-generating means, which are connected to the activating means.","Under water the speed of sound is many times higher than it is in air.","The result of this is that under water the human ear is not capable of determining the direction of sound.","It is therefore preferable to use light-signal-generating means, such as, for example, a stroboscopic lamp or a stroboscopic LED.","Such lighting means are known in the prior art.","Another advantage of using light-generating means is that a diver in distress can be located easily even when diving at night or in murky water.","Of course, the advantages also apply to a victim who is unconscious, who can be found more easily in this way.","The activating means can be designed in many different ways according to the invention.","The activating means preferably comprise a relatively large push-button.","The push-button is so large that it can be pressed with certainty by an arbitrary uncontrolled striking movement.","The surface of the push-button is advantageously at least 2 cm2.More advantageously, the push-button also projects from the surface of the diving aid.","In the case of a large push-button it is advantageous if said button is provided with passages for water, so that the water pressure of the environment under the button can be evened out.","This ensures that during diving the diving aid is not activated undesirably by the pressure of the water.","In particular, a striking colour, for example red or yellow, is selected for such a push-button.","The activating means advantageously comprise an acceleration detector.","An example of this is a so-called tilt detector.","Such detectors are switches that are known in the prior art and can be activated by a shock load, without the direction of the shock load being important.","The diving aid can comprise an ON/OFF button, for switching on the diving aid just before diving, and for switching it off after diving.","The diving aid preferably comprises water detection means for switching on the diving aid as soon as it comes into contact with water.","This has the advantage that the diving aid is therefore always switched on under water, even if the diver forgets to switch on the diving aid.","Such water detection means are known in the prior art.","An example of these means is the detection electrodes that are also used on diving computers.","In this context the term switching on means putting the diving aid into a state ready for use.","In other words, when the diving aid is not switched on operation of the activating means will not result in the generation of an alarm sound.","In particular, the diving aid is designed in such a way that it can be worn on the wrist.","In practice, it has been found that the wrist is a suitable place for attachment of the diving aid, since in practice said diving aid can be reached easily with the other hand, so that in a panic situation the diving aid can be activated by an uncoordinated movement.","It will be clear that the diving aid can likewise be attached to the diver's equipment, provided that this attachment involves an unmistakable orientation, so that the diver can always reach the diving aid.","As a particular example of this, the means for attaching the diving aid to the diver's body or the diver's equipment comprise a clip.","In a special embodiment the diving aid forms part of an ordinary diving computer.","An ordinary diving computer is a device from which, inter alia, the diving depth, the diving time that has elapsed and the like can be read.","The invention will be explained in greater detail below with reference to the appended drawing, in which: FIG.","1 shows a top view of an embodiment of a diving aid according to the invention; FIG.","2 shows a perspective view of the embodiment according to FIG.","1; FIG.","3 shows a top view of a different embodiment of a diving aid according to the invention; and FIG.","4 shows a perspective view of the embodiment according to FIG.","3.FIG.","1 shows an embodiment of a diving aid according to the invention, which is suitable for attachment around the wrist.","The diving aid comprises a housing 1 on which a large push-button 2 and alarm sound-generating means 3 are situated.","Reference numeral 5 indicates two water detection electrodes, which serve to activate the diving aid on contact with water.","A power supply is not shown in the figures, but it will be clear that in principle any power supply that is suitable for use under water can be used.","An example in this case is a power supply for a diving computer or the like.","Reference numeral 6 indicates a wristband for attachment of the diving aid to a diver's wrist.","Finally, reference numeral 7 indicates a reset button, by means of which the diving aid can be switched off after being activated, for example when the diver in distress or danger has been rescued.","For testing the functioning of the diving aid according to the invention above water, the diving aid can be switched on by touching the electrodes with moistened fingertips.","It will be clear that the diving aid is certain to be activated when in a panic situation a striking movement with the hand is made in an uncontrolled way in the direction of the diving aid according to the invention.","All this has been confirmed by experiments.","The alarm sound-generating means in this case are in the form of a piezoelectric sound-generating device.","FIG.","2 shows the diving aid according to FIG.","1 in perspective view.","It can be seen clearly in this figure that the large push-button 2 projects slightly from the surface of the device, so that said push-button is easy to press.","FIGS.","3 and 4 show a different preferred embodiment of a diving aid according to the invention, in which similar parts are indicated by the same reference numerals.","Apart from the alarm sound-generating means, this diving aid also comprises a stroboscopic xenon gas discharge lamp 8, for generating a light alarm.","When a diver in distress/danger uses this diving aid, he will be able to indicate his position not only by means of an audible sound signal, but also by means of light signals.","This means that the diver can be found even more quickly by co-divers."]],"string":"[\n [\n \"Diving aid\",\n \"Diving aid for divers, at least comprising a power supply, alarm sound-generating means (3) and activating means (2) for activating the sound-generating means (3), which activating means (2) are designed in such a way that when the alarm device is in use said activating means can be activated by an uncontrolled shock load, and that the device comprises means (6) for attaching to the divers's body or the divers's equipment in an unmistakable orientation.\",\n \"The diving aid according to the invention is preferably designed in the form to a wristwatch.\"\n ],\n [\n \"1.Diving aid for reducing the dive stress of divers, in particular pre-dive stress, at least comprising a power supply, alarm sound-generating means and activating means for activating the alarm sound-generating means, which activating means are designed in such a way that when the alarm device is in use said activating means can be activated by an uncontrolled shock load, and which device also comprises means for attaching to the diver's body or the diver's equipment in an unmistakable orientation.\",\n \"2.Diving aid according to claim 1, wherein the diving aid also comprises light-signal-generating means that are connected to the activating means.\",\n \"3.Diving aid according to claim 1, wherein the activating means comprise a push-button.\",\n \"4.Diving aid according to claim 1, wherein the activating means comprise an acceleration detector.\",\n \"5.Diving aid according claim 1, wherein the diving aid comprises water detection means by means of which the device can be activated.\",\n \"6.Diving aid according to claim 1, wherein the diving aid is designed in the form of a wristwatch.\",\n \"7.Diving aid according to claim 1, wherein the means for attaching the diving aid to the diver's body or the diver's equipment comprise a clip.\",\n \"8.Diving aid according to claim 1, wherein the diving aid forms part of an ordinary diving computer.\"\n ],\n [\n \"The present invention relates to a diving aid.\",\n \"A major problem encountered by both experienced and inexperienced divers during diving is so-called dive stress, in particular pre-dive stress.\",\n \"This dive stress increases the likelihood of mistakes being made prior to and during diving, mistakes which can have fatal consequences.\",\n \"The chance of a panic situation under water is also increased considerably by this dive stress.\",\n \"In practice, it is found that most divers dive only once or twice a year.\",\n \"These are the divers at greatest risk.\",\n \"It has been found that when such an inexperienced diver is in distress or in danger he has only three seconds in which it is possible to take action.\",\n \"In the first second the distress or danger is perceived, in the second second the diver becomes aware of the problem and his position, and in the third second the diver is in a panic.\",\n \"The most common reaction of such divers is to swim as fast as possible towards the surface, with all the risks this involves.\",\n \"In a panic situation inexperienced divers are found to be incapable of any coordinated movement, for example in order to activate an alarm device.\",\n \"The known alarm devices for use as diving aids are therefore found to be inadequate.\",\n \"In this connection reference is made to FR-A-2,695,747.This French patent application discloses an alarm device for attaching to the wrist.\",\n \"This device comprises two parts that are rotatable relative to each other and can be locked relative to each other by means of a locking pin.\",\n \"This locking pin has to be removed in order to activate the alarm device.\",\n \"FR-A-2,755,023 also discloses an alarm device, which comprises a housing with a battery and sound-generating means.\",\n \"The housing comprises a push-button for activating the alarm device.\",\n \"This alarm device is attached to an eye present on the housing.\",\n \"It has been found that a diver who is in a panic is incapable of removing a locking pin in a controlled manner or locating and pressing a push-button on a trailing device.\",\n \"Divers also very often wear gloves during diving, which makes such actions even more difficult.\",\n \"Furthermore, a fully equipped diver has very limited freedom of movement.\",\n \"There are also alarm devices that are operated by compressed air and are therefore connected to the compressed air system of the diver.\",\n \"A common problem is that the diver is not getting any air and there is a fault in the compressed air system.\",\n \"Such devices are therefore not sufficiently reliable, even if the diver were capable of activating the activating means.\",\n \"In other words, all devices known in the prior art require coordinated movements from the diver.\",\n \"These devices are therefore found to be inadequate in practice.\",\n \"Knowledge of this fact increases both pre-dive stress and dive stress among divers.\",\n \"There is consequently a great demand for a diving aid that can give the diver a relaxed and safe feeling.\",\n \"The object of the present invention is to meet this demand, and to that end the invention provides a diving aid at least comprising a power supply, alarm sound-generating means and activating means for activating the alarm sound-generating means, which activating means are designed in such a way that when the alarm device is in use said activating means can be activated by an uncontrolled shock load, and that the device comprises means for attaching to the diver's body or the diver's equipment in an unmistakable orientation.\",\n \"The diving aid according to the invention is found to be extremely satisfactory, even in a situation where an inexperienced diver panics.\",\n \"It appears that an inexperienced diver in a panic is in fact only capable of making an uncoordinated striking movement with one hand in the direction of the alarm device.\",\n \"For the device according to the invention, this is sufficient to activate the latter and make co-divers aware of the distress or danger.\",\n \"It has been proved in practice that knowledge of the substantially increased safety under water through use of the diving aid according to the invention substantially reduces dive stress even in the case of very experienced divers.\",\n \"It is pointed out that alarm devices that can be activated by an uncontrolled shock load are known for use on dry land.\",\n \"These devices are not, however, suitable for use under water.\",\n \"In this connection; reference is made to GB-A-2,267,373, GB-A-2,295,910 and GB-A-2,316,784.Apart from alarm sound-generating means, other means can, of course, also be used for alerting co-divers.\",\n \"The diving aid also preferably comprises light-signal-generating means, which are connected to the activating means.\",\n \"Under water the speed of sound is many times higher than it is in air.\",\n \"The result of this is that under water the human ear is not capable of determining the direction of sound.\",\n \"It is therefore preferable to use light-signal-generating means, such as, for example, a stroboscopic lamp or a stroboscopic LED.\",\n \"Such lighting means are known in the prior art.\",\n \"Another advantage of using light-generating means is that a diver in distress can be located easily even when diving at night or in murky water.\",\n \"Of course, the advantages also apply to a victim who is unconscious, who can be found more easily in this way.\",\n \"The activating means can be designed in many different ways according to the invention.\",\n \"The activating means preferably comprise a relatively large push-button.\",\n \"The push-button is so large that it can be pressed with certainty by an arbitrary uncontrolled striking movement.\",\n \"The surface of the push-button is advantageously at least 2 cm2.More advantageously, the push-button also projects from the surface of the diving aid.\",\n \"In the case of a large push-button it is advantageous if said button is provided with passages for water, so that the water pressure of the environment under the button can be evened out.\",\n \"This ensures that during diving the diving aid is not activated undesirably by the pressure of the water.\",\n \"In particular, a striking colour, for example red or yellow, is selected for such a push-button.\",\n \"The activating means advantageously comprise an acceleration detector.\",\n \"An example of this is a so-called tilt detector.\",\n \"Such detectors are switches that are known in the prior art and can be activated by a shock load, without the direction of the shock load being important.\",\n \"The diving aid can comprise an ON/OFF button, for switching on the diving aid just before diving, and for switching it off after diving.\",\n \"The diving aid preferably comprises water detection means for switching on the diving aid as soon as it comes into contact with water.\",\n \"This has the advantage that the diving aid is therefore always switched on under water, even if the diver forgets to switch on the diving aid.\",\n \"Such water detection means are known in the prior art.\",\n \"An example of these means is the detection electrodes that are also used on diving computers.\",\n \"In this context the term switching on means putting the diving aid into a state ready for use.\",\n \"In other words, when the diving aid is not switched on operation of the activating means will not result in the generation of an alarm sound.\",\n \"In particular, the diving aid is designed in such a way that it can be worn on the wrist.\",\n \"In practice, it has been found that the wrist is a suitable place for attachment of the diving aid, since in practice said diving aid can be reached easily with the other hand, so that in a panic situation the diving aid can be activated by an uncoordinated movement.\",\n \"It will be clear that the diving aid can likewise be attached to the diver's equipment, provided that this attachment involves an unmistakable orientation, so that the diver can always reach the diving aid.\",\n \"As a particular example of this, the means for attaching the diving aid to the diver's body or the diver's equipment comprise a clip.\",\n \"In a special embodiment the diving aid forms part of an ordinary diving computer.\",\n \"An ordinary diving computer is a device from which, inter alia, the diving depth, the diving time that has elapsed and the like can be read.\",\n \"The invention will be explained in greater detail below with reference to the appended drawing, in which: FIG.\",\n \"1 shows a top view of an embodiment of a diving aid according to the invention; FIG.\",\n \"2 shows a perspective view of the embodiment according to FIG.\",\n \"1; FIG.\",\n \"3 shows a top view of a different embodiment of a diving aid according to the invention; and FIG.\",\n \"4 shows a perspective view of the embodiment according to FIG.\",\n \"3.FIG.\",\n \"1 shows an embodiment of a diving aid according to the invention, which is suitable for attachment around the wrist.\",\n \"The diving aid comprises a housing 1 on which a large push-button 2 and alarm sound-generating means 3 are situated.\",\n \"Reference numeral 5 indicates two water detection electrodes, which serve to activate the diving aid on contact with water.\",\n \"A power supply is not shown in the figures, but it will be clear that in principle any power supply that is suitable for use under water can be used.\",\n \"An example in this case is a power supply for a diving computer or the like.\",\n \"Reference numeral 6 indicates a wristband for attachment of the diving aid to a diver's wrist.\",\n \"Finally, reference numeral 7 indicates a reset button, by means of which the diving aid can be switched off after being activated, for example when the diver in distress or danger has been rescued.\",\n \"For testing the functioning of the diving aid according to the invention above water, the diving aid can be switched on by touching the electrodes with moistened fingertips.\",\n \"It will be clear that the diving aid is certain to be activated when in a panic situation a striking movement with the hand is made in an uncontrolled way in the direction of the diving aid according to the invention.\",\n \"All this has been confirmed by experiments.\",\n \"The alarm sound-generating means in this case are in the form of a piezoelectric sound-generating device.\",\n \"FIG.\",\n \"2 shows the diving aid according to FIG.\",\n \"1 in perspective view.\",\n \"It can be seen clearly in this figure that the large push-button 2 projects slightly from the surface of the device, so that said push-button is easy to press.\",\n \"FIGS.\",\n \"3 and 4 show a different preferred embodiment of a diving aid according to the invention, in which similar parts are indicated by the same reference numerals.\",\n \"Apart from the alarm sound-generating means, this diving aid also comprises a stroboscopic xenon gas discharge lamp 8, for generating a light alarm.\",\n \"When a diver in distress/danger uses this diving aid, he will be able to indicate his position not only by means of an audible sound signal, but also by means of light signals.\",\n \"This means that the diver can be found even more quickly by co-divers.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10451113"}}},{"rowIdx":295,"cells":{"text":{"kind":"list like","value":[["Composition comprising an oxidizing and complexing compound","The present invention is related to a composition comprising an oxidizing compound and a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.","The oxidizing compound can be in the form of an aqueous solution.","The complexing compound is for complexing metal ions.","Metal ions can be present in the solution or in an external medium being contacted with the solution.","The present invention can be used for cleaning a semiconductor substrate."],["1.A composition comprising an aqueous solution, said aqueous solution comprising an oxidizing compound and a complexing compound, said complexing compound comprising a chemical formula wherein R1, R2, R3, and R4 are each independently selected from the group consisting of H and an organic side chain.","2.The composition as recited in claim 1, further comprising an alkaline compound.","3.The composition as recited in claim 1, wherein said organic side chain is selected from the group consisting of an aliphatic side chain, a heterocyclic side chain, and an aromatic side chain.","4.The composition as recited in claim 1, wherein R3 and R4 each comprise hydrogen and R1 and R2 each comprise a functionalized aliphatic side chain.","5.The composition as recited in claim 1, wherein said complexing compound is selected from the group consisting of 1,2-diethyl-3-hydroxy-4(1H)-pyridinone, 1-ethyl-2-methyl-3-hydroxy-4(1H)-pyridinone, 1-methyl-2-ethyl-3-hydroxy-4(1H)-pyridinone 1,2-dimethyl-3-hydroxy-4(1H)pyridinone, 1-propyl-2-ethyl-3-hydroxy-4(1H)-pyridinone, 1-propyl-2-methyl-3-hydroxy-4(1H)-pyridinone, 1-(2-carboxyethyl)-2-methyl-3-hydroxy-4( 1H)-pyridinone, and 1-(2-carboxyethyl)-2-ethyl-3-hydroxy-4(1H)-pyridinone.","6.The composition as recited in claim 1, wherein said oxidizing compound comprises hydrogen peroxide.","7.The composition as recited in recited in claim 2, wherein said alkaline compound comprises an inorganic basic compound or an organic basic compound.","8.The composition as recited in claim 7, wherein said alkaline compound is selected from the group consisting of ammonia and organic amine compounds.","9.The composition as recited in claim 2, wherein an amount of oxidizing compound comprises from 0.001 to 30 weight %.","10.The composition as recited in claim 1, wherein an amount of the complexing agent comprises from 0.1 to 1000 ppm of said solution.","11.The composition as recited in claim 7, wherein an amount of the inorganic basic compound or the organic basic compound comprises from 0.001 to 30 weight %.","12.The composition as recited in claim 1, wherein an amount of said oxidizing compound comprises from 0.001 to 95 weight %.","13.A method of treating a semiconductor substrate, wherein said method comprises treating a semiconductor substrate with a composition comprising an aqueous solution, said aqueous solution comprising a complexing compound comprising the chemical formula wherein R1, R2, R3, and R4 are each independently selected from the group consisting of H and an organic side chain.","14.The method as recited in claim 13, wherein said composition further comprises an oxidizing compound.","15.The method as recited in claim 13, wherein said composition further comprises an alkaline compound.","16.The method as recited in claim 13, wherein said organic side chain is selected from the group consisting of an aliphatic side chain, a heterocyclic side chain, and an aromatic side chain.","17.The method as recited in claim 13, wherein R3 and R4 each comprise hydrogen and R1 and R2 each comprise a functionalized aliphatic side chain.","18.The method as recited in claim 13, wherein said complexing compound is selected from the group consisting of 1,2-diethyl-3-hydroxy-4(1H)-pyridinone, 1-ethyl-2-methyl-3-hydroxy-4(1H)-pyridinone, 1-methyl-2-ethyl-3-hydroxy-4(1H)-pyridinone, 1,2-dimethyl-3-hydroxy-4(1H)pyridinone, 1-propyl-2-ethyl-3-hydroxy-4(1H)-pyridinone, 1-propyl-2-methyl-3-hydroxy-4(1H)-pyridinone, 1-(2-carboxyethyl)-2-methyl-3-hydroxy-4(1H)-pyridinone, and 1-(2-carboxyethyl)-2-ethyl-3-hydroxy-4(1H)-pyridinone.","19.The method as recited in claim 14, wherein said oxidizing compound comprises hydrogen peroxide.","20.The method as recited in claim 15, wherein the alkaline compound comprises an inorganic basic compound or an organic basic compound.","21.The method as recited in claim 20, wherein said inorganic basic compound or said organic basic compound is selected from the group consisting of ammonia and organic amine compounds.","22.The method as recited in claim 14, wherein an amount of the oxidizing compound comprises from 0.001 to 30 weight %.","23.The method as recited in claim 15, wherein an amount of the alkaline compound comprises from 0.001 to 30 weight %.","24.The method as recited in claim 13, wherein an amount of said complexing compound comprises from 0.1 ppm to 1000 ppm.","25.Use of a composition as recited in claim 1 in a field selected from the group consisting of soil remediation, metal etch bath, denture bleaching, bleaching of fats, bleaching of oils, bleaching of waxes, wastewater treatment, paper pulp bleaching, and textile bleaching.","26.The composition as recited in claim 8, wherein said organic amine is selected from the group consisting of tetraalkylammoniumhydroxide compounds, alkanolamine compounds, choline(hydroxyltrialkylammoniumhydroxide) compounds, and guanidine compounds.","27.The method as recited in claim 21, wherein said organic amine is selected from the group consisting of tetraalkylammoniumhydroxide compounds, alkanolamine compounds, choline(hydroxyltrialkylammoniumhydroxide) compounds, and guanidine compounds."],["<SOH> FIELD OF THE INVENTION <EOH>This invention is situated in the field of products and methods for the stabilizing a composition comprising an oxidizing compound."],["<SOH> SUMMARY OF THE INVENTION <EOH>In a first aspect of this invention, a composition is disclosed comprising an oxidizing compound and a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.","The oxidizing compound can be in the form of an aqueous solution.","The complexing compound is for complexing metal ions.","Metal ions can be present in the solution or in an external medium being contacted with the solution.","Depending on the metal ion being complexed, one or more complexing molecules/metal ion are required.","In an embodiment of this first aspect, said composition as recited in the first aspect of this invention can further comprise an alkaline compound.","In a further embodiment of this first aspect, said organic side chain can comprise an aliphatic side chain, a heterocyclic side chain or an aromatic side chain.","In a further embodiment of the first aspect of this invention, R3 and R4 can be hydrogen while R1 and R2 can be a functionalized aliphatic side chain.","Preferably, said complexing compound is one of the group consisting of DEHP, 1-EMHP, 2-EMHP, DMHP, PEPH, PMHP, ECMHP and ECEHP.","In a further embodiment of this first aspect, a solution as recited in the first aspect of this invention is disclosed characterized in that said oxidizing compound is hydrogen peroxide.","Hydrogen peroxide will be stabilized by the addition of the complexing compound, such that decomposition is substantially inhibited.","In a further embodiment of this first aspect of the invention, a composition as disclosed wherein said oxidizing compound is hydrogen peroxide.","In a further embodiment of this first aspect, a composition as recited in the first embodiment of the first aspect of the invention is disclosed wherein alkaline compound comprises an inorganic or organic basic compound.","Said alkaline compound can be ammonia or an organic amine, preferably an organic amine chosen from the group consisting of tetraalkylammoniumhydroxide, alkanolamine, choline(hydroxyltrialkylammoniumhydroxide) and guanidine compounds.","In a second aspect of this invention, a method for treating a semiconductor substrate is disclosed.","Said semicondcutor substrate is treated with a composition comprising an aqueous solution of a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.","Said composition can further comprise an oxidizing compound.","Said composition can be used for treating a substrate, such that particles are oxidized and metallic contamination is removed.","The complexing molecule is for complexing metallic residues being present on the substrate and in the solution.","Additionally, the lifetime of the solution is increased since de decomposition of the oxidizing compound is substantially inhibited.","Said solution is particularly suitable for cleaning a semiconductor substrate.","Said composition for cleaning a semiconductor substrate can be any composition described in the first aspect of this invention.","In an embodiment of this second aspect of this invention, said R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.","Said organic side chain can comprise an aliphatic side chain, a heterocyclic side chain or an aromatic side chain.","In a further embodiment of this second aspect of the invention, R3 and R4 can be hydrogen while R1 and R1 can be a functionalized aliphatic side chain.","In a further embodiment, said complexing compound is one of the group consisting of DEHP, 1-EMHP, 2-EMHP, DMHP, PEPH, PMHP, ECMHP and ECEHP.","In another embodiment of the second aspect of the present invention, said oxidizing compound can be hydrogen peroxide.","In an embodiment of the second aspect of this invention, said composition can further comprise an alkaline compound.","Said alkaline compound can comprise an inorganic or organic basic compound.","Said basic compound can be chosen from the group consisting of ammonia and organic amine, preferably an organic amine chosen from the group consisting of tetraalkylammoniumhydroxide, alkanolamine, choline(hydroxyltrialkylammoniumhydroxide) and guanidine compounds.","In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed wherein the concentration of hydrogen peroxide in the solution lies between 0.001 to 30 weight %.","In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed in which the amount of the complexing agent lies between 0.1 and 1000 ppm of said solution.","In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed wherein the amount of the alkaline compound lies between 0.001 and 30 weight %.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["FIELD OF THE INVENTION This invention is situated in the field of products and methods for the stabilizing a composition comprising an oxidizing compound.","STATE OF THE ART Chemical solutions comprising an oxidizing compound such as hydrogen peroxide are used in a wide area of applications, for example, as bleaching agent for paper pulp applications, dental bleaching composition or as cleaning agents.","Chemical solutions comprising an oxidizing compound have often problems related to the stability of the solution.","In pure form, aqueous solution are stable over extended periods of time.","However, the presence of certain metal ions in the solution causes decomposition of the oxidizing compound.","Consequently, stabilizers to prevent such decomposition should be added.","Stabilizers can be e.g.","a complexing compound, such that the complexing compound will bind to the metal and consequently, the metal is not available for reaction with the oxidizing compound.","Thus, the decomposition of the oxidizing compound is substantially inhibited and the lifetime of the solution is increased.","Very stringent specifications must be met by oxidizing solutions for specialized applications such as semiconductor applications or reagent chemicals.","An overview of stabilizing oxidizing compound, and more specifically hydrogen peroxide solutions, is given in Kirk-Othmer Encyclopedia of Chemical Technology (4th edition), vol.","13 p. 965.U.S.","Pat.","No.","4,239,643 discloses an aqueous peroxide-containing solutions used in bleaching of cellulose fiber, wherein the stability of the peroxide is very greatly increased by inclusion of alkali metal polyphosphate and alkali metal diethylene triamine penta(methylene phosphonate).","U.S. Pat.","No.","3,903,244 describes a hydrogen peroxide concentrate containing up to 50% hydrogen peroxide, 1 to 3% of a soluble amino(methyl phosphonic acid) or a salt thereof, and 0.05 to 0.5% of phenol.","The concentrate is useful in the preparation of highly acid metal pickling baths.","Stabilizing compounds are useful for a wide area of application, such as soil remediation, metal etch bath, denture bleaching, bleaching of fats, oils, waxes,, wastewater treatment, paper pulp bleaching, textile bleaching, CMP applications or semiconductor applications.","In cleaning application, for example in semiconductor application, oxidizing solutions have been investigated extensively.","Since the invention of the RCA cleaning by Kern et al.","in 1965 (W. Kern and D. A. Pautinen, RCA Review 31, 187, 1970) this cleaning cycle has become the most used for semiconductors.","The conventional RCA cleaning consists of two steps: an alkaline solution, the so called SC1 solution and an acidic solution, SC2.The SC1 solution is composed of 1 part ammonia (NH4OH), 1 part hydrogen peroxide (H2O2) and 5 parts ultra pure water (H2O) and is often referred to as APM-cleaning (i.e.","Ammonia Peroxide Mixture).","Originally it was used to remove organic residues by oxidation.","Later it has been proven to be very efficient to remove particles.","A drawback of the SC1 solution is that metals precipitate on silicon surfaces; especially aluminum, iron and zinc have been shown to adsorb strongly on the wafer surface (Mertens et al., Proc.","of the 8th Internat.","Symp.","On Silicon Materials Science and Technology PV98-1 (1998)).","In addition, especially Fe and Cu are found to catalyze the decomposition reaction of the peroxide (Mertens et al., Proc.","of the 5th Internat.","Symp.","on Cleaning Technology in Semiconductor Device Manufacturing PV97-35 (1997)) leading to a decrease in the bath lifetime.","In order to remove the metallic surface contamination, the SC2 solution consisting of 1 part hydrochloric acid, 1 part hydrogen peroxide and 6 parts ultra-pure water is used.","However, it is expensive to get hydrochloric acid of sufficient quality for the usage in SC2 solution.","There is also a risk of re-contaminating the surface with particles.","Problems also occur in spray tools due the corrosive behavior of hydrochloric acid.","With the progress in semiconductor manufacturing the requirements concerning particle and metal contamination as well as roughness of the silicon surfaces became more stringent.","This led to a number of variations of the RCA clean.","The potential problems related to the SC2 and the consideration to reduce process time and equipment by leaving out this acidic step led to the development of single-stage cleaning procedures.","This can be done by using chemicals with reduced amount of metallic impurities.","For that purpose, advanced purification procedures are established for obtaining ultra-pure water, ammonia and hydrogen peroxide.","However, these chemicals are very expensive and the purity is not always assured when they are used in a cleaning bath.","Moreover, the cleaning solution is not very robust with respect to metal contamination from the semiconductor substrate and from the hardware.","Besides this, having an extra step in the cleaning cycle to remove residual metallic contamination implies extra hardware, e.g.","a SC2-tank and a rinse tank need to be used, and more chemicals.","Leaving out this extra step will results in a reduction of the hardware cost and a reduction of the amount of chemicals used in the cleaning cycle.","EP 528053 describes a method for treating a surface of a substrate with a surface treatment composition.","The surface treatment composition comprises a liquid medium containing a complexing agent as a metal deposition preventive.","The surface treatment composition is improved by incorporating at least two complexing agents.","A first complexing agent is preferably an aromatic hydrocarbon ring with at least an OH or O− group bonded to a carbon atom constituting the ring.","A second complexing agent is compound having a donor atom, such as heterocyclic amines.","Since this cleaning composition comprises two complexing agents, drawbacks such as higher cost and increased waste treatment are observed.","Moreover, aromatic hydrocarbons such as Tiron, Catechol derivatives are hazardous for the environment and for humans.","U.S. Pat.","No.","5,290,361 and U.S. Pat.","No.","5,302,311 describes an aqueous hydrogen peroxide solution further comprising a complexing compound containing phosphonic acid groups and showing complexing ability.","Cleaning solution comprising phosphonic acid goups are not effective in removing or suppressing Al from the substrate.","Moreover, enhanced deposition of Cu has been measured.","This makes the cleaning solutions less suitable.","In the present invention, the problems related to removal of metals as mentioned in the prior art, are avoided.","AIMS OF THE INVENTION It is an aim of the invention to provide a stable composition comprising an oxidizing compound and a complexing compound.","It is a further aim of the invention to provide a new composition for treating a surface which is stable and provokes less or no metal precipitation on the surface.","Another aim of the invention is to provide a new cleaning solution.","Another aim of the present invention is to provide an efficient APM-cleaning solution having a good robustness with respect to metal contamination.","A further aim is to provide a new single-step method for cleaning semiconductor surfaces.","FIGURE CAPTIONS FIG.","1 chemical structure of complexing molecules FIG.","2 bath age (minutes) as function of the normalized H2O2 concentration, 1=no complexing agent; 30 2=DEHP; 3=10×DEHP; 4=1-EMHP; 5=10×1-EMHP; 6-DMHP and 7=10×DMHP.","SUMMARY OF THE INVENTION In a first aspect of this invention, a composition is disclosed comprising an oxidizing compound and a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.","The oxidizing compound can be in the form of an aqueous solution.","The complexing compound is for complexing metal ions.","Metal ions can be present in the solution or in an external medium being contacted with the solution.","Depending on the metal ion being complexed, one or more complexing molecules/metal ion are required.","In an embodiment of this first aspect, said composition as recited in the first aspect of this invention can further comprise an alkaline compound.","In a further embodiment of this first aspect, said organic side chain can comprise an aliphatic side chain, a heterocyclic side chain or an aromatic side chain.","In a further embodiment of the first aspect of this invention, R3 and R4 can be hydrogen while R1 and R2 can be a functionalized aliphatic side chain.","Preferably, said complexing compound is one of the group consisting of DEHP, 1-EMHP, 2-EMHP, DMHP, PEPH, PMHP, ECMHP and ECEHP.","In a further embodiment of this first aspect, a solution as recited in the first aspect of this invention is disclosed characterized in that said oxidizing compound is hydrogen peroxide.","Hydrogen peroxide will be stabilized by the addition of the complexing compound, such that decomposition is substantially inhibited.","In a further embodiment of this first aspect of the invention, a composition as disclosed wherein said oxidizing compound is hydrogen peroxide.","In a further embodiment of this first aspect, a composition as recited in the first embodiment of the first aspect of the invention is disclosed wherein alkaline compound comprises an inorganic or organic basic compound.","Said alkaline compound can be ammonia or an organic amine, preferably an organic amine chosen from the group consisting of tetraalkylammoniumhydroxide, alkanolamine, choline(hydroxyltrialkylammoniumhydroxide) and guanidine compounds.","In a second aspect of this invention, a method for treating a semiconductor substrate is disclosed.","Said semicondcutor substrate is treated with a composition comprising an aqueous solution of a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.","Said composition can further comprise an oxidizing compound.","Said composition can be used for treating a substrate, such that particles are oxidized and metallic contamination is removed.","The complexing molecule is for complexing metallic residues being present on the substrate and in the solution.","Additionally, the lifetime of the solution is increased since de decomposition of the oxidizing compound is substantially inhibited.","Said solution is particularly suitable for cleaning a semiconductor substrate.","Said composition for cleaning a semiconductor substrate can be any composition described in the first aspect of this invention.","In an embodiment of this second aspect of this invention, said R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.","Said organic side chain can comprise an aliphatic side chain, a heterocyclic side chain or an aromatic side chain.","In a further embodiment of this second aspect of the invention, R3 and R4 can be hydrogen while R1 and R1 can be a functionalized aliphatic side chain.","In a further embodiment, said complexing compound is one of the group consisting of DEHP, 1-EMHP, 2-EMHP, DMHP, PEPH, PMHP, ECMHP and ECEHP.","In another embodiment of the second aspect of the present invention, said oxidizing compound can be hydrogen peroxide.","In an embodiment of the second aspect of this invention, said composition can further comprise an alkaline compound.","Said alkaline compound can comprise an inorganic or organic basic compound.","Said basic compound can be chosen from the group consisting of ammonia and organic amine, preferably an organic amine chosen from the group consisting of tetraalkylammoniumhydroxide, alkanolamine, choline(hydroxyltrialkylammoniumhydroxide) and guanidine compounds.","In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed wherein the concentration of hydrogen peroxide in the solution lies between 0.001 to 30 weight %.","In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed in which the amount of the complexing agent lies between 0.1 and 1000 ppm of said solution.","In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed wherein the amount of the alkaline compound lies between 0.001 and 30 weight %.","DETAILED DESCRIPTION OF THE INVENTION In relation to the appended drawings the present invention is described in detail in the sequel.","It is apparent however that a person skilled in the art can imagine several other equivalent embodiments or other ways of executing the present invention.","In an aspect of this invention, said composition comprises an oxidizing compound and a complexing compound.","Said oxidizing compound can be in the form of an aqueous solution.","Said complexing agent can have a chemical formula as given in FIG.","1.R1, R2, R3 and R4 are independently selected from the group comprising hydrogen (H) or any organic group.","R1, R2, R3,or R4 can have a different chemical structure.","For the purpose of this invention, said complexing agent as mentioned above is generally referred to as pyridinone.","Said organic group can be every possible sequence of C, N, O or S atoms linked to each other by single, double or triple bonds such that the complexing properties of the final complexing agent are assured.","Said organic group can be selected from the group comprising aliphatic side chains, heterocycles and aromatic structures.","Said organic side chain is every possible sequence of carbon, atoms linked to each other by a single, double or triple bound and optionally characterised by the presence of functional groups linked to the carbon atoms.","Functional groups can be alcohol, carboxyl, carbonyl, aldehyde, keton, ether, ester, amine, amide, halogen containing groups.","Said heterocycle can be one of the group comprising a crown ether, a cryptant, a calixarene, .",".",".","In a preferred embodiment, R3 and R4 are H atoms, while R1 and R2 are a methyl, ethyl, (iso)propyl or butyl group.","Said methyl, ethyl, (iso) propyl or butyl group can functionalized, e.g.","with a carboxyl group (COOH or COO−).","Preferably, the complexing molecules as described in FIG.","2 are used.","DEHP=1,2-Diethyl-3-hydroxy-4(1H)-pyridinone 1-EMHP=1-Ethyl-2-methyl-3-hydroxy-4(1H)-pyridinone 2-EMHP=1-Methyl-2-ethyl-3-hydroxy-4(1H)-pyridinone DMHP=1,2-Dimethyl-3-hydroxy-4(1H)-pyridinone PEPH=1-Propyl-2-ethyl-3-hydroxy-4(1H)-pyridinone PMHP=1-Propyl-2-methyl-3-hydroxy-4(1H)-pyridinone ECMHP=1- (2′Carboxyethyl)-methyl-3-hydroxy-4(1H)-pyridinone ECEHP=1-(2′Carboxyethyl)-2-ethyl-3-hydroxy-4(1H)-pyridinone.","Although the addition of the amount of complexing agent in the composition in this invention is not particularly limited, it depends on the degree of metal contamination -and on the kind of other compounds being present in the solution.","Furtheron, the amount of complexing agent depends on the specific chemical structure of the complexing compound.","The amount of the complexing agent lies 0.01 and 10000 ppm, between 0.1 and 1000 ppm of said composition.","For this application, ppm should be understood as parts per million in the composition.","The amount of complexing compound depends on the specific complexing compound.","Since said complexing agents do not form a 1:1 metal/complexing agent complex, higher concentrations of complexing agents are required, compared to other complexing agents such as EDTA.","Typical values for the stoichiometry CA/metal for Fe, Al, Cu and Zn are given in the table: Complex Stability Constants: Fe1 Al2 Cu Zn Fe2 Al2 Cu Zn2 DMHP DMHP DMHP DMHP DEHP DEHP DEHP DEHP [ML]/[M] [L] 15.10 12.20 10.62 7.19 15.2 13.42 10.74 7.70 [ML2]/[ML] 11.51 11.05 8.99 6.34 11.76 11.64 9.07 6.09 [L] [ML3]/[ML2] 9.27 9.37 9.78 8.48 5.12 [L] [MLx]/[M] 35.88 32.62 19.61 13.53 36.8 33.54 19.81 18.91 [L]x 2 DMHP 1-EMHP DEHP [MLx]/[M] 37.2/36.4 37.7 36.8 [L]x PFe 19.4 19.7 1X = 3 2Values have been taken from different authors and determined by different techniques.","Said oxidizing compound can be every chemical compound having oxidizing properties.","E.g.","organic species, metallic compounds, inorganic particles, silicon, etc.","can be oxidized.","The oxidizing compound is a compound selected from the group comprising hydrogen peroxide or oxidizing anions.","The oxidizing anions can be e.g.","nitric acid and its salts, nitrate, persulfate, periodate, perbromate, perchlorate, iodate, bromate and chlorate salts of ammonium.","Preferably, the oxidizing compound is hydrogen peroxide.","The concentration of the oxidizing compound can be, but is not limited hereto, between 0.0001 and 99 weight %, between 0.001 and 90 weight % and preferebly between 0.001 to 30 weight %.","In this application, weight % should be understood as the percentage of weight of the specified compound in the composition.","Said composition can further comprise an alkaline compound.","The alkaline compound or base can be every chemical compound with a pH higher than 7.The alkaline compound can be an organic or inorganic compound.","The alkaline compound can be an organic base, ammonia, ammoniumhydroxide, or an alkaline solution containing metal ions such as potassium or sodium.","Said organic base can be a quaternary ammonium hydroxide such as tetraalkyl ammonium hydroxide in which the alkyl groups can contain hydroxy- and alkoxy-containing groups with 1 to 4 carbon atoms in the alkyl or alkoxy group.","Said organic base can further be an organic amine such as an alkanol amine.","Alkanol amines can be 2-aminoethanol, 1-amino 2-propanol, 1-amino 3-propanol.","Preferably, the alkaline compounds are tetramethyl ammonium hydroxide, and trimethyl 2-hydroxy ethyl ammonium hydroxide (choline) and ammonium hydroxide.","The amount of the alkaline compound lies between 0.0001 and 90 weight %, between 0.001 and 50 weight %, between 0.001 and 30 weight %.","Said composiiton can further comprise a surfactant.","In a second aspect of this invention, a method for treating a semiconductor substrate is disclosed.","Said semiconductor substrate is treated with a composition comprising an aqueous solution of a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.","Said composition can further comprise an oxidizing compound.","Said composition can be, but is not limited hereto, the composition described in the first aspect of this invention.","Said composition is particularly usefull for cleaning a substrate such that particles are oxidized and metallic contamination is removed.","The complexing compound is for complexing metals being present on the substrate and in the solution.","Additionally, the lifetime of the solution is increased since de decomposition of the oxidizing compound is substantially inhibited.","A substrate can be, but is not limited hereto, a substrate such as semiconducting material, glass, quartz, ceramics, metal, plastic, magnetic material, superconductor and the like.","Preferably, said substrate is a semiconductor substrate.","Semiconductor substrate can be every possible substrate used in semiconductor processing.","Said semiconductor substrate can be a substrate selected from the group, but not limited hereto, comprising a substrate made of silicon, germanium, gallium arsenide, indium phosphide, etc.","The semiconductor substrate be e.g.","the substrates as mentioned above covered entirely op partially with a thin film of e.g.","an oxide, a nitride, a metal, a polymeric insulating layer, an anti-reflecting coating, a barrier, a photoresist layer, etc.","The present invention is particularly relevant for cleaning or etching a semiconductor substrate of which the surface should be highly clean.","When the composition is used for treating a substrate, the weight concentration range of the alkaline compound in the-cleaning solution are typically but not limited to 0.001-100%, 0.1-20% and preferably 0.1-5% by weight.","For ammonium hydroxide, the weight concentration range of the alkaline compound in the cleaning solution are typically but not limited to 0.001-30%, 0.1-20% and preferably 0.1-5% by weight.","For other alkaline compounds, the weight concentration range is equivalent, and function of the strength of the alkaline compound.","For peroxide, the weight concentration the hydrogenperoxide is typically but not limited to 0.001-100%, 0.1-20% and preferably 0.1-5% by weight.","In the preferred embodiment of this invention, a composition for treating a semiconductor surface comprises ammonium hydroxide, hydrogen peroxide, water (hereafter called APM mictures) and additionally a complexing agent.","Said complexing agent is one of the group consisting of the molecules as described in FIG.","2.APM-cleaning mixtures comprising a complexing agent according to the present invention are robust with respect to metal contamination coming from the fresh chemicals as well as with respect to metal contamination introduced in the course of its use for cleaning.","The robustness of the basic APM process can be improved by the addition of complexing agents which keep the metals in solution and prevent the above mentioned catalysis of the peroxide decomposition.","The volume mixing ratio of NH4OH(296)/H2O2(30%)/H2O is typically, but not limited hereto, 0.25/1/5.The cleaning solution is prepared with the amounts as described above and afterwards the semiconductor substrate is treated with the cleaning solution.","In the best mode known to the applicant, molecule DMHP is selected and added in the amounts described above.","The complexing agent can be added as the pure compound to the cleaning solution.","Alternatively, the complexing agent can be dissolved in either water, ammonia or peroxide or a dilution of the two latter chemicals and added as such to the cleaning solution.","It is a further aim of the invention to disclose a process for treating a semiconductor substrate comprising the steps of treating said semiconductor substrate with the cleaning solution as described above and drying said semiconductor substrate.","An optional step: rinsing said semiconductor substrate can be performed after said treating said semiconductor substrate with the cleaning solution as described above.","In the step of treating said semiconductor substrate with said cleaning solution, the semiconductor substrate can be immersed in a bath containing the cleaning solution.","Alternatively, the cleaning solution can be dispensed or sprayed onto the semiconductor substrate for instance by using a spray processor.","In all cases, the cleaning performance of the solution can be enhanced by using a megasonic transducer.","The temperature range for treating the semiconductor substrate with the cleaning solution is typically but not limited to 0-95 degrees Celcius, 10-80 degrees Celcius and preferably between 20-70 degrees Celcius.","In the step of drying the semiconductor substrate, several techniques known in the art can be used, e.g.","spin-drying, Maragoni-drying, drying techniques using organic vapours.","The step of rinsing the semiconductor substrate comprises treating the semiconductor substrate with DI water or treating the semiconductor substrate with a diluted acidic solution or with DI water containing the complexing agent in an amount of 1 to 100000 ppm, 10 to 10000 ppm and by preference 100 to 1000 ppm.","It is a further aim of the invention to describe a process for treating a semiconductor substrate comprising the step of: treating said semiconductor substrate with any cleaning solution and/or treating said semiconductor substrate with any rinsing solution Said any cleaning solution can be any cleaning solution, not being limited to the compositions described in this application.","Said rinsing solution comprises said complexing agent and water.","Said complexing agent can be any complexing agent described in this application.","The amount of the complexing agent in the composition can be between 1 and 100000 ppm, 10 and 10000 ppm and by preference between 100 and 1000 ppm.","This rinsing solution can also comprise a surfactant in an amount of 0.1 ppm to 10 w %.","A surfactant is a surface-active agent comprising a lyophobic group and a lyophilic group.","The lyophobic group can be a straight-chain alkyl group or a branched-chain alkyl group (C8-C20), an long-chain (C8-C20) alkyl benzene residue, an alkylnaphtalene residue (C3 and greater-length alkyl groups), high-molecular-weight propylene oxide polymers (polyoxypropylene glycol derivatives), long-chain perfluoroalkyl or polysiloxane groups.","Depending upon the lyophilic group, the surfactant can be an anionic, cationic, nonionic or zwitterionic surfactant.","Anionic surfactants can be carboxylic acids or carboxylic acid salts (such as sodium and potassium salts of straight-chain fatty acids), sulfonic acids or sulfonic acid salts (such as linear alkylbenzenesulfonates, higher alkylbenzenesulfonates, benzene-, toluene-, xylene- and cumenesulfonates, ligninsulfonates, petroleum sulfonates, N-acyl-n-alkyltaureates, paraffin sulfonates, secondary n-alkanesulfonates, α-olefin sulfonates, sulfosuccinate esters, alkylnaphtalenesulfonates or isethionates), sulfuric acid ester salts (such as sulfated linear primary alcohols, sulfated polyoxyethylenated straight-chain alcohols or sulfated triglyceride oils), phosphoric and polyphosphoric acid esters.","Cationic surfactants can be primary amines and their salts, diamines and polyamines and their salts, quaternary ammonium salts (such as tetralkylammonium salts or imidazolinium salts), polyoxyethylenated long-chain amines (RN(CH2CH2O)xH]2), quaternized polyoxyethylenated long-chain amines or amine oxides (such as N-alkyldimethylamine oxides).","Nonionic surfactants can be polyoxyethylenated alkylphenols, polyoxyethylenated straight-chain alcohols, polyoxyethylenated polyoxypropylene glycols, polyoxyethylenated mercaptans, long-chain carboxylic acid esters (such as glyceryl and polyglyceryl esters of natural fatty acids, propylene glycol, sorbitol or polyoxyethylenated sorbitol esters, polyoxyethylene glycol esters and polyoxyethylenated fatty acids), alkanolamides, tertiary acetylenic glycols, polyoxyethylenated silicones, N-alkylpyrrolidones or alkylpolyglycosides.","Zwitterionic surfactants have both anionic and cationic charges present in the lyophilic portion (such as β-N-alkylaminopropionic acids, N-alkyl-β-iminodipropionic acids, imidazoline carboxylates, N-alkylbetaines, amine oxides, sulfobetaines or sultaines) (M. J. Rosen, Surfactants and Interfacial phenomena, 2nd Edition, John Wiley and Sons, New York, 1989]) No additional alkaline compound should need to be added to the said rinsing solution.","The pH range of said rinsing solution can typically be, but not limited to, between 5 and 8.Said Rinse solution can be dispensed or sprayed onto the semiconductor surface as described above.","During rinsing the performance can also be enhanced by using a megasonic transducer.","The process of treating a semiconductor substrate with a cleaning solution comprising the above mentioned steps can be performed for a certain number of semiconductor substrates.","After treating at least one substrate, but preferably after treating more substrates, the composition of the cleaning solution can be modified by e.g.","adding extra alkaline compound, adding extra complexing compound, adding oxidizing compound such that the initial composition of the cleaning solution is kept constant as function of the process time.","COMPARATIVE EXAMPLES The present invention will be further described using non-limiting examples and drawings.","The effectiveness of the new class of complexing agents on the inhibition of the metal catalyzed decomposition of peroxide and the prevention of metal outplating on silicon wafers in metal contaminated APM cleaning solutions is described next.","For complexing agents as disclosed in this invention, different model compounds are selected (1-EMHP, 2-EMHP, DMHP, DEHP, PEHP, PMHP, ECMHP and ECEHP) and added to the bath at a concentration of 2.67×10−5 M and at a 10×, 40×, or 50× higher concentration (namely 2.65×10−4 10.6×10−4 and 13.25×10−4 M).","A comparison is made with other types of complexing agents containing as functional groups either phosphonic acids, such as diethylene (kleine letter) triamine penta-methylenephosphonic acid (DTPMP), carboxylic acids, such as ethylene diamino tetra acetic acid (EDTA).","An overview of the different chemicals used for the experiments is given in Table 1.All experiments were done in a class 1000 clean room environment or better.","TABLE 1 Chemicals used for preparation of APM baths.","Chemical Vendor Grade H2O2 30 (w/w) % Ashland TB(*) NH4OH 29 (w/w) % Ashland TB(*) EDTA Fluka DTPMP Monsanto Dequest 2060S DMHP Aldrich (*)TB-grade corresponds with a specification of maximal 100 ppt of metal ions in the chemical.","Example 1 Metal Outplating from APM Mixtures in Presence of Different Complexing Agents The efficiency of complexing agents to suppress the deposition of metallic contamination onto wafer surfaces was studied.","This was done through intentionally spiking well controlled trace amounts of metallic contamination to cleaning solutions.","For these metal deposition tests, p-type monitor wafers with a diameter of 150 mm and <100> orientation were used.","The wafers were pre-cleaned using IMEC Clean® in an automated Steag wet bench (i.e.","SOM+dHF+O3-rinse rendering a perfectly clean hydrophilic surface).","The metal deposition experiments were performed in a static quartz tank with a quartz cover plate.","This tank was not equipped with a megasonic transducer.","APM mixtures were prepared containing 1 w-ppb of different metals of interest with/without the complexing agent added.","The metals spiked to the APM bath were added from AAS-standard solutions (Merck).","After a bath age of 5 minutes, three wafers were immersed for 10 minutes, rinsed for 10 minutes in an overflow rinse tank and dried with a commercially available Marangoni drier (STEAG).","The resulting metal contamination was measured with straight TXRF or VPD-DSE-DC-TXRF (Vapor Phase Decomposition-Droplet Surface Etching-Droplet Collection Total X-Ray Fluorescence).","Determination of Al water surface concentration was done using VPD-DC GF-AAS (Graphite Furnace Atomic Absorption Spectroscopy).","An overview of the resulting metal surface contamination after dipping a clean wafer in an APM spiked with metals and different complexing agents is given in Table 2.TABLE 2 Measured metal surface concentrations after immersion in metal contaminated 0.25/1/5 APM solution at 20-50° C. with different complexing agents present.","Complexing Conc.","CA Conc.","Mt Al (1010 Fe agent (2.7 × 10−5 M) (w-ppb) at/cm2) (1010 at/cm2) Pre-clean only NA NA 2.34 <DL None 0 0 39.3 ± 6 1.5 ± 0.4 None 0 1 360 ± 21 109 ± 14 EDTA 1 1 NM 25.1 ± 0.1 10 1 272 ± 16 NM 100 1 274 2.78 EDTA + 1 + 2 1 257 ± 2 <DL DTPMP 1-EMHP (1) 1 1 356 ± 32 0.51 ± 0.02 10 1 142 ± 68 0.23 1-EMHP (2) 1 1 292 ± 1 0.20 ± 0.00 10 1 27 ± 33 NA 50 1 0.5 ± 0.2 0.26 2.4 ± 1.4 0.16 2-EMHP 1 1 404 0.35 ± 0.01 10 1 248 ± 69 0.22 50 1 0.8 ± 0.2 0.15 DEHP 1 1 369 ± 0 0.33 ± 0.01 10 1 150 ± 41 0.92 50 1 2.6 ± 0.6 0.15 DMHP 1 1 392 ± 0 0.45 ± 0.01 (Aldrich) 10 1 101 ± 2 0.21 50 1 1.2 ± 0.1 NM DMHP 1 1 387 ± 0 0.40 ± 0.02 10 1 230 ± 5 0.18 PEHP 40 1 4.6 ± 0.2 0.14 ± 0.0 PMHP 40 1 1.13 0.14 ± 0.14 ECMHP 40 1 15.0 ± 0.5 0.08 ± 0.04 ECEHP 40 1 14.2 ± 1.7 0.15 ± 0.05 These data show that the addition of EDTA results in a decrease of the final Fe contamination levels but has no effect on the reduction of Al contamination.","The use of DTPMP in a 1 w-ppb metal spiked bath, is very efficient to reduce the final Fe surface concentration but has no effect on suppression of Al deposition.","The new class of pyridinone complexing agents are able to reduce the final Fe and Al surface concentration on the wafer.","Higher concentrations of complexing agent are needed because the pyridones do not form a 1:1 metal/chelate complex.","When using the pyridinones at a concentration of 13.25×10−4 M in the bath, the resulting Fe and Al surface contamination levels are below the values measured on a wafer treated with a clean, i.e.","no metals spiked, APM solution.","Example 2 Decomposition of Peroxide in APM Cleaning Mixtures in Presence of Trace Metal Contamination and Metal Complexing Agents The effect of the addition of a complexing agent to APM cleaning solutions on the kinetics of the decomposition reaction of H2O2 has been investigated.","Well controlled amounts of metallic contamination were added to the cleaning mixture under study.","As hydrogen peroxide decomposes, an amount of oxygen gas is liberated following the overall reaction 2 H2O2⇄O2+2 H2O The decay of the total peroxide concentration in the APM mixture can be monitored by measuring the time-dependent increase of the pressure due to the O2-evolution in a dedicated set-up as described by Schmidt.","Numerical integration over time yields the actual peroxide concentration in the bath.","It is convenient to use peroxide concentrations normalized to its initial value [H2O2]i as [ H 2 O ] n = [ H 2 O 2 ] [ H 2 O 2 ] i Since the decomposition reaction is mainly catalyzed by Fe and in a lesser content Cu (Mertens et al.","Proc.","of the 5th Internat.","Symp.","on Cleaning Technology in Semiconductor Device Manufacturing PV97-35 (1997), the decay of peroxide concentration in a metal contaminated bath and in presence of a CA, illustrates the ability of complexing primarily Fe in the APM bath.","The decomposition rate as function of bath age is determined in APM mixtures (0.25/1/5 29%NH4OH/30% H2O2/H2O) spiked with 1 w-ppb of the metals of interest with and without different complexing agents.","The effect of different additives on the inhibition of the metal catalyzed decomposition reaction of peroxide in APM cleaning mixtures is shown in FIG.","2.This graph shows the normalized H2O2 concentration as function of bath age for an APM mixture at 50° C. spiked with different complexing agents and the metals of interest.","All complexing agents are found to suppress to some extent the decomposition reaction, at least when the mixture is fresh.","For some CA, the suppression action vanishes over time.","This may be attributed to the destruction of the complexing agent or more specifically of the metal-complex in the hot APM.","The time over which they remain active in complexing metals is summarized in Table 3 and can be used to establish the effective lifetime.","In example 1 it was demonstrated that higher CA concentrations have to be used in the cleaning solution.","FIG.","1 and Table 3 show that with increasing complexing agent concentration, the effective lifetime of the bath can be increased substantially.","Increasing the complexing agent concentration even further would result in a more prolonged lifetime.","TABLE 3 Effective lifetime of complexing agents in 0.25/1/5 metal spiked APM-cleaning mixtures at 50° C. Conc.","CA Effective lifetime Complexing agent (2.7 × 10−5 M) (min) None 0 40 DEHP 1 40 10 120 1-EMHP (1) 1 40 10 50 1-EMHP (2) 1 60 10 90 2-EMHP 1 30 10 40 DMHP FfU 1 40 10 100 DMHP Aldrich 1 20 10 60"]],"string":"[\n [\n \"Composition comprising an oxidizing and complexing compound\",\n \"The present invention is related to a composition comprising an oxidizing compound and a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.\",\n \"The oxidizing compound can be in the form of an aqueous solution.\",\n \"The complexing compound is for complexing metal ions.\",\n \"Metal ions can be present in the solution or in an external medium being contacted with the solution.\",\n \"The present invention can be used for cleaning a semiconductor substrate.\"\n ],\n [\n \"1.A composition comprising an aqueous solution, said aqueous solution comprising an oxidizing compound and a complexing compound, said complexing compound comprising a chemical formula wherein R1, R2, R3, and R4 are each independently selected from the group consisting of H and an organic side chain.\",\n \"2.The composition as recited in claim 1, further comprising an alkaline compound.\",\n \"3.The composition as recited in claim 1, wherein said organic side chain is selected from the group consisting of an aliphatic side chain, a heterocyclic side chain, and an aromatic side chain.\",\n \"4.The composition as recited in claim 1, wherein R3 and R4 each comprise hydrogen and R1 and R2 each comprise a functionalized aliphatic side chain.\",\n \"5.The composition as recited in claim 1, wherein said complexing compound is selected from the group consisting of 1,2-diethyl-3-hydroxy-4(1H)-pyridinone, 1-ethyl-2-methyl-3-hydroxy-4(1H)-pyridinone, 1-methyl-2-ethyl-3-hydroxy-4(1H)-pyridinone 1,2-dimethyl-3-hydroxy-4(1H)pyridinone, 1-propyl-2-ethyl-3-hydroxy-4(1H)-pyridinone, 1-propyl-2-methyl-3-hydroxy-4(1H)-pyridinone, 1-(2-carboxyethyl)-2-methyl-3-hydroxy-4( 1H)-pyridinone, and 1-(2-carboxyethyl)-2-ethyl-3-hydroxy-4(1H)-pyridinone.\",\n \"6.The composition as recited in claim 1, wherein said oxidizing compound comprises hydrogen peroxide.\",\n \"7.The composition as recited in recited in claim 2, wherein said alkaline compound comprises an inorganic basic compound or an organic basic compound.\",\n \"8.The composition as recited in claim 7, wherein said alkaline compound is selected from the group consisting of ammonia and organic amine compounds.\",\n \"9.The composition as recited in claim 2, wherein an amount of oxidizing compound comprises from 0.001 to 30 weight %.\",\n \"10.The composition as recited in claim 1, wherein an amount of the complexing agent comprises from 0.1 to 1000 ppm of said solution.\",\n \"11.The composition as recited in claim 7, wherein an amount of the inorganic basic compound or the organic basic compound comprises from 0.001 to 30 weight %.\",\n \"12.The composition as recited in claim 1, wherein an amount of said oxidizing compound comprises from 0.001 to 95 weight %.\",\n \"13.A method of treating a semiconductor substrate, wherein said method comprises treating a semiconductor substrate with a composition comprising an aqueous solution, said aqueous solution comprising a complexing compound comprising the chemical formula wherein R1, R2, R3, and R4 are each independently selected from the group consisting of H and an organic side chain.\",\n \"14.The method as recited in claim 13, wherein said composition further comprises an oxidizing compound.\",\n \"15.The method as recited in claim 13, wherein said composition further comprises an alkaline compound.\",\n \"16.The method as recited in claim 13, wherein said organic side chain is selected from the group consisting of an aliphatic side chain, a heterocyclic side chain, and an aromatic side chain.\",\n \"17.The method as recited in claim 13, wherein R3 and R4 each comprise hydrogen and R1 and R2 each comprise a functionalized aliphatic side chain.\",\n \"18.The method as recited in claim 13, wherein said complexing compound is selected from the group consisting of 1,2-diethyl-3-hydroxy-4(1H)-pyridinone, 1-ethyl-2-methyl-3-hydroxy-4(1H)-pyridinone, 1-methyl-2-ethyl-3-hydroxy-4(1H)-pyridinone, 1,2-dimethyl-3-hydroxy-4(1H)pyridinone, 1-propyl-2-ethyl-3-hydroxy-4(1H)-pyridinone, 1-propyl-2-methyl-3-hydroxy-4(1H)-pyridinone, 1-(2-carboxyethyl)-2-methyl-3-hydroxy-4(1H)-pyridinone, and 1-(2-carboxyethyl)-2-ethyl-3-hydroxy-4(1H)-pyridinone.\",\n \"19.The method as recited in claim 14, wherein said oxidizing compound comprises hydrogen peroxide.\",\n \"20.The method as recited in claim 15, wherein the alkaline compound comprises an inorganic basic compound or an organic basic compound.\",\n \"21.The method as recited in claim 20, wherein said inorganic basic compound or said organic basic compound is selected from the group consisting of ammonia and organic amine compounds.\",\n \"22.The method as recited in claim 14, wherein an amount of the oxidizing compound comprises from 0.001 to 30 weight %.\",\n \"23.The method as recited in claim 15, wherein an amount of the alkaline compound comprises from 0.001 to 30 weight %.\",\n \"24.The method as recited in claim 13, wherein an amount of said complexing compound comprises from 0.1 ppm to 1000 ppm.\",\n \"25.Use of a composition as recited in claim 1 in a field selected from the group consisting of soil remediation, metal etch bath, denture bleaching, bleaching of fats, bleaching of oils, bleaching of waxes, wastewater treatment, paper pulp bleaching, and textile bleaching.\",\n \"26.The composition as recited in claim 8, wherein said organic amine is selected from the group consisting of tetraalkylammoniumhydroxide compounds, alkanolamine compounds, choline(hydroxyltrialkylammoniumhydroxide) compounds, and guanidine compounds.\",\n \"27.The method as recited in claim 21, wherein said organic amine is selected from the group consisting of tetraalkylammoniumhydroxide compounds, alkanolamine compounds, choline(hydroxyltrialkylammoniumhydroxide) compounds, and guanidine compounds.\"\n ],\n [\n \"<SOH> FIELD OF THE INVENTION <EOH>This invention is situated in the field of products and methods for the stabilizing a composition comprising an oxidizing compound.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>In a first aspect of this invention, a composition is disclosed comprising an oxidizing compound and a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.\",\n \"The oxidizing compound can be in the form of an aqueous solution.\",\n \"The complexing compound is for complexing metal ions.\",\n \"Metal ions can be present in the solution or in an external medium being contacted with the solution.\",\n \"Depending on the metal ion being complexed, one or more complexing molecules/metal ion are required.\",\n \"In an embodiment of this first aspect, said composition as recited in the first aspect of this invention can further comprise an alkaline compound.\",\n \"In a further embodiment of this first aspect, said organic side chain can comprise an aliphatic side chain, a heterocyclic side chain or an aromatic side chain.\",\n \"In a further embodiment of the first aspect of this invention, R3 and R4 can be hydrogen while R1 and R2 can be a functionalized aliphatic side chain.\",\n \"Preferably, said complexing compound is one of the group consisting of DEHP, 1-EMHP, 2-EMHP, DMHP, PEPH, PMHP, ECMHP and ECEHP.\",\n \"In a further embodiment of this first aspect, a solution as recited in the first aspect of this invention is disclosed characterized in that said oxidizing compound is hydrogen peroxide.\",\n \"Hydrogen peroxide will be stabilized by the addition of the complexing compound, such that decomposition is substantially inhibited.\",\n \"In a further embodiment of this first aspect of the invention, a composition as disclosed wherein said oxidizing compound is hydrogen peroxide.\",\n \"In a further embodiment of this first aspect, a composition as recited in the first embodiment of the first aspect of the invention is disclosed wherein alkaline compound comprises an inorganic or organic basic compound.\",\n \"Said alkaline compound can be ammonia or an organic amine, preferably an organic amine chosen from the group consisting of tetraalkylammoniumhydroxide, alkanolamine, choline(hydroxyltrialkylammoniumhydroxide) and guanidine compounds.\",\n \"In a second aspect of this invention, a method for treating a semiconductor substrate is disclosed.\",\n \"Said semicondcutor substrate is treated with a composition comprising an aqueous solution of a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.\",\n \"Said composition can further comprise an oxidizing compound.\",\n \"Said composition can be used for treating a substrate, such that particles are oxidized and metallic contamination is removed.\",\n \"The complexing molecule is for complexing metallic residues being present on the substrate and in the solution.\",\n \"Additionally, the lifetime of the solution is increased since de decomposition of the oxidizing compound is substantially inhibited.\",\n \"Said solution is particularly suitable for cleaning a semiconductor substrate.\",\n \"Said composition for cleaning a semiconductor substrate can be any composition described in the first aspect of this invention.\",\n \"In an embodiment of this second aspect of this invention, said R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.\",\n \"Said organic side chain can comprise an aliphatic side chain, a heterocyclic side chain or an aromatic side chain.\",\n \"In a further embodiment of this second aspect of the invention, R3 and R4 can be hydrogen while R1 and R1 can be a functionalized aliphatic side chain.\",\n \"In a further embodiment, said complexing compound is one of the group consisting of DEHP, 1-EMHP, 2-EMHP, DMHP, PEPH, PMHP, ECMHP and ECEHP.\",\n \"In another embodiment of the second aspect of the present invention, said oxidizing compound can be hydrogen peroxide.\",\n \"In an embodiment of the second aspect of this invention, said composition can further comprise an alkaline compound.\",\n \"Said alkaline compound can comprise an inorganic or organic basic compound.\",\n \"Said basic compound can be chosen from the group consisting of ammonia and organic amine, preferably an organic amine chosen from the group consisting of tetraalkylammoniumhydroxide, alkanolamine, choline(hydroxyltrialkylammoniumhydroxide) and guanidine compounds.\",\n \"In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed wherein the concentration of hydrogen peroxide in the solution lies between 0.001 to 30 weight %.\",\n \"In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed in which the amount of the complexing agent lies between 0.1 and 1000 ppm of said solution.\",\n \"In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed wherein the amount of the alkaline compound lies between 0.001 and 30 weight %.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"FIELD OF THE INVENTION This invention is situated in the field of products and methods for the stabilizing a composition comprising an oxidizing compound.\",\n \"STATE OF THE ART Chemical solutions comprising an oxidizing compound such as hydrogen peroxide are used in a wide area of applications, for example, as bleaching agent for paper pulp applications, dental bleaching composition or as cleaning agents.\",\n \"Chemical solutions comprising an oxidizing compound have often problems related to the stability of the solution.\",\n \"In pure form, aqueous solution are stable over extended periods of time.\",\n \"However, the presence of certain metal ions in the solution causes decomposition of the oxidizing compound.\",\n \"Consequently, stabilizers to prevent such decomposition should be added.\",\n \"Stabilizers can be e.g.\",\n \"a complexing compound, such that the complexing compound will bind to the metal and consequently, the metal is not available for reaction with the oxidizing compound.\",\n \"Thus, the decomposition of the oxidizing compound is substantially inhibited and the lifetime of the solution is increased.\",\n \"Very stringent specifications must be met by oxidizing solutions for specialized applications such as semiconductor applications or reagent chemicals.\",\n \"An overview of stabilizing oxidizing compound, and more specifically hydrogen peroxide solutions, is given in Kirk-Othmer Encyclopedia of Chemical Technology (4th edition), vol.\",\n \"13 p. 965.U.S.\",\n \"Pat.\",\n \"No.\",\n \"4,239,643 discloses an aqueous peroxide-containing solutions used in bleaching of cellulose fiber, wherein the stability of the peroxide is very greatly increased by inclusion of alkali metal polyphosphate and alkali metal diethylene triamine penta(methylene phosphonate).\",\n \"U.S. Pat.\",\n \"No.\",\n \"3,903,244 describes a hydrogen peroxide concentrate containing up to 50% hydrogen peroxide, 1 to 3% of a soluble amino(methyl phosphonic acid) or a salt thereof, and 0.05 to 0.5% of phenol.\",\n \"The concentrate is useful in the preparation of highly acid metal pickling baths.\",\n \"Stabilizing compounds are useful for a wide area of application, such as soil remediation, metal etch bath, denture bleaching, bleaching of fats, oils, waxes,, wastewater treatment, paper pulp bleaching, textile bleaching, CMP applications or semiconductor applications.\",\n \"In cleaning application, for example in semiconductor application, oxidizing solutions have been investigated extensively.\",\n \"Since the invention of the RCA cleaning by Kern et al.\",\n \"in 1965 (W. Kern and D. A. Pautinen, RCA Review 31, 187, 1970) this cleaning cycle has become the most used for semiconductors.\",\n \"The conventional RCA cleaning consists of two steps: an alkaline solution, the so called SC1 solution and an acidic solution, SC2.The SC1 solution is composed of 1 part ammonia (NH4OH), 1 part hydrogen peroxide (H2O2) and 5 parts ultra pure water (H2O) and is often referred to as APM-cleaning (i.e.\",\n \"Ammonia Peroxide Mixture).\",\n \"Originally it was used to remove organic residues by oxidation.\",\n \"Later it has been proven to be very efficient to remove particles.\",\n \"A drawback of the SC1 solution is that metals precipitate on silicon surfaces; especially aluminum, iron and zinc have been shown to adsorb strongly on the wafer surface (Mertens et al., Proc.\",\n \"of the 8th Internat.\",\n \"Symp.\",\n \"On Silicon Materials Science and Technology PV98-1 (1998)).\",\n \"In addition, especially Fe and Cu are found to catalyze the decomposition reaction of the peroxide (Mertens et al., Proc.\",\n \"of the 5th Internat.\",\n \"Symp.\",\n \"on Cleaning Technology in Semiconductor Device Manufacturing PV97-35 (1997)) leading to a decrease in the bath lifetime.\",\n \"In order to remove the metallic surface contamination, the SC2 solution consisting of 1 part hydrochloric acid, 1 part hydrogen peroxide and 6 parts ultra-pure water is used.\",\n \"However, it is expensive to get hydrochloric acid of sufficient quality for the usage in SC2 solution.\",\n \"There is also a risk of re-contaminating the surface with particles.\",\n \"Problems also occur in spray tools due the corrosive behavior of hydrochloric acid.\",\n \"With the progress in semiconductor manufacturing the requirements concerning particle and metal contamination as well as roughness of the silicon surfaces became more stringent.\",\n \"This led to a number of variations of the RCA clean.\",\n \"The potential problems related to the SC2 and the consideration to reduce process time and equipment by leaving out this acidic step led to the development of single-stage cleaning procedures.\",\n \"This can be done by using chemicals with reduced amount of metallic impurities.\",\n \"For that purpose, advanced purification procedures are established for obtaining ultra-pure water, ammonia and hydrogen peroxide.\",\n \"However, these chemicals are very expensive and the purity is not always assured when they are used in a cleaning bath.\",\n \"Moreover, the cleaning solution is not very robust with respect to metal contamination from the semiconductor substrate and from the hardware.\",\n \"Besides this, having an extra step in the cleaning cycle to remove residual metallic contamination implies extra hardware, e.g.\",\n \"a SC2-tank and a rinse tank need to be used, and more chemicals.\",\n \"Leaving out this extra step will results in a reduction of the hardware cost and a reduction of the amount of chemicals used in the cleaning cycle.\",\n \"EP 528053 describes a method for treating a surface of a substrate with a surface treatment composition.\",\n \"The surface treatment composition comprises a liquid medium containing a complexing agent as a metal deposition preventive.\",\n \"The surface treatment composition is improved by incorporating at least two complexing agents.\",\n \"A first complexing agent is preferably an aromatic hydrocarbon ring with at least an OH or O− group bonded to a carbon atom constituting the ring.\",\n \"A second complexing agent is compound having a donor atom, such as heterocyclic amines.\",\n \"Since this cleaning composition comprises two complexing agents, drawbacks such as higher cost and increased waste treatment are observed.\",\n \"Moreover, aromatic hydrocarbons such as Tiron, Catechol derivatives are hazardous for the environment and for humans.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,290,361 and U.S. Pat.\",\n \"No.\",\n \"5,302,311 describes an aqueous hydrogen peroxide solution further comprising a complexing compound containing phosphonic acid groups and showing complexing ability.\",\n \"Cleaning solution comprising phosphonic acid goups are not effective in removing or suppressing Al from the substrate.\",\n \"Moreover, enhanced deposition of Cu has been measured.\",\n \"This makes the cleaning solutions less suitable.\",\n \"In the present invention, the problems related to removal of metals as mentioned in the prior art, are avoided.\",\n \"AIMS OF THE INVENTION It is an aim of the invention to provide a stable composition comprising an oxidizing compound and a complexing compound.\",\n \"It is a further aim of the invention to provide a new composition for treating a surface which is stable and provokes less or no metal precipitation on the surface.\",\n \"Another aim of the invention is to provide a new cleaning solution.\",\n \"Another aim of the present invention is to provide an efficient APM-cleaning solution having a good robustness with respect to metal contamination.\",\n \"A further aim is to provide a new single-step method for cleaning semiconductor surfaces.\",\n \"FIGURE CAPTIONS FIG.\",\n \"1 chemical structure of complexing molecules FIG.\",\n \"2 bath age (minutes) as function of the normalized H2O2 concentration, 1=no complexing agent; 30 2=DEHP; 3=10×DEHP; 4=1-EMHP; 5=10×1-EMHP; 6-DMHP and 7=10×DMHP.\",\n \"SUMMARY OF THE INVENTION In a first aspect of this invention, a composition is disclosed comprising an oxidizing compound and a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.\",\n \"The oxidizing compound can be in the form of an aqueous solution.\",\n \"The complexing compound is for complexing metal ions.\",\n \"Metal ions can be present in the solution or in an external medium being contacted with the solution.\",\n \"Depending on the metal ion being complexed, one or more complexing molecules/metal ion are required.\",\n \"In an embodiment of this first aspect, said composition as recited in the first aspect of this invention can further comprise an alkaline compound.\",\n \"In a further embodiment of this first aspect, said organic side chain can comprise an aliphatic side chain, a heterocyclic side chain or an aromatic side chain.\",\n \"In a further embodiment of the first aspect of this invention, R3 and R4 can be hydrogen while R1 and R2 can be a functionalized aliphatic side chain.\",\n \"Preferably, said complexing compound is one of the group consisting of DEHP, 1-EMHP, 2-EMHP, DMHP, PEPH, PMHP, ECMHP and ECEHP.\",\n \"In a further embodiment of this first aspect, a solution as recited in the first aspect of this invention is disclosed characterized in that said oxidizing compound is hydrogen peroxide.\",\n \"Hydrogen peroxide will be stabilized by the addition of the complexing compound, such that decomposition is substantially inhibited.\",\n \"In a further embodiment of this first aspect of the invention, a composition as disclosed wherein said oxidizing compound is hydrogen peroxide.\",\n \"In a further embodiment of this first aspect, a composition as recited in the first embodiment of the first aspect of the invention is disclosed wherein alkaline compound comprises an inorganic or organic basic compound.\",\n \"Said alkaline compound can be ammonia or an organic amine, preferably an organic amine chosen from the group consisting of tetraalkylammoniumhydroxide, alkanolamine, choline(hydroxyltrialkylammoniumhydroxide) and guanidine compounds.\",\n \"In a second aspect of this invention, a method for treating a semiconductor substrate is disclosed.\",\n \"Said semicondcutor substrate is treated with a composition comprising an aqueous solution of a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.\",\n \"Said composition can further comprise an oxidizing compound.\",\n \"Said composition can be used for treating a substrate, such that particles are oxidized and metallic contamination is removed.\",\n \"The complexing molecule is for complexing metallic residues being present on the substrate and in the solution.\",\n \"Additionally, the lifetime of the solution is increased since de decomposition of the oxidizing compound is substantially inhibited.\",\n \"Said solution is particularly suitable for cleaning a semiconductor substrate.\",\n \"Said composition for cleaning a semiconductor substrate can be any composition described in the first aspect of this invention.\",\n \"In an embodiment of this second aspect of this invention, said R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.\",\n \"Said organic side chain can comprise an aliphatic side chain, a heterocyclic side chain or an aromatic side chain.\",\n \"In a further embodiment of this second aspect of the invention, R3 and R4 can be hydrogen while R1 and R1 can be a functionalized aliphatic side chain.\",\n \"In a further embodiment, said complexing compound is one of the group consisting of DEHP, 1-EMHP, 2-EMHP, DMHP, PEPH, PMHP, ECMHP and ECEHP.\",\n \"In another embodiment of the second aspect of the present invention, said oxidizing compound can be hydrogen peroxide.\",\n \"In an embodiment of the second aspect of this invention, said composition can further comprise an alkaline compound.\",\n \"Said alkaline compound can comprise an inorganic or organic basic compound.\",\n \"Said basic compound can be chosen from the group consisting of ammonia and organic amine, preferably an organic amine chosen from the group consisting of tetraalkylammoniumhydroxide, alkanolamine, choline(hydroxyltrialkylammoniumhydroxide) and guanidine compounds.\",\n \"In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed wherein the concentration of hydrogen peroxide in the solution lies between 0.001 to 30 weight %.\",\n \"In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed in which the amount of the complexing agent lies between 0.1 and 1000 ppm of said solution.\",\n \"In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed wherein the amount of the alkaline compound lies between 0.001 and 30 weight %.\",\n \"DETAILED DESCRIPTION OF THE INVENTION In relation to the appended drawings the present invention is described in detail in the sequel.\",\n \"It is apparent however that a person skilled in the art can imagine several other equivalent embodiments or other ways of executing the present invention.\",\n \"In an aspect of this invention, said composition comprises an oxidizing compound and a complexing compound.\",\n \"Said oxidizing compound can be in the form of an aqueous solution.\",\n \"Said complexing agent can have a chemical formula as given in FIG.\",\n \"1.R1, R2, R3 and R4 are independently selected from the group comprising hydrogen (H) or any organic group.\",\n \"R1, R2, R3,or R4 can have a different chemical structure.\",\n \"For the purpose of this invention, said complexing agent as mentioned above is generally referred to as pyridinone.\",\n \"Said organic group can be every possible sequence of C, N, O or S atoms linked to each other by single, double or triple bonds such that the complexing properties of the final complexing agent are assured.\",\n \"Said organic group can be selected from the group comprising aliphatic side chains, heterocycles and aromatic structures.\",\n \"Said organic side chain is every possible sequence of carbon, atoms linked to each other by a single, double or triple bound and optionally characterised by the presence of functional groups linked to the carbon atoms.\",\n \"Functional groups can be alcohol, carboxyl, carbonyl, aldehyde, keton, ether, ester, amine, amide, halogen containing groups.\",\n \"Said heterocycle can be one of the group comprising a crown ether, a cryptant, a calixarene, .\",\n \".\",\n \".\",\n \"In a preferred embodiment, R3 and R4 are H atoms, while R1 and R2 are a methyl, ethyl, (iso)propyl or butyl group.\",\n \"Said methyl, ethyl, (iso) propyl or butyl group can functionalized, e.g.\",\n \"with a carboxyl group (COOH or COO−).\",\n \"Preferably, the complexing molecules as described in FIG.\",\n \"2 are used.\",\n \"DEHP=1,2-Diethyl-3-hydroxy-4(1H)-pyridinone 1-EMHP=1-Ethyl-2-methyl-3-hydroxy-4(1H)-pyridinone 2-EMHP=1-Methyl-2-ethyl-3-hydroxy-4(1H)-pyridinone DMHP=1,2-Dimethyl-3-hydroxy-4(1H)-pyridinone PEPH=1-Propyl-2-ethyl-3-hydroxy-4(1H)-pyridinone PMHP=1-Propyl-2-methyl-3-hydroxy-4(1H)-pyridinone ECMHP=1- (2′Carboxyethyl)-methyl-3-hydroxy-4(1H)-pyridinone ECEHP=1-(2′Carboxyethyl)-2-ethyl-3-hydroxy-4(1H)-pyridinone.\",\n \"Although the addition of the amount of complexing agent in the composition in this invention is not particularly limited, it depends on the degree of metal contamination -and on the kind of other compounds being present in the solution.\",\n \"Furtheron, the amount of complexing agent depends on the specific chemical structure of the complexing compound.\",\n \"The amount of the complexing agent lies 0.01 and 10000 ppm, between 0.1 and 1000 ppm of said composition.\",\n \"For this application, ppm should be understood as parts per million in the composition.\",\n \"The amount of complexing compound depends on the specific complexing compound.\",\n \"Since said complexing agents do not form a 1:1 metal/complexing agent complex, higher concentrations of complexing agents are required, compared to other complexing agents such as EDTA.\",\n \"Typical values for the stoichiometry CA/metal for Fe, Al, Cu and Zn are given in the table: Complex Stability Constants: Fe1 Al2 Cu Zn Fe2 Al2 Cu Zn2 DMHP DMHP DMHP DMHP DEHP DEHP DEHP DEHP [ML]/[M] [L] 15.10 12.20 10.62 7.19 15.2 13.42 10.74 7.70 [ML2]/[ML] 11.51 11.05 8.99 6.34 11.76 11.64 9.07 6.09 [L] [ML3]/[ML2] 9.27 9.37 9.78 8.48 5.12 [L] [MLx]/[M] 35.88 32.62 19.61 13.53 36.8 33.54 19.81 18.91 [L]x 2 DMHP 1-EMHP DEHP [MLx]/[M] 37.2/36.4 37.7 36.8 [L]x PFe 19.4 19.7 1X = 3 2Values have been taken from different authors and determined by different techniques.\",\n \"Said oxidizing compound can be every chemical compound having oxidizing properties.\",\n \"E.g.\",\n \"organic species, metallic compounds, inorganic particles, silicon, etc.\",\n \"can be oxidized.\",\n \"The oxidizing compound is a compound selected from the group comprising hydrogen peroxide or oxidizing anions.\",\n \"The oxidizing anions can be e.g.\",\n \"nitric acid and its salts, nitrate, persulfate, periodate, perbromate, perchlorate, iodate, bromate and chlorate salts of ammonium.\",\n \"Preferably, the oxidizing compound is hydrogen peroxide.\",\n \"The concentration of the oxidizing compound can be, but is not limited hereto, between 0.0001 and 99 weight %, between 0.001 and 90 weight % and preferebly between 0.001 to 30 weight %.\",\n \"In this application, weight % should be understood as the percentage of weight of the specified compound in the composition.\",\n \"Said composition can further comprise an alkaline compound.\",\n \"The alkaline compound or base can be every chemical compound with a pH higher than 7.The alkaline compound can be an organic or inorganic compound.\",\n \"The alkaline compound can be an organic base, ammonia, ammoniumhydroxide, or an alkaline solution containing metal ions such as potassium or sodium.\",\n \"Said organic base can be a quaternary ammonium hydroxide such as tetraalkyl ammonium hydroxide in which the alkyl groups can contain hydroxy- and alkoxy-containing groups with 1 to 4 carbon atoms in the alkyl or alkoxy group.\",\n \"Said organic base can further be an organic amine such as an alkanol amine.\",\n \"Alkanol amines can be 2-aminoethanol, 1-amino 2-propanol, 1-amino 3-propanol.\",\n \"Preferably, the alkaline compounds are tetramethyl ammonium hydroxide, and trimethyl 2-hydroxy ethyl ammonium hydroxide (choline) and ammonium hydroxide.\",\n \"The amount of the alkaline compound lies between 0.0001 and 90 weight %, between 0.001 and 50 weight %, between 0.001 and 30 weight %.\",\n \"Said composiiton can further comprise a surfactant.\",\n \"In a second aspect of this invention, a method for treating a semiconductor substrate is disclosed.\",\n \"Said semiconductor substrate is treated with a composition comprising an aqueous solution of a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.\",\n \"Said composition can further comprise an oxidizing compound.\",\n \"Said composition can be, but is not limited hereto, the composition described in the first aspect of this invention.\",\n \"Said composition is particularly usefull for cleaning a substrate such that particles are oxidized and metallic contamination is removed.\",\n \"The complexing compound is for complexing metals being present on the substrate and in the solution.\",\n \"Additionally, the lifetime of the solution is increased since de decomposition of the oxidizing compound is substantially inhibited.\",\n \"A substrate can be, but is not limited hereto, a substrate such as semiconducting material, glass, quartz, ceramics, metal, plastic, magnetic material, superconductor and the like.\",\n \"Preferably, said substrate is a semiconductor substrate.\",\n \"Semiconductor substrate can be every possible substrate used in semiconductor processing.\",\n \"Said semiconductor substrate can be a substrate selected from the group, but not limited hereto, comprising a substrate made of silicon, germanium, gallium arsenide, indium phosphide, etc.\",\n \"The semiconductor substrate be e.g.\",\n \"the substrates as mentioned above covered entirely op partially with a thin film of e.g.\",\n \"an oxide, a nitride, a metal, a polymeric insulating layer, an anti-reflecting coating, a barrier, a photoresist layer, etc.\",\n \"The present invention is particularly relevant for cleaning or etching a semiconductor substrate of which the surface should be highly clean.\",\n \"When the composition is used for treating a substrate, the weight concentration range of the alkaline compound in the-cleaning solution are typically but not limited to 0.001-100%, 0.1-20% and preferably 0.1-5% by weight.\",\n \"For ammonium hydroxide, the weight concentration range of the alkaline compound in the cleaning solution are typically but not limited to 0.001-30%, 0.1-20% and preferably 0.1-5% by weight.\",\n \"For other alkaline compounds, the weight concentration range is equivalent, and function of the strength of the alkaline compound.\",\n \"For peroxide, the weight concentration the hydrogenperoxide is typically but not limited to 0.001-100%, 0.1-20% and preferably 0.1-5% by weight.\",\n \"In the preferred embodiment of this invention, a composition for treating a semiconductor surface comprises ammonium hydroxide, hydrogen peroxide, water (hereafter called APM mictures) and additionally a complexing agent.\",\n \"Said complexing agent is one of the group consisting of the molecules as described in FIG.\",\n \"2.APM-cleaning mixtures comprising a complexing agent according to the present invention are robust with respect to metal contamination coming from the fresh chemicals as well as with respect to metal contamination introduced in the course of its use for cleaning.\",\n \"The robustness of the basic APM process can be improved by the addition of complexing agents which keep the metals in solution and prevent the above mentioned catalysis of the peroxide decomposition.\",\n \"The volume mixing ratio of NH4OH(296)/H2O2(30%)/H2O is typically, but not limited hereto, 0.25/1/5.The cleaning solution is prepared with the amounts as described above and afterwards the semiconductor substrate is treated with the cleaning solution.\",\n \"In the best mode known to the applicant, molecule DMHP is selected and added in the amounts described above.\",\n \"The complexing agent can be added as the pure compound to the cleaning solution.\",\n \"Alternatively, the complexing agent can be dissolved in either water, ammonia or peroxide or a dilution of the two latter chemicals and added as such to the cleaning solution.\",\n \"It is a further aim of the invention to disclose a process for treating a semiconductor substrate comprising the steps of treating said semiconductor substrate with the cleaning solution as described above and drying said semiconductor substrate.\",\n \"An optional step: rinsing said semiconductor substrate can be performed after said treating said semiconductor substrate with the cleaning solution as described above.\",\n \"In the step of treating said semiconductor substrate with said cleaning solution, the semiconductor substrate can be immersed in a bath containing the cleaning solution.\",\n \"Alternatively, the cleaning solution can be dispensed or sprayed onto the semiconductor substrate for instance by using a spray processor.\",\n \"In all cases, the cleaning performance of the solution can be enhanced by using a megasonic transducer.\",\n \"The temperature range for treating the semiconductor substrate with the cleaning solution is typically but not limited to 0-95 degrees Celcius, 10-80 degrees Celcius and preferably between 20-70 degrees Celcius.\",\n \"In the step of drying the semiconductor substrate, several techniques known in the art can be used, e.g.\",\n \"spin-drying, Maragoni-drying, drying techniques using organic vapours.\",\n \"The step of rinsing the semiconductor substrate comprises treating the semiconductor substrate with DI water or treating the semiconductor substrate with a diluted acidic solution or with DI water containing the complexing agent in an amount of 1 to 100000 ppm, 10 to 10000 ppm and by preference 100 to 1000 ppm.\",\n \"It is a further aim of the invention to describe a process for treating a semiconductor substrate comprising the step of: treating said semiconductor substrate with any cleaning solution and/or treating said semiconductor substrate with any rinsing solution Said any cleaning solution can be any cleaning solution, not being limited to the compositions described in this application.\",\n \"Said rinsing solution comprises said complexing agent and water.\",\n \"Said complexing agent can be any complexing agent described in this application.\",\n \"The amount of the complexing agent in the composition can be between 1 and 100000 ppm, 10 and 10000 ppm and by preference between 100 and 1000 ppm.\",\n \"This rinsing solution can also comprise a surfactant in an amount of 0.1 ppm to 10 w %.\",\n \"A surfactant is a surface-active agent comprising a lyophobic group and a lyophilic group.\",\n \"The lyophobic group can be a straight-chain alkyl group or a branched-chain alkyl group (C8-C20), an long-chain (C8-C20) alkyl benzene residue, an alkylnaphtalene residue (C3 and greater-length alkyl groups), high-molecular-weight propylene oxide polymers (polyoxypropylene glycol derivatives), long-chain perfluoroalkyl or polysiloxane groups.\",\n \"Depending upon the lyophilic group, the surfactant can be an anionic, cationic, nonionic or zwitterionic surfactant.\",\n \"Anionic surfactants can be carboxylic acids or carboxylic acid salts (such as sodium and potassium salts of straight-chain fatty acids), sulfonic acids or sulfonic acid salts (such as linear alkylbenzenesulfonates, higher alkylbenzenesulfonates, benzene-, toluene-, xylene- and cumenesulfonates, ligninsulfonates, petroleum sulfonates, N-acyl-n-alkyltaureates, paraffin sulfonates, secondary n-alkanesulfonates, α-olefin sulfonates, sulfosuccinate esters, alkylnaphtalenesulfonates or isethionates), sulfuric acid ester salts (such as sulfated linear primary alcohols, sulfated polyoxyethylenated straight-chain alcohols or sulfated triglyceride oils), phosphoric and polyphosphoric acid esters.\",\n \"Cationic surfactants can be primary amines and their salts, diamines and polyamines and their salts, quaternary ammonium salts (such as tetralkylammonium salts or imidazolinium salts), polyoxyethylenated long-chain amines (RN(CH2CH2O)xH]2), quaternized polyoxyethylenated long-chain amines or amine oxides (such as N-alkyldimethylamine oxides).\",\n \"Nonionic surfactants can be polyoxyethylenated alkylphenols, polyoxyethylenated straight-chain alcohols, polyoxyethylenated polyoxypropylene glycols, polyoxyethylenated mercaptans, long-chain carboxylic acid esters (such as glyceryl and polyglyceryl esters of natural fatty acids, propylene glycol, sorbitol or polyoxyethylenated sorbitol esters, polyoxyethylene glycol esters and polyoxyethylenated fatty acids), alkanolamides, tertiary acetylenic glycols, polyoxyethylenated silicones, N-alkylpyrrolidones or alkylpolyglycosides.\",\n \"Zwitterionic surfactants have both anionic and cationic charges present in the lyophilic portion (such as β-N-alkylaminopropionic acids, N-alkyl-β-iminodipropionic acids, imidazoline carboxylates, N-alkylbetaines, amine oxides, sulfobetaines or sultaines) (M. J. Rosen, Surfactants and Interfacial phenomena, 2nd Edition, John Wiley and Sons, New York, 1989]) No additional alkaline compound should need to be added to the said rinsing solution.\",\n \"The pH range of said rinsing solution can typically be, but not limited to, between 5 and 8.Said Rinse solution can be dispensed or sprayed onto the semiconductor surface as described above.\",\n \"During rinsing the performance can also be enhanced by using a megasonic transducer.\",\n \"The process of treating a semiconductor substrate with a cleaning solution comprising the above mentioned steps can be performed for a certain number of semiconductor substrates.\",\n \"After treating at least one substrate, but preferably after treating more substrates, the composition of the cleaning solution can be modified by e.g.\",\n \"adding extra alkaline compound, adding extra complexing compound, adding oxidizing compound such that the initial composition of the cleaning solution is kept constant as function of the process time.\",\n \"COMPARATIVE EXAMPLES The present invention will be further described using non-limiting examples and drawings.\",\n \"The effectiveness of the new class of complexing agents on the inhibition of the metal catalyzed decomposition of peroxide and the prevention of metal outplating on silicon wafers in metal contaminated APM cleaning solutions is described next.\",\n \"For complexing agents as disclosed in this invention, different model compounds are selected (1-EMHP, 2-EMHP, DMHP, DEHP, PEHP, PMHP, ECMHP and ECEHP) and added to the bath at a concentration of 2.67×10−5 M and at a 10×, 40×, or 50× higher concentration (namely 2.65×10−4 10.6×10−4 and 13.25×10−4 M).\",\n \"A comparison is made with other types of complexing agents containing as functional groups either phosphonic acids, such as diethylene (kleine letter) triamine penta-methylenephosphonic acid (DTPMP), carboxylic acids, such as ethylene diamino tetra acetic acid (EDTA).\",\n \"An overview of the different chemicals used for the experiments is given in Table 1.All experiments were done in a class 1000 clean room environment or better.\",\n \"TABLE 1 Chemicals used for preparation of APM baths.\",\n \"Chemical Vendor Grade H2O2 30 (w/w) % Ashland TB(*) NH4OH 29 (w/w) % Ashland TB(*) EDTA Fluka DTPMP Monsanto Dequest 2060S DMHP Aldrich (*)TB-grade corresponds with a specification of maximal 100 ppt of metal ions in the chemical.\",\n \"Example 1 Metal Outplating from APM Mixtures in Presence of Different Complexing Agents The efficiency of complexing agents to suppress the deposition of metallic contamination onto wafer surfaces was studied.\",\n \"This was done through intentionally spiking well controlled trace amounts of metallic contamination to cleaning solutions.\",\n \"For these metal deposition tests, p-type monitor wafers with a diameter of 150 mm and <100> orientation were used.\",\n \"The wafers were pre-cleaned using IMEC Clean® in an automated Steag wet bench (i.e.\",\n \"SOM+dHF+O3-rinse rendering a perfectly clean hydrophilic surface).\",\n \"The metal deposition experiments were performed in a static quartz tank with a quartz cover plate.\",\n \"This tank was not equipped with a megasonic transducer.\",\n \"APM mixtures were prepared containing 1 w-ppb of different metals of interest with/without the complexing agent added.\",\n \"The metals spiked to the APM bath were added from AAS-standard solutions (Merck).\",\n \"After a bath age of 5 minutes, three wafers were immersed for 10 minutes, rinsed for 10 minutes in an overflow rinse tank and dried with a commercially available Marangoni drier (STEAG).\",\n \"The resulting metal contamination was measured with straight TXRF or VPD-DSE-DC-TXRF (Vapor Phase Decomposition-Droplet Surface Etching-Droplet Collection Total X-Ray Fluorescence).\",\n \"Determination of Al water surface concentration was done using VPD-DC GF-AAS (Graphite Furnace Atomic Absorption Spectroscopy).\",\n \"An overview of the resulting metal surface contamination after dipping a clean wafer in an APM spiked with metals and different complexing agents is given in Table 2.TABLE 2 Measured metal surface concentrations after immersion in metal contaminated 0.25/1/5 APM solution at 20-50° C. with different complexing agents present.\",\n \"Complexing Conc.\",\n \"CA Conc.\",\n \"Mt Al (1010 Fe agent (2.7 × 10−5 M) (w-ppb) at/cm2) (1010 at/cm2) Pre-clean only NA NA 2.34 <DL None 0 0 39.3 ± 6 1.5 ± 0.4 None 0 1 360 ± 21 109 ± 14 EDTA 1 1 NM 25.1 ± 0.1 10 1 272 ± 16 NM 100 1 274 2.78 EDTA + 1 + 2 1 257 ± 2 <DL DTPMP 1-EMHP (1) 1 1 356 ± 32 0.51 ± 0.02 10 1 142 ± 68 0.23 1-EMHP (2) 1 1 292 ± 1 0.20 ± 0.00 10 1 27 ± 33 NA 50 1 0.5 ± 0.2 0.26 2.4 ± 1.4 0.16 2-EMHP 1 1 404 0.35 ± 0.01 10 1 248 ± 69 0.22 50 1 0.8 ± 0.2 0.15 DEHP 1 1 369 ± 0 0.33 ± 0.01 10 1 150 ± 41 0.92 50 1 2.6 ± 0.6 0.15 DMHP 1 1 392 ± 0 0.45 ± 0.01 (Aldrich) 10 1 101 ± 2 0.21 50 1 1.2 ± 0.1 NM DMHP 1 1 387 ± 0 0.40 ± 0.02 10 1 230 ± 5 0.18 PEHP 40 1 4.6 ± 0.2 0.14 ± 0.0 PMHP 40 1 1.13 0.14 ± 0.14 ECMHP 40 1 15.0 ± 0.5 0.08 ± 0.04 ECEHP 40 1 14.2 ± 1.7 0.15 ± 0.05 These data show that the addition of EDTA results in a decrease of the final Fe contamination levels but has no effect on the reduction of Al contamination.\",\n \"The use of DTPMP in a 1 w-ppb metal spiked bath, is very efficient to reduce the final Fe surface concentration but has no effect on suppression of Al deposition.\",\n \"The new class of pyridinone complexing agents are able to reduce the final Fe and Al surface concentration on the wafer.\",\n \"Higher concentrations of complexing agent are needed because the pyridones do not form a 1:1 metal/chelate complex.\",\n \"When using the pyridinones at a concentration of 13.25×10−4 M in the bath, the resulting Fe and Al surface contamination levels are below the values measured on a wafer treated with a clean, i.e.\",\n \"no metals spiked, APM solution.\",\n \"Example 2 Decomposition of Peroxide in APM Cleaning Mixtures in Presence of Trace Metal Contamination and Metal Complexing Agents The effect of the addition of a complexing agent to APM cleaning solutions on the kinetics of the decomposition reaction of H2O2 has been investigated.\",\n \"Well controlled amounts of metallic contamination were added to the cleaning mixture under study.\",\n \"As hydrogen peroxide decomposes, an amount of oxygen gas is liberated following the overall reaction 2 H2O2⇄O2+2 H2O The decay of the total peroxide concentration in the APM mixture can be monitored by measuring the time-dependent increase of the pressure due to the O2-evolution in a dedicated set-up as described by Schmidt.\",\n \"Numerical integration over time yields the actual peroxide concentration in the bath.\",\n \"It is convenient to use peroxide concentrations normalized to its initial value [H2O2]i as [ H 2 O ] n = [ H 2 O 2 ] [ H 2 O 2 ] i Since the decomposition reaction is mainly catalyzed by Fe and in a lesser content Cu (Mertens et al.\",\n \"Proc.\",\n \"of the 5th Internat.\",\n \"Symp.\",\n \"on Cleaning Technology in Semiconductor Device Manufacturing PV97-35 (1997), the decay of peroxide concentration in a metal contaminated bath and in presence of a CA, illustrates the ability of complexing primarily Fe in the APM bath.\",\n \"The decomposition rate as function of bath age is determined in APM mixtures (0.25/1/5 29%NH4OH/30% H2O2/H2O) spiked with 1 w-ppb of the metals of interest with and without different complexing agents.\",\n \"The effect of different additives on the inhibition of the metal catalyzed decomposition reaction of peroxide in APM cleaning mixtures is shown in FIG.\",\n \"2.This graph shows the normalized H2O2 concentration as function of bath age for an APM mixture at 50° C. spiked with different complexing agents and the metals of interest.\",\n \"All complexing agents are found to suppress to some extent the decomposition reaction, at least when the mixture is fresh.\",\n \"For some CA, the suppression action vanishes over time.\",\n \"This may be attributed to the destruction of the complexing agent or more specifically of the metal-complex in the hot APM.\",\n \"The time over which they remain active in complexing metals is summarized in Table 3 and can be used to establish the effective lifetime.\",\n \"In example 1 it was demonstrated that higher CA concentrations have to be used in the cleaning solution.\",\n \"FIG.\",\n \"1 and Table 3 show that with increasing complexing agent concentration, the effective lifetime of the bath can be increased substantially.\",\n \"Increasing the complexing agent concentration even further would result in a more prolonged lifetime.\",\n \"TABLE 3 Effective lifetime of complexing agents in 0.25/1/5 metal spiked APM-cleaning mixtures at 50° C. Conc.\",\n \"CA Effective lifetime Complexing agent (2.7 × 10−5 M) (min) None 0 40 DEHP 1 40 10 120 1-EMHP (1) 1 40 10 50 1-EMHP (2) 1 60 10 90 2-EMHP 1 30 10 40 DMHP FfU 1 40 10 100 DMHP Aldrich 1 20 10 60\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10451230"}}},{"rowIdx":296,"cells":{"text":{"kind":"list like","value":[["Level sensor","A level sensor (2) comprising a support (6) with a bearing (11) for a lever arm (3) with a float (4) and a guide rail (16) pointing in the direction of the bearing (11).","The lever arm (3) has a stirrup (5) made of plastic and a lever wire (7) clipped thereto.","The stirrup has two guide elements (12,13) which are arranged next to each other and which engage with the guide rail (16), thereby preventing the dual contact (10) of the stirrup (5) from rising above the slideways (9) of a potentiometer (8) which are disposed on the support (6)."],["1.A filling-level sensor for generating electrical signals as a function of a pivoting angle of a float-carrying lever arm for a tank of a motor vehicle, comprising: a carrier for fastening to a lateral wall in the tank; a yoke made of plastic and pivotable in a mounting of the carrier; a lever wire holding a float and fastened to the yoke; and a guide rail, arranged on the carrier, for guiding a guide element of the yoke in an intended axial position in relation to the carrier, wherein the guide rail is arranged so as to point in the direction of the mounting, and the yoke has a fork-shaped guide element which is open at an end facing away from the mounting and which is in engagement with the guide rail.","2.The filling-level sensor as claimed in claim 1, wherein the guide rail has a web-shaped configuration and is arranged parallel to the carrier.","3.The filling-level sensor as claimed in claim 1, wherein the fork-shaped guide element is arranged at the end of the yoke which faces away from the mounting.","4.The filling-level sensor as claimed in claim 1, wherein the yoke is designed to engage around at least part of the carrier in the region of the mounting.","5.The filling-level sensor as claimed in claim 1, wherein two fork-shaped guide elements are arranged next to one another.","6.The filling-level sensor as claimed in claim 1, further comprising a spring elements prestressing a wiper contact against a wiping track of a potentiometer fastened between the guide elements arranged next to one another.","7.The filling-level sensor as claimed in claim 1, wherein the guide rail has a projecting edge.","8.The filling-level sensor as claimed in claim 1, wherein the guide rail is at a greater or equal distance from the carrier at its end facing away from the mounting than at its end facing the mounting, and in that surfaces of the carrier and of the guide rail which face one another in each case have a plane configuration.","9.The filling-level sensor as claimed in claim 1, wherein the guide rail is held at its ends by holding parts in front of the carrier."],["<SOH> BACKGROUND OF THE INVENTION <EOH>Prior art filling-level sensors usually have a potentiometer with a wiping track arranged on the carrier and with wiper contacts, fastened to the yoke, for generating the electrical signals.","Alternatively, the electrical signals are often also generated by means of a magnetically active position sensor.","Magnetically active position sensors mostly have a resistance network which is to be fastened to the carrier and which is switchable by a magnet arranged on the yoke.","The tank may be, for example, a fuel tank or a washing-water tank of a windshield cleaning system.","The guide rail of the known filling-level sensor is designed as part of the carrier having a plane surface and is oriented away from the mounting.","A U-shaped guide element arranged on the yoke engages behind the guide rail.","This, in the case of lateral forces acting on the yoke and the lever wire, prevents the yoke from being bent away from the carrier and therefore the wiper contacts from being lifted off from the wiping track or the magnet from being removed from the resistance network.","One disadvantage of the known filling-level sensor is that, because of the guide element, the yoke has very large dimensions and is therefore heavy.","As a result, the center of gravity of the yoke is at a very great distance from the mounting, and therefore the filling-level sensor is very slow-acting.","Consequently, minor variations in the filling level are detected by the potentiometer only with a delay or abruptly.","Moreover, because of the high weight of the yoke, the latter requires a large quantity of material."],["<SOH> SUMMARY OF THE INVENTION <EOH>The invention is directed a filling-level sensor, with a carrier provided for fastening to a lateral wall in the tank, with a yoke made from plastic and pivotable in the mounting of the carrier, with a lever wire holding the float and fastened to the yoke, and with a guide rail, arranged on the carrier, for guiding a guide element of the yoke in an intended axial position in relation to the carrier.","The filling-level sensor according to the invention detects a variation in the filling level particularly quickly and is constructed cost-effectively.","According to an aspect of the invention, the guide rail is arranged so as to point in the direction of the mounting, and the yoke has a fork-shaped guide element which is open at its end facing away from the mounting and engages with a guide rail.","By virtue of this design, the yoke has very small dimensions and a particularly low weight.","Moreover, the center of gravity of the yoke is arranged very near the mounting, so that the yoke has particularly low inertia.","The yoke can therefore follow a variation in the filling level in the tank particularly quickly.","Due to the low weight of the yoke, the latter necessitates the use of a correspondingly small amount of material, so that it can be produced particularly cost-effectively.","The guide rail could, for example, have a round cross section.","However, according to an aspect of the invention, the carrier can be manufactured at a particularly low outlay in one piece with the guide rail from plastic by the injection molding method if the guide rail has a web-shaped configuration and is arranged parallel to the carrier.","By virtue of this configuration, the guide rail has a sliding track for the guide element on each of the two sides.","The fork-shaped guide element could, for example, be arranged in a middle region of the yoke.","However, a contribution to further reducing the dimensions of the yoke is made when the fork-shaped guide element is arranged at that end of the yoke which faces away from the mounting.","The yoke is particularly reliably mounted on the carrier axially and can nevertheless be mounted on the carrier very simply when it is designed to engage around at least part of the carrier in the region of the mounting.","For mounting, the yoke can simply be pushed over the carrier from the mounting.","The yoke is subsequently guided axially in the region of the mounting and on the guide rail.","To prevent the yoke from tilting, the guide element may be made particularly wide.","However, because of this, the guide element has a high weight and a large bearing surface on the guide rail.","The large bearing surface leads to undesirable frictional losses during the pivoting movement of the yoke.","According to another aspect of the invention, a tilting of the yoke can be prevented in a simple way by two fork-shaped guide elements arranged next to one another, without the weight of the yoke being appreciably increased thereby.","According to another aspect of the invention, the yoke is mounted in a particularly simple way when a spring element prestressing the wiper contact against the wiping track of the potentiometer is fastened between the guide elements arranged next to one another.","A deformation of the guide rail could lead to the wiper contact being lifted off from the wiping track or to a rubbing of the guide element.","According to another aspect of the invention, the guide rail has very high stability when it has a projecting edge.","The filling-level sensor according to the invention is manufactured particularly cost-effectively when the guide rail is at a greater or equal distance from the carrier at its end facing away from the mounting than at its end facing the mounting, and when surfaces of the carrier and of the guide rail which face one another in each case have a plane configuration.","By virtue of this configuration, the carrier can be manufactured, together with the guide rail, in a very simple way from plastic by the injection molding method in an injection mold from which removal can easily take place axially.","According to another aspect of the invention, the carrier requires the use of a particularly small amount of material when the guide rail is held at its ends by holding parts in front of the carrier."],["CLAIM FOR PRIORITY This application claims priority to International Application No.","PCT/DE01/04729 which was published in the German language on Jul.","4, 2002.TECHNICAL FIELD OF THE INVENTION The invention relates to a filling-level sensor for generating electrical signals as a function of a pivoting angle of a float-carrying lever arm for a tank of a motor vehicle.","BACKGROUND OF THE INVENTION Prior art filling-level sensors usually have a potentiometer with a wiping track arranged on the carrier and with wiper contacts, fastened to the yoke, for generating the electrical signals.","Alternatively, the electrical signals are often also generated by means of a magnetically active position sensor.","Magnetically active position sensors mostly have a resistance network which is to be fastened to the carrier and which is switchable by a magnet arranged on the yoke.","The tank may be, for example, a fuel tank or a washing-water tank of a windshield cleaning system.","The guide rail of the known filling-level sensor is designed as part of the carrier having a plane surface and is oriented away from the mounting.","A U-shaped guide element arranged on the yoke engages behind the guide rail.","This, in the case of lateral forces acting on the yoke and the lever wire, prevents the yoke from being bent away from the carrier and therefore the wiper contacts from being lifted off from the wiping track or the magnet from being removed from the resistance network.","One disadvantage of the known filling-level sensor is that, because of the guide element, the yoke has very large dimensions and is therefore heavy.","As a result, the center of gravity of the yoke is at a very great distance from the mounting, and therefore the filling-level sensor is very slow-acting.","Consequently, minor variations in the filling level are detected by the potentiometer only with a delay or abruptly.","Moreover, because of the high weight of the yoke, the latter requires a large quantity of material.","SUMMARY OF THE INVENTION The invention is directed a filling-level sensor, with a carrier provided for fastening to a lateral wall in the tank, with a yoke made from plastic and pivotable in the mounting of the carrier, with a lever wire holding the float and fastened to the yoke, and with a guide rail, arranged on the carrier, for guiding a guide element of the yoke in an intended axial position in relation to the carrier.","The filling-level sensor according to the invention detects a variation in the filling level particularly quickly and is constructed cost-effectively.","According to an aspect of the invention, the guide rail is arranged so as to point in the direction of the mounting, and the yoke has a fork-shaped guide element which is open at its end facing away from the mounting and engages with a guide rail.","By virtue of this design, the yoke has very small dimensions and a particularly low weight.","Moreover, the center of gravity of the yoke is arranged very near the mounting, so that the yoke has particularly low inertia.","The yoke can therefore follow a variation in the filling level in the tank particularly quickly.","Due to the low weight of the yoke, the latter necessitates the use of a correspondingly small amount of material, so that it can be produced particularly cost-effectively.","The guide rail could, for example, have a round cross section.","However, according to an aspect of the invention, the carrier can be manufactured at a particularly low outlay in one piece with the guide rail from plastic by the injection molding method if the guide rail has a web-shaped configuration and is arranged parallel to the carrier.","By virtue of this configuration, the guide rail has a sliding track for the guide element on each of the two sides.","The fork-shaped guide element could, for example, be arranged in a middle region of the yoke.","However, a contribution to further reducing the dimensions of the yoke is made when the fork-shaped guide element is arranged at that end of the yoke which faces away from the mounting.","The yoke is particularly reliably mounted on the carrier axially and can nevertheless be mounted on the carrier very simply when it is designed to engage around at least part of the carrier in the region of the mounting.","For mounting, the yoke can simply be pushed over the carrier from the mounting.","The yoke is subsequently guided axially in the region of the mounting and on the guide rail.","To prevent the yoke from tilting, the guide element may be made particularly wide.","However, because of this, the guide element has a high weight and a large bearing surface on the guide rail.","The large bearing surface leads to undesirable frictional losses during the pivoting movement of the yoke.","According to another aspect of the invention, a tilting of the yoke can be prevented in a simple way by two fork-shaped guide elements arranged next to one another, without the weight of the yoke being appreciably increased thereby.","According to another aspect of the invention, the yoke is mounted in a particularly simple way when a spring element prestressing the wiper contact against the wiping track of the potentiometer is fastened between the guide elements arranged next to one another.","A deformation of the guide rail could lead to the wiper contact being lifted off from the wiping track or to a rubbing of the guide element.","According to another aspect of the invention, the guide rail has very high stability when it has a projecting edge.","The filling-level sensor according to the invention is manufactured particularly cost-effectively when the guide rail is at a greater or equal distance from the carrier at its end facing away from the mounting than at its end facing the mounting, and when surfaces of the carrier and of the guide rail which face one another in each case have a plane configuration.","By virtue of this configuration, the carrier can be manufactured, together with the guide rail, in a very simple way from plastic by the injection molding method in an injection mold from which removal can easily take place axially.","According to another aspect of the invention, the carrier requires the use of a particularly small amount of material when the guide rail is held at its ends by holding parts in front of the carrier.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 shows a perspective illustration of a filling-level sensor according to the invention, FIG.","2 shows a sectional illustration through the filling-level sensor from FIG.","1 along line II-II, FIG.","3 shows a further embodiment of the filling-level sensor according to the invention in a longitudinal section through a yoke.","DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG.","1 shows a filling-level sensor 2 according to the invention, mounted on a vertical wall 1 within a fuel tank.","The filling-level sensor 2 has a float 4 fastened to a lever arm 3.The lever arm 3 is mounted pivotably on a carrier 6 by a yoke 5 made from plastic and has a lever wire 7, fastened to the yoke 5, for holding the float 4.The float 4 follows a fuel level in the fuel tank and at the same time pivots the lever arm 3.The pivoting angle of the lever arm 3 is detected by a potentiometer 8.The potentiometer 8 has two wiping tracks 9 arranged on the carrier 6 and a wiper contact 10 fastened to the yoke 5.The wiper contact 10 is designed as a double contact for bridging the wiping tracks 9.To guide the yoke 5 in an intended axial position in relation to the carrier 6, the yoke 5 engages behind the carrier 6 in the region of a mounting 11.The yoke 5 has, at its end facing away from the mounting 11, two fork-shaped guide elements 12, 13 which are arranged next to one another and which engage with a guide rail 16 fastened laterally to the carrier 6 by holding parts 14, 15.By the yoke 5 being guided in the intended axial position in relation to the carrier 6, the wiper contact 10 is reliably prevented from being lifted off from the wiping tracks 9.FIG.","2 shows a filling-level sensor 2 according to the invention from FIG.","1 in a sectional illustration along the line can be seen here that the II-II.","FIG.","2 illustrates that the lever wire 7 forms, in the region of the mounting 11 of the yoke 5, an angled portion 17 functioning as a bearing axle for the lever arm 3.The carrier 6 has a bearing bore 18 comprising two parts 19, 20.A free end of an engaging-around portion 21 of the yoke 5 is arranged between the two parts 19, 20 of the bearing bore.","The wiper contact 10 is prestressed against the wiping tracks 9 by a tongue-shaped spring element 22.The tongue-shaped spring element 22 is fastened to the yoke 5 between to two fork-shaped guide elements 12, 13.To increase the stability of the guide rail 16, the latter has two vertically projecting edges 23, 24.FIG.","3 shows a further embodiment of the filling-level sensor according to the invention, with a lever arm 26 pivotable on a carrier 25 in a mounting 31.The lever arm 26 has a yoke 28 snapped together with a lever wire 27.The carrier 25, here, has a one-part bearing bore 29, behind which an angled portion 30 of the yoke 28 is engaged.","At the end facing away from the mounting 31, the yoke 28 has a fork-shaped guide element 33 which engages with a guide rail 32 of the carrier 25.The guide rail 32 and a region of the carrier 25 which faces the guide rail 32 has plane surfaces 34, 35 arranged parallel to one another."]],"string":"[\n [\n \"Level sensor\",\n \"A level sensor (2) comprising a support (6) with a bearing (11) for a lever arm (3) with a float (4) and a guide rail (16) pointing in the direction of the bearing (11).\",\n \"The lever arm (3) has a stirrup (5) made of plastic and a lever wire (7) clipped thereto.\",\n \"The stirrup has two guide elements (12,13) which are arranged next to each other and which engage with the guide rail (16), thereby preventing the dual contact (10) of the stirrup (5) from rising above the slideways (9) of a potentiometer (8) which are disposed on the support (6).\"\n ],\n [\n \"1.A filling-level sensor for generating electrical signals as a function of a pivoting angle of a float-carrying lever arm for a tank of a motor vehicle, comprising: a carrier for fastening to a lateral wall in the tank; a yoke made of plastic and pivotable in a mounting of the carrier; a lever wire holding a float and fastened to the yoke; and a guide rail, arranged on the carrier, for guiding a guide element of the yoke in an intended axial position in relation to the carrier, wherein the guide rail is arranged so as to point in the direction of the mounting, and the yoke has a fork-shaped guide element which is open at an end facing away from the mounting and which is in engagement with the guide rail.\",\n \"2.The filling-level sensor as claimed in claim 1, wherein the guide rail has a web-shaped configuration and is arranged parallel to the carrier.\",\n \"3.The filling-level sensor as claimed in claim 1, wherein the fork-shaped guide element is arranged at the end of the yoke which faces away from the mounting.\",\n \"4.The filling-level sensor as claimed in claim 1, wherein the yoke is designed to engage around at least part of the carrier in the region of the mounting.\",\n \"5.The filling-level sensor as claimed in claim 1, wherein two fork-shaped guide elements are arranged next to one another.\",\n \"6.The filling-level sensor as claimed in claim 1, further comprising a spring elements prestressing a wiper contact against a wiping track of a potentiometer fastened between the guide elements arranged next to one another.\",\n \"7.The filling-level sensor as claimed in claim 1, wherein the guide rail has a projecting edge.\",\n \"8.The filling-level sensor as claimed in claim 1, wherein the guide rail is at a greater or equal distance from the carrier at its end facing away from the mounting than at its end facing the mounting, and in that surfaces of the carrier and of the guide rail which face one another in each case have a plane configuration.\",\n \"9.The filling-level sensor as claimed in claim 1, wherein the guide rail is held at its ends by holding parts in front of the carrier.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>Prior art filling-level sensors usually have a potentiometer with a wiping track arranged on the carrier and with wiper contacts, fastened to the yoke, for generating the electrical signals.\",\n \"Alternatively, the electrical signals are often also generated by means of a magnetically active position sensor.\",\n \"Magnetically active position sensors mostly have a resistance network which is to be fastened to the carrier and which is switchable by a magnet arranged on the yoke.\",\n \"The tank may be, for example, a fuel tank or a washing-water tank of a windshield cleaning system.\",\n \"The guide rail of the known filling-level sensor is designed as part of the carrier having a plane surface and is oriented away from the mounting.\",\n \"A U-shaped guide element arranged on the yoke engages behind the guide rail.\",\n \"This, in the case of lateral forces acting on the yoke and the lever wire, prevents the yoke from being bent away from the carrier and therefore the wiper contacts from being lifted off from the wiping track or the magnet from being removed from the resistance network.\",\n \"One disadvantage of the known filling-level sensor is that, because of the guide element, the yoke has very large dimensions and is therefore heavy.\",\n \"As a result, the center of gravity of the yoke is at a very great distance from the mounting, and therefore the filling-level sensor is very slow-acting.\",\n \"Consequently, minor variations in the filling level are detected by the potentiometer only with a delay or abruptly.\",\n \"Moreover, because of the high weight of the yoke, the latter requires a large quantity of material.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The invention is directed a filling-level sensor, with a carrier provided for fastening to a lateral wall in the tank, with a yoke made from plastic and pivotable in the mounting of the carrier, with a lever wire holding the float and fastened to the yoke, and with a guide rail, arranged on the carrier, for guiding a guide element of the yoke in an intended axial position in relation to the carrier.\",\n \"The filling-level sensor according to the invention detects a variation in the filling level particularly quickly and is constructed cost-effectively.\",\n \"According to an aspect of the invention, the guide rail is arranged so as to point in the direction of the mounting, and the yoke has a fork-shaped guide element which is open at its end facing away from the mounting and engages with a guide rail.\",\n \"By virtue of this design, the yoke has very small dimensions and a particularly low weight.\",\n \"Moreover, the center of gravity of the yoke is arranged very near the mounting, so that the yoke has particularly low inertia.\",\n \"The yoke can therefore follow a variation in the filling level in the tank particularly quickly.\",\n \"Due to the low weight of the yoke, the latter necessitates the use of a correspondingly small amount of material, so that it can be produced particularly cost-effectively.\",\n \"The guide rail could, for example, have a round cross section.\",\n \"However, according to an aspect of the invention, the carrier can be manufactured at a particularly low outlay in one piece with the guide rail from plastic by the injection molding method if the guide rail has a web-shaped configuration and is arranged parallel to the carrier.\",\n \"By virtue of this configuration, the guide rail has a sliding track for the guide element on each of the two sides.\",\n \"The fork-shaped guide element could, for example, be arranged in a middle region of the yoke.\",\n \"However, a contribution to further reducing the dimensions of the yoke is made when the fork-shaped guide element is arranged at that end of the yoke which faces away from the mounting.\",\n \"The yoke is particularly reliably mounted on the carrier axially and can nevertheless be mounted on the carrier very simply when it is designed to engage around at least part of the carrier in the region of the mounting.\",\n \"For mounting, the yoke can simply be pushed over the carrier from the mounting.\",\n \"The yoke is subsequently guided axially in the region of the mounting and on the guide rail.\",\n \"To prevent the yoke from tilting, the guide element may be made particularly wide.\",\n \"However, because of this, the guide element has a high weight and a large bearing surface on the guide rail.\",\n \"The large bearing surface leads to undesirable frictional losses during the pivoting movement of the yoke.\",\n \"According to another aspect of the invention, a tilting of the yoke can be prevented in a simple way by two fork-shaped guide elements arranged next to one another, without the weight of the yoke being appreciably increased thereby.\",\n \"According to another aspect of the invention, the yoke is mounted in a particularly simple way when a spring element prestressing the wiper contact against the wiping track of the potentiometer is fastened between the guide elements arranged next to one another.\",\n \"A deformation of the guide rail could lead to the wiper contact being lifted off from the wiping track or to a rubbing of the guide element.\",\n \"According to another aspect of the invention, the guide rail has very high stability when it has a projecting edge.\",\n \"The filling-level sensor according to the invention is manufactured particularly cost-effectively when the guide rail is at a greater or equal distance from the carrier at its end facing away from the mounting than at its end facing the mounting, and when surfaces of the carrier and of the guide rail which face one another in each case have a plane configuration.\",\n \"By virtue of this configuration, the carrier can be manufactured, together with the guide rail, in a very simple way from plastic by the injection molding method in an injection mold from which removal can easily take place axially.\",\n \"According to another aspect of the invention, the carrier requires the use of a particularly small amount of material when the guide rail is held at its ends by holding parts in front of the carrier.\"\n ],\n [\n \"CLAIM FOR PRIORITY This application claims priority to International Application No.\",\n \"PCT/DE01/04729 which was published in the German language on Jul.\",\n \"4, 2002.TECHNICAL FIELD OF THE INVENTION The invention relates to a filling-level sensor for generating electrical signals as a function of a pivoting angle of a float-carrying lever arm for a tank of a motor vehicle.\",\n \"BACKGROUND OF THE INVENTION Prior art filling-level sensors usually have a potentiometer with a wiping track arranged on the carrier and with wiper contacts, fastened to the yoke, for generating the electrical signals.\",\n \"Alternatively, the electrical signals are often also generated by means of a magnetically active position sensor.\",\n \"Magnetically active position sensors mostly have a resistance network which is to be fastened to the carrier and which is switchable by a magnet arranged on the yoke.\",\n \"The tank may be, for example, a fuel tank or a washing-water tank of a windshield cleaning system.\",\n \"The guide rail of the known filling-level sensor is designed as part of the carrier having a plane surface and is oriented away from the mounting.\",\n \"A U-shaped guide element arranged on the yoke engages behind the guide rail.\",\n \"This, in the case of lateral forces acting on the yoke and the lever wire, prevents the yoke from being bent away from the carrier and therefore the wiper contacts from being lifted off from the wiping track or the magnet from being removed from the resistance network.\",\n \"One disadvantage of the known filling-level sensor is that, because of the guide element, the yoke has very large dimensions and is therefore heavy.\",\n \"As a result, the center of gravity of the yoke is at a very great distance from the mounting, and therefore the filling-level sensor is very slow-acting.\",\n \"Consequently, minor variations in the filling level are detected by the potentiometer only with a delay or abruptly.\",\n \"Moreover, because of the high weight of the yoke, the latter requires a large quantity of material.\",\n \"SUMMARY OF THE INVENTION The invention is directed a filling-level sensor, with a carrier provided for fastening to a lateral wall in the tank, with a yoke made from plastic and pivotable in the mounting of the carrier, with a lever wire holding the float and fastened to the yoke, and with a guide rail, arranged on the carrier, for guiding a guide element of the yoke in an intended axial position in relation to the carrier.\",\n \"The filling-level sensor according to the invention detects a variation in the filling level particularly quickly and is constructed cost-effectively.\",\n \"According to an aspect of the invention, the guide rail is arranged so as to point in the direction of the mounting, and the yoke has a fork-shaped guide element which is open at its end facing away from the mounting and engages with a guide rail.\",\n \"By virtue of this design, the yoke has very small dimensions and a particularly low weight.\",\n \"Moreover, the center of gravity of the yoke is arranged very near the mounting, so that the yoke has particularly low inertia.\",\n \"The yoke can therefore follow a variation in the filling level in the tank particularly quickly.\",\n \"Due to the low weight of the yoke, the latter necessitates the use of a correspondingly small amount of material, so that it can be produced particularly cost-effectively.\",\n \"The guide rail could, for example, have a round cross section.\",\n \"However, according to an aspect of the invention, the carrier can be manufactured at a particularly low outlay in one piece with the guide rail from plastic by the injection molding method if the guide rail has a web-shaped configuration and is arranged parallel to the carrier.\",\n \"By virtue of this configuration, the guide rail has a sliding track for the guide element on each of the two sides.\",\n \"The fork-shaped guide element could, for example, be arranged in a middle region of the yoke.\",\n \"However, a contribution to further reducing the dimensions of the yoke is made when the fork-shaped guide element is arranged at that end of the yoke which faces away from the mounting.\",\n \"The yoke is particularly reliably mounted on the carrier axially and can nevertheless be mounted on the carrier very simply when it is designed to engage around at least part of the carrier in the region of the mounting.\",\n \"For mounting, the yoke can simply be pushed over the carrier from the mounting.\",\n \"The yoke is subsequently guided axially in the region of the mounting and on the guide rail.\",\n \"To prevent the yoke from tilting, the guide element may be made particularly wide.\",\n \"However, because of this, the guide element has a high weight and a large bearing surface on the guide rail.\",\n \"The large bearing surface leads to undesirable frictional losses during the pivoting movement of the yoke.\",\n \"According to another aspect of the invention, a tilting of the yoke can be prevented in a simple way by two fork-shaped guide elements arranged next to one another, without the weight of the yoke being appreciably increased thereby.\",\n \"According to another aspect of the invention, the yoke is mounted in a particularly simple way when a spring element prestressing the wiper contact against the wiping track of the potentiometer is fastened between the guide elements arranged next to one another.\",\n \"A deformation of the guide rail could lead to the wiper contact being lifted off from the wiping track or to a rubbing of the guide element.\",\n \"According to another aspect of the invention, the guide rail has very high stability when it has a projecting edge.\",\n \"The filling-level sensor according to the invention is manufactured particularly cost-effectively when the guide rail is at a greater or equal distance from the carrier at its end facing away from the mounting than at its end facing the mounting, and when surfaces of the carrier and of the guide rail which face one another in each case have a plane configuration.\",\n \"By virtue of this configuration, the carrier can be manufactured, together with the guide rail, in a very simple way from plastic by the injection molding method in an injection mold from which removal can easily take place axially.\",\n \"According to another aspect of the invention, the carrier requires the use of a particularly small amount of material when the guide rail is held at its ends by holding parts in front of the carrier.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 shows a perspective illustration of a filling-level sensor according to the invention, FIG.\",\n \"2 shows a sectional illustration through the filling-level sensor from FIG.\",\n \"1 along line II-II, FIG.\",\n \"3 shows a further embodiment of the filling-level sensor according to the invention in a longitudinal section through a yoke.\",\n \"DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG.\",\n \"1 shows a filling-level sensor 2 according to the invention, mounted on a vertical wall 1 within a fuel tank.\",\n \"The filling-level sensor 2 has a float 4 fastened to a lever arm 3.The lever arm 3 is mounted pivotably on a carrier 6 by a yoke 5 made from plastic and has a lever wire 7, fastened to the yoke 5, for holding the float 4.The float 4 follows a fuel level in the fuel tank and at the same time pivots the lever arm 3.The pivoting angle of the lever arm 3 is detected by a potentiometer 8.The potentiometer 8 has two wiping tracks 9 arranged on the carrier 6 and a wiper contact 10 fastened to the yoke 5.The wiper contact 10 is designed as a double contact for bridging the wiping tracks 9.To guide the yoke 5 in an intended axial position in relation to the carrier 6, the yoke 5 engages behind the carrier 6 in the region of a mounting 11.The yoke 5 has, at its end facing away from the mounting 11, two fork-shaped guide elements 12, 13 which are arranged next to one another and which engage with a guide rail 16 fastened laterally to the carrier 6 by holding parts 14, 15.By the yoke 5 being guided in the intended axial position in relation to the carrier 6, the wiper contact 10 is reliably prevented from being lifted off from the wiping tracks 9.FIG.\",\n \"2 shows a filling-level sensor 2 according to the invention from FIG.\",\n \"1 in a sectional illustration along the line can be seen here that the II-II.\",\n \"FIG.\",\n \"2 illustrates that the lever wire 7 forms, in the region of the mounting 11 of the yoke 5, an angled portion 17 functioning as a bearing axle for the lever arm 3.The carrier 6 has a bearing bore 18 comprising two parts 19, 20.A free end of an engaging-around portion 21 of the yoke 5 is arranged between the two parts 19, 20 of the bearing bore.\",\n \"The wiper contact 10 is prestressed against the wiping tracks 9 by a tongue-shaped spring element 22.The tongue-shaped spring element 22 is fastened to the yoke 5 between to two fork-shaped guide elements 12, 13.To increase the stability of the guide rail 16, the latter has two vertically projecting edges 23, 24.FIG.\",\n \"3 shows a further embodiment of the filling-level sensor according to the invention, with a lever arm 26 pivotable on a carrier 25 in a mounting 31.The lever arm 26 has a yoke 28 snapped together with a lever wire 27.The carrier 25, here, has a one-part bearing bore 29, behind which an angled portion 30 of the yoke 28 is engaged.\",\n \"At the end facing away from the mounting 31, the yoke 28 has a fork-shaped guide element 33 which engages with a guide rail 32 of the carrier 25.The guide rail 32 and a region of the carrier 25 which faces the guide rail 32 has plane surfaces 34, 35 arranged parallel to one another.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10451269"}}},{"rowIdx":297,"cells":{"text":{"kind":"list like","value":[["Inks for in-mould decoration","An energy-core ink or varnish composition is provided for use in an in-mould decoration (IMD) process, comprising an energy-curable resin, additional reactive monomers and/or oligomers and, optionally and in an amount not exceeding 10% by weight, a solvent, wherein the resin comprises a urethane acrylate oligomer having an aromatic or aliphatic polycarbonate backbone.","For inks, the composition additionally includes a pigment or dye.","For photocure compositions, a photoinitiator is also included.","Also provided is an in-mould decoration (IMD) process employing the ink or varnish composition."],["1.An energy-cure ink or varnish composition for use in an in-mould decoration (IMD) process, comprising an energy-curable resin, additional reactive monomers and/or oligomers and optionally and in an amount not exceeding 10% by weight of the composition, a solvent, wherein the energy-curable resin comprises a urethane acrylate oligomer having an aromatic or aliphatic polycarbonate backbone.","2.A composition according to claim 1, wherein the composition is an ink and further comprises a pigment or dye.","3.A composition according to claim 2, wherein the pigment or dye is present in an amount of from 1 to 40% by weight of the composition.","4.A composition according to any preceding claim wherein the composition is a photocure composition and further comprises a photoinitiator.","5.A composition according to claim 4, wherein the photoinitiator is present in an amount of from 1 to 14% by weight of the composition.","6.A composition according to any preceding claim wherein the polycarbonate backbone is aliphatic.","7.A composition according to any preceding claim, wherein the polycarbonate backbone is linear.","8.A composition according to any preceding claim, wherein the polycarbonate-backboned urethane acrylate oligomer has a molecular weight of from 1,000 to 30,000, preferably from 3,000 to 15,000, and more preferably from 6,500 to 10,000.9.A composition according to any preceding claim, wherein the polycarbonate-backboned urethane acrylate component is present in an amount of from 5 to 70% by weight of the composition.","10.A composition according to any preceding claim, wherein the polycarbonate-backboned urethane acrylate oligomer is of the general formula (I) or (II): wherein: R2 and R3 are such that OCN—R—NCO (where R=R2 or R3) represents a diisocyanate; Y represents a hydrogen atom or a methyl group; R1 and R4 together with their attached (meth)acrylate group represent the residue of a hydroxy (meth)acrylate functional unsaturated monomer.","11.A composition according to any preceding claim, wherein the composition comprises additional acrylate monomer and/or oligomer and the acrylate monomer and/or oligomer is present in an amount of from 0 to 80% by weight of the composition.","12.A composition according to claim 11, wherein the acrylate monomer and/or oligomer is monofunctional.","13.A composition according to any preceding claim, wherein the composition comprises the reactive monomer N-vinyl-2-pyrrolidone (NVP), in an amount not exceeding 10%, preferably not exceeding 5%, by weight of the composition.","14.A composition according to any one of claims 1 to 13, wherein the composition comprises an organic solvent in an amount not exceeding 5% by weight of the composition.","15.A composition according to any one of claims 1 to 13, wherein the composition is free of organic solvent.","16.A method of in-mould decoration employing an ink or varnish composition as defined in any of the preceding claims.","17.A method according to claim 16, wherein the ink is printed onto a polycarbonate or polycarbonate/polyester substrate.","18.A method according to claim 16 or claim 17, wherein the ink is printed onto a substrate and the ink on the substrate is subjected to polycarbonate or polycarbonate/polyester blend resin injection to provide a polycarbonate or a polycarbonate/polyester blend backfill adhered to the printed substrate."],["The present invention relates to a new ink composition for in-mould decorative use and to an in-mould decorative process employing this ink.","In its broadest sense, in-mould decorating (IMD) simply means applying a decoration in the course of moulding the decorated part.","A particular IMD process referred to as insert moulding or insert IMD has been known for many years, but has seen little practical uses except in the automotive industry, until recently.","In insert IMD processes, a film substrate is printed with the desired decoration this is preferably a second surface printing process in which the decoration is printed in reverse on the reverse side of a transparent or translucent (or partially transparent or translucent) film substrate, so that it shows through and is protected by the substrate.","Then, in one or more steps, the substrate is, if necessary, formed into shape and further resin or plastics material is injection moulded to give the final product.","It will be seen readily that this process can give rise to several difficulties.","First, if the printed substrate is to be moulded after printing, the printing ink must have the necessary mechanical properties to wild the strains of moulding.","Thus, it must be flexible, and preferably have at least a similar flexibility to that of the substrate, so as to stretch with the substrate as the substrate is moulded.","It must also have sufficient adhesion to the substrate and abrasion resistance to withstand any abrading action in the course of moulding.","In the final step, where a liquid resin or resin precursor is injected onto the formed decorated substrate, the printing ink must be able to resist the heat, pressure and shear imparted to it by the resin injection, or it has to be protected with an additional layer.","Otherwise, the ink will be caused to degrade or smudge from its printed location by the injection process, resulting in so-called “wash-out”.","Finally, the ink has to be compatible with the injected resin and in particular must provide a good adhesion with the injected resin so as to prevent delamination of the printed substrate from the resin backfill.","One way of solving the problems of avoiding wash-out of the ink and ensuring good adhesive bond strength to the resin backfill has been to provide the printed substrate with an additional coating over the irk which additional coating is typically an aqueous laminating adhesive that is applied over the graphic inks.","This coating then serves to bond or laminate an additional sheet of substrate such that the ink is sandwiched between the substrate and the additional sheet of substrate.","In this twin-layer construction, the laminated substrate serves to protect the ink against wash-out by the backfill injection and provides good adhesion to the injected resin.","However, this construction method requires additional processing steps and material cost.","DE-A-19832570 discloses a solvent-based ink system comprising a blend of a polycarbonate and a thermoplastic polyester polyurethane.","U.S. Pat.","No.","5,648,414 discloses a solvent-based ink suitable for insert IMD, containing a polycarbonate based on geminally disubstituted, dihydroxydiphenyl cycloalkanes as binder.","One such ink is commercially available as Noriphan™ (ex Proll under licence from Bayer AG).","Whilst this solvent-based ink permits a single-layer printed substrate construction to be used in IMD processes, it is associated with certain disadvantages.","For example, because it is a solvent-based ink, it is relatively difficult or inconvenient to screen print due to its poor press stability and difficulties in washing up.","Also, the printed substrate has been found to be susceptible to curling, and it is often therefore difficult to place the printed substrate in registration in the mould tool prior to injection with the resin backfill.","Furthermore, it is essential to ensure that all solvent is completely removed before the printed substrate is formed and injection moulded, in order to prevent wash-out, and blister or bubble formation, leading to delamination.","Another disadvantage is that the range of colours available is limited due to the fact that certain pigments can break down the polymer resin.","Finally, the need for a solvent in the ink has obvious environmental and health hazard implications.","These deficiencies associated with solvent-based systems have led to the development of photocurable ink systems, for example inks curable by UV-energy, for use in IMD processes, which require less or no solvent.","However, existing UV-curable inks are not wholly satisfactory, particularly in terms of formability, washout resistance and adhesion to injected resin backfill.","It will be appreciated that inks suitable for IMD must have very good adhesion to the substrate, good flexibility and elongation to permit forming with the substrate, good mechanical resistance to abrasion by the moulds, good washout resistance to the resin injection step, and good adhesive bonding to the injected resin backfill.","Because UV-curable inks undergo cross-linking, it is far more difficult to achieve this combination of properties in a UV-curable ink than in a solvent-based ink.","Although these UV-curable ink systems have eliminated or reduced the solvent, and hence the problems associated with the use of solvent in a solvent-based ink system the need for flexibility and elongation has meant that the inks have tended to be too so* to withstand the mechanical impact of the forming and injection processes.","In addition adhesion to the resin backfill was often not satisfactory.","Therefore, it was generally still found necessary to provide the printed substrate with a protective or tie coating, in order to ensure adequate resistance to the resin injection process and to provide good bonding to the backfill resin, so as to avoid wash-out, delamination, or both.","In addition to boost adhesion to the substrate and avoid delamination at the ink:substrate interface, it was generally still necessary to include aggressive monomers, such as N-vinyl-2-pyrrolidone (NVP), in the ink, which in turn have associated drawbacks, for example they may lead to delamination due to unreacted material, and to health and safety concerns.","We have now found, surprisingly, that it is possible to provide an energy-curable composition, preferably a UV-curable ink, suitable for use in IMD processes, that can be printed onto a substrate, that allows the printed substrate to be formed using conventional forming techniques, and backfilled with a resin injection without undergoing wash-out, even in the absence of a protective coating.","Furthermore, the ink can provide excellent adhesion to the resin backfill without the need for a tie coating.","Because the claimed energy-curable resin composition gives improved adhesion the requirement for NVP is reduced or eliminated.","A fisher advantage is that solvent may be significantly reduced or even eliminated from the IMD process.","The reduction or removal of solvent or NVP, or both, may lead to further advantages, apart from obvious environmental and health considerations, such as reduced delamination of the final part especially in environmental testing, caused by poor removal or poor full reaction of potentially volatile materials.","In addition, we have found that ink compositions according to the present invention can provide a harder, tougher surface, when cured, than conventional insert IMD inks that contain high molecular weight urge acrylate resins, whilst exhibiting excellent flexibility and forming characteristics.","It will be appreciated that hardness and toughness are desirable in an insert IMD ink in order to avoid accidental marking or damage caused by forming or moulding tools.","Broadly speaking, this has been achieved by using a new urethane acrylate resin having a polycarbonate backbone as carrier for the pigment or dye.","Accordingly, in a first aspect, the present invention provides an energy-cure ink or varnish composition for use in an in-mould decoration (IMD) process, comprising an energy-curable resin, additional reactive monomers and/or oligomers and, optionally and in an amount not exceeding 10% by weight of the composition, a solvent, wherein the energy-curable resin comprises a urethane acrylate, oligomer having an aromatic or aliphatic-polycarbonate backbone.","In a second aspect, the invention provides a method of in-mould decoration employing the ink or varnish composition.","By “energy-curable resin” or “energy-cure compositions”, as used herein, is meant a resin or composition that is curable by exposure to a source of radiation of an appropriate wavelength or intensity, i.e.","is photocurable by exposure to a source of electromagnetic radiation of an appropriate wavelength, such as ultraviolet (UV) radiation, or is electron beam (EB) curable by exposure to an electron beam of an appropriate intensity.","Preferably, the energy-cure compositions according to the present invention are at least UV-curable.","The energy-cure compositions according to the invention contain an energy-curable resin, additional reactive monomers and/or oligomers, additives and, in the case of an ink composition, pigments or dyes.","In the case of photocure compositions such as UV-cure compositions, a photoinitiator is also included in the composition.","Energy-Curable Resin: The energy-curable resin at least comprises a mono-, di- or trifunctional urethane acrylate oligomer that has a polycarbonate backbone and is obtainable by reaction of a diisocyanate, a hydroxy (meth)acrylate functional unsaturated monomer and a polycarbonate polyol.","Preferably, the resin at least comprises a mono- or difunctional urethane acrylate oligomer, as represented by the general formula (I) or (II): wherein: R2 and R3 are such that OCN—R—NCO (where R=R2 or R3) represents the diisocyanate used in the synthesis; Y represents a hydrogen atom or a methyl group; R1 and R4 together with their attached (meth)acrylate group represent the residue of the hydroxy (meth)acrylate functional unsaturated monomer used in the synthesis.","Examples of the diisocyanate compounds include, but are not limited to, hexamethylene diisocyanate (HMDI), isophorone diisocyanate (IPDI), 4,4-dicyclohexylmethane diisocyanate (H12MDI), 2,2,4-trimethyl hexamethylene diisocyanate 2,4-tolylene diisocyanate (TDI), 2,6-tolylene diisocyanate (TDI), trimethylhexamethylene diisocyanate (TMDI), diphenylmethane diisocyanate (MDI), tetramethylxylene diisocyanate (TMXDI), and xylene diisocyanate (XDI).","A preferred isocyanate is isophorone diisocyanate (IPDI) because of improved resistance and selective reactivities of the isocyanate groups enabling preparation of an ethylenically unsaturated monoisocyanate.","The above diisocyanate compounds may be used individually or in combination.","Examples of the hydroxy (meth)acrylate functional unsaturated monomers include, but are not limited to, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, 2-hydroxypropyl acrylate, 1,4-butanediol monoacrylate and glyceryl diacrylate, trimethylolethane di(meth)acrylate.","Suitable polycarbonate polyols are represented by the general formula (III): wherein: R5 and R6 represent the same or different aliphatic or aromatic groups; and n is an integer of 1 to 60.The polycarbonate polyols can be produced through, for example, an ester interchange reaction or alcoholysis of diethyl carbonate or diphenyl carbonate with a polyol, preferably a diol such as an alkylene diol, e.g.","1,4-butane diol, 1,6-hexane diol, or an alkylene ether diol.","e.g.","triethylene glycol, tripropylene glycol.","Other suitable diols include 2,2-bis-[4-(2-hydroxypropoxy)phenyl]propane and 1,1-bis-(4-hydroxyphenyl)-3,3,5-trimethyl cyclohexane.","Polyols with three or more hydroxyl groups, such as trimethylol propane, glycerine and pentaerythritol, can be incorporated for preparing polycarbonate polyols suitable for synthesising trifunctional polycarbonate urethane acrylates.","Mixtures of the polycarbonate polyols may also be utilised in the synthesis.","Other polyols such as polyether- or polyester-polyols may also be included, although to achieve the desired IMD properties the backbone should be substantially polycarbonate in nature.","The polycarbonate backbone may be aliphatic or aromatic but preferably is aliphatic in nature.","It may be linear or branched, and is preferably linear.","Suitable polycarbonate backboned urethane acrylates are available as NTX 4711 and NTX 4867 (ex Sartomer).","RD2/105.RD2/106.RD3/101, RD3/102, RD4/103 and RD4/104 (ex UCB Chemicals SA), and RXX-01-344 (ex Rahn).","We prefer that the polycarbonate urethane acrylate (PCUA) oligomer has a average molecular weight in the range of from 1,000 to 30,000, more preferably from 3,000 to 15,000, still more preferably from 4,000 to 10,000, and in particular from 6,500 to 10,000.For the purposes of providing improved flexibility and elongation, higher molecular weight polycarbonate backbones would be desirable.","However, higher molecular weights can have an adverse effect on the viscosity and printability of the ink.","Moreover, higher molecular weights tend to produce unduly soft ink coatings, which are more susceptible to marking by mould parts or during handling.","Thus, it will be appreciated that the particular molecular weight chosen will represent a compromise between these conflicting factors, on the one hand providing acceptable flexibility and elongation whilst on the other hand, ensuring satisfactory viscosity, printability and hardness.","If desired, the flexibility, elongation and hardness properties of the ink can be tailored by blends of monofunctional, difunctional or trifunctional PCUA oligomer.","It will be appreciated that the higher degrees of cross-linking afforded by inclusion of di- and trifunctional oligomers will tend to reduce the overall flexibility and elongation of the ink, and increase its hardness.","Thus, the energy-curable resin may comprise a mono-, di- or trifunctional polycarbonate urethane acrylate oligomer, or a blend of two or more of mono-, di- and trifunctional polycarbonate urethane acrylate oligomers.","Preferably, the energy-curable resin at least comprises a mono- or difunctional polycarbonate urethane acrylate oligomer, or a blend thereof.","We prefer that the average functionality of the energy-curable resin is less than 2.5, more preferably less than 2.2, and most preferably less than 2.1.The monofunctional polycarbonate urethane acrylate may be present in the composition in an amount of from 0 to 70% by weight.","Difunctional polycarbonate urethane acrylate may be included in an amount from 0 to 50% by weight.","Trifunctional polycarbonate urethane acrylate may be included in an amount from 0 to 5% by weight.","The total polycarbonate urethane acrylate component is present in an amount of from 5 to 70%, preferably from 20 to 60%, by weight of the composition.","Photoinitiators: Preferably the compositions according to the invention are at least photocurable, and most preferably are UV-curable, and accordingly contain a photoinitiator.","However, it will be appreciated that EB-cure compositions in accordance with the invention can be formulated without the need for a photoinitiator.","For photocure compositions such as UV-cure compositions, a wide range of commercially available photoinitiators can be incorporated to initiate the photocure mechanism.","Preferably these photoinitiators have low potential to migration and low volatility, so as to avoid problems such as delamination of the injection moulded part.","The photoinitiator package should also have low UV thermal yellowing, important to meet typical heat and environmental cycling tests typically used on the final IMD part.","The photoinitiator package is typically present in an amount of from 0.5 to 20% for example from 1 to 14%, by weight of the composition.","Suitable photoinitiators include, for example, 2,4,6-trimethylbenzyl diphenyl phosphine oxide (Lucerin™ TPO ex BASF), bis(2,4,6-trimethylbenzoyl)phenyl phosphine oxide (Irgacure 819, ex Ciba Geigy), bis(2,6-dimethoxybenzoyl)-2,4,4-trimethylpentyl phosphine oxide, ethyl-2,4,6-trimethylbenzoylphenyl-phosphinate (Lucerin™.","TPO-L, ex BASF), 2-benzyl-2-dimethylamino-1-(4-morpholinphenyl) butan-1-one (Irgacure™ 369, ex Ciba Geigy), 1-hydroxycyclohexyl acetophenone (Irgacure™ 184, ex Ciba Geigy), iso-propyl thioxanthone (Quantacure™ ITX, ex IBIS or Speedcure™ ITX ex Lambson; 2-chloro thioxanthone (Kayacure™ CTX, ex Nippon Kayaku), oligo[2-hydroxy-2-methyl-1-[4-(1-methylvinyl)phenyl]propanone and 2-hydroxy-2-methyl-1-phenyl-1-propanone (Esacure™ KIP100F, ex Lamberti), methyl benzoyl formate (Genocure MBF ex Rahn), benzophenone, 2-methyl-1-[4-(methylthio)phenyl]-2-morpholinopropan-1-one (Irgacure™ 907, ex Ciba Geigy).","Of these, 2,4,6-trimethylbenzoyl diphenyl phosphine oxide (Lucerin™ TPO, ex BASF), bis(2,4,6-trimethylbenzoyl)-phenyl phosphine oxide (Irgacure 819, ex Ciba Geigy), and bis(2,6-dimethoxybenzoyl)-2,4,4-trimethylpentyl phosphine oxide are preferred photoinitiators.","Additional Reactive Monomers and/or Oligomers: Additional reactive energy-cure monomers and/or oligomers may be present in an amount of from 0 to 80%, preferably from 0 to 60%, by weight of the composition.","These reactive monomers and/or oligomers are preferably monofunctional.","Suitable acrylate monomers or oligomers include, but are not limited to, isobornyl acrylate (IBOA), 2-phenoxy ethyl acrylate (2PEA), 2-(2-ethoxyethoxy) ethyl acrylate (EOEOEA).","CTF acrylate, 4-t-butylcyclohexyl acrylate.","THF-acrylate, alkoxylated acrylates, diethylene glycol diacrylate, dipropylene glycol diacrylate, 1,6-hexanediol diacrylate, low molecular weight monofunctional urethane acrylates, polyether acrylates, polyester acrylates and low molecular weight epoxy acrylates.","Non-acrylated reactive diluents that may be incorporated include, but are not limited to, acryloyl morpholine (ACMO), n-vinylformamide (NVF), n-vinylformamide derivatives and n-vinyl caprolactam (NVC).","The compositions of the invention can provide improved adhesion to the substrate and backfill, thereby advantageously allowing the incorporation of N-vinyl-2-pyrrolidone (NVP) to be significantly reduced or even eliminates NVP is conventionally used to boost adhesion to the substrate, but concerns about health and safety, and its effect due to the volatility of unreacted material on delamination, means that it would be advantageous to eliminate, or at least reduce, NVP.","Nevertheless, in certain cases it will still be desirable to include monomers such as NVP in the ink and varnish compositions of the invention, to improve ‘key’ to substrate.","If used, NVP can be incorporated in an amount not exceeding 30%, preferably not exceeding 15%, more preferably not exceeding 10%, and in particular not exceeding 5%, by weight of the composition.","Most preferably, the composition is free of NVP.","Additives: If desired, inert or passive resins such as acrylics, styrene acrylates, polyesters, polycarbonates or celluloses may be included in the ink in small amounts, in order to improve the adhesion of the ink coating.","However, these inert or passive resins tend to adversely affect the resistance of the ink to injection of the backfill resin, and thus increase the likelihood of wash-out.","If included, therefore, we that only a small amount is used, for example an amount not exceeding 10%, and preferably not exceeding 7%, by weight of the composition.","Additives such as wetting agents, silicone and non-silicone antifoams may be incorporated to improved print properties such as substrate wetting and flow-out, and may be included in an amount of from 0 to 5%, preferably from 0 to 2%, by weight of the composition.","It should be noted that some additives, typically with low molecular weights, may have a tendency to migrate to the print surface in the cured coating.","This can affect the IMD properties, and therefore the total additive content should be kept to a minimum and additives with a high migration potential avoided.","Where the composition is intended for use as an ink, instead of a varnish, a pigment or dye is included in the composition.","Organic and/or inorganic pigments or dyes can be incorporated in an amount of from 0 to 50%, preferably from 0 to 40%, by weight of the composition.","The pigments or dyes should be selected to have good resistance to thermal decomposition and change, and resistance to sublimation.","Suitable pigments include, but not limited to, titanium dioxide white, zinc sulphide, carbon black, azo diarylide yellow, isoindoline yellow, diarylide orange, quinacridone magneta, diketo-pyrrolo-pyrrol red, copper phthalocyanine blue, copper phthalocyanine green, dioxazine violet, diketometal oxides.","Speciality effect pigments, such as metal oxide-coated mica pigments and aluminium metallic pigments, can also be included.","Fillers may be included to control the viscosity and rheology of the composition typically to improve printing characteristics, and may be present in an amount of from 0 to 40%, preferably from 0 to 30%, by weight of the composition.","Suitable fillers include, but not limited to, calcium carbonate, china clay, aluminium hydrate, talc, barium sulphate, aluminium silicate, and silica.","It will be appreciated that the compositions according to the invention will be substantially free of organic solvent.","However, small amounts of organic solvent may be included, if needed, in amounts not exceeding 10%, and preferably not exceeding 5%, by weight of the composition.","Most preferred is that the composition is free of organic solvent.","In-Mould Decorating (IMD) Process: The energy-cure compositions of the invention are particularly suitable for use in IMD processes, and especially in insert IMD or insert moulding processes.","The application of the compositions to the substrate may be effected using conventional printing techniques.","Preferred techniques include flexographic, lithographic, digital and screen print processes, but other methods may be used as appropriate.","Application by screen printing is particularly preferred.","Suitable substrate materials onto which the ink may be printed or the varnish applied as the case may be, include print-receptive polyester, polycarbonate, ABS.","PMMA, polycarbonate/polyester blends, polycarbonate/ABS blends materials such as those supplied by Bayer AG (Bayfol®, Makrolon®, Marofol®, Bayblend®), GE Structured Products (Lexan®) and Autotype (Autoflex Hiform™, Autoflex XtraForm™).","Preferably, the substrate is of a polycarbonate or polycarbonate/polyester blend resin material.","Similarly, suitable backfill materials which may be injected onto the printed substrate include the following or blends of the following: polyesters polycarbonate, styrenes ABS and PMMA resin materials.","Preferably, the injected backfill is of polycarbonate or a polycarbonate/polyester blend resin material.","The invention will be further illustrated by reference to the following non-limiting Examples: EXAMPLES A) Formulations: Example 1 Comparative Solvent-Based IMD Technology A sample of Noriphan™ black HTR952 was obtained (ex Proll) for comparison to UV IMD ink technology.","Prints of Noriphan™ were prepared in accordance to the Noriphan™ product data sheet supplied by Proll.","The prints were examined for adhesion, surface hardness, print curl, forming, and IMD properties.","Example 2 Comparative Standard Flexible Urethane Acrylate Technology The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.","Urethane acrylate, Actilane ™ 290 24.0 IBOA, SR506 ex Sartomer 31.8 NVP ex BASF or ISP 12.0 Silicone antifoam 0.7 UV Stabiliser, Genorad ™ 16 ex Rahn 0.5 Lucerin ™ TPO ex BASF 4.0 Irgacure ™ 184 ex Ciba 3.0 Filler 20.0 Carbon Black 4.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).","The prints were examined for adhesion, surface hardness, forming, and IMD properties.","Example 3 Invention Polycarbonate Urethane Acrylate Technology The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.","PCUA, NTX 4867 34.3 IBOA, SR506 ex Sartomer 23.5 UV Stabiliser, Genorad ™ 16 ex Rahn 0.5 Silicone antifoam 0.7 Lucerin ™ TPO ex BASF 4.0 Quantacure ™ ITX ex IBlS 1.0 NVP ex BASF 12.0 Filler 20.0 Carbon Black 4.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).","The prints were examined for adhesion, surface hardness, print curl, forming, and IMD properties.","Example 4 Invention Polycarbonate Urethane Acrylate Technology (NVP Free) The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.","PCUA, NTX 4867 ex Sartomer 36.4 EOEOEA, S256 ex Sartomer 8.7 IBOA, SR506 ex Sartomer 11.9 NVC, ex BASF or ISP 18.1 Polyester urethane acrylate ex Rahn 3.8 UV stabiliser, Genorad ™ 16 ex Rahn 0.4 Lucerin ™ TPO ex BASF 4.7 Irgacure ™ 184 ex Ciba 2.6 Silicone antifoam 0.7 Carbon black 3.6 Filler 9.1 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).","The prints were examined for adhesion, surface hardness, forming, and IMD properties.","Example 5 Invention Polycarbonate Urethane Acrylate Technology (NVP Free) The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.","PCUA, NTX 4867 40.0 IBOA, SR506 ex Sartomer 11.8 UV Stabiliser, Genorad ™ 16 ex Rahn 0.5 Silicone antifoam 0.7 Lucerin ™ TPO ex BASF 4.0 Irgacure ™ 184 ex Ciba Geigy 1.0 ACMO ex Rahn 18.0 EOEOEA, SR256 ex Sartomer 8.0 Filler 10.0 Red 6.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).","The prints were examined for adhesion, surface hardness, forming and IMD properties.","Example 6 Invention Polycarbonate Urethane Acrylate Technology (NVP Free) The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.","PCUA, RD2/105 ex UCB 25.8 EOEOEA, S256 ex Sartomer 6.0 IBOA, SR506 ex Sartomer 37.2 ACMO ex Rahn 7.0 UV stabiliser, Genorad ™ 16 ex Rahn 0.3 Lucerin ™ TPO ex BASF 4.0 Genocure ™ MBF ex Rahn 2.0 Speedcure ™ ITX ex Lambson 1.0 Silicone antifoam 0.7 Phthalocyanine blue 6.0 Filler 10.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).","The prints were examined for adhesion, surface hardness, forming, and IMD properties.","Example 7 Invention Polycarbonate Urethane Acrylate Technology The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.","PCUA, RXX0] 344 ex Rahn 26.0 IBOA, SR506 ex Sartomer 27.0 NVP ex BASF or ISP 12.0 UV stabiliser, Genorad ™ 16 ex Rahn 0.3 Lucerin ™ TPO ex BASF 4.0 Silicone antifoam 0.7 Titanium dioxide white pigment 30.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).","The prints were examined for adhesion, surface hardness, forming, and IMD properties.","Example 8 Invention Polycarbonate Urethane Acrylate Technology (NVP Free) The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μM) was achieved.","PCUA, RD3/101 ex UCB 40.0 EOEOEA, S256 ex Sartomer 6.0 IBOA, SR506 ex Sartomer 15.0 ACMO ex Rahn 7.0 UV stabiliser, Genorad ™ 16 ex Rahn 0.3 Lucerin ™ TPO ex BASF 4.0 Genocure ™ MBF ex Rahn 2.0 Speedcure ™ ITX ex Lambson 1.0 Silicone antifoam 0.7 Red 6.0 Filler 18.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).","The print were examined for adhesion, surface hardness, forming, and IMD properties.","B) Experimental Results: 1) Physical Properties TABLE 1 Pencil Hardness Ink system Hardness* Comments Example 1 H Can be marked, but good scratch resistance Example 2 HB Moderate scratch, easily marked Example 3 2H Difficult to scratch, excellent adhesion Example 4 H-2H Difficult to scratch, excellent adhesion Example 5 H-2H Difficult to scratch, excellent adhesion Example 6 H Difficult to scratch, excellent adhesion Example 7 H Difficult to scratch, excellent adhesion Example 8 H Difficult to scratch, excellent adhesion *according to ASTM D3363, BS 3900-E19, ISO 15184 TABLE 2 Cross-Hatch Adhesion Ink system Ranking** Comments Example 1 4-5 No removal Example 2 4 Slight/no removal Example 3 5 No removal Example 4 5 No removal Example 5 5 No removal Example 6 5 No removal Example 7 5 No removal Example 8 5 No removal **1 = poor (total ink removal).","5 = excellent (no ink removal) according to BS 3900-E6.ISO 2409 2) Forming Properties The printed substrates were examined for forming properties using the most commonly used techniques (vacuum thermoform.","HPF-Niebling, hydroform, matched metal): TABLE 3 Vacuum Thermoform Ink system Ranking*** Comments Example 1 4 Good forming, suitable for medium to deep draw Example 2 3-4 Mould marking can be a problem, inferior at high temps/long cycle times Example 3 4 Suitable for medium to deep draw, improved resistance to mould marking Example 4 4 Suitable for medium to deep draw, improved resistance to mould marking Example 5 4 Suitable for medium to deep draw, improved resistance to mould marking Example 6 4 Suitable for medium to deep draw, improved resistance to mould marking Example 7 4 Suitable for medium to deep draw, improved resistance to mould marking Example 8 4 Suitable for medium to deep draw, improved resistance to mould marking TABLE 4 HPF-Niebling Ink system Ranking*** Comments Example 1 4 Good forming suitable for medium to deep draw Example 2 4 Good forming suitable for medium to deep draw Example 3 4 Good forming suitable for medium to deep draw Example 4 4 Good forming suitable for medium to deep draw Example 5 4 Good forming suitable for medium to deep draw Example 6 4 Good forming suitable for medium to deep draw Example 7 4 Good forming suitable for medium to deep draw Example 8 4 Good forming suitable for medium to deep draw TABLE 5 Hydroform Ink system Ranking*** Comments Example 1 3-4 Moderate forming suitable for medium draw Example 2 4 Good forming suitable for medium to deep draw Example 3 4 Good forming suitable for medium to deep draw Example 4 4 Good forming suitable for medium to deep draw Example 5 4 Good forming suitable for medium to deep draw Example 6 4 Good forming suitable for medium to deep draw Example 7 4 Good forming suitable for medium to deep draw Example 8 4 Good forming suitable for medium to deep draw TABLE 6 Matched Metal Ink system Ranking*** Comments Example 1 5 Superior resistance to marking from mould tool Example 2 2 Composition too soft.","marked by mould tool Example 3 5 Superior resistance to marking from mould tool Example 4 5 Slightly softer than Example 3 Example 5 5 Superior resistance to marking from mould tool Example 6 5 Superior resistance to marking from mould tool Example 7 5 Superior resistance to marking from mould tool Example 8 5 Superior resistance to marking from mould tool ***1 = poor (not formable), 5 = excellent (deep draw possible within limits of forming technique) 3) Resistance to Wash-Out The printed substrates were subjected to direct injection with a sprue gate, of a polycarbonate backfill.","The degree of ink movement (wash-out) was visually assessed and ranked: TABLE 7 Resistance to PC injection Ink system Ranking**** Comments Example 1 5 No wash-out, but total solvent removal critical Example 2 2 Wash-out, can be improved by use of tie/protective coat e.g.","Aqualam ™ (ex Coates) Example 3 5 No wash-out Example 4 5 No wash-out Example 5 5 No wash-out Example 6 5 No wash-out Example 7 5 No wash-out Example 8 4-5 Slight wash-out directly around injection point ****1 = poor (ink wash-out), 5 = excellent (no ink wash-out) 4) Adhesion or Bond to Injected Backfill Resin The printed substrates were subjected to injection of polycarbonate resin using a plaque tool with a wide fan gate injection port.","The bond strength between the backfill resin and the printed substrate was assessed by peel strength analysis: TABLE 8 Peel Strength Ink system Typical value (N) Range (N) Example 1 50 50.0-58.0 Example 2 6.7 0.0-8.8 Example 3 51.1 37.8-64.4 Example 4 52.3 34.2-68.0 Example 5 53 33.8-69.2 Example 6 48.8 30.0-60.2 5) Resistance to Heat Ageing and Environmental Cycling Heat ageing and environmental cycling test were undertaken on full IMD parts to ensure that there were no changes to e.g.","laminate strength, colouristic properties after the heat/environmental cycles: TABLE 9 Heat Ageing/Environmental Cycling Delamination Ink system test Comments Example 1 Pass Thorough drying critical Example 2 Fail Poor bond strength prior to testing Example 3 Pass No appreciable loss of properties Example 4 Pass No appreciable loss of properties Example 5 Pass No appreciable loss of properties Example 6 Pass No appreciable loss of properties TABLE 10 Heat Ageing/Environmental Cycling Ink system Colouristic test Comments Example 1 Pass — Example 2 Fail — Example 3 Pass No appreciable loss of properties Example 4 Pass No appreciable loss of properties Example 5 Pass No appreciable loss of properties Example 6 Pass No appreciable loss of properties 6) Print Curl Single and multilayer prints of Example 1 and Example 3 were prepared on 125 micron (μm) Bayfol® and assessed for degree of substrate curl or distortion after 24 hours.","TABLE 11 Ink film Assessment Number of thickness***** of curl Ink system print layers (μm) (mm) Comment Example 1 1 5 >45 Very poor 2 12 >47 Very poor 3 17 >47 Very poor 4 22 49 Very poor Example 3 1 10 2 — 2 16 2-3 — 3 22 3 — 4 29 3-6 Very slight curl *****Ink film thickness determined using digital micrometer and comparing printed (various number of ink deposits) and unprinted areas of the substrate."]],"string":"[\n [\n \"Inks for in-mould decoration\",\n \"An energy-core ink or varnish composition is provided for use in an in-mould decoration (IMD) process, comprising an energy-curable resin, additional reactive monomers and/or oligomers and, optionally and in an amount not exceeding 10% by weight, a solvent, wherein the resin comprises a urethane acrylate oligomer having an aromatic or aliphatic polycarbonate backbone.\",\n \"For inks, the composition additionally includes a pigment or dye.\",\n \"For photocure compositions, a photoinitiator is also included.\",\n \"Also provided is an in-mould decoration (IMD) process employing the ink or varnish composition.\"\n ],\n [\n \"1.An energy-cure ink or varnish composition for use in an in-mould decoration (IMD) process, comprising an energy-curable resin, additional reactive monomers and/or oligomers and optionally and in an amount not exceeding 10% by weight of the composition, a solvent, wherein the energy-curable resin comprises a urethane acrylate oligomer having an aromatic or aliphatic polycarbonate backbone.\",\n \"2.A composition according to claim 1, wherein the composition is an ink and further comprises a pigment or dye.\",\n \"3.A composition according to claim 2, wherein the pigment or dye is present in an amount of from 1 to 40% by weight of the composition.\",\n \"4.A composition according to any preceding claim wherein the composition is a photocure composition and further comprises a photoinitiator.\",\n \"5.A composition according to claim 4, wherein the photoinitiator is present in an amount of from 1 to 14% by weight of the composition.\",\n \"6.A composition according to any preceding claim wherein the polycarbonate backbone is aliphatic.\",\n \"7.A composition according to any preceding claim, wherein the polycarbonate backbone is linear.\",\n \"8.A composition according to any preceding claim, wherein the polycarbonate-backboned urethane acrylate oligomer has a molecular weight of from 1,000 to 30,000, preferably from 3,000 to 15,000, and more preferably from 6,500 to 10,000.9.A composition according to any preceding claim, wherein the polycarbonate-backboned urethane acrylate component is present in an amount of from 5 to 70% by weight of the composition.\",\n \"10.A composition according to any preceding claim, wherein the polycarbonate-backboned urethane acrylate oligomer is of the general formula (I) or (II): wherein: R2 and R3 are such that OCN—R—NCO (where R=R2 or R3) represents a diisocyanate; Y represents a hydrogen atom or a methyl group; R1 and R4 together with their attached (meth)acrylate group represent the residue of a hydroxy (meth)acrylate functional unsaturated monomer.\",\n \"11.A composition according to any preceding claim, wherein the composition comprises additional acrylate monomer and/or oligomer and the acrylate monomer and/or oligomer is present in an amount of from 0 to 80% by weight of the composition.\",\n \"12.A composition according to claim 11, wherein the acrylate monomer and/or oligomer is monofunctional.\",\n \"13.A composition according to any preceding claim, wherein the composition comprises the reactive monomer N-vinyl-2-pyrrolidone (NVP), in an amount not exceeding 10%, preferably not exceeding 5%, by weight of the composition.\",\n \"14.A composition according to any one of claims 1 to 13, wherein the composition comprises an organic solvent in an amount not exceeding 5% by weight of the composition.\",\n \"15.A composition according to any one of claims 1 to 13, wherein the composition is free of organic solvent.\",\n \"16.A method of in-mould decoration employing an ink or varnish composition as defined in any of the preceding claims.\",\n \"17.A method according to claim 16, wherein the ink is printed onto a polycarbonate or polycarbonate/polyester substrate.\",\n \"18.A method according to claim 16 or claim 17, wherein the ink is printed onto a substrate and the ink on the substrate is subjected to polycarbonate or polycarbonate/polyester blend resin injection to provide a polycarbonate or a polycarbonate/polyester blend backfill adhered to the printed substrate.\"\n ],\n [\n \"The present invention relates to a new ink composition for in-mould decorative use and to an in-mould decorative process employing this ink.\",\n \"In its broadest sense, in-mould decorating (IMD) simply means applying a decoration in the course of moulding the decorated part.\",\n \"A particular IMD process referred to as insert moulding or insert IMD has been known for many years, but has seen little practical uses except in the automotive industry, until recently.\",\n \"In insert IMD processes, a film substrate is printed with the desired decoration this is preferably a second surface printing process in which the decoration is printed in reverse on the reverse side of a transparent or translucent (or partially transparent or translucent) film substrate, so that it shows through and is protected by the substrate.\",\n \"Then, in one or more steps, the substrate is, if necessary, formed into shape and further resin or plastics material is injection moulded to give the final product.\",\n \"It will be seen readily that this process can give rise to several difficulties.\",\n \"First, if the printed substrate is to be moulded after printing, the printing ink must have the necessary mechanical properties to wild the strains of moulding.\",\n \"Thus, it must be flexible, and preferably have at least a similar flexibility to that of the substrate, so as to stretch with the substrate as the substrate is moulded.\",\n \"It must also have sufficient adhesion to the substrate and abrasion resistance to withstand any abrading action in the course of moulding.\",\n \"In the final step, where a liquid resin or resin precursor is injected onto the formed decorated substrate, the printing ink must be able to resist the heat, pressure and shear imparted to it by the resin injection, or it has to be protected with an additional layer.\",\n \"Otherwise, the ink will be caused to degrade or smudge from its printed location by the injection process, resulting in so-called “wash-out”.\",\n \"Finally, the ink has to be compatible with the injected resin and in particular must provide a good adhesion with the injected resin so as to prevent delamination of the printed substrate from the resin backfill.\",\n \"One way of solving the problems of avoiding wash-out of the ink and ensuring good adhesive bond strength to the resin backfill has been to provide the printed substrate with an additional coating over the irk which additional coating is typically an aqueous laminating adhesive that is applied over the graphic inks.\",\n \"This coating then serves to bond or laminate an additional sheet of substrate such that the ink is sandwiched between the substrate and the additional sheet of substrate.\",\n \"In this twin-layer construction, the laminated substrate serves to protect the ink against wash-out by the backfill injection and provides good adhesion to the injected resin.\",\n \"However, this construction method requires additional processing steps and material cost.\",\n \"DE-A-19832570 discloses a solvent-based ink system comprising a blend of a polycarbonate and a thermoplastic polyester polyurethane.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,648,414 discloses a solvent-based ink suitable for insert IMD, containing a polycarbonate based on geminally disubstituted, dihydroxydiphenyl cycloalkanes as binder.\",\n \"One such ink is commercially available as Noriphan™ (ex Proll under licence from Bayer AG).\",\n \"Whilst this solvent-based ink permits a single-layer printed substrate construction to be used in IMD processes, it is associated with certain disadvantages.\",\n \"For example, because it is a solvent-based ink, it is relatively difficult or inconvenient to screen print due to its poor press stability and difficulties in washing up.\",\n \"Also, the printed substrate has been found to be susceptible to curling, and it is often therefore difficult to place the printed substrate in registration in the mould tool prior to injection with the resin backfill.\",\n \"Furthermore, it is essential to ensure that all solvent is completely removed before the printed substrate is formed and injection moulded, in order to prevent wash-out, and blister or bubble formation, leading to delamination.\",\n \"Another disadvantage is that the range of colours available is limited due to the fact that certain pigments can break down the polymer resin.\",\n \"Finally, the need for a solvent in the ink has obvious environmental and health hazard implications.\",\n \"These deficiencies associated with solvent-based systems have led to the development of photocurable ink systems, for example inks curable by UV-energy, for use in IMD processes, which require less or no solvent.\",\n \"However, existing UV-curable inks are not wholly satisfactory, particularly in terms of formability, washout resistance and adhesion to injected resin backfill.\",\n \"It will be appreciated that inks suitable for IMD must have very good adhesion to the substrate, good flexibility and elongation to permit forming with the substrate, good mechanical resistance to abrasion by the moulds, good washout resistance to the resin injection step, and good adhesive bonding to the injected resin backfill.\",\n \"Because UV-curable inks undergo cross-linking, it is far more difficult to achieve this combination of properties in a UV-curable ink than in a solvent-based ink.\",\n \"Although these UV-curable ink systems have eliminated or reduced the solvent, and hence the problems associated with the use of solvent in a solvent-based ink system the need for flexibility and elongation has meant that the inks have tended to be too so* to withstand the mechanical impact of the forming and injection processes.\",\n \"In addition adhesion to the resin backfill was often not satisfactory.\",\n \"Therefore, it was generally still found necessary to provide the printed substrate with a protective or tie coating, in order to ensure adequate resistance to the resin injection process and to provide good bonding to the backfill resin, so as to avoid wash-out, delamination, or both.\",\n \"In addition to boost adhesion to the substrate and avoid delamination at the ink:substrate interface, it was generally still necessary to include aggressive monomers, such as N-vinyl-2-pyrrolidone (NVP), in the ink, which in turn have associated drawbacks, for example they may lead to delamination due to unreacted material, and to health and safety concerns.\",\n \"We have now found, surprisingly, that it is possible to provide an energy-curable composition, preferably a UV-curable ink, suitable for use in IMD processes, that can be printed onto a substrate, that allows the printed substrate to be formed using conventional forming techniques, and backfilled with a resin injection without undergoing wash-out, even in the absence of a protective coating.\",\n \"Furthermore, the ink can provide excellent adhesion to the resin backfill without the need for a tie coating.\",\n \"Because the claimed energy-curable resin composition gives improved adhesion the requirement for NVP is reduced or eliminated.\",\n \"A fisher advantage is that solvent may be significantly reduced or even eliminated from the IMD process.\",\n \"The reduction or removal of solvent or NVP, or both, may lead to further advantages, apart from obvious environmental and health considerations, such as reduced delamination of the final part especially in environmental testing, caused by poor removal or poor full reaction of potentially volatile materials.\",\n \"In addition, we have found that ink compositions according to the present invention can provide a harder, tougher surface, when cured, than conventional insert IMD inks that contain high molecular weight urge acrylate resins, whilst exhibiting excellent flexibility and forming characteristics.\",\n \"It will be appreciated that hardness and toughness are desirable in an insert IMD ink in order to avoid accidental marking or damage caused by forming or moulding tools.\",\n \"Broadly speaking, this has been achieved by using a new urethane acrylate resin having a polycarbonate backbone as carrier for the pigment or dye.\",\n \"Accordingly, in a first aspect, the present invention provides an energy-cure ink or varnish composition for use in an in-mould decoration (IMD) process, comprising an energy-curable resin, additional reactive monomers and/or oligomers and, optionally and in an amount not exceeding 10% by weight of the composition, a solvent, wherein the energy-curable resin comprises a urethane acrylate, oligomer having an aromatic or aliphatic-polycarbonate backbone.\",\n \"In a second aspect, the invention provides a method of in-mould decoration employing the ink or varnish composition.\",\n \"By “energy-curable resin” or “energy-cure compositions”, as used herein, is meant a resin or composition that is curable by exposure to a source of radiation of an appropriate wavelength or intensity, i.e.\",\n \"is photocurable by exposure to a source of electromagnetic radiation of an appropriate wavelength, such as ultraviolet (UV) radiation, or is electron beam (EB) curable by exposure to an electron beam of an appropriate intensity.\",\n \"Preferably, the energy-cure compositions according to the present invention are at least UV-curable.\",\n \"The energy-cure compositions according to the invention contain an energy-curable resin, additional reactive monomers and/or oligomers, additives and, in the case of an ink composition, pigments or dyes.\",\n \"In the case of photocure compositions such as UV-cure compositions, a photoinitiator is also included in the composition.\",\n \"Energy-Curable Resin: The energy-curable resin at least comprises a mono-, di- or trifunctional urethane acrylate oligomer that has a polycarbonate backbone and is obtainable by reaction of a diisocyanate, a hydroxy (meth)acrylate functional unsaturated monomer and a polycarbonate polyol.\",\n \"Preferably, the resin at least comprises a mono- or difunctional urethane acrylate oligomer, as represented by the general formula (I) or (II): wherein: R2 and R3 are such that OCN—R—NCO (where R=R2 or R3) represents the diisocyanate used in the synthesis; Y represents a hydrogen atom or a methyl group; R1 and R4 together with their attached (meth)acrylate group represent the residue of the hydroxy (meth)acrylate functional unsaturated monomer used in the synthesis.\",\n \"Examples of the diisocyanate compounds include, but are not limited to, hexamethylene diisocyanate (HMDI), isophorone diisocyanate (IPDI), 4,4-dicyclohexylmethane diisocyanate (H12MDI), 2,2,4-trimethyl hexamethylene diisocyanate 2,4-tolylene diisocyanate (TDI), 2,6-tolylene diisocyanate (TDI), trimethylhexamethylene diisocyanate (TMDI), diphenylmethane diisocyanate (MDI), tetramethylxylene diisocyanate (TMXDI), and xylene diisocyanate (XDI).\",\n \"A preferred isocyanate is isophorone diisocyanate (IPDI) because of improved resistance and selective reactivities of the isocyanate groups enabling preparation of an ethylenically unsaturated monoisocyanate.\",\n \"The above diisocyanate compounds may be used individually or in combination.\",\n \"Examples of the hydroxy (meth)acrylate functional unsaturated monomers include, but are not limited to, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, 2-hydroxypropyl acrylate, 1,4-butanediol monoacrylate and glyceryl diacrylate, trimethylolethane di(meth)acrylate.\",\n \"Suitable polycarbonate polyols are represented by the general formula (III): wherein: R5 and R6 represent the same or different aliphatic or aromatic groups; and n is an integer of 1 to 60.The polycarbonate polyols can be produced through, for example, an ester interchange reaction or alcoholysis of diethyl carbonate or diphenyl carbonate with a polyol, preferably a diol such as an alkylene diol, e.g.\",\n \"1,4-butane diol, 1,6-hexane diol, or an alkylene ether diol.\",\n \"e.g.\",\n \"triethylene glycol, tripropylene glycol.\",\n \"Other suitable diols include 2,2-bis-[4-(2-hydroxypropoxy)phenyl]propane and 1,1-bis-(4-hydroxyphenyl)-3,3,5-trimethyl cyclohexane.\",\n \"Polyols with three or more hydroxyl groups, such as trimethylol propane, glycerine and pentaerythritol, can be incorporated for preparing polycarbonate polyols suitable for synthesising trifunctional polycarbonate urethane acrylates.\",\n \"Mixtures of the polycarbonate polyols may also be utilised in the synthesis.\",\n \"Other polyols such as polyether- or polyester-polyols may also be included, although to achieve the desired IMD properties the backbone should be substantially polycarbonate in nature.\",\n \"The polycarbonate backbone may be aliphatic or aromatic but preferably is aliphatic in nature.\",\n \"It may be linear or branched, and is preferably linear.\",\n \"Suitable polycarbonate backboned urethane acrylates are available as NTX 4711 and NTX 4867 (ex Sartomer).\",\n \"RD2/105.RD2/106.RD3/101, RD3/102, RD4/103 and RD4/104 (ex UCB Chemicals SA), and RXX-01-344 (ex Rahn).\",\n \"We prefer that the polycarbonate urethane acrylate (PCUA) oligomer has a average molecular weight in the range of from 1,000 to 30,000, more preferably from 3,000 to 15,000, still more preferably from 4,000 to 10,000, and in particular from 6,500 to 10,000.For the purposes of providing improved flexibility and elongation, higher molecular weight polycarbonate backbones would be desirable.\",\n \"However, higher molecular weights can have an adverse effect on the viscosity and printability of the ink.\",\n \"Moreover, higher molecular weights tend to produce unduly soft ink coatings, which are more susceptible to marking by mould parts or during handling.\",\n \"Thus, it will be appreciated that the particular molecular weight chosen will represent a compromise between these conflicting factors, on the one hand providing acceptable flexibility and elongation whilst on the other hand, ensuring satisfactory viscosity, printability and hardness.\",\n \"If desired, the flexibility, elongation and hardness properties of the ink can be tailored by blends of monofunctional, difunctional or trifunctional PCUA oligomer.\",\n \"It will be appreciated that the higher degrees of cross-linking afforded by inclusion of di- and trifunctional oligomers will tend to reduce the overall flexibility and elongation of the ink, and increase its hardness.\",\n \"Thus, the energy-curable resin may comprise a mono-, di- or trifunctional polycarbonate urethane acrylate oligomer, or a blend of two or more of mono-, di- and trifunctional polycarbonate urethane acrylate oligomers.\",\n \"Preferably, the energy-curable resin at least comprises a mono- or difunctional polycarbonate urethane acrylate oligomer, or a blend thereof.\",\n \"We prefer that the average functionality of the energy-curable resin is less than 2.5, more preferably less than 2.2, and most preferably less than 2.1.The monofunctional polycarbonate urethane acrylate may be present in the composition in an amount of from 0 to 70% by weight.\",\n \"Difunctional polycarbonate urethane acrylate may be included in an amount from 0 to 50% by weight.\",\n \"Trifunctional polycarbonate urethane acrylate may be included in an amount from 0 to 5% by weight.\",\n \"The total polycarbonate urethane acrylate component is present in an amount of from 5 to 70%, preferably from 20 to 60%, by weight of the composition.\",\n \"Photoinitiators: Preferably the compositions according to the invention are at least photocurable, and most preferably are UV-curable, and accordingly contain a photoinitiator.\",\n \"However, it will be appreciated that EB-cure compositions in accordance with the invention can be formulated without the need for a photoinitiator.\",\n \"For photocure compositions such as UV-cure compositions, a wide range of commercially available photoinitiators can be incorporated to initiate the photocure mechanism.\",\n \"Preferably these photoinitiators have low potential to migration and low volatility, so as to avoid problems such as delamination of the injection moulded part.\",\n \"The photoinitiator package should also have low UV thermal yellowing, important to meet typical heat and environmental cycling tests typically used on the final IMD part.\",\n \"The photoinitiator package is typically present in an amount of from 0.5 to 20% for example from 1 to 14%, by weight of the composition.\",\n \"Suitable photoinitiators include, for example, 2,4,6-trimethylbenzyl diphenyl phosphine oxide (Lucerin™ TPO ex BASF), bis(2,4,6-trimethylbenzoyl)phenyl phosphine oxide (Irgacure 819, ex Ciba Geigy), bis(2,6-dimethoxybenzoyl)-2,4,4-trimethylpentyl phosphine oxide, ethyl-2,4,6-trimethylbenzoylphenyl-phosphinate (Lucerin™.\",\n \"TPO-L, ex BASF), 2-benzyl-2-dimethylamino-1-(4-morpholinphenyl) butan-1-one (Irgacure™ 369, ex Ciba Geigy), 1-hydroxycyclohexyl acetophenone (Irgacure™ 184, ex Ciba Geigy), iso-propyl thioxanthone (Quantacure™ ITX, ex IBIS or Speedcure™ ITX ex Lambson; 2-chloro thioxanthone (Kayacure™ CTX, ex Nippon Kayaku), oligo[2-hydroxy-2-methyl-1-[4-(1-methylvinyl)phenyl]propanone and 2-hydroxy-2-methyl-1-phenyl-1-propanone (Esacure™ KIP100F, ex Lamberti), methyl benzoyl formate (Genocure MBF ex Rahn), benzophenone, 2-methyl-1-[4-(methylthio)phenyl]-2-morpholinopropan-1-one (Irgacure™ 907, ex Ciba Geigy).\",\n \"Of these, 2,4,6-trimethylbenzoyl diphenyl phosphine oxide (Lucerin™ TPO, ex BASF), bis(2,4,6-trimethylbenzoyl)-phenyl phosphine oxide (Irgacure 819, ex Ciba Geigy), and bis(2,6-dimethoxybenzoyl)-2,4,4-trimethylpentyl phosphine oxide are preferred photoinitiators.\",\n \"Additional Reactive Monomers and/or Oligomers: Additional reactive energy-cure monomers and/or oligomers may be present in an amount of from 0 to 80%, preferably from 0 to 60%, by weight of the composition.\",\n \"These reactive monomers and/or oligomers are preferably monofunctional.\",\n \"Suitable acrylate monomers or oligomers include, but are not limited to, isobornyl acrylate (IBOA), 2-phenoxy ethyl acrylate (2PEA), 2-(2-ethoxyethoxy) ethyl acrylate (EOEOEA).\",\n \"CTF acrylate, 4-t-butylcyclohexyl acrylate.\",\n \"THF-acrylate, alkoxylated acrylates, diethylene glycol diacrylate, dipropylene glycol diacrylate, 1,6-hexanediol diacrylate, low molecular weight monofunctional urethane acrylates, polyether acrylates, polyester acrylates and low molecular weight epoxy acrylates.\",\n \"Non-acrylated reactive diluents that may be incorporated include, but are not limited to, acryloyl morpholine (ACMO), n-vinylformamide (NVF), n-vinylformamide derivatives and n-vinyl caprolactam (NVC).\",\n \"The compositions of the invention can provide improved adhesion to the substrate and backfill, thereby advantageously allowing the incorporation of N-vinyl-2-pyrrolidone (NVP) to be significantly reduced or even eliminates NVP is conventionally used to boost adhesion to the substrate, but concerns about health and safety, and its effect due to the volatility of unreacted material on delamination, means that it would be advantageous to eliminate, or at least reduce, NVP.\",\n \"Nevertheless, in certain cases it will still be desirable to include monomers such as NVP in the ink and varnish compositions of the invention, to improve ‘key’ to substrate.\",\n \"If used, NVP can be incorporated in an amount not exceeding 30%, preferably not exceeding 15%, more preferably not exceeding 10%, and in particular not exceeding 5%, by weight of the composition.\",\n \"Most preferably, the composition is free of NVP.\",\n \"Additives: If desired, inert or passive resins such as acrylics, styrene acrylates, polyesters, polycarbonates or celluloses may be included in the ink in small amounts, in order to improve the adhesion of the ink coating.\",\n \"However, these inert or passive resins tend to adversely affect the resistance of the ink to injection of the backfill resin, and thus increase the likelihood of wash-out.\",\n \"If included, therefore, we that only a small amount is used, for example an amount not exceeding 10%, and preferably not exceeding 7%, by weight of the composition.\",\n \"Additives such as wetting agents, silicone and non-silicone antifoams may be incorporated to improved print properties such as substrate wetting and flow-out, and may be included in an amount of from 0 to 5%, preferably from 0 to 2%, by weight of the composition.\",\n \"It should be noted that some additives, typically with low molecular weights, may have a tendency to migrate to the print surface in the cured coating.\",\n \"This can affect the IMD properties, and therefore the total additive content should be kept to a minimum and additives with a high migration potential avoided.\",\n \"Where the composition is intended for use as an ink, instead of a varnish, a pigment or dye is included in the composition.\",\n \"Organic and/or inorganic pigments or dyes can be incorporated in an amount of from 0 to 50%, preferably from 0 to 40%, by weight of the composition.\",\n \"The pigments or dyes should be selected to have good resistance to thermal decomposition and change, and resistance to sublimation.\",\n \"Suitable pigments include, but not limited to, titanium dioxide white, zinc sulphide, carbon black, azo diarylide yellow, isoindoline yellow, diarylide orange, quinacridone magneta, diketo-pyrrolo-pyrrol red, copper phthalocyanine blue, copper phthalocyanine green, dioxazine violet, diketometal oxides.\",\n \"Speciality effect pigments, such as metal oxide-coated mica pigments and aluminium metallic pigments, can also be included.\",\n \"Fillers may be included to control the viscosity and rheology of the composition typically to improve printing characteristics, and may be present in an amount of from 0 to 40%, preferably from 0 to 30%, by weight of the composition.\",\n \"Suitable fillers include, but not limited to, calcium carbonate, china clay, aluminium hydrate, talc, barium sulphate, aluminium silicate, and silica.\",\n \"It will be appreciated that the compositions according to the invention will be substantially free of organic solvent.\",\n \"However, small amounts of organic solvent may be included, if needed, in amounts not exceeding 10%, and preferably not exceeding 5%, by weight of the composition.\",\n \"Most preferred is that the composition is free of organic solvent.\",\n \"In-Mould Decorating (IMD) Process: The energy-cure compositions of the invention are particularly suitable for use in IMD processes, and especially in insert IMD or insert moulding processes.\",\n \"The application of the compositions to the substrate may be effected using conventional printing techniques.\",\n \"Preferred techniques include flexographic, lithographic, digital and screen print processes, but other methods may be used as appropriate.\",\n \"Application by screen printing is particularly preferred.\",\n \"Suitable substrate materials onto which the ink may be printed or the varnish applied as the case may be, include print-receptive polyester, polycarbonate, ABS.\",\n \"PMMA, polycarbonate/polyester blends, polycarbonate/ABS blends materials such as those supplied by Bayer AG (Bayfol®, Makrolon®, Marofol®, Bayblend®), GE Structured Products (Lexan®) and Autotype (Autoflex Hiform™, Autoflex XtraForm™).\",\n \"Preferably, the substrate is of a polycarbonate or polycarbonate/polyester blend resin material.\",\n \"Similarly, suitable backfill materials which may be injected onto the printed substrate include the following or blends of the following: polyesters polycarbonate, styrenes ABS and PMMA resin materials.\",\n \"Preferably, the injected backfill is of polycarbonate or a polycarbonate/polyester blend resin material.\",\n \"The invention will be further illustrated by reference to the following non-limiting Examples: EXAMPLES A) Formulations: Example 1 Comparative Solvent-Based IMD Technology A sample of Noriphan™ black HTR952 was obtained (ex Proll) for comparison to UV IMD ink technology.\",\n \"Prints of Noriphan™ were prepared in accordance to the Noriphan™ product data sheet supplied by Proll.\",\n \"The prints were examined for adhesion, surface hardness, print curl, forming, and IMD properties.\",\n \"Example 2 Comparative Standard Flexible Urethane Acrylate Technology The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.\",\n \"Urethane acrylate, Actilane ™ 290 24.0 IBOA, SR506 ex Sartomer 31.8 NVP ex BASF or ISP 12.0 Silicone antifoam 0.7 UV Stabiliser, Genorad ™ 16 ex Rahn 0.5 Lucerin ™ TPO ex BASF 4.0 Irgacure ™ 184 ex Ciba 3.0 Filler 20.0 Carbon Black 4.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).\",\n \"The prints were examined for adhesion, surface hardness, forming, and IMD properties.\",\n \"Example 3 Invention Polycarbonate Urethane Acrylate Technology The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.\",\n \"PCUA, NTX 4867 34.3 IBOA, SR506 ex Sartomer 23.5 UV Stabiliser, Genorad ™ 16 ex Rahn 0.5 Silicone antifoam 0.7 Lucerin ™ TPO ex BASF 4.0 Quantacure ™ ITX ex IBlS 1.0 NVP ex BASF 12.0 Filler 20.0 Carbon Black 4.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).\",\n \"The prints were examined for adhesion, surface hardness, print curl, forming, and IMD properties.\",\n \"Example 4 Invention Polycarbonate Urethane Acrylate Technology (NVP Free) The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.\",\n \"PCUA, NTX 4867 ex Sartomer 36.4 EOEOEA, S256 ex Sartomer 8.7 IBOA, SR506 ex Sartomer 11.9 NVC, ex BASF or ISP 18.1 Polyester urethane acrylate ex Rahn 3.8 UV stabiliser, Genorad ™ 16 ex Rahn 0.4 Lucerin ™ TPO ex BASF 4.7 Irgacure ™ 184 ex Ciba 2.6 Silicone antifoam 0.7 Carbon black 3.6 Filler 9.1 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).\",\n \"The prints were examined for adhesion, surface hardness, forming, and IMD properties.\",\n \"Example 5 Invention Polycarbonate Urethane Acrylate Technology (NVP Free) The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.\",\n \"PCUA, NTX 4867 40.0 IBOA, SR506 ex Sartomer 11.8 UV Stabiliser, Genorad ™ 16 ex Rahn 0.5 Silicone antifoam 0.7 Lucerin ™ TPO ex BASF 4.0 Irgacure ™ 184 ex Ciba Geigy 1.0 ACMO ex Rahn 18.0 EOEOEA, SR256 ex Sartomer 8.0 Filler 10.0 Red 6.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).\",\n \"The prints were examined for adhesion, surface hardness, forming and IMD properties.\",\n \"Example 6 Invention Polycarbonate Urethane Acrylate Technology (NVP Free) The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.\",\n \"PCUA, RD2/105 ex UCB 25.8 EOEOEA, S256 ex Sartomer 6.0 IBOA, SR506 ex Sartomer 37.2 ACMO ex Rahn 7.0 UV stabiliser, Genorad ™ 16 ex Rahn 0.3 Lucerin ™ TPO ex BASF 4.0 Genocure ™ MBF ex Rahn 2.0 Speedcure ™ ITX ex Lambson 1.0 Silicone antifoam 0.7 Phthalocyanine blue 6.0 Filler 10.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).\",\n \"The prints were examined for adhesion, surface hardness, forming, and IMD properties.\",\n \"Example 7 Invention Polycarbonate Urethane Acrylate Technology The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.\",\n \"PCUA, RXX0] 344 ex Rahn 26.0 IBOA, SR506 ex Sartomer 27.0 NVP ex BASF or ISP 12.0 UV stabiliser, Genorad ™ 16 ex Rahn 0.3 Lucerin ™ TPO ex BASF 4.0 Silicone antifoam 0.7 Titanium dioxide white pigment 30.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).\",\n \"The prints were examined for adhesion, surface hardness, forming, and IMD properties.\",\n \"Example 8 Invention Polycarbonate Urethane Acrylate Technology (NVP Free) The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μM) was achieved.\",\n \"PCUA, RD3/101 ex UCB 40.0 EOEOEA, S256 ex Sartomer 6.0 IBOA, SR506 ex Sartomer 15.0 ACMO ex Rahn 7.0 UV stabiliser, Genorad ™ 16 ex Rahn 0.3 Lucerin ™ TPO ex BASF 4.0 Genocure ™ MBF ex Rahn 2.0 Speedcure ™ ITX ex Lambson 1.0 Silicone antifoam 0.7 Red 6.0 Filler 18.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).\",\n \"The print were examined for adhesion, surface hardness, forming, and IMD properties.\",\n \"B) Experimental Results: 1) Physical Properties TABLE 1 Pencil Hardness Ink system Hardness* Comments Example 1 H Can be marked, but good scratch resistance Example 2 HB Moderate scratch, easily marked Example 3 2H Difficult to scratch, excellent adhesion Example 4 H-2H Difficult to scratch, excellent adhesion Example 5 H-2H Difficult to scratch, excellent adhesion Example 6 H Difficult to scratch, excellent adhesion Example 7 H Difficult to scratch, excellent adhesion Example 8 H Difficult to scratch, excellent adhesion *according to ASTM D3363, BS 3900-E19, ISO 15184 TABLE 2 Cross-Hatch Adhesion Ink system Ranking** Comments Example 1 4-5 No removal Example 2 4 Slight/no removal Example 3 5 No removal Example 4 5 No removal Example 5 5 No removal Example 6 5 No removal Example 7 5 No removal Example 8 5 No removal **1 = poor (total ink removal).\",\n \"5 = excellent (no ink removal) according to BS 3900-E6.ISO 2409 2) Forming Properties The printed substrates were examined for forming properties using the most commonly used techniques (vacuum thermoform.\",\n \"HPF-Niebling, hydroform, matched metal): TABLE 3 Vacuum Thermoform Ink system Ranking*** Comments Example 1 4 Good forming, suitable for medium to deep draw Example 2 3-4 Mould marking can be a problem, inferior at high temps/long cycle times Example 3 4 Suitable for medium to deep draw, improved resistance to mould marking Example 4 4 Suitable for medium to deep draw, improved resistance to mould marking Example 5 4 Suitable for medium to deep draw, improved resistance to mould marking Example 6 4 Suitable for medium to deep draw, improved resistance to mould marking Example 7 4 Suitable for medium to deep draw, improved resistance to mould marking Example 8 4 Suitable for medium to deep draw, improved resistance to mould marking TABLE 4 HPF-Niebling Ink system Ranking*** Comments Example 1 4 Good forming suitable for medium to deep draw Example 2 4 Good forming suitable for medium to deep draw Example 3 4 Good forming suitable for medium to deep draw Example 4 4 Good forming suitable for medium to deep draw Example 5 4 Good forming suitable for medium to deep draw Example 6 4 Good forming suitable for medium to deep draw Example 7 4 Good forming suitable for medium to deep draw Example 8 4 Good forming suitable for medium to deep draw TABLE 5 Hydroform Ink system Ranking*** Comments Example 1 3-4 Moderate forming suitable for medium draw Example 2 4 Good forming suitable for medium to deep draw Example 3 4 Good forming suitable for medium to deep draw Example 4 4 Good forming suitable for medium to deep draw Example 5 4 Good forming suitable for medium to deep draw Example 6 4 Good forming suitable for medium to deep draw Example 7 4 Good forming suitable for medium to deep draw Example 8 4 Good forming suitable for medium to deep draw TABLE 6 Matched Metal Ink system Ranking*** Comments Example 1 5 Superior resistance to marking from mould tool Example 2 2 Composition too soft.\",\n \"marked by mould tool Example 3 5 Superior resistance to marking from mould tool Example 4 5 Slightly softer than Example 3 Example 5 5 Superior resistance to marking from mould tool Example 6 5 Superior resistance to marking from mould tool Example 7 5 Superior resistance to marking from mould tool Example 8 5 Superior resistance to marking from mould tool ***1 = poor (not formable), 5 = excellent (deep draw possible within limits of forming technique) 3) Resistance to Wash-Out The printed substrates were subjected to direct injection with a sprue gate, of a polycarbonate backfill.\",\n \"The degree of ink movement (wash-out) was visually assessed and ranked: TABLE 7 Resistance to PC injection Ink system Ranking**** Comments Example 1 5 No wash-out, but total solvent removal critical Example 2 2 Wash-out, can be improved by use of tie/protective coat e.g.\",\n \"Aqualam ™ (ex Coates) Example 3 5 No wash-out Example 4 5 No wash-out Example 5 5 No wash-out Example 6 5 No wash-out Example 7 5 No wash-out Example 8 4-5 Slight wash-out directly around injection point ****1 = poor (ink wash-out), 5 = excellent (no ink wash-out) 4) Adhesion or Bond to Injected Backfill Resin The printed substrates were subjected to injection of polycarbonate resin using a plaque tool with a wide fan gate injection port.\",\n \"The bond strength between the backfill resin and the printed substrate was assessed by peel strength analysis: TABLE 8 Peel Strength Ink system Typical value (N) Range (N) Example 1 50 50.0-58.0 Example 2 6.7 0.0-8.8 Example 3 51.1 37.8-64.4 Example 4 52.3 34.2-68.0 Example 5 53 33.8-69.2 Example 6 48.8 30.0-60.2 5) Resistance to Heat Ageing and Environmental Cycling Heat ageing and environmental cycling test were undertaken on full IMD parts to ensure that there were no changes to e.g.\",\n \"laminate strength, colouristic properties after the heat/environmental cycles: TABLE 9 Heat Ageing/Environmental Cycling Delamination Ink system test Comments Example 1 Pass Thorough drying critical Example 2 Fail Poor bond strength prior to testing Example 3 Pass No appreciable loss of properties Example 4 Pass No appreciable loss of properties Example 5 Pass No appreciable loss of properties Example 6 Pass No appreciable loss of properties TABLE 10 Heat Ageing/Environmental Cycling Ink system Colouristic test Comments Example 1 Pass — Example 2 Fail — Example 3 Pass No appreciable loss of properties Example 4 Pass No appreciable loss of properties Example 5 Pass No appreciable loss of properties Example 6 Pass No appreciable loss of properties 6) Print Curl Single and multilayer prints of Example 1 and Example 3 were prepared on 125 micron (μm) Bayfol® and assessed for degree of substrate curl or distortion after 24 hours.\",\n \"TABLE 11 Ink film Assessment Number of thickness***** of curl Ink system print layers (μm) (mm) Comment Example 1 1 5 >45 Very poor 2 12 >47 Very poor 3 17 >47 Very poor 4 22 49 Very poor Example 3 1 10 2 — 2 16 2-3 — 3 22 3 — 4 29 3-6 Very slight curl *****Ink film thickness determined using digital micrometer and comparing printed (various number of ink deposits) and unprinted areas of the substrate.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10451279"}}},{"rowIdx":298,"cells":{"text":{"kind":"list like","value":[["Map kinase integrin-binding domain","Dislosed is the integrin-binding domain of MAP kinase.","Interaction between MAP kinase and integrin via this domain activates MAP kinase and initiates cellular activity.","Methods of modulating cellular activity by inhibiting direct MAP kinase-integrin binding are provided.","These methods have particular use in inhibiting growth of cancer cells."],["1.A fragment of a MAP kinase wherein the fragment consists of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin.","2.A fragment of a MAP kinase wherein the fragment comprises a binding domain of the MAP kinase for an integrin or a partial amino acid sequence of the binding domain that is capable of binding to the integrin and has a length of 100 amino acids or less, or an analog or derivative of the fragment that is capable of binding to the integrin.","3.A fragment according to claim 2 wherein the fragment has a length of about 50 amino acids or less.","4.A fragment according to claim 3 wherein the fragment has a length of about 40 amino acids or less.","5.A fragment according to claim 4 wherein the fragment has a length of about 30 amino acids or less.","6.A fragment according to claim 5 wherein the fragment has a length of about 20 amino acids or less.","7.A fragment according to claim 2 comprising the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or a contiguous amino acid sequence thereof.","8.A fragment according to claim 2 comprising the amino acid sequence PSNLLLNTTCDLKIC or a contiguous amino acid sequence thereof.","9.A fragment according to claim 1 wherein the MAP kinase is an ERK or a JNK family member.","10.A fragment according to claim 9 wherein the MAP kinase is ERK2.11.A fragment according to claim 1 wherein the integrin is selected from the group consisting of β3, β5 and β6.12.An isolated or purified polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase.","13.A polypeptide according to claim 12 comprising a core amino acid sequence of a binding domain of the MAP kinase for the integrin.","14.A polypeptide according to claim 12 wherein the MAP kinase is a member of the ERK family or the JNK family.","15.A polypeptide according to claim 14 wherein the MAP kinase is ERK2.16.A polypeptide according to claim 12 wherein the integrin is selected from the group consisting of β3, β5 and β6.17.A fusion protein incorporating a binding moiety capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin.","18.A fusion protein according to claim 17 adapted for passing across a cell membrane.","19.A fusion protein according to claim 18 further incorporating a carrier moiety for facilitating passage across the cell membrane.","20.A fusion protein according to claim 17 wherein the binding moiety comprises a fragment of a MAP kinase consisting of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin or a polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase.","21.A fusion protein according to claim 18 adapted for releasing the binding moiety after passing across the cell membrane.","22.An agent for inhibiting binding of a MAP kinase to an integrin, comprising: a targeting moiety for targeting a cell expressing the integrin; a binding moiety for binding to a binding domain of the integrin for the MAP kinase to thereby inhibit the binding of the MAP kinase to the integrin; and a carrier moiety for facilitating passage of the binding moiety across the cell membrane of the cell; wherein the binding moiety is other than an antibody or a binding fragment thereof.","23.An agent according to claim 22 wherein the binding and carrier moieties are capable of being released from the targeting moiety at the cell for passage of the carrier moiety and the binding moiety across the cell membrane of the cell.","24.An agent according to claim 23 wherein the agent incorporates an enzyme cleavage site for being cleaved to thereby release the carrier moiety and the inhibitor moiety at the cell.","25.An agent according to claim 24 wherein the enzyme cleavage site is a cleavage site for matrix-metalloproteinase-9 (MMP-9).","26.An agent according to claim 22 adapted for release of the inhibitor moiety from the carrier moiety after passage of the inhibitor moiety across the cell membrane of the cell.","27.An agent according to claim 22 wherein the targeting moiety is an antibody or a binding fragment of an antibody.","28.An agent according to claim 27 wherein the antibody or the binding fragment is specific for an extracellular region of the integrin.","29.An agent according to claim 22 wherein the targeting moiety is an integrin receptor targeted peptide for binding to the integrin.","30.An agent according to claim 22 wherein the binding moiety is capable of binding to a binding domain of the integrin for the MAP kinase.","31.An agent according to claim 30 wherein the binding moiety comprises a fragment consisting of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin or a polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase, or an analog or derivative of the fragment or polypeptide that is capable of binding to the binding domain of the integrin for the MAP kinase.","32.An agent according to claim 22 wherein the binding moiety is an analog of a binding domain of a MAP kinase for an integrin.","33.An agent capable of inhibiting binding of a MAP kinase to an integrin, wherein the agent comprises an analog of a binding domain of the MAP kinase for the integrin.","34.An agent according to claim 33 wherein the analog comprises a mimetic of the binding domain of the MAP kinase.","35.An isolated nucleic acid sequence encoding a fragment as defined in claim 1 or an analog or derivative thereof capable of binding to the binding domain of the integrin for the MAP kinase.","36.An isolated nucleic acid sequence encoding a polypeptide as defined in claim 12 or an analog or derivative thereof capable of binding to the binding domain of the integrin for the MAP kinase.","37.An isolated nucleic acid sequence encoding a fusion protein as defined in claim 17.38.An isolated antisense nucleic acid sequence complementary to a nucleic acid sequence as defined in claim 35.39.An isolated antisense nucleic acid sequence according to claim 38 wherein the antisense nucleic acid sequence is labelled.","40.An oligonucleotide comprising from 14 to 45 contiguous nucleotides of a nucleic acid sequence as defined in claim 35.41.An oligonucleotide comprising from 14 to 45 nucleotides and which is capable of hybridising with a nucleic acid sequence as defined in claim 35.42.A vector incorporating a nucleic acid sequence as defined in claim 35.43.An expression vector incorporating a nucleic acid sequence as defined in claim 35.44.A host cell transformed with a vector as defined in claim 42.45.A host cell transformed according to claim 44 wherein the host cell is selected from the group consisting of a mammalian cell, an epithelial cell, a neoplastic cell and a cancer cell.","46.A host cell according to claim 45 wherein the host cell is a colon cancer cell.","47.A pharmaceutical composition comprising a fragment as defined in claim 1 or an analog or derivative thereof capable of binding to the binding domain of the integrin for the MAP kinase, together with a pharmaceutically acceptable carrier or diluent.","48.A pharmaceutical composition comprising a polypeptide as defined in claim 12 or an analog or derivative thereof capable of binding to the binding domain of the integrin of the MAP kinase, together with a pharmaceutically acceptable carrier or diluent.","49.A pharmaceutical composition comprising fusion protein as defined in claim 17 together with a pharmaceutically acceptable carrier or diluent.","50.A pharmaceutical composition comprising an agent for inhibiting binding of a MAP kinase with an integrin as defined in claim 22, together with a pharmaceutically acceptable carrier or diluent.","51.A pharmaceutical composition comprising a vector as defined in claim 43 together with a pharmaceutically acceptable carrier or diluent.","52.A pharmaceutical composition comprising an antisense nucleic acid sequence as defined in claim 38 together with a pharmaceutically acceptable carrier or diluent.","53.A pharmaceutical composition comprising an oligonucleotide as defined in claim 40 together with a pharmaceutically acceptable carrier or diluent.","54.An antibody specific for a binding domain of a MAP kinase for an integrin, or a binding fragment of the antibody.","55.An antibody specific for a fragment as defined in claim 1 or an analog or derivative of the fragment that is capable of binding to the binding domain of the integrin for the MAP kinase.","56.An antibody specific for a polypeptide as defined in claim 12 or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin in the MAP kinase.","57.An antibody according to claim 54 wherein the antibody is a monoclonal antibody.","58.A method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin comprising: (a) testing a number of agents for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if any said agent is capable of binding to the binding domain of the MAP kinase on the basis of the testing.","59.A method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing a number of agents for ability to bind to the MAP kinase; (b) selecting an agent or agents identified as being able to bind to the MAP kinase on the basis of the testing; and (c) utilising the selected said agent or agents in an assay for indicating whether the or any of the selected said agents is capable of binding to the binding domain of the MAP kinase for the integrin.","60.A method of evaluating whether an agent is capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing the agent for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if the agent is capable of binding to the binding domain of the MAP kinase on the basis of the testing.","61.A method according to claim 60 wherein the integrin is selected from the group consisting of β3, β5 and β6.62.A method according to claim 61 wherein the MAP kinase is an ERK family member or a JNK family member.","63.A method according to claim 62 wherein the MAP kinase is ERK2.64.An agent identified to be capable of binding to a binding domain of a MAP kinase for an integrin by a method as defined in claim 61.65.A method of isolating an agent from a sample utilizing a molecule immobilised on a solid support and which is capable of binding to a binding domain of an integrin for a MAP kinase, comprising: (a) contacting the molecule immobilised on the solid support with the sample under conditions suitable for binding of the agent to the molecule; (b) eluting the agent from the solid support; and (c) collecting the eluted agent; wherein the molecule is other than an antibody or binding fragment of an antibody.","66.A method according to claim 65 wherein the molecule is a MAP kinase or fragment thereof, or an analog or derivative of a fragment of a MAP kinase.","67.A method according to claim 65 wherein the molecule is a fragment consisting of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin, or an analog or derivative thereof.","68.A method according to claim 65 wherein the molecule is a polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase, or an analog or derivative thereof.","69.A method according to claim 65 wherein the integrin is selected from the group consisting of β3, β5 and β6.70.A method according to claim 65 wherein the MAP kinase is an ERK family member or a JNK family member.","71.A method according to claim 70 wherein the MAP kinase is ERK2.72.An agent isolated from a sample by a method as defined in claim 61.73.A method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an antisense oligonucleotide for hybridising with at least part of a nucleic acid sequence coding for the binding domain of the MAP kinase for the integrin to thereby cause expression of the MAP kinase to be down-regulated.","74.A method according to claim 73 wherein the oligonucleotide is about 50 nucleotides or less in length.","75.A method according to claim 74 wherein the oligonucleotide is 45 nucleotides in length or less.","76.A method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising treating the cell with an antisense oligonucleotide for hybridising with at least part of a nucleic acid sequence coding for the binding domain of the MAP kinase for the integrin to thereby cause expression of the MAP kinase to be down-regulated, wherein the antisense oligonucleotide is an oligonucleotide as defined in claim 38.77.A method according to claim 73 wherein the cell is transfected with the antisense oligonucleotide or the antisense oligonucleotide is transcribed from a vector introduced into the cell.","78.A method according to claim 73 wherein the cell is a cancer cell.","79.A method according to claim 73 wherein the MAP kinase is an ERK family member or a JNK family member.","80.A method according to claim 79 wherein the MAP kinase is ERK2.81.A method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: transfecting the cell with an expression vector incorporating a nucleic acid sequence encoding a MAP kinase of a fragment thereof, or a homolog, analog or variant of the MAP kinase or fragment, with a mutagenised binding domain for the integrin or in which the binding domain has been deleted whereby binding to the integrin is inhibited or prevented.","82.A method according to claim 81 wherein the binding domain is mutagenised.","83.A method according to claim 81 wherein the MAP kinase is an ERK family member of a JNK family member.","84.A method according to claim 81 wherein the MAP kinase is ERK2.85.A method according to claim 81 wherein the integrin subunit is selected from the group consisting of β3, β5 and β6.86.A method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the integrin for the MAP kinase, wherein the agent is other than an antibody or a binding fragment thereof.","87.A method according to claim 86 wherein the agent is a fragment consisting of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin, or an analog or derivative thereof capable of binding to the binding domain of the integrin.","88.A method according to claim 86 wherein the agent is a polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase, or an analog or derivative thereof capable of binding to the binding domain of the integrin.","89.A method according to claim 86 wherein the agent is a fusion protein incorporating a binding moiety capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin.","90.A method according to claim 86 wherein the agent is an agent comprising: a targeting moiety for targeting a cell expressing the integrin; a binding moiety for binding to a binding domain of the integrin for the MAP kinase to thereby inhibit the binding of the MAP kinase to the integrin; and a carrier moiety for facilitating passage of the binding moiety across the cell membrane of the cell; wherein the binding moiety is other than an antibody or a binding fragment thereof.","91.A method according to claim 86 wherein the activity of the cell is growth, migration, spreading, invasion, matrix-degrading enzyme secretion, differentiation and/or apoptosis of the cell.","92.A method according to claim 86 wherein the cell is a cancer cell.","93.A method according to claim 92 wherein the cancer cell is a colon cancer cell.","94.A method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the MAP kinase, wherein the agent is other than an agent comprising a binding domain of the integrin for the MAP kinase or an analog or derivative thereof.","95.A method according to claim 94 wherein the agent comprises an antibody or binding fragment thereof.","96.A method according to claim 94 wherein the activity of the cell is growth, migration, spreading, invasion, matrix-degrading enzyme secretion, differentiation and/or apoptosis.","97.A method according to claim 94 wherein the cell is a cancer cell.","98.A method according to claim 94 wherein the cancer cell is a colon cancer cell.","99.A method according to claim 73 comprising treating a mammal in need of such treatment.","100.A method according to claim 73 wherein the method comprises treatment or prophylaxis of cancer or a condition associated with a predisposition to cancer.","101.A method according to claim 100 wherein the cancer is selected from the group consisting of cancer of the lip, tongue, salivary glands, gums, floor and other areas of the mouth, oropharynx, nasopharynx, hypopharynx and other oral cavities, oesophagus, stomach, small intestine, duodenum colon, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung, breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid and skin.","102.A method according to claim 101 wherein the cancer is colon cancer.","103.A method of prophylaxis or treatment of cancer in a mammal, comprising: treating the mammal with an effective amount of an antisense nucleic acid for hybridising with a nucleic acid encoding a MAP kinase to cause expression of the MAP kinase to be down-regulated wherein the MAP kinase has a binding domain for an integrin.","104.A method according to claim 103 comprising administering an effective amount of the antisense nucleic acid to the mammal.","105.A method according to claim 103 comprising administering to the mammal an effective amount of a vector incorporating an nucleic acid sequence complementary to the antisense nucleic acid for generation of the antisense nucleic sequence in vivo.","106.A method according to claim 103 wherein the antisense nucleic acid is an antisense oligonucleotide for hybridising with at least part of the sequence of the nucleic acid encoding the binding domain of the MAP kinase.","107.A method according to claim 106 wherein the oligonucleotide has a length of 150 nucleotides or less.","108.A method according to claim 107 wherein the oligonucleotide has a length of 90 nucleotides or less.","109.A method according to claim 108 wherein the oligonucleotide has a length of 50 nucleotides or less.","110.A method according to claim 109 wherein the oligonucleotide has a length of from 15 to 30 nucleotides.","111.A method according to claim 103 wherein the integrin subunit is selected from the group consisting of β3, β5 and β6.112.A method according to claim 103 wherein the cancer is colon cancer.","113.A fragment according to claim 9 wherein the integrin is selected from the group consisting of β3, β5 and β6.114.A polypeptide according to claim 15 wherein the integrin is selected from the group consisting of β3, β5 and β6.115.A vector incorporating an oligonucleotide as defined in claim 40.116.An expression vector incorporating an oligonucleotide as defined in claim 41 for being expressed in a cell.","117.A host cell transformed with a vector as defined in claim 43.118.A pharmaceutical composition comprising an antisense nucleic acid sequence as defined in claim 39 together with a pharmaceutically acceptable carrier or diluent.","119.A pharmaceutical composition comprising an oligonucleotide as defined in claim 41 together with a pharmaceutically acceptable carrier or diluent.","120.An antibody according to claim 55 wherein the antibody is a monoclonal antibody.","121.An antibody according to claim 56 wherein the antibody is a monoclonal antibody.","122.A method according to claim 69 wherein the MAP kinase is an ERK family member or a JNK family member.","123.A method according to claim 86 comprising treating a mammal in need of such treatment.","124.A method according to claim 86 wherein the method comprises treatment or prophylaxis of cancer or a condition associated with a predisposition to cancer."],["<SOH> BACKGROUND OF THE INVENTION <EOH>Colorectal cancer is the commonest internal malignancy affecting men and women in Australia.","About 4% of individuals develop this disease during the course of their lifetime and it was responsible for 14% of cancer deaths in that country in 1990.In 1995 there were 10,615 cases of colorectal cancer and 4508 deaths in Australia.","Worldwide, an estimated 875,000 cases of colorectal cancer occurred in 1996, accounting for 8.5% of all new cases of cancer.","Incidence rates vary approximately 20-fold around the world, with the highest rates seen in the developed world and lowest in India.","Australian incidence rates are towards the higher end of the scale internationally alongside those for North America and New Zealand.","Five-year survival following diagnosis of colon cancer is around 55% in the developed world and has altered little during the past few decades despite advances in chemo-, immuno- and radiotherapy.","Colorectal cancer is a malignant tumour that starts in the bowel wall and is confined locally for a relatively long period before spreading through the bowel wall and metastasising to lymph nodes and other parts of the body.","Survival rates are significantly improved where the disease is detected and treated early.","The aetiology of colorectal cancer is complex and appears to involve interactions between inherited susceptibility and environmental factors.","Recognition of the genetic component of colorectal cancer is growing.","Mutations are present as inherited germline defects or arise in somatic cells secondary to environmental insult.","There are two main inherited predisposition syndromes: Familial Adenomatous Polyposis (FAP) and Hereditary Non-Polyposis Colorectal Cancer (HNPCC); the remaining cases are attributed to so-called sporadic colorectal cancer.","FAP and HNPCC contribute to approximately 1% and 4%, respectively, of all colorectal cancers and a strong family history of bowel cancer in first-degree relatives is obtained in another 10-15% of patients.","However, in the vast majority of patients the aetiology of large bowel cancer remains unknown.","Most colon cancer arises within pre-existing benign precursor lesions or adenomas.","Adenomas are classified by histological architecture as tubular, tubulovillous or villous.","Villous change is associated with a higher malignant potential, as are large and high-grade epithelial dysplasia.","Environmental risk factors for development of colorectal cancer include diets low in fibre and vegetables and high in fat, red meat and alcohol and cigarette smoking which may induce mutations in somatic cells.","Studies have shown that persistent genetic instability and accumulation of mutations in several genes that are mainly concerned with cell growth or DNA repair, may be critical for the development of all colorectal cancers.","For example, a normal mucosal cell with inactivation of tumour suppressor genes such as the Adenomatous Polyposis Coli (APC) gene or Mutated in Colon Cancer (MCC) gene can proliferate and become a small adenomatous polyp.","Mutations in oncogenes such as k ras and in tumour suppressor genes such as p53 and the Deleted in Colon Cancer gene (DCC) may then occur and lead to the transformation of the polyp into a large adenoma, from which a carcinoma can eventually arise.","The uncontrolled cell growth that leads to the development of neoplasia is believed to result, therefore, from a series of inherited and acquired accumulated genetic changes.","This multistep process confers on cells the capacity to survive and proliferate in a manner freed from the constraints imposed on normal cell growth.","Spread of cancer cells involves tumour cell migration through the extracellular matrix scaffold, invasion of basement membranes, arrest of circulating tumour cells, and tumour cell extravasation and proliferation at metastatic sites.","Detachment of cells from the primary tumour mass and modification of the peri-cellular environment aid penetration of tumour cells into blood and lymphatic vessels.","It is the invasive and metastatic potential of tumour cells that ultimately dictates the fate of most patients suffering from malignant diseases.","Hence, tumourigenesis can be viewed as a tissue remodelling process that reflects the ability of cancer cells to proliferate and digest surrounding matrix barriers.","These events are thought to be regulated, at least in part, by cell adhesion molecules and matrix-degrading enzymes.","Cell adhesion receptors on the surface of colon cancer cells are involved in complex cell signalling which may regulate cell proliferation, migration, invasion and metastasis and several families of adhesion molecules have now been identified including integrins, cadherins, the immunoglobulin superfamily, hyaluronate receptors, and mucins (Agrez, 1996.)","In general, these cell surface molecules mediate both cell-cell and cell-matrix binding, the latter involving attachment of tumour cells to extracellular scaffolding molecules such as collagen, fibronectin and laminin.","It is now clear that multiple and varied cell adhesion receptors exist on colon cancer cells at any one time and an understanding of the role of individual receptors in promoting growth and spread of colon cancer is only just beginning to be elucidated.","Of all the families of cell adhesion molecules, the best-characterised at the present time is the family known as integrins.","Integrins are involved in several fundamental processes including leucocyte recruitment, immune activation, thrombosis, wound healing, embryogenesis, virus internalisation and tumourigenesis.","Integrins are transmembrane glycoproteins consisting of an alpha (α) and beta (β) chain in close association that provide a structural and functional bridge between extracellular matrix molecules and cytoskeletal components with the cell.","The integrin family comprises 17 different α and 8β subunits and the αβ combinations are subsumed under 3 subfamilies.","Excluding the leucocyte integrin subfamily that is designated by the β2 nomenclature, the remaining integrins are arranged into two major subgroups, designated β1 and αv based on sharing common chains.","In the β1 subfamily, the β1 chain combines with any one of nine a chain members (α1-9), and the a chain which associates with β1 determines the matrix-binding specificity of that receptor.","For example, α2β1 binds collagen and laminin, α3β1 binds collagen, laminin and fibronectin, and α5β1 binds fibronectin.","In the αv subfamily of receptors, the abundant and promiscuous αv chain combines with any one of five β chains, and a distinguishing feature of αv integrins is that they all recognise and bind with high affinity to arginine-glycine-aspartate (RGD) sequences present in the matrix molecules to which they adhere (Hynes, 1992).","The current picture of integrins is that the N-terminal domains of α and β subunits combine to form a ligand-binding head on each integrin.","This head, containing the cation binding domains, is connected by two stalks representing both subunits, to the membrane-spanning segments and thus to the two cytoplasmic domains.","The β subunits all show considerable similarity at the amino acid level (Loftus et al, 1994).","All have a molecular mass between 90 and 110 kDa, with the exception of β4 which is larger at 210 kDa.","Similarly, they all contain 56 conserved cysteine residues, except for β4 which has 48.These cysteines are arranged in four repeating patterns which are thought to be linked internally by disulphide bonds.","The α-subunits have a molecular mass ranging from 150-200 kDa.","They exhibit a lower degree of similarity than the β chains, although all contain seven repeating amino acid sequences interspaced with non-repeating domains.","The β subunit cytoplasmic domain is required for linking integrins to the cytoskeleton (Hynes, 1992).","In many cases, this linkage is reflected in localisation to focal contacts, which is believed to lead to the assembly of signalling complexes that include α-actinin, talin, and focal adhesion kinase (FAK) (Otey et al, 1990; Guan and Shalloway, 1992; Kornberg et al, 1992).","At least three different regions that are required for focal contact localisation of β1 integrins have been delineated (Reszka et al, 1992).","These regions contain conserved sequences that are also found in the cytoplasmic domains of the β2, β3, β5, β6 and β7 integrin subunits.","The functional differences between these cytoplasmic domains with regard to their signalling capacity have not yet been established.","Ligation of integrins by their extracellular matrix protein ligands induces a cascade of intracellular signals that include tyrosine phosphorylation of focal adhesion kinase, increases in intracellular Ca 2+ levels, inositol lipid synthesis, synthesis of cyclins and expression of immediate early genes.","In contrast, prevention of integrin-ligand interactions suppresses cellular growth or induces apoptotic cell death (Meredith et al, 1993; Montgomery et al, 1994; Brooks et al, 1994; Varner et al, 1995; Boudreau et al, 1995).","Thus, integrins play roles in a number of cellular processes that impact on the development of tumours, including the regulation of proliferation and apoptosis.","The integrin β6 subunit was first identified in cultured epithelial cells as part of the αvβ6 heterodimer, and the αvβ6 complex was shown to bind fibronectin in an arginine-glycine-aspartate (RGD)-dependent manner in human pancreatic carcinoma cells (Sheppard et al, 1990; Busk et al, 1992).","The β6 subunit is composed of 788 amino acids and shares 34-51% sequence homology with other β integrin subunits β1-β5.The β6 subunit also contains 9 potential glycosylation sites on the extracellular domain (Sheppard et al, 1990).","The cytoplasmic domain differs from other β subunits in that it is composed of a 41 amino acid region that is highly conserved among integrin β subunits, and a unique 11 amino acid carboxy-terminal extension.","The 11 amino acid extension has been shown not to be necessary for localisation of β6 to focal contacts; in fact, its removal appears to increase receptor localisation.","However, removal of any of the three conserved regions previously identified as important for the localisation of β1 integrins to focal contacts (Reszka et al, 1992) has been shown to eliminate recruitment of β6 to focal contacts (Cone et al, 1994).","The integrin αvβ6 has previously been shown to enhance growth of colon cancer cells in vitro and in vivo, and this growth-enhancing effect is due, at least in part, to αvβ6-mediated gelatinase B secretion (Agrez et al, 1994; Niu et al, 1998 Agrez et al, 1999).","What makes this epithelial-restricted integrin of particular interest in cancer is that it is not expressed on normal epithelial cells but is highly expressed during would healing and tumourigenesis, particularly at the invading edge of tumour cell islands (Breuss et al, 1995; Agrez et al, 1996).","Further more, αvβ6 has been found to induce its own expression in an autocrine manner with cell crowding and a self-perpetuating model of colon cancer progression regulated by αvβ6-mediated gelatinase B secretion has been proposed (Niu et al, 2000).","Invasion of the extracellular matrix and metastatic spread of colon cancer is also likely to reflect the ability of tumour cells to digest their surrounding matrix scaffold through secretion of matrix-degrading enzymes such as matrix metalloproteinases (MMPs).","The mechanisms whereby human colon cancer cells escape the constraints on growth imposed on normal cells by cell crowding and dense pericellular matrices is unclear.","However, even colon cancer cells are subject to relative growth inhibition in vitro in a dense extracellular matrix environment (Agrez, 1989).","Integrins can signal through the cell membrane in either direction.","The extracellular binding activity of integrins can be regulated from the cell interior as, for example, by phosphorylation of integrin cytolasmic domains (inside-out signalling), while the binding of the extracellular matrix (ECM) elicits signals that are transmitted into the cell (outside-in signalling) (Giancotti and Ruoslahti, 1999).","Outside-in signalling can be roughly divided into two descriptive categories.","The first is ‘direct signalling’ in which ligation and clustering of integrins is the only extracellular stimulus.","Thus, adhesion to ECM proteins can activate cytoplasmic tyrosine kinases (e.g.","focal adhesion kinase FAK) and serine/threonine kinases (such as those in the mitogen-activated protein kinase (MAPK) cascade) and stimulate lipid metabolism (eg phosphatidylinositol-4,5-biphosphate (P 1 P 2 ) synthesis.","The second category of integrin signalling is ‘collaborative signalling’, in which integrin-mediated cell adhesion modulates signalling events initiated through other types of receptors, particularly receptor tyrosine kinases that are activated by polypeptide growth factors (Howe et al, 1998).","In all cases, however, integrin-mediated adhesion seems to be required for efficient transduction of signals into the cytosol or nucleus.","MAP kinases behave as a convergence point for diverse receptor-initiated signalling events at the plasma membrane.","The core unit of MAP kinase pathways is a three-member protein kinase cascade in which MAP kinases are phosphorylated by MAP kinase kinases (MEKs) which are in turn phosphorylated by MAP kinase kinases (e.g.","Raf-1) (Garrington and Johnson, 1999).","Amongst the 12 member proteins of the MAP kinase family are the extracellular signal-regulated kinases (ERKs) (Boulton et al, 1991) activated by phosphorylation of tyrosine and threonine residues (Payne et al, 1991) which is the type of activation common to all known MAP kinase isoforms.","ERK 1/2 (44 kD and 42 kD MAPks, respectively) share 90% amino acid identity and are ubiquitous components of signal transduction pathways (Boulton et al, 1991).","These serine/threonine kinases phosphorylate and modulate the function of many proteins with regulatory functions including other protein kinases (such as p90 rsk ) cytoskeletal proteins (such as microtubule-associated phospholipase A 2 ), upstream regulators (such as the epidermal growth factor receptor and Ras exchange factor) and transcription factors (such as C-Myc and Elk-1).","ERKs play a major role in growth-promoting events, especially when the concentration of growth factors available to a cell is limited (Giancotti and Ruoslahti, 1999).","MAP kinases can be activated through non-receptor tyrosine kinases such as focal adhesion kinase (FAK), cytoplasmic tyrosine kinase (pp60 c-srk) (Schlaepher and Hunter, 1998), and growth factors acting through membrane-associated receptor tyrosine kinases.","The FAK pathway is activated by most integrins.","In addition to activating FAK, some β1 and αv integrins also activate the tyrosine kinase Fynu and through it, the adaptor protein Shc (Wang et al, 1996).","It is likely that both FAK and Shc contribute to the activation of Ras and thence to the downstream kinase cascade of Raf-1, MEK, and MAP kinases (Schlaepfer et al, 1994; 1997).","It is now generally accepted that the activation of ERK in response to integrin ligation requires Ras signalling (Wary et al, 1996; Schlaepfer and Hunter, 1997).","The laminin receptor α6β4, the laminin/collagen receptor α1β1, the fibronectin receptor α5β1 and the RGD binding receptor αvβ3 are linked to the Ras-Raf-MEK-ERK signalling pathway and control of immediate early gene expression (Wary et al, 1996; Maniero et al, 1995; 1997).","The ability of integrins to activate ERK may be especially important when the concentration of growth factors available to the cell is limited.","In this setting, proliferation is likely to require co-stimulation of ERK through integrins and growth factor receptors (Giancotti and Ruoslahti, 1999).","Moreover, activation of ERK in response to integrin ligation may play a role in regulating cell migration (Klemke et al, 1997) possibly by initiating matrix-degrading enzyme secretion.","While there is a good deal of evidence in support of a key role for FAK and the phosphotyrosine-domain-containing adaptor protein Shc (Howe et al, 1998; Giancotti & Ruoslahti, 1999) in the Ras-Raf MEK-MAP kinase activation pathway there are also data implicating alternate pathways independent of MEKs.","For example, MEK-independent regulation of MAP kinase activation in NIH3T3 fibroblasts has been shown to be mediated by phosphatidylinositol-3-kinases and the conventional protein kinase C (PKC) isoforms and is thought to be due to inactivation of a MAP kinase inhibitor (Grammer and Blenis, 1997).","Although the mechanism by which PKC regulates integrin function is not known, PKC has been shown to regulate integrin-induced activation of the MAP kinase pathway upstream of Shc.","For example, PKC inhibition has been shown to inhibit ERK2 activation by fibronectin receptors without any effect on integrin-induced FAK or paxillin tyrosine phosphorylation (Miranti et al, 1999).","Hence, MAP kinase activation is more complicated than a simple linear pathway, and the mechanistic basis for the commonly observed integrin-mediated activation of MAP kinases remains controversial.","The importance of the MAP kinase pathway in promoting colon cancer growth in vivo is now no longer in question.","Although relatively little is known about the role of MAP kinase activation in for instance, colon tumour progression, MAP kinase pathways have been shown to be highly activated during the late progression of colorectal cancer (Licato et al, 1997).","In a recent breakthrough in this field, a highly potent inhibitor of MAP kinase activation has been identified which is capable of inhibiting human colon cancer growth in immune-deficient mice raising hops for the clinical application of MAP kinase inhibitors in the treatment of colon cancer (Sebolt-Leopold et at, 1999).","Various intracellular proteins may be linked directly or spatially to integrin cytoplasmic domains.","Direct interactions have been identified between cytoskeletal proteins such as α-actinin and talin and β1 and β3 integrin tails (Horwitz et al, 1986; Otey et al, 1990; Knezevic et al, 1996; Pfaff et al, 1998).","A direct association between FAK and the β1 integrin tail has been suggested based on in vitro β1 peptide studies, but this remains to be confirmed (Schaller et al, 1995).","More recently, the cytoplasmic domain of the α4 subunit has been found to be physically associated with the signalling adaptor protein paxillin in Jurkat T cells, and this binding event regulates the kinetics of FAK tyrosine phosphorylation (Liu et al, 1999).","Direct integrin links with the intracellular calcium-binding protein, calreticulin, and integrin-linked kinase (ILK) (Hannigan et al, 1996) have been shown to regulate “inside-out” integrin signalling.","For example, calreticulin has been shown to bind to a chain cytoplasmic domains (Rojiani et al, 1991) and modify α2β1 integrin activation by phorbol esters and anti-integrin antibodies (Coppolino et al, 1995).","Newly identified integrin-binding molecules include the serine/threonine integrin-linked kinase, ILK, which can associate with the β1, β2 and β3 subunits.","When over-expressed, ILK has been shown to reduce anchorage-independent growth and tumourigenicity in nude mice (Hannigan et al, 1996).","Co-immunoprecipitation strategies have also demonstrated interactions between integrins and the integral plasma membrane protein IAP, and members of the four transmembrane domain protein family (tetraspans).","The extracellular Ig region of the IAP molecule mediates association with αvβ3 and is required for cell binding to vitronectin-coated particles (Lindberg et al, 1996).","An emerging model for tetraspans is that they recruit signalling enzymes such as phosphatidylinositol-4-kinase and PKC into complexes with integrins (Hemler, 1998).","Integrins have also been shown to be physically linked with matrix-degrading enzymes and growth factors.","For example, the integrin αvβ6 has been shown to bind and activate latent TGFβ1 in keratinocytes (Munger et al, 1999) which is thought to be important in modulating the inflammatory process following epithelial injury.","In melanoma cells, αvβ3 binds activated gelatinase A (Brooks et al, 1996), and both insulin and platelet-derived growth factor (PDGF) co-immunoprecipitate with this integrin in NIH3T3 mouse fibroblasts (Schneller et al, 1997).","Synergism between integrin-mediated signalling processes and growth factor responses is now well-recognised and Schneller et al (1997) showed that a small subset of each of the insulin receptor and PDGF β-receptor is tyrosine phosphorylated upon growth factor stimulation.","Interestingly, this subset can associate with the αvβ3 integrin, and PDGF activity is enhanced in association with increased MAP kinase activity in cells plated on the αvβ3 ligand, vitronectin."],["<SOH> SUMMARY OF THE INVENTION <EOH>Broadly stated, the present invention relates to the surprising finding that members of the mitogen activated protein (MAP) kinase family can associate with the cytoplasmic domain of an integrin molecule.","It is believed that no member of the MAP kinase family has previously been found to directly associate with any integrin.","The identification of this functional relationship permits the rational design of agents for therapeutically or prophylactically modulating cellular activity mediated by the MAP kinase and integrin interaction.","In an aspect of the present invention there is provided an agent capable of binding to a binding domain of an integrin for a MAP kinase.","In another aspect of the present invention there is provided an isolated or purified polypeptide capable of binding to a binding domain of an integrin for a MAP kinase or a homolog, analog, variant or derivative of the polypeptide.","The polypeptide may consist of or comprise the binding domain of the MAP kinase to which the integrin binds or sufficient core amino acid sequence of the binding domain of the MAP kinase to enable binding of the polypeptide to the integrin.","Preferably, the polypeptide will be a fragment of a MAP kinase.","Most preferably, the polypeptide will comprise the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or PSNLLLNTTCDLKIC or a region or regions of such sequences.","Accordingly, in a further aspect of the present invention there is provided a fragment of a MAP kinase wherein the fragment is capable of binding with an integrin, or a homolog, analog, variant or derivative of the fragment.","In another aspect of the present invention there is provided a fragment of a MAP kinase wherein the fragment consists of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin.","In another aspect of the present invention there is provided an isolated or purified polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase.","Preferably, a polypeptide or fragment of the invention will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less and more usually about 40 amino acids or less.","Typically, the length will be between about 5 to about 30 amino acids.","In yet another aspect of the present invention there is provided a fusion protein a fusion protein incorporating a binding moiety capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin.","Preferably, the binding moiety will comprise a fragment or polypeptide of the invention.","Most preferably, the fusion protein will incorporate a carrier moiety for facilitating passage across the cell membrane.","The carrier moiety may be penetratin.","In still another aspect of the present invention there is provided an agent for inhibiting binding of a MAP kinase to an integrin, comprising: a targeting moiety for targeting a cell expressing the integrin; a binding moiety for binding to a binding domain of the integrin for the MAP kinase to thereby inhibit the binding of the MAP kinase to the integrin; and a carrier moiety for facilitating passage of the binding moiety across the cell membrane of the cell; wherein the binding moiety is other than an antibody or an binding fragment thereof.","In another aspect of the present invention there is provided a MAP kinase or a fragment thereof with a mutagenised binding domain for an integrin or in which the binding domain is deleted, or a homolog, analog or variant of the MAP kinase or fragment, wherein capability to bind with the integrin is thereby reduced.","In another aspect of the present invention there is provided an isolated nucleic acid sequence encoding a fragment or polypeptide of the invention or a homolog, analog, variant or derivative of the fragment or polypeptide.","In another aspect of the invention there is provided a nucleic acid sequence coding for a MAP kinase or fragment thereof having a mutagenised binding domain for an integrin or in which the binding domain is deleted or a homolog, analog or variant of the MAP kinase or fragment, wherein capability to bind with the integrin is thereby reduced.","In a still further aspect there is provided an isolated nucleic acid sequence encoding a fusion protein or agent of the invention.","There are also provided antisense nucleic acid sequences complimentary to the sense nucleic sequences of the invention.","Such antisense sequences find application in antisense therapy of cells in which down regulation of cellular activity is desired, and include oligonucleotides.","Sense oligonucleotides coding for all or part of the binding domain of a MAP kinase for an integrin, or that of a homolog, analog or variant thereof, and complimentary antisense oligonucleotides find particular application as primers or probes.","A nucleic acid primer or probe of the invention may be labelled with a suitable reporter molecule for enabling detection of hybridisation of the primer or probe to a target nucleic acid sequence.","In yet another aspect of the present invention there is provided a vector incorporating a nucleic acid sequence of the invention.","Typically, the vector will be an expression vector and the nucleic acid sequence will be capable of being transcribed.","In a further aspect of the present invention there is provided a host cell transformed with a vector of the invention.","Preferably, the host cell will be selected from the group consisting of a mammalian cell, an epithelial cell, a neoplastic cell, and a cancer cell.","Preferably, the host cell will be a mammalian cell and most preferably, a colon cancer cell.","In yet another aspect of the present invention there is provided an antibody generated with the use of a polypeptide, fragment, agent or fusion protein of the invention.","In a still further aspect of the invention there is provided an antibody and binding fragment thereof capable of binding to a binding domain of a MAP kinase for an integrin.","The antibody may be a polyclonal or monoclonal antibody.","Preferably, the antibody is a monoclonal antibody.","In still another aspect of the present invention there is provided a method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing a number of agents for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if any said agent is capable of binding to the binding domain of the MAP kinase on the basis of the testing.","In yet another aspect of the invention there is provided a method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing a number of agents for ability to bind to the MAP kinase; (b) selecting an agent or agents identified as being able to bind to the MAP kinase on the basis of the testing; and (c) utilising the selected said agent or agents in an assay for indicating whether the or any of the selected said agents is capable of binding to the binding domain of the MAP kinase for the integrin.","In another aspect of the present invention there is provided a method of evaluating whether an agent is capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing the agent for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if the agent is capable of binding to the binding domain on the basis of the testing.","Preferably, a polypeptide or fragment of the invention comprising or consisting of the binding domain of the MAP kinase or core amino acid sequence of the binding domain or a homolog, analog, variant or derivative of the polypeptide or fragment or core amino acid sequence is used in the testing or assaying for whether an agent is capable of binding to the binding domain of the MAP kinase.","Most preferably, the polypeptide or fragment will comprise or consist of the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or PSNLLLNTTCDLKIC, core amino acid sequence of such sequences capable of binding to an integrin, or a homolog variant, analog or derivative of such sequences.","Testing or assaying of an agent for ability to bind to the binding domain of the MAP kinase for the integrin and thereby inhibit binding of the MAP kinase to the integrin, may comprise exposing the MAP kinase to the agent(s) to enable binding of the agent(s) to the MAP kinase to occur either in the presence of the integrin or prior to the addition of the integrin.","Rather than using the MAP kinase, a polypeptide, fragment of a MAP kinase or other molecule capable of binding to the binding site on the integrin for the MAP kinase may be used in such testing or assaying.","Similarly, rather than utilising an intact integrin, a polypeptide or integrin fragment comprising or incorporating the binding domain of the integrin for the MAP kinase, or other molecule capable of binding with the binding domain on the MAP kinase for the integrin, may be utilised.","In still another aspect of the present invention there is provided a method of isolating an agent from a sample utilising a molecule immobilised on a solid support and which is capable of binding to a binding domain of an integrin for a MAP kinase, comprising: (a) contacting the molecule immobilised on the solid support with the sample under conditions suitable for binding of the agent to the molecule; (b) eluting the agent from the solid support; and (c) collecting the eluted agent; wherein the molecule is other than an antibody or binding fragment of an antibody.","The molecule may be the MAP kinase, or a fusion protein, a polypeptide, or a fragment of the invention to which the integrin is capable of binding, or for instance a homolog, analog, variant or derivative of such a polypeptide or fragment.","In yet another aspect of the present invention there is provided the agent so isolated.","An agent of the invention will usually be provided in the form of a pharmaceutical composition.","Accordingly, in another aspect of the present invention there is provided a pharmaceutical composition comprising an agent of the invention capable of binding to a binding domain of an integrin for a MAP kinase, and a pharmaceutically acceptable carrier or diluent.","The agent may or not be proteinaceous in nature.","Preferably, the agent will comprise a fusion protein, or polypeptide.","Most preferably, the agent will be coupled to a carrier molecule for facilitating entry of the agent into a cell.","In a further aspect of the invention there is provided a pharmaceutical composition comprising a nucleic acid sequence of the invention and a pharmaceutically acceptable carrier or diluent.","Preferably, the nucleic acid sequence is incorporated into a vector as described herein.","Alternatively, the nucleic acid sequence may be joined to a carrier molecule such as penetratin for facilitating entry of the nucleic acid sequence into a target cell.","In another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: transfecting the cell with an expression vector incorporating a nucleic acid sequence encoding a MAP kinase of a fragment thereof, or a homolog, analog or variant of the MAP kinase or fragment, for being expressed by the cell and having a mutagenised binding domain for the integrin or in which the binding domain has been deleted whereby binding to the integrin is inhibited or prevented.","In still another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: transfecting the cell with a nucleic acid sequence encoding a fragment of a MAP kinase, or a homolog, analog or variant of the fragment, for being expressed by the cell and capable of binding to the binding domain of an integrin for the MAP kinase.","In yet another aspect of the present invention there is provided a method of modulating activity of a cell, comprising: transfecting the cell with a nucleic acid encoding a polypeptide for being expressed by the cell wherein the polypeptide is capable of binding to a binding domain of an integrin for a MAP kinase.","In still another aspect of the invention there is provided a method of modulating activity of a cell, comprising causing the expression of a MAP kinase to which an integrin is able to bind to be down-regulated.","Preferably, down-regulation of the expression of the MAP kinase is achieved using an antisense nucleic acid sequence that inhibits expression of gene coding for the MAP kinase.","The antisense nucleic acid sequence may be administered to the cell or generated within the cell.","Preferably, the cell will be transformed with a vector of the invention for generation of the antisense nucleic acid sequence.","Preferably, the antisense nucleic acid sequence will specifically hybridise with sense nucleic acid sequence coding for at least part of the binding domain of the MAP kinase for the integrin and/or intron sequence between such coding sequence.","Accordingly, in another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an antisense oligonucleotide for hybridising with at least part of a nucleic acid sequence coding for the binding domain of a MAP kinase for an integrin to thereby cause expression of the MAP kinase to be down-regulated.","In another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the integrin for the MAP kinase, wherein the agent is other than an antibody or a binding fragment thereof.","In a further aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the MAP kinase, wherein the agent is other than an agent comprising a binding domain of the integrin for the MAP kinase or an analog or derivative thereof.","In yet another aspect of the invention there is provided a method of modulating the activity of a cell, comprising: contacting the cell with an effective amount of a fusion protein, antibody or other molecule of the invention capable of binding to the binding domain of an integrin for a MAP kinase.","Preferably, modulation of the activity of a cell by a method as described herein will comprise treating a mammal in need of such treatment.","Accordingly, in a further aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid sequence encoding a MAP kinase or a fragment thereof or a homolog, analog or variant of the MAP kinase or fragment, with a mutagenised binding domain for an integrin or in which the binding domain has been deleted and wherein the nucleic acid sequence is capable of being expressed.","In another aspect of the invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid capable of causing the expression of a MAP kinase to which an integrin is able to bind to be down regulated.","In a further aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal a nucleic acid sequence encoding a fragment of a MAP kinase, or a homolog, analog or variant of the fragment, for being expressed by the cell and capable of binding to the binding domain of an integrin for the MAP kinase.","In still another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid sequence for expression of a polypeptide or a fragment of a MAP kinase within the cell, wherein the polypeptide or fragment is capable of binding to a binding domain of an integrin for the MAP kinase to thereby modulate the cellular activity.","In another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of an agent capable of binding to the binding domain of an integrin for a MAP kinase, wherein the agent is other than an antibody or a binding fragment thereof.","In another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of an agent capable of binding to the binding domain of a MAP kinase for an integrin wherein the agent is other than an agent comprising the binding domain of the integrin for the MAP kinase or an analog or derivative thereof.","In another aspect of there is provided a method of treatment or prophylaxis of a disease or condition in a mammal, wherein the disease or condition is responsive to an agent capable of binding to a binding domain of an integrin for a MAP kinase and wherein the agent comprises a polypeptide or fragment of the invention or a homolog, variant, analog or derivative of such a polypeptide or fragment, and the method comprises administering an effective amount of the agent to the mammal.","In yet another aspect of the present invention there is provided a method of treatment or prophylaxis of a disease or condition in a mammal, wherein said condition is responsive to an agent capable of binding to a binding domain of an integrin for a MAP kinase wherein the agent is a fusion protein of the invention.","In another aspect there is provided use of a nucleic acid of the invention in the manufacture of a medicament for administration to a mammal in the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable.","In a still further aspect of the invention there is provided the use of an agent capable of binding to a binding domain of an integrin for a MAP kinase in the manufacture of a medicament for administration to a mammal for the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable, wherein the agent is other than an antibody or binding fragment thereof.","The agent may for instance be a polypeptide, fragment or the like as described herein.","In another aspect of the present invention there is provided the use of an agent capable of binding to the binding domain of a MAP kinase for an integrin wherein the agent is other than an agent comprising the binding domain of the integrin for the MAP kinase or an analog or derivative thereof.","In another aspect there is provided the use of fusion protein of the invention in the manufacture of a medicament for administration to a mammal for the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable.","Methods as described above will typically comprise treatment or prophylaxis of cancer or a condition associated with a predisposition to cancer.","The cancer may for instance be selected from the group consisting of cancer of the lip, tongue, salivary glands, gums, floor and other areas of the mouth, oropharynx, nasopharynx, hypopharynx and other oral cavities, oesophagus, stomach, small intestine, duodenum, colon, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung, breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid and skin.","In still another aspect of the present invention there is provided a method of causing an activity of a cell to be upregulated, comprising: contacting the cell with an effective amount of a nucleic acid capable of being expressed within the cell and coding for an agent capable of binding to a binding domain of a MAP kinase for an integrin or a binding domain of the integrin for the MAP kinase.","In a still further aspect of the invention there is provided a method of causing an activity of a cell to be upregulated, comprising contacting the cell with an effective amount of an agent capable of binding to a binding domain of a MAP kinase for an integrin or a binding domain of the integrin for the MAP kinase.","Typically, the agent will be a polypeptide or fragment of an integrin comprising or consisting of the binding domain of an integrin for a MAP kinase or comprising or consisting of the core amino acid sequence of the binding domain directly involved in the binding to the MAP kinase, or a homolog, variant, analog or derivative of such polypeptide or fragment.","Preferably, the polypeptide or fragment will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less and more usually about 40 amino acids or less.","Typically, the length will be between about 5 to about 30 amino acids.","Most preferably, the polypeptide will comprise or consist of the amino acid sequence RSKAKWQTGTNPLYR or RSKAKNPLYR.","Up regulation of cellular activity is particularly desirable for instance in wound healing, re-endothelialisation within natural or artificial blood vessels, nerve growth and for instance induction of labour.","The cellular activity desired to be modulated will typically but not exclusively, be cell growth or proliferation.","Indeed, any activity mediated by MAP kinase signalling is expressly included within the scope of the invention.","The cell may be any cell type in which functional activity arising from signalling mediated by a MAP kinase may occur.","Preferably, the cell will be an epithelial cell and usually, a neoplastic cell.","Usually, the MAP kinase will be selected from the group consisting of an extracellular signal-regulated kinase (ERK) and a JNK MAP kinase.","Preferably, the MAP kinase is ERK2 or JNK-1.Most preferably, the MAP kinase is ERK2.The mammal may be any mammal treatable with a method of the invention.","For instance, the mammal may be a member of the bovine, porcine, ovine or equine families, a laboratory test animal such as a mouse, rabbit, guinea pig, a cat or dog, a primate or a human being.","Preferably, the mammal will be a human being.","As will be understood, modulation of cellular activity within the context of the invention may be up regulation or down regulation of cellular activity.","Reference to “down modulation” or “down-regulating” or the like should be understood to include preventing, reducing or otherwise inhibiting one or more aspects of the activity of the cell.","Conversely, reference to “up regulation” or like terms should be understood to include enhancing or increasing one or more aspects of the activity of the cell.","In the broadest sense, the term “integrin” unless otherwise specified, is to be taken to encompass an integrin family member or a homolog, derivative, variant or analog of an integrin subunit, or an integrin family member incorporating at least one such homolog, derivative, variant or analog of an integrin subunit.","Usually, the integrin will be a member of the αv subfamily.","Preferably, the integrin is or incorporates an integrin subunit selected from the group consisting of β3, β5 and β6.Most preferably, the integrin comprises β6.By “binding domain” is meant the minimum length of contiguous amino acid sequence required for binding.","By “core amino acid sequence” is meant regions or amino acids of the binding domain that directly participate in the binding as distinct from any amino acids that do not directly participate in the binding interaction.","Typically, the core amino acid sequence of the binding domain will comprise regions of the binding domain linked together by a number of intervening amino acids which do not directly participate in the binding.","The term “homolog” is to be taken to mean a molecule that has amino acid sequence similarity.","The homology between amino acid sequences can be determined by comparing amino acids at each position in the sequences when optimally aligned for the purpose of comparison.","The sequences are considered homologous at a position if the amino acids at that position are the same.","Typically, a homolog will have an overall amino acid sequence homology of at least about 30% more preferably at least about 50% or 70% and most preferably, greater than about 80%, 90% or 98% sequence homology.","Homology with a binding domain may be greater than the overall amino acid sequence homology of the homolog, and will usually be greater than about 60% or 80%, and more usually greater than about 90%, 95% or 98%.","The term “analog” is to be taken to mean a molecule that has one or more aspects of biological function characteristic of the molecule on which at least part of the analog is based or which was otherwise utilised in the design or preparation of the analog.","An analog may have substantial overall structural similarity with the molecule or only structural similarity with one or more regions or domains thereof responsible for the desired characteristic biological function.","By “structural” similarity is meant similarity in shape, conformation and/or other structural features responsible for the provision of the biological function or which otherwise have involvement in the provision of the biological function.","Alternatively, it will be understood that with knowledge of the region(s) or domain(s) of a molecule that provide(s) the characteristic biological function, analogs may be designed that while differing in structure nevertheless possess such biological function.","Indeed, it is not necessary that an analog have amino acid sequence homology, and an analog may not be proteinaceous at all.","An analog may for instance be a mimetic of a molecule.","By the term “variant” is meant an isoform, an allelic variant of a gene or region thereof, a naturally occurring mutant form of a gene or region thereof, or a polypeptide or fragment having an amino acid sequence that differs in one or more amino acids but which retains one or more aspects of desired characteristic biological function.","This may be achieved by the addition of one or more amino acids to an amino acid sequence, deletion of one or more amino acids from an amino acid sequence and/or the substitution of one or more amino acids with another amino acid or amino acids.","Inversion of amino acids and any other mutational change that results in alteration of an amino acid sequence are also encompassed.","A variant may be prepared by introducing nucleotide changes in a nucleic acid sequence such that the desired amino acid changes are achieved upon expression of the mutagenised nucleic acid sequence, or for instance by synthesising an amino acid sequence incorporating the desired amino acid changes which possibility is well within the capability of the skilled addressee.","Substitution of an amino acid may involve a conservative or non-conservative amino acid substitution.","By conservative amino acid substitution is meant replacing an amino acid residue with another amino acid having similar stereochemical properties (eg.","structure, charge, acidity or basicity characteristics) and which does not substantially effect conformation or the desired aspect or aspects of characteristic biological function.","Preferred variants include ones having amino acid sequences in which one or more amino acids have been substituted with alanine or other neutrally charged amino acid residue(s), or to which one or more such residues have been added.","A variant may also incorporate an amino acid or amino acids that are not encoded by the genetic code.","By the term “derivative” is meant a molecule that is derived or obtained from another molecule and which retains one or more aspects of characteristic biological function of that molecule.","A derivative may for instance arise as a result of the cleavage of the parent molecule, cyclisation and/or coupling with one or more additional moieties that improve solubility, lipophilic characteristics to enhance uptake by cells, stability or biological half-life, decreased cellular toxicity, or for instance to act as a label for subsequent detection or the like.","A derivative may also result from post-translational or post-synthesis modification such as the attachment of carbohydrate moieties or chemical reaction(s) resulting in structural modification(s) such as the alkylation or acetylation of amino acid residues or other changes involving the formation of chemical bonds.","The term “polypeptide” is used interchangeably herein with “peptide” and encompasses amino acid sequences incorporating only a few amino acid residues or many amino acid residues coupled by peptide bonds.","The term “neoplastic cell” is to be taken to mean a cell exhibiting abnormal growth and may or may not be a malignant cell.","“Growth” is to be taken in its broadest sense and includes proliferation of the cell.","In this regard, an example of abnormal cell growth is the uncontrolled proliferation of a cell.","Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.","The features and advantages of the present invention will become further apparent from the following detailed description of preferred embodiments and the accompanying drawings."],["FIELD OF THE INVENTION The present invention relates to a method of modulating cell activity mediated by a mitogen activated protein kinase (MAP) and more particularly, modulation of the growth and/or the proliferation of a cell such as a neoplastic cell.","In particular, the invention finds use inter alia in applications requiring modulation of cell activity.","The present invention also provides agents for use in the above.","BACKGROUND OF THE INVENTION Colorectal cancer is the commonest internal malignancy affecting men and women in Australia.","About 4% of individuals develop this disease during the course of their lifetime and it was responsible for 14% of cancer deaths in that country in 1990.In 1995 there were 10,615 cases of colorectal cancer and 4508 deaths in Australia.","Worldwide, an estimated 875,000 cases of colorectal cancer occurred in 1996, accounting for 8.5% of all new cases of cancer.","Incidence rates vary approximately 20-fold around the world, with the highest rates seen in the developed world and lowest in India.","Australian incidence rates are towards the higher end of the scale internationally alongside those for North America and New Zealand.","Five-year survival following diagnosis of colon cancer is around 55% in the developed world and has altered little during the past few decades despite advances in chemo-, immuno- and radiotherapy.","Colorectal cancer is a malignant tumour that starts in the bowel wall and is confined locally for a relatively long period before spreading through the bowel wall and metastasising to lymph nodes and other parts of the body.","Survival rates are significantly improved where the disease is detected and treated early.","The aetiology of colorectal cancer is complex and appears to involve interactions between inherited susceptibility and environmental factors.","Recognition of the genetic component of colorectal cancer is growing.","Mutations are present as inherited germline defects or arise in somatic cells secondary to environmental insult.","There are two main inherited predisposition syndromes: Familial Adenomatous Polyposis (FAP) and Hereditary Non-Polyposis Colorectal Cancer (HNPCC); the remaining cases are attributed to so-called sporadic colorectal cancer.","FAP and HNPCC contribute to approximately 1% and 4%, respectively, of all colorectal cancers and a strong family history of bowel cancer in first-degree relatives is obtained in another 10-15% of patients.","However, in the vast majority of patients the aetiology of large bowel cancer remains unknown.","Most colon cancer arises within pre-existing benign precursor lesions or adenomas.","Adenomas are classified by histological architecture as tubular, tubulovillous or villous.","Villous change is associated with a higher malignant potential, as are large and high-grade epithelial dysplasia.","Environmental risk factors for development of colorectal cancer include diets low in fibre and vegetables and high in fat, red meat and alcohol and cigarette smoking which may induce mutations in somatic cells.","Studies have shown that persistent genetic instability and accumulation of mutations in several genes that are mainly concerned with cell growth or DNA repair, may be critical for the development of all colorectal cancers.","For example, a normal mucosal cell with inactivation of tumour suppressor genes such as the Adenomatous Polyposis Coli (APC) gene or Mutated in Colon Cancer (MCC) gene can proliferate and become a small adenomatous polyp.","Mutations in oncogenes such as k ras and in tumour suppressor genes such as p53 and the Deleted in Colon Cancer gene (DCC) may then occur and lead to the transformation of the polyp into a large adenoma, from which a carcinoma can eventually arise.","The uncontrolled cell growth that leads to the development of neoplasia is believed to result, therefore, from a series of inherited and acquired accumulated genetic changes.","This multistep process confers on cells the capacity to survive and proliferate in a manner freed from the constraints imposed on normal cell growth.","Spread of cancer cells involves tumour cell migration through the extracellular matrix scaffold, invasion of basement membranes, arrest of circulating tumour cells, and tumour cell extravasation and proliferation at metastatic sites.","Detachment of cells from the primary tumour mass and modification of the peri-cellular environment aid penetration of tumour cells into blood and lymphatic vessels.","It is the invasive and metastatic potential of tumour cells that ultimately dictates the fate of most patients suffering from malignant diseases.","Hence, tumourigenesis can be viewed as a tissue remodelling process that reflects the ability of cancer cells to proliferate and digest surrounding matrix barriers.","These events are thought to be regulated, at least in part, by cell adhesion molecules and matrix-degrading enzymes.","Cell adhesion receptors on the surface of colon cancer cells are involved in complex cell signalling which may regulate cell proliferation, migration, invasion and metastasis and several families of adhesion molecules have now been identified including integrins, cadherins, the immunoglobulin superfamily, hyaluronate receptors, and mucins (Agrez, 1996.)","In general, these cell surface molecules mediate both cell-cell and cell-matrix binding, the latter involving attachment of tumour cells to extracellular scaffolding molecules such as collagen, fibronectin and laminin.","It is now clear that multiple and varied cell adhesion receptors exist on colon cancer cells at any one time and an understanding of the role of individual receptors in promoting growth and spread of colon cancer is only just beginning to be elucidated.","Of all the families of cell adhesion molecules, the best-characterised at the present time is the family known as integrins.","Integrins are involved in several fundamental processes including leucocyte recruitment, immune activation, thrombosis, wound healing, embryogenesis, virus internalisation and tumourigenesis.","Integrins are transmembrane glycoproteins consisting of an alpha (α) and beta (β) chain in close association that provide a structural and functional bridge between extracellular matrix molecules and cytoskeletal components with the cell.","The integrin family comprises 17 different α and 8β subunits and the αβ combinations are subsumed under 3 subfamilies.","Excluding the leucocyte integrin subfamily that is designated by the β2 nomenclature, the remaining integrins are arranged into two major subgroups, designated β1 and αv based on sharing common chains.","In the β1 subfamily, the β1 chain combines with any one of nine a chain members (α1-9), and the a chain which associates with β1 determines the matrix-binding specificity of that receptor.","For example, α2β1 binds collagen and laminin, α3β1 binds collagen, laminin and fibronectin, and α5β1 binds fibronectin.","In the αv subfamily of receptors, the abundant and promiscuous αv chain combines with any one of five β chains, and a distinguishing feature of αv integrins is that they all recognise and bind with high affinity to arginine-glycine-aspartate (RGD) sequences present in the matrix molecules to which they adhere (Hynes, 1992).","The current picture of integrins is that the N-terminal domains of α and β subunits combine to form a ligand-binding head on each integrin.","This head, containing the cation binding domains, is connected by two stalks representing both subunits, to the membrane-spanning segments and thus to the two cytoplasmic domains.","The β subunits all show considerable similarity at the amino acid level (Loftus et al, 1994).","All have a molecular mass between 90 and 110 kDa, with the exception of β4 which is larger at 210 kDa.","Similarly, they all contain 56 conserved cysteine residues, except for β4 which has 48.These cysteines are arranged in four repeating patterns which are thought to be linked internally by disulphide bonds.","The α-subunits have a molecular mass ranging from 150-200 kDa.","They exhibit a lower degree of similarity than the β chains, although all contain seven repeating amino acid sequences interspaced with non-repeating domains.","The β subunit cytoplasmic domain is required for linking integrins to the cytoskeleton (Hynes, 1992).","In many cases, this linkage is reflected in localisation to focal contacts, which is believed to lead to the assembly of signalling complexes that include α-actinin, talin, and focal adhesion kinase (FAK) (Otey et al, 1990; Guan and Shalloway, 1992; Kornberg et al, 1992).","At least three different regions that are required for focal contact localisation of β1 integrins have been delineated (Reszka et al, 1992).","These regions contain conserved sequences that are also found in the cytoplasmic domains of the β2, β3, β5, β6 and β7 integrin subunits.","The functional differences between these cytoplasmic domains with regard to their signalling capacity have not yet been established.","Ligation of integrins by their extracellular matrix protein ligands induces a cascade of intracellular signals that include tyrosine phosphorylation of focal adhesion kinase, increases in intracellular Ca2+ levels, inositol lipid synthesis, synthesis of cyclins and expression of immediate early genes.","In contrast, prevention of integrin-ligand interactions suppresses cellular growth or induces apoptotic cell death (Meredith et al, 1993; Montgomery et al, 1994; Brooks et al, 1994; Varner et al, 1995; Boudreau et al, 1995).","Thus, integrins play roles in a number of cellular processes that impact on the development of tumours, including the regulation of proliferation and apoptosis.","The integrin β6 subunit was first identified in cultured epithelial cells as part of the αvβ6 heterodimer, and the αvβ6 complex was shown to bind fibronectin in an arginine-glycine-aspartate (RGD)-dependent manner in human pancreatic carcinoma cells (Sheppard et al, 1990; Busk et al, 1992).","The β6 subunit is composed of 788 amino acids and shares 34-51% sequence homology with other β integrin subunits β1-β5.The β6 subunit also contains 9 potential glycosylation sites on the extracellular domain (Sheppard et al, 1990).","The cytoplasmic domain differs from other β subunits in that it is composed of a 41 amino acid region that is highly conserved among integrin β subunits, and a unique 11 amino acid carboxy-terminal extension.","The 11 amino acid extension has been shown not to be necessary for localisation of β6 to focal contacts; in fact, its removal appears to increase receptor localisation.","However, removal of any of the three conserved regions previously identified as important for the localisation of β1 integrins to focal contacts (Reszka et al, 1992) has been shown to eliminate recruitment of β6 to focal contacts (Cone et al, 1994).","The integrin αvβ6 has previously been shown to enhance growth of colon cancer cells in vitro and in vivo, and this growth-enhancing effect is due, at least in part, to αvβ6-mediated gelatinase B secretion (Agrez et al, 1994; Niu et al, 1998 Agrez et al, 1999).","What makes this epithelial-restricted integrin of particular interest in cancer is that it is not expressed on normal epithelial cells but is highly expressed during would healing and tumourigenesis, particularly at the invading edge of tumour cell islands (Breuss et al, 1995; Agrez et al, 1996).","Further more, αvβ6 has been found to induce its own expression in an autocrine manner with cell crowding and a self-perpetuating model of colon cancer progression regulated by αvβ6-mediated gelatinase B secretion has been proposed (Niu et al, 2000).","Invasion of the extracellular matrix and metastatic spread of colon cancer is also likely to reflect the ability of tumour cells to digest their surrounding matrix scaffold through secretion of matrix-degrading enzymes such as matrix metalloproteinases (MMPs).","The mechanisms whereby human colon cancer cells escape the constraints on growth imposed on normal cells by cell crowding and dense pericellular matrices is unclear.","However, even colon cancer cells are subject to relative growth inhibition in vitro in a dense extracellular matrix environment (Agrez, 1989).","Integrins can signal through the cell membrane in either direction.","The extracellular binding activity of integrins can be regulated from the cell interior as, for example, by phosphorylation of integrin cytolasmic domains (inside-out signalling), while the binding of the extracellular matrix (ECM) elicits signals that are transmitted into the cell (outside-in signalling) (Giancotti and Ruoslahti, 1999).","Outside-in signalling can be roughly divided into two descriptive categories.","The first is ‘direct signalling’ in which ligation and clustering of integrins is the only extracellular stimulus.","Thus, adhesion to ECM proteins can activate cytoplasmic tyrosine kinases (e.g.","focal adhesion kinase FAK) and serine/threonine kinases (such as those in the mitogen-activated protein kinase (MAPK) cascade) and stimulate lipid metabolism (eg phosphatidylinositol-4,5-biphosphate (P1P2) synthesis.","The second category of integrin signalling is ‘collaborative signalling’, in which integrin-mediated cell adhesion modulates signalling events initiated through other types of receptors, particularly receptor tyrosine kinases that are activated by polypeptide growth factors (Howe et al, 1998).","In all cases, however, integrin-mediated adhesion seems to be required for efficient transduction of signals into the cytosol or nucleus.","MAP kinases behave as a convergence point for diverse receptor-initiated signalling events at the plasma membrane.","The core unit of MAP kinase pathways is a three-member protein kinase cascade in which MAP kinases are phosphorylated by MAP kinase kinases (MEKs) which are in turn phosphorylated by MAP kinase kinases (e.g.","Raf-1) (Garrington and Johnson, 1999).","Amongst the 12 member proteins of the MAP kinase family are the extracellular signal-regulated kinases (ERKs) (Boulton et al, 1991) activated by phosphorylation of tyrosine and threonine residues (Payne et al, 1991) which is the type of activation common to all known MAP kinase isoforms.","ERK 1/2 (44 kD and 42 kD MAPks, respectively) share 90% amino acid identity and are ubiquitous components of signal transduction pathways (Boulton et al, 1991).","These serine/threonine kinases phosphorylate and modulate the function of many proteins with regulatory functions including other protein kinases (such as p90rsk) cytoskeletal proteins (such as microtubule-associated phospholipase A2), upstream regulators (such as the epidermal growth factor receptor and Ras exchange factor) and transcription factors (such as C-Myc and Elk-1).","ERKs play a major role in growth-promoting events, especially when the concentration of growth factors available to a cell is limited (Giancotti and Ruoslahti, 1999).","MAP kinases can be activated through non-receptor tyrosine kinases such as focal adhesion kinase (FAK), cytoplasmic tyrosine kinase (pp60 c-srk) (Schlaepher and Hunter, 1998), and growth factors acting through membrane-associated receptor tyrosine kinases.","The FAK pathway is activated by most integrins.","In addition to activating FAK, some β1 and αv integrins also activate the tyrosine kinase Fynu and through it, the adaptor protein Shc (Wang et al, 1996).","It is likely that both FAK and Shc contribute to the activation of Ras and thence to the downstream kinase cascade of Raf-1, MEK, and MAP kinases (Schlaepfer et al, 1994; 1997).","It is now generally accepted that the activation of ERK in response to integrin ligation requires Ras signalling (Wary et al, 1996; Schlaepfer and Hunter, 1997).","The laminin receptor α6β4, the laminin/collagen receptor α1β1, the fibronectin receptor α5β1 and the RGD binding receptor αvβ3 are linked to the Ras-Raf-MEK-ERK signalling pathway and control of immediate early gene expression (Wary et al, 1996; Maniero et al, 1995; 1997).","The ability of integrins to activate ERK may be especially important when the concentration of growth factors available to the cell is limited.","In this setting, proliferation is likely to require co-stimulation of ERK through integrins and growth factor receptors (Giancotti and Ruoslahti, 1999).","Moreover, activation of ERK in response to integrin ligation may play a role in regulating cell migration (Klemke et al, 1997) possibly by initiating matrix-degrading enzyme secretion.","While there is a good deal of evidence in support of a key role for FAK and the phosphotyrosine-domain-containing adaptor protein Shc (Howe et al, 1998; Giancotti & Ruoslahti, 1999) in the Ras-Raf MEK-MAP kinase activation pathway there are also data implicating alternate pathways independent of MEKs.","For example, MEK-independent regulation of MAP kinase activation in NIH3T3 fibroblasts has been shown to be mediated by phosphatidylinositol-3-kinases and the conventional protein kinase C (PKC) isoforms and is thought to be due to inactivation of a MAP kinase inhibitor (Grammer and Blenis, 1997).","Although the mechanism by which PKC regulates integrin function is not known, PKC has been shown to regulate integrin-induced activation of the MAP kinase pathway upstream of Shc.","For example, PKC inhibition has been shown to inhibit ERK2 activation by fibronectin receptors without any effect on integrin-induced FAK or paxillin tyrosine phosphorylation (Miranti et al, 1999).","Hence, MAP kinase activation is more complicated than a simple linear pathway, and the mechanistic basis for the commonly observed integrin-mediated activation of MAP kinases remains controversial.","The importance of the MAP kinase pathway in promoting colon cancer growth in vivo is now no longer in question.","Although relatively little is known about the role of MAP kinase activation in for instance, colon tumour progression, MAP kinase pathways have been shown to be highly activated during the late progression of colorectal cancer (Licato et al, 1997).","In a recent breakthrough in this field, a highly potent inhibitor of MAP kinase activation has been identified which is capable of inhibiting human colon cancer growth in immune-deficient mice raising hops for the clinical application of MAP kinase inhibitors in the treatment of colon cancer (Sebolt-Leopold et at, 1999).","Various intracellular proteins may be linked directly or spatially to integrin cytoplasmic domains.","Direct interactions have been identified between cytoskeletal proteins such as α-actinin and talin and β1 and β3 integrin tails (Horwitz et al, 1986; Otey et al, 1990; Knezevic et al, 1996; Pfaff et al, 1998).","A direct association between FAK and the β1 integrin tail has been suggested based on in vitro β1 peptide studies, but this remains to be confirmed (Schaller et al, 1995).","More recently, the cytoplasmic domain of the α4 subunit has been found to be physically associated with the signalling adaptor protein paxillin in Jurkat T cells, and this binding event regulates the kinetics of FAK tyrosine phosphorylation (Liu et al, 1999).","Direct integrin links with the intracellular calcium-binding protein, calreticulin, and integrin-linked kinase (ILK) (Hannigan et al, 1996) have been shown to regulate “inside-out” integrin signalling.","For example, calreticulin has been shown to bind to a chain cytoplasmic domains (Rojiani et al, 1991) and modify α2β1 integrin activation by phorbol esters and anti-integrin antibodies (Coppolino et al, 1995).","Newly identified integrin-binding molecules include the serine/threonine integrin-linked kinase, ILK, which can associate with the β1, β2 and β3 subunits.","When over-expressed, ILK has been shown to reduce anchorage-independent growth and tumourigenicity in nude mice (Hannigan et al, 1996).","Co-immunoprecipitation strategies have also demonstrated interactions between integrins and the integral plasma membrane protein IAP, and members of the four transmembrane domain protein family (tetraspans).","The extracellular Ig region of the IAP molecule mediates association with αvβ3 and is required for cell binding to vitronectin-coated particles (Lindberg et al, 1996).","An emerging model for tetraspans is that they recruit signalling enzymes such as phosphatidylinositol-4-kinase and PKC into complexes with integrins (Hemler, 1998).","Integrins have also been shown to be physically linked with matrix-degrading enzymes and growth factors.","For example, the integrin αvβ6 has been shown to bind and activate latent TGFβ1 in keratinocytes (Munger et al, 1999) which is thought to be important in modulating the inflammatory process following epithelial injury.","In melanoma cells, αvβ3 binds activated gelatinase A (Brooks et al, 1996), and both insulin and platelet-derived growth factor (PDGF) co-immunoprecipitate with this integrin in NIH3T3 mouse fibroblasts (Schneller et al, 1997).","Synergism between integrin-mediated signalling processes and growth factor responses is now well-recognised and Schneller et al (1997) showed that a small subset of each of the insulin receptor and PDGF β-receptor is tyrosine phosphorylated upon growth factor stimulation.","Interestingly, this subset can associate with the αvβ3 integrin, and PDGF activity is enhanced in association with increased MAP kinase activity in cells plated on the αvβ3 ligand, vitronectin.","SUMMARY OF THE INVENTION Broadly stated, the present invention relates to the surprising finding that members of the mitogen activated protein (MAP) kinase family can associate with the cytoplasmic domain of an integrin molecule.","It is believed that no member of the MAP kinase family has previously been found to directly associate with any integrin.","The identification of this functional relationship permits the rational design of agents for therapeutically or prophylactically modulating cellular activity mediated by the MAP kinase and integrin interaction.","In an aspect of the present invention there is provided an agent capable of binding to a binding domain of an integrin for a MAP kinase.","In another aspect of the present invention there is provided an isolated or purified polypeptide capable of binding to a binding domain of an integrin for a MAP kinase or a homolog, analog, variant or derivative of the polypeptide.","The polypeptide may consist of or comprise the binding domain of the MAP kinase to which the integrin binds or sufficient core amino acid sequence of the binding domain of the MAP kinase to enable binding of the polypeptide to the integrin.","Preferably, the polypeptide will be a fragment of a MAP kinase.","Most preferably, the polypeptide will comprise the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or PSNLLLNTTCDLKIC or a region or regions of such sequences.","Accordingly, in a further aspect of the present invention there is provided a fragment of a MAP kinase wherein the fragment is capable of binding with an integrin, or a homolog, analog, variant or derivative of the fragment.","In another aspect of the present invention there is provided a fragment of a MAP kinase wherein the fragment consists of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin.","In another aspect of the present invention there is provided an isolated or purified polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase.","Preferably, a polypeptide or fragment of the invention will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less and more usually about 40 amino acids or less.","Typically, the length will be between about 5 to about 30 amino acids.","In yet another aspect of the present invention there is provided a fusion protein a fusion protein incorporating a binding moiety capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin.","Preferably, the binding moiety will comprise a fragment or polypeptide of the invention.","Most preferably, the fusion protein will incorporate a carrier moiety for facilitating passage across the cell membrane.","The carrier moiety may be penetratin.","In still another aspect of the present invention there is provided an agent for inhibiting binding of a MAP kinase to an integrin, comprising: a targeting moiety for targeting a cell expressing the integrin; a binding moiety for binding to a binding domain of the integrin for the MAP kinase to thereby inhibit the binding of the MAP kinase to the integrin; and a carrier moiety for facilitating passage of the binding moiety across the cell membrane of the cell; wherein the binding moiety is other than an antibody or an binding fragment thereof.","In another aspect of the present invention there is provided a MAP kinase or a fragment thereof with a mutagenised binding domain for an integrin or in which the binding domain is deleted, or a homolog, analog or variant of the MAP kinase or fragment, wherein capability to bind with the integrin is thereby reduced.","In another aspect of the present invention there is provided an isolated nucleic acid sequence encoding a fragment or polypeptide of the invention or a homolog, analog, variant or derivative of the fragment or polypeptide.","In another aspect of the invention there is provided a nucleic acid sequence coding for a MAP kinase or fragment thereof having a mutagenised binding domain for an integrin or in which the binding domain is deleted or a homolog, analog or variant of the MAP kinase or fragment, wherein capability to bind with the integrin is thereby reduced.","In a still further aspect there is provided an isolated nucleic acid sequence encoding a fusion protein or agent of the invention.","There are also provided antisense nucleic acid sequences complimentary to the sense nucleic sequences of the invention.","Such antisense sequences find application in antisense therapy of cells in which down regulation of cellular activity is desired, and include oligonucleotides.","Sense oligonucleotides coding for all or part of the binding domain of a MAP kinase for an integrin, or that of a homolog, analog or variant thereof, and complimentary antisense oligonucleotides find particular application as primers or probes.","A nucleic acid primer or probe of the invention may be labelled with a suitable reporter molecule for enabling detection of hybridisation of the primer or probe to a target nucleic acid sequence.","In yet another aspect of the present invention there is provided a vector incorporating a nucleic acid sequence of the invention.","Typically, the vector will be an expression vector and the nucleic acid sequence will be capable of being transcribed.","In a further aspect of the present invention there is provided a host cell transformed with a vector of the invention.","Preferably, the host cell will be selected from the group consisting of a mammalian cell, an epithelial cell, a neoplastic cell, and a cancer cell.","Preferably, the host cell will be a mammalian cell and most preferably, a colon cancer cell.","In yet another aspect of the present invention there is provided an antibody generated with the use of a polypeptide, fragment, agent or fusion protein of the invention.","In a still further aspect of the invention there is provided an antibody and binding fragment thereof capable of binding to a binding domain of a MAP kinase for an integrin.","The antibody may be a polyclonal or monoclonal antibody.","Preferably, the antibody is a monoclonal antibody.","In still another aspect of the present invention there is provided a method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing a number of agents for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if any said agent is capable of binding to the binding domain of the MAP kinase on the basis of the testing.","In yet another aspect of the invention there is provided a method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing a number of agents for ability to bind to the MAP kinase; (b) selecting an agent or agents identified as being able to bind to the MAP kinase on the basis of the testing; and (c) utilising the selected said agent or agents in an assay for indicating whether the or any of the selected said agents is capable of binding to the binding domain of the MAP kinase for the integrin.","In another aspect of the present invention there is provided a method of evaluating whether an agent is capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing the agent for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if the agent is capable of binding to the binding domain on the basis of the testing.","Preferably, a polypeptide or fragment of the invention comprising or consisting of the binding domain of the MAP kinase or core amino acid sequence of the binding domain or a homolog, analog, variant or derivative of the polypeptide or fragment or core amino acid sequence is used in the testing or assaying for whether an agent is capable of binding to the binding domain of the MAP kinase.","Most preferably, the polypeptide or fragment will comprise or consist of the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or PSNLLLNTTCDLKIC, core amino acid sequence of such sequences capable of binding to an integrin, or a homolog variant, analog or derivative of such sequences.","Testing or assaying of an agent for ability to bind to the binding domain of the MAP kinase for the integrin and thereby inhibit binding of the MAP kinase to the integrin, may comprise exposing the MAP kinase to the agent(s) to enable binding of the agent(s) to the MAP kinase to occur either in the presence of the integrin or prior to the addition of the integrin.","Rather than using the MAP kinase, a polypeptide, fragment of a MAP kinase or other molecule capable of binding to the binding site on the integrin for the MAP kinase may be used in such testing or assaying.","Similarly, rather than utilising an intact integrin, a polypeptide or integrin fragment comprising or incorporating the binding domain of the integrin for the MAP kinase, or other molecule capable of binding with the binding domain on the MAP kinase for the integrin, may be utilised.","In still another aspect of the present invention there is provided a method of isolating an agent from a sample utilising a molecule immobilised on a solid support and which is capable of binding to a binding domain of an integrin for a MAP kinase, comprising: (a) contacting the molecule immobilised on the solid support with the sample under conditions suitable for binding of the agent to the molecule; (b) eluting the agent from the solid support; and (c) collecting the eluted agent; wherein the molecule is other than an antibody or binding fragment of an antibody.","The molecule may be the MAP kinase, or a fusion protein, a polypeptide, or a fragment of the invention to which the integrin is capable of binding, or for instance a homolog, analog, variant or derivative of such a polypeptide or fragment.","In yet another aspect of the present invention there is provided the agent so isolated.","An agent of the invention will usually be provided in the form of a pharmaceutical composition.","Accordingly, in another aspect of the present invention there is provided a pharmaceutical composition comprising an agent of the invention capable of binding to a binding domain of an integrin for a MAP kinase, and a pharmaceutically acceptable carrier or diluent.","The agent may or not be proteinaceous in nature.","Preferably, the agent will comprise a fusion protein, or polypeptide.","Most preferably, the agent will be coupled to a carrier molecule for facilitating entry of the agent into a cell.","In a further aspect of the invention there is provided a pharmaceutical composition comprising a nucleic acid sequence of the invention and a pharmaceutically acceptable carrier or diluent.","Preferably, the nucleic acid sequence is incorporated into a vector as described herein.","Alternatively, the nucleic acid sequence may be joined to a carrier molecule such as penetratin for facilitating entry of the nucleic acid sequence into a target cell.","In another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: transfecting the cell with an expression vector incorporating a nucleic acid sequence encoding a MAP kinase of a fragment thereof, or a homolog, analog or variant of the MAP kinase or fragment, for being expressed by the cell and having a mutagenised binding domain for the integrin or in which the binding domain has been deleted whereby binding to the integrin is inhibited or prevented.","In still another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: transfecting the cell with a nucleic acid sequence encoding a fragment of a MAP kinase, or a homolog, analog or variant of the fragment, for being expressed by the cell and capable of binding to the binding domain of an integrin for the MAP kinase.","In yet another aspect of the present invention there is provided a method of modulating activity of a cell, comprising: transfecting the cell with a nucleic acid encoding a polypeptide for being expressed by the cell wherein the polypeptide is capable of binding to a binding domain of an integrin for a MAP kinase.","In still another aspect of the invention there is provided a method of modulating activity of a cell, comprising causing the expression of a MAP kinase to which an integrin is able to bind to be down-regulated.","Preferably, down-regulation of the expression of the MAP kinase is achieved using an antisense nucleic acid sequence that inhibits expression of gene coding for the MAP kinase.","The antisense nucleic acid sequence may be administered to the cell or generated within the cell.","Preferably, the cell will be transformed with a vector of the invention for generation of the antisense nucleic acid sequence.","Preferably, the antisense nucleic acid sequence will specifically hybridise with sense nucleic acid sequence coding for at least part of the binding domain of the MAP kinase for the integrin and/or intron sequence between such coding sequence.","Accordingly, in another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an antisense oligonucleotide for hybridising with at least part of a nucleic acid sequence coding for the binding domain of a MAP kinase for an integrin to thereby cause expression of the MAP kinase to be down-regulated.","In another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the integrin for the MAP kinase, wherein the agent is other than an antibody or a binding fragment thereof.","In a further aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the MAP kinase, wherein the agent is other than an agent comprising a binding domain of the integrin for the MAP kinase or an analog or derivative thereof.","In yet another aspect of the invention there is provided a method of modulating the activity of a cell, comprising: contacting the cell with an effective amount of a fusion protein, antibody or other molecule of the invention capable of binding to the binding domain of an integrin for a MAP kinase.","Preferably, modulation of the activity of a cell by a method as described herein will comprise treating a mammal in need of such treatment.","Accordingly, in a further aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid sequence encoding a MAP kinase or a fragment thereof or a homolog, analog or variant of the MAP kinase or fragment, with a mutagenised binding domain for an integrin or in which the binding domain has been deleted and wherein the nucleic acid sequence is capable of being expressed.","In another aspect of the invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid capable of causing the expression of a MAP kinase to which an integrin is able to bind to be down regulated.","In a further aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal a nucleic acid sequence encoding a fragment of a MAP kinase, or a homolog, analog or variant of the fragment, for being expressed by the cell and capable of binding to the binding domain of an integrin for the MAP kinase.","In still another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid sequence for expression of a polypeptide or a fragment of a MAP kinase within the cell, wherein the polypeptide or fragment is capable of binding to a binding domain of an integrin for the MAP kinase to thereby modulate the cellular activity.","In another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of an agent capable of binding to the binding domain of an integrin for a MAP kinase, wherein the agent is other than an antibody or a binding fragment thereof.","In another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of an agent capable of binding to the binding domain of a MAP kinase for an integrin wherein the agent is other than an agent comprising the binding domain of the integrin for the MAP kinase or an analog or derivative thereof.","In another aspect of there is provided a method of treatment or prophylaxis of a disease or condition in a mammal, wherein the disease or condition is responsive to an agent capable of binding to a binding domain of an integrin for a MAP kinase and wherein the agent comprises a polypeptide or fragment of the invention or a homolog, variant, analog or derivative of such a polypeptide or fragment, and the method comprises administering an effective amount of the agent to the mammal.","In yet another aspect of the present invention there is provided a method of treatment or prophylaxis of a disease or condition in a mammal, wherein said condition is responsive to an agent capable of binding to a binding domain of an integrin for a MAP kinase wherein the agent is a fusion protein of the invention.","In another aspect there is provided use of a nucleic acid of the invention in the manufacture of a medicament for administration to a mammal in the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable.","In a still further aspect of the invention there is provided the use of an agent capable of binding to a binding domain of an integrin for a MAP kinase in the manufacture of a medicament for administration to a mammal for the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable, wherein the agent is other than an antibody or binding fragment thereof.","The agent may for instance be a polypeptide, fragment or the like as described herein.","In another aspect of the present invention there is provided the use of an agent capable of binding to the binding domain of a MAP kinase for an integrin wherein the agent is other than an agent comprising the binding domain of the integrin for the MAP kinase or an analog or derivative thereof.","In another aspect there is provided the use of fusion protein of the invention in the manufacture of a medicament for administration to a mammal for the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable.","Methods as described above will typically comprise treatment or prophylaxis of cancer or a condition associated with a predisposition to cancer.","The cancer may for instance be selected from the group consisting of cancer of the lip, tongue, salivary glands, gums, floor and other areas of the mouth, oropharynx, nasopharynx, hypopharynx and other oral cavities, oesophagus, stomach, small intestine, duodenum, colon, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung, breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid and skin.","In still another aspect of the present invention there is provided a method of causing an activity of a cell to be upregulated, comprising: contacting the cell with an effective amount of a nucleic acid capable of being expressed within the cell and coding for an agent capable of binding to a binding domain of a MAP kinase for an integrin or a binding domain of the integrin for the MAP kinase.","In a still further aspect of the invention there is provided a method of causing an activity of a cell to be upregulated, comprising contacting the cell with an effective amount of an agent capable of binding to a binding domain of a MAP kinase for an integrin or a binding domain of the integrin for the MAP kinase.","Typically, the agent will be a polypeptide or fragment of an integrin comprising or consisting of the binding domain of an integrin for a MAP kinase or comprising or consisting of the core amino acid sequence of the binding domain directly involved in the binding to the MAP kinase, or a homolog, variant, analog or derivative of such polypeptide or fragment.","Preferably, the polypeptide or fragment will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less and more usually about 40 amino acids or less.","Typically, the length will be between about 5 to about 30 amino acids.","Most preferably, the polypeptide will comprise or consist of the amino acid sequence RSKAKWQTGTNPLYR or RSKAKNPLYR.","Up regulation of cellular activity is particularly desirable for instance in wound healing, re-endothelialisation within natural or artificial blood vessels, nerve growth and for instance induction of labour.","The cellular activity desired to be modulated will typically but not exclusively, be cell growth or proliferation.","Indeed, any activity mediated by MAP kinase signalling is expressly included within the scope of the invention.","The cell may be any cell type in which functional activity arising from signalling mediated by a MAP kinase may occur.","Preferably, the cell will be an epithelial cell and usually, a neoplastic cell.","Usually, the MAP kinase will be selected from the group consisting of an extracellular signal-regulated kinase (ERK) and a JNK MAP kinase.","Preferably, the MAP kinase is ERK2 or JNK-1.Most preferably, the MAP kinase is ERK2.The mammal may be any mammal treatable with a method of the invention.","For instance, the mammal may be a member of the bovine, porcine, ovine or equine families, a laboratory test animal such as a mouse, rabbit, guinea pig, a cat or dog, a primate or a human being.","Preferably, the mammal will be a human being.","As will be understood, modulation of cellular activity within the context of the invention may be up regulation or down regulation of cellular activity.","Reference to “down modulation” or “down-regulating” or the like should be understood to include preventing, reducing or otherwise inhibiting one or more aspects of the activity of the cell.","Conversely, reference to “up regulation” or like terms should be understood to include enhancing or increasing one or more aspects of the activity of the cell.","In the broadest sense, the term “integrin” unless otherwise specified, is to be taken to encompass an integrin family member or a homolog, derivative, variant or analog of an integrin subunit, or an integrin family member incorporating at least one such homolog, derivative, variant or analog of an integrin subunit.","Usually, the integrin will be a member of the αv subfamily.","Preferably, the integrin is or incorporates an integrin subunit selected from the group consisting of β3, β5 and β6.Most preferably, the integrin comprises β6.By “binding domain” is meant the minimum length of contiguous amino acid sequence required for binding.","By “core amino acid sequence” is meant regions or amino acids of the binding domain that directly participate in the binding as distinct from any amino acids that do not directly participate in the binding interaction.","Typically, the core amino acid sequence of the binding domain will comprise regions of the binding domain linked together by a number of intervening amino acids which do not directly participate in the binding.","The term “homolog” is to be taken to mean a molecule that has amino acid sequence similarity.","The homology between amino acid sequences can be determined by comparing amino acids at each position in the sequences when optimally aligned for the purpose of comparison.","The sequences are considered homologous at a position if the amino acids at that position are the same.","Typically, a homolog will have an overall amino acid sequence homology of at least about 30% more preferably at least about 50% or 70% and most preferably, greater than about 80%, 90% or 98% sequence homology.","Homology with a binding domain may be greater than the overall amino acid sequence homology of the homolog, and will usually be greater than about 60% or 80%, and more usually greater than about 90%, 95% or 98%.","The term “analog” is to be taken to mean a molecule that has one or more aspects of biological function characteristic of the molecule on which at least part of the analog is based or which was otherwise utilised in the design or preparation of the analog.","An analog may have substantial overall structural similarity with the molecule or only structural similarity with one or more regions or domains thereof responsible for the desired characteristic biological function.","By “structural” similarity is meant similarity in shape, conformation and/or other structural features responsible for the provision of the biological function or which otherwise have involvement in the provision of the biological function.","Alternatively, it will be understood that with knowledge of the region(s) or domain(s) of a molecule that provide(s) the characteristic biological function, analogs may be designed that while differing in structure nevertheless possess such biological function.","Indeed, it is not necessary that an analog have amino acid sequence homology, and an analog may not be proteinaceous at all.","An analog may for instance be a mimetic of a molecule.","By the term “variant” is meant an isoform, an allelic variant of a gene or region thereof, a naturally occurring mutant form of a gene or region thereof, or a polypeptide or fragment having an amino acid sequence that differs in one or more amino acids but which retains one or more aspects of desired characteristic biological function.","This may be achieved by the addition of one or more amino acids to an amino acid sequence, deletion of one or more amino acids from an amino acid sequence and/or the substitution of one or more amino acids with another amino acid or amino acids.","Inversion of amino acids and any other mutational change that results in alteration of an amino acid sequence are also encompassed.","A variant may be prepared by introducing nucleotide changes in a nucleic acid sequence such that the desired amino acid changes are achieved upon expression of the mutagenised nucleic acid sequence, or for instance by synthesising an amino acid sequence incorporating the desired amino acid changes which possibility is well within the capability of the skilled addressee.","Substitution of an amino acid may involve a conservative or non-conservative amino acid substitution.","By conservative amino acid substitution is meant replacing an amino acid residue with another amino acid having similar stereochemical properties (eg.","structure, charge, acidity or basicity characteristics) and which does not substantially effect conformation or the desired aspect or aspects of characteristic biological function.","Preferred variants include ones having amino acid sequences in which one or more amino acids have been substituted with alanine or other neutrally charged amino acid residue(s), or to which one or more such residues have been added.","A variant may also incorporate an amino acid or amino acids that are not encoded by the genetic code.","By the term “derivative” is meant a molecule that is derived or obtained from another molecule and which retains one or more aspects of characteristic biological function of that molecule.","A derivative may for instance arise as a result of the cleavage of the parent molecule, cyclisation and/or coupling with one or more additional moieties that improve solubility, lipophilic characteristics to enhance uptake by cells, stability or biological half-life, decreased cellular toxicity, or for instance to act as a label for subsequent detection or the like.","A derivative may also result from post-translational or post-synthesis modification such as the attachment of carbohydrate moieties or chemical reaction(s) resulting in structural modification(s) such as the alkylation or acetylation of amino acid residues or other changes involving the formation of chemical bonds.","The term “polypeptide” is used interchangeably herein with “peptide” and encompasses amino acid sequences incorporating only a few amino acid residues or many amino acid residues coupled by peptide bonds.","The term “neoplastic cell” is to be taken to mean a cell exhibiting abnormal growth and may or may not be a malignant cell.","“Growth” is to be taken in its broadest sense and includes proliferation of the cell.","In this regard, an example of abnormal cell growth is the uncontrolled proliferation of a cell.","Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.","The features and advantages of the present invention will become further apparent from the following detailed description of preferred embodiments and the accompanying drawings.","BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS FIG.","1: Surface biotinylation and immunoprecipitation of integrin subunits β5 and β6 in HT29 colon cancer cells stably transfected with either vector alone (mock transfectants) or antisense β6 construct; FIG.","2: Amplification of β6 and glyceraldehyde dehydrogenase (GAPDHu) mRNA: Ethidium-stained agarose gels with amplification products following RT-PCR from total RNA extracted from transfected HT29 and WiDr cell lines.","Equal amounts of PCR product obtained from RT-PCR reactions were loaded on each lane and the β6 (141 basepairs) and GAPDH (216 basepairs) bands are indicated (WT, wild-type; S, sense β6; A/S, antisense β6; mock, vector alone); FIG.","3: Non-transfected HT29 cells (wild) and cells transfected with vector alone (mock), sense β6 and antisense β6 analysed by FACScan for expression of the β6 subunit.","White and black histograms represent cells stained in the absence and presence, respectively, of mAb E7P6 (anti-β6); FIG.","4: WiDr cells transfected with vector alone (mock) or antisense β6 and analysed by FACScan for expression of the β6 subunit.","White and black histograms represent cells stained in the absence and presence, respectively, of mAb E7P6 (anti-β6); FIG.","5: Tumor cell proliferation in vitro assessed by (3H)-thymidine uptake for WiDr wild-type cells and transfectants (mock and antisense β6) cultured on plastic for the times indicated.","FIG.","6: Tumor cell proliferation in vitro assessed by (3H)-thymidine uptake for HT29 wild-type cells and transfectants (mock, sense β6 and antisense β6) cultured on plastic for the times indicated.","FIG.","7: Tumour growth after 6 weeks following subcutaneous inoculation of 106 viable cells of HT29 mock (vector alone) and antisense β6 transfectants; FIG.","8: Graph showing average tumour size at weekly intervals for each subgroup of 10 animals following subcutaneous inoculation of clone 1 from WiDr mock (vector alone) and antisense β6 transfectants; FIG.","9: Graph showing average tumour size at weekly intervals for each subgroup of 10 animals following subcutaneous inoculation of HT29 mock (vector alone) and antisense β6 transfectants; FIG.","10: β6-associated ERK identified by immunoprecipitations of integrin subunits from SW480 β6 transfectants.","(Top) Cell lysates were immunoprecipitated with mAbs QE2E5 (anti-β1), L230 (anti-αv), P1F6 (anti-β5), R6G9 (anti-β6), or matched isotype control Abs (IgG1 and IgG2A), and (bottom) blotted with anti-ERK1/2 mAb, SC-1647, which recognises total ERK (phosphorylated and non-phosphorylated).","Purified, non-phosphorylated ERK2 is shown in the left hand lane; FIGS.","11(A) and 11(B): (A) Western blotting: equal protein loads of cell lysates from one representative clone each from WiDr mock and antisense β6 blotted with anti-ERK mAb (SC-1647) against total ERK.","Purified non-phosphorylated ERK2 is shown in the left hand lane.","(B) β6 immunoprecipitates (mAb R6G9) from equal protein loads of the cell lysates in (A) probed with anti-ERK mAb (E10).","Purified phosphorylated ERK2 is shown in the left hand lane; FIG.","12: β6-bound ERK shown for the high and low SW480 β6-expressing clones by probing β6 immunoprecipitates with anti-ERK mAb (E10) against phosphorylated forms of ERK1/2.Purified, phosphorylated ERK2 is shown in the left hand lane; FIGS.","13(A) and 13(B): (A) Surface biotinylation of WiDr wild-type cells and β6 immunodepletion of the cell lysates by three successive rounds of β6 and β5 immunoprecipitations using mAb R6G9 and P1F6, respectively or control mAb (IgG2A).","The β6 and partner αv bands are arrowed.","(B) β6-immunodepleted WiDr cell lysates after 3 successive rounds of β6-immunoprecipitations probed with anti-ERK mAb SC-1647 recognising both phosphorylated and non-phosphorylated forms of ERK1/2 and compared with non-β6 immunodepleted lysates and control lysates sequentially immunoprecipitated 3 times with either isotype matched control mAb (IgG2A) or mAb P1F6 (anti-β5); FIG.","14: Non-transformed (HaCaT) and Ras-transformed (HaRas) human keratinocytes: β6 immunoprecipitation and ERK western blots probed with monoclonal antibody E10 (against Phosphorylated ERK 1/2) and monoclonal antibody SC1647 (against total ERK 1/2), respectively.","FIGS.","15(A) and 15(B): (A) Depletion of β6 with sequential rounds of ERK immunodepletion of cell lysates using anti-ERK mAb (SC-1647).","(B) MAP kinase activity in cell lysates before and after 5 rounds of sequential β6 immunodepletion from SW480 β6 and SW480 mock transfectants (full grey and hatched bars, respectively).","MAP kinase activity is shown as the mean of three independent experiments.","The reduction in MAP kinase activity following β6 immunodepletion was highly significant (P≦0.005, students T test).","FIG.","16: Shows the amino acid sequence of the cytoplasmic domain of the β6 subunit as well as the amino acid sequences for the β1 to β3 subunits, respectively; FIG.","17: Graph showing binding of non-phosphorylated ERK2 (GST.ERK2) to overlapping fragments corresponding to different regions of the cytoplasmic domain of the β6 subunit; FIG.","18: Graph showing binding of ERK2 (GST.ERK2) to the β6 cytoplasmic domain and the peptide fragments indicated in FIG.","16 over a range of concentrations of ERK2; FIG.","19: Graph showing binding of ERK2 (GST.ERK2) to the β6 cytoplasmic domain and fragments thereof; FIG.","20: Graph showing binding of ERK2 (GST.ERK2) to a 15 mer fragment of the β6 cytoplasmic domain and which has the amino acid sequence RSKAKWQTGTNPLYR; and FIG.","21: Shows regions of the cytoplasmic domain of the β6 subunit corresponding to synthesised fragments thereof evaluated for capacity to be bound by ERK2.FIG.","22: Graph showing assay results for ERK2 (GST.ERK) binding to synthesised 10 mer fragments identified in FIG.","21.FIG.","23: Graph showing binding of ERK2 (thrombin cleaved) to synthesised peptide having the amino acid sequence RSKAKNPLYR compared to the 15 mer RSKAKWQTGTNPLYR fragment of the cytoplasmic domain of the β6 subunit.","FIG.","24: Graph showing binding of JNK-1 to the β6 cytoplasmic domain.","FIG.","25: Location of β6 Δ746-764, β6(770t) and β6(777t) deletions in the cytoplasmic domain of the β6 subunit.","FIG.","26: SW480 cells transfected with wild-type full length coding sequence for β6 or β6 Δ746-764 deletion mutant analysed by FACScan for expression of wild-type or mutant β6.White and black histograms represent cells stained in the absence and presence of the integrin subunit, respectively.","FIGS.","27(A) and 27(B): (A) Western blotting: equal protein loads of cell lyates from SW480 cells expressing wild-type β6 or β6 Δ746-764 deletion mutant.","(B) β6 immunoprecipitates (mAb R6G9) from equal protein loads of cell lysates (A) probed with anti-ERK mAb (E10).","FIG.","28: Proliferation of HT29 colon cancer cells cultured for 48 hours and treated with penetratin, the fragment of β6 cytoplasmic domain having amino acid sequence RSKAKWQTGTNPLYR alone or the fragment coupled to penetratin for the final 24 hours of the incubation period.","FIG.","29: Proliferation of SW480 cells expressing wild-type β6 cultured on plastic for 48 hours and treated with penetratin, the RSKAKWQTGTNPLYR peptide alone or the peptide coupled to penetratin for the final 24 hours of the incubation period.","FIGS.","30(A) to 29 (C): (A) SW480 cells cultured with control additive for 24 hours; (B) SW480 cells cultured with penetratin for 24 hours; (C) SW480 cells cultured with RSKAKWQTGTNPLYR bound to penetratin for 24 hours.","FIGS.","31(A) and 31 (B): (A) SW480 mock (−β6) and SW480 β6 transfectants (+β6) cultured in presence of the RSKAKWQTGTNPLYR peptide coupled to penetratin.","(B) Photomicrographs of cells shown in (A) cultured in the presence/absence of the peptide penetratin complex.","FIGS.","32(A) to 32(C): (A) SW480 cells cultured in a 3-dimensional collagen type I matrix photographed in the gel at the end of 10 days.","(B) The SW480 cells following dissolution of the collagen with collagenase.","(C) Graph showing numbers of colonies of the SW480 cells having a diameter exceeding 200μ.","FIGS.","33(A) and 33(B): Graphs showing inhibition of proliferation of SW480 cells expressing full length wild-type β6 in the presence of RSKAKWQTGTNPLYR peptide bound to penetratin.","FIG.","34: Graph showing binding of ERK2 to RSKAKWQTGTNPLYR peptide and peptides corresponding to regions of the cytoplasmic domain of β3 and β5.FIG.","35(A) to 35(C): (A) Immunoprecipitations of β5 and β6 subunits followed by immunoblotting with anti-biotin antibody.","αv/β5/β6 bands are shown for the SW480 cell line: wild-type cells, mock transfectants (vector alone) or cells expressing either full length β6 or a Δ746-764 deletion mutant.","(B) Indentical amounts of precipitated proteins shown in (A) were immunoblotted with anti-ERK mAb (E10) against phosphorylated ERK1/2.Purified phosphorylated ERK2 is shown in the left hand lane.","(C) Comparative binding of ERK2 (1 μm) to synthetic peptides comprising either RSKAKWQTGTNPLYR or peptide sequences derived from β5 and β1 cytoplasmic domains that correspond to the binding domain on β6 for ERK.","FIG.","36: In vitro phosphorylation of GST-Elk-1 by immunoprecipitated ERK from cell lysates prepared from human umbilical vein endothelial cells (HUVECs) cultured in the absence/presence of epidermal growth factor (EGF) after a 24 hour culture period in serum-free and growth factor free medium.","FIGS.","37 and 38: Overlapping peptides derived from ERK2 sequence tested for ability to inhibit the β6-ERK2 interaction.","FIG.","39: Inhibition of β6-ERK2 interaction using ERK2 peptide HRDLKPSNLLLNTTCDLK (0.25 μm) and β6 cytoplasmic tail peptide.","FIGS.","40(A) to 40(D): Illustrate a dose-dependent wound repair process for peptide-agonist complexed with penetratin as a delivery vehicle.","FIG.","41: Nucleotide and amino acid sequence for ERK2.DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE PRESENT INVENTION The distribution of β6 integrin subunit within various tissues has been assessed by both in situ hybridisation and immunostaining and reported in the art.","For instance, β6 mRNA in adult primate tissues was detected only in epithelial cells and at very low or undetectable levels in most normal tissues (Breuss et al, 1993).","High-level expression of β6 has been observed in secretory endometrial glands while low-level expression was detected in the ductal epithelia of salivary gland, mammary gland and epididymis, in gall and urinary bladder, and in the digestive tract.","Immunostaining data has also shown that β6 expression is restricted to epithelia and is up-regulated in parallel with morphogenetic events, tumourigenesis and epithelial repair (Breuss et al, 1993; 1995).","During development of the kidney, lung and skin, β6 is expressed by specific types of epithelial cells, whereas it is mostly undetectable in normal adult kidney, lung and skin.","In contrast, high level expression of β6 has been observed in several types of carcinoma.","For example, β6 is almost invariably neo-expressed in squamous cell carcinomas derived from the oral mucosa, and often focally localised at the infiltrating edges of tumour cell islands (Breuss et al, 1995; Thomas et al, 1997).","Moreover, expression of the β6 subunit has been observed in renal cell carcinoma and testicular tumour cell lines (Takiuchi et al, 1994) and 50% of lung cancers have been shown to express this subunit (Smythe et al, 1995).","Recent studies have shown that αvβ6 is a major fibronectin-binding receptor in colorectal cancer (Agrez et al, 1996).","Moreover, normal colonic epithelium from cancer patients does not express αvβ6 in immunostaining studies, and as with squamous cell carcinomas from the oral mucosa (Thomas et al, 1997) maximal β6 expression in colon cancer has been observed at the invading edges of tumour cell islands (Agrez et al, 1996).","Importantly, heterologous expression of the β6 subunit in colon cancer cells lacking constitutive expression of this receptor has been shown to stimulate tumour cell proliferation in vitro and tumour growth in athymic immune-deficient mice, and this growth-promoting effect is mediated through the 11 amino acid C-terminal cytoplasmic extension unique to the β6 subunit (Agrez et al, 1994).","These findings suggest that one mechanism of the enhanced growth effect involves induced secretion of matrix-degrading enzymes as has been previously suggested for invasive melanoma cells.","Support for this arises from findings that the proliferative capacity of colon cancer cells within 3-dimensional collagen matrices is inversely related to the density of the extracellular matrix (Agrez, 1989), and the observation that induced expression of αVβ6 in colon cancer cells markedly enhances gelatinase B secretion (Agrez et al, 1999).","Indeed, the β6 subunit is widely observed in cancers of various origins (Breuss et al, 1995).","As described above, β6 is detected in at least 50% of bowel cancer tumours.","Others have reported its presence in oropharyngeal cancers where it is also present and strongly expressed in the invading margins of the cancer cell islands as is commonly found in bowel cancer.","In the oropharyngeal mucosa, no β6 is observed in the normal lining cells of the mouth but in both primary and metastatic tumours from the oropharyngeal mucosa, strong β6 expression is seen which does not correlate with degree of differentiation and in particular, is restricted to the basal layer of epithelial cells.","In colon cancer, β6 expression is similarly maximal at the advancing edges of tumour cell islands (Agrez et al, 1996).","Hence, modulation of MAP kinase interaction with the β6 subunit in epithelial cells is of interest in the prophylaxis or treatment of cancer of the lip, tongue, salivary glands, gums, floor and other areas of the mouth, oropharynx, nasopharynx, hypopharynx and other oral cavities, oesophagus, stomach, small intestine, duodenum, colon, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung, female and male breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid and skin.","In terms of prophylactic use in relation to cancer, modulation of the MAP kinase β6 interaction and specifically down regulation of the interaction may find application in protecting against ultraviolet-induced skin cancer, lung cancer in smokers, cancer of the gut where polyps are present as in polyposis coli or other inheritable disease where a pre-disposition to the development of polyps exists, breast cancer in high risk patients with a familial history of breast cancer or otherwise identified as carrying known mutations of the breast cancer susceptibility gene BRCA1 or BRCA2 associated with breast cancer, transitional cell cancers arising from bladder papillomas, and cancer of the cervix in individuals deemed to be at high risk.","Expression of β6 is also up-regulated in migrating keratinocytes at the wound edge during experimental epidermal wound healing.","αvβ6 is not expressed in normal epithelium (Jones et al, 1997).","However, following experimental wounding, αv appears to switch its heterodimeric association from β5 to β6 subunit during re-epithelialisation.","At day 3 after wounding, β6 is absent but then appears around the perimeter of the basal cells of the migrating epidermis (Clark et al, 1996).","By day 14 after wounding, when re-epithelialisation is complete, all suprabasalar cells overlying the wound express β6 but not β5.In human mucosal wounds, maximal expression of β6 has been observed relatively late when epithelial sheets are fused and granulation tissue is present (Haapasalmi et al, 1996).","Furthermore, those investigators observed maximal expression of tenascin with αvβ6 expression.","Interestingly, freshly isolated keratinocytes have not been found to express β6 but begin to express this after subculturing (Haapasalmi et al, 1996).","Moreover, TGF-β1 has been shown to induce the de novo expression of αvβ6 at the cell surface on keratinocytes (Zambruno et al, 1995).","This is particularly relevant in view of the recent observation that αvβ6 binds and activates latent TGF-β1 which may be a means of locally regulating TGF-β1 function in vivo during tissue response to injury (Munger et al, 1999).","In contrast to persistent αvβ6 expression observed in colon cancer cells, new expression of αvβ6 in migrating keratinocytes is down-regulated to undetectable levels once re-epithelialisation is complete (Happasalami et al, 1996; Cass et al, 1998).","While in the normal unwounded skin, just as in other normal epithelia, αvβ6 expression is absent indicating that this MAP kinase activation pathway is normally suppressed.","However, alterations in cell shape trigger biochemical signalling by MAP kinases in human keratinocytes and it is likely that alterations of skin mechanics in vivo during wound formation and healing trigger physiological responses via the MAP kinase pathway (Krippenberg et al, 2000).","In addition, gelatinase B secretion by keratinocytes which facilitates the process of cell migration during normal re-epithelialisation is MAP kinase-dependent (McCawley et al, 1999).","As outlined above, MAP kinases behave as a convergence point for diverse receptor-initiated signalling events at the plasma membrane which regulate cell proliferation, differentiation, migration and invasion.","In particular, extracellular signal-regulated kinases (ERKs) become activated upon integrin ligation and, thereby, regulate cell migration possibly by initiating matrix degrading enzyme secretion (Klemke et al, 1997) such as gelatinase B. Gelatinase B production has been shown to be dependent on endogenous ERK signalling in growth factor-stimulated human kertinocytes (McCawley et al, 1999; Zeigler et all, 1999).","Aside from integrins, growth factors such as foetal calf serum or epidermal growth factor (EGF) have been shown to stimulate ERK activity via the Ras-Raf-Mek-MAPk cascade resulting in directed cell migration through a porous membrane (Giehl et al, 2000).","There is also abundant data indicating that the MAP kinase cascade plays a major role in alterations of cell shape and migration for a range of normal cell types.","For example, the MAP kinase signalling pathway has been shown to mediate neurite outgrowth by cerebellar neurons (Schmid et al, 2000) implying a role for MAP kinase in nerve repair, while growth factor-induced migration of endothelial cells has been shown to be dependant on MAP kinase signalling and this dependency for new blood vessel formation (angiogenesis) on sustained ERK activity is regulated by the ligation state of both growth factor receptors and integrins (Eliceiri et al, 1998).","In addition, ERK activation has been demonstrated to be essential for up-regulation of vascular endothelial growth factor (VEGF) in other cell types (Jung et al, 1999), and induction of endothelial tube formation by human umbilical vein endothelial cells (HUVECs) dispersed within a 3-dimensional collagen gel has been shown to be dependent on MAP kinase activation (Ilan et al, 1998).","Growth factors and their receptors are also known to play important roles in tissue repair and ulcer healing.","For example, EGF accelerates gastroduodenal ulcer healing by stimulating cell proliferation, and this event is regulated through the Ras/Raf/MAP kinase pathway (Tarnawski and Jones, 1998).","Moreover, in the healing process of gastric ulcers, ERKs have been shown to translocate to cell nuclei with activation of the c-fos gene which triggers cell proliferation (Tarnawski et al, 1998).","Cutaneous wound re-epithelialisation and keratinocyte migration requires ordered gene expression and the fibroblast growth factor (FGF)-inducible response element (FiRE) required for migrating (but no proliferating) keratinocytes has been shown to be induced by treatment of cells with okadaic acid, the inhibitor of protein phosphatases which inactivate ERKs (Jaakkola et al, 1998).","The MAP kinase cascade is also be implicated in events relating to parturition.","For example, induction of labour is commonly achieved with oxytocin and oxytocin induction of prostaglandin synthesis has been shown to be MAP kinase-dependent (Strakova et al, 1998).","In fact, oxytocin causes rapid activation of MAP kinases in both human and rat puerperal uterine myometrial cultured cells (Nohara et al, 1996).","Moreover, MAP kinase inhibition has been shown to inhibit prostaglandin-induced uterine contraction in animal models (Ohmichi et al, 1997).","In addition, gelatinase B has been identified in amniotic fluid association with normal labour (Vadillo-Ortega et al, 1996) and gelatinase B production has been shown to be dependent on endogenous ERK signalling in other cell types such as keratinocytes (Zeigler et al, 1999; McCawley et al, 1999).","Taken together, these date suggest that MAP kinase signalling may be an important regulator of parturition.","αvβ6 expression is also upregulated in type II alveolar epithelial cells during lung injury caused by injection of live bacteria and αvβ6 mRNA is induced within 5 hours of acute injury (Breuss et al, 1995).","Interestingly, αvβ6 has been shown to be expressed on proximal airway epithelial cells in 50% of smokers undergoing lung resection (Weinacker et al, 1995).","Just as in human keratinocytes, in primary cultures of human airway epithelial cells, TGF-β1 has been shown to dramatically increase expression of αvβ6 without affecting surface expression of any other integrin.","Moreover, epidermal growth factor (EGF) also induces αvβ6 in these cells, and the effect of both growth factors on β6 expression has been shown to be additive (Wang et al, 1996).","αvβ6 expression has also been observed in adult lungs and kidneys at focal sites of sub-clinical inflammation as well as in a variety of clinical specimens from patients with chronic or acute inflammation of the lungs or kidneys (Breuss et al, 1995).","Taken together, this data and studies outlined above indicate that αvβ6 has a role in inflammation and affects for instance cell spreading, migration and growth during re-organisation of epithelial in development, tissue repair and neoplasia.","Hence, the application of agents, on either unwounded epithelial tissues (such as before surgery) or upon wounded epithelium, which up-relate MAP kinase activity may enhance cell migration and wound re-epithelialisation and thereby, hasten the healing process.","Such agents may be based upon the binding domain on β6 for ERK2 and act therefore as β6 agonists (in the absence of this integrin) leading to MAP kinase activation.","Examples of the application of such agents may include cell migration in wound healing, re-endothelialisation within natural or artificial blood vessels, or renewed nerve outgrowth following injury or disease.","In addition, use of such agonists may for instance be useful in promoting induction of labour.","The epithelial restricted integrin subunit β6 is shown herein to interact with at least MAP kinases ERK2 and JNK-1, and its down-regulation dramatically suppresses growth of colon cancer.","Hence, therapeutic strategies to inhibit growth and proliferation of colon cancer and growth of other malignant cancer cells by switching off either the permissive β6 integrin and/or inhibiting the β6 MAP kinase interaction are of particular interest.","In particular, the fact that β6 expression may be significantly upregulated on tumour cells compared to normal cells offers the potential for tumour cell specificity substantially without impairment of normal cellular function.","Down-regulation of the functional activity of an integrin or MAP kinase may be achieved by preventing or down-regulating the expression of the integrin molecule or MAP kinase or by inhibiting the signalling function of the integrin or MAP kinase.","Gene therapy is one strategy for treating cancers of different types.","The use of recombinant adenoviruses has for instance been utilised for restoring expression of polypeptide encoded by a wild-type p53 tumour suppressor gene (Bookstein et al, 1996), and adenoviral vectors for expression of wild-type p53 have been shown to suppress growth of human colon cancers by as much as 60% in animal models following intra-tumoural injection of recombinant virus (Spitz et al, 1996).","Rather than replacing a defective gene with one encoding a polypeptide the expression of which restores normal function of the cell as in the above examples, the possibility exists to achieve down regulation of cancer cells such as colon cells by introducing a gene that codes for an integrin subunit in which the binding domain for a MAP kinase has been rendered defective by mutagenesis, or in which the binding domain has been wholly or partially deleted, to thereby achieve down regulation through the inhibition of the MAP kinase integrin interaction.","The defective integrin subunit will nevertheless usually be able to associate with its normal partner integrin subunit and be expressed on the cell membrane.","Preferably, the defective integrin subunit will be expressed at a higher level than the corresponding wild-type integrin subunit such that down regulation is achieved by a dominant negative effect.","Alternatively, such therapy may involve the introduction and expression of a nucleic acid sequence that encodes a fragment or truncated form of an integrin subunit that excludes the binding domain for the MAP kinase or in which the binding domain has been partially or wholly deleted or otherwise mutagenised so as to be defective.","Similarly, a gene coding for a MAP kinase or a nucleic acid sequence coding for a fragment of a MAP kinase comprising the binding domain for an integrin may also be modified such that the binding domain of the MAP kinase is mutagenised or deleted.","Another option is to introduce a nucleic acid sequence encoding a polypeptide capable of binding to the binding domain on the integrin for the MAP kinase or to the binding site on the MAP kinase for the integrin upon being expressed within the cell to thereby inhibit intracellular binding of the MAP kinase to the integrin and thereby achieve down regulation of cellular activity.","A gene or nucleic acid sequence encoding an integrin subunit or MAP kinase may also be modified such that although the encoded binding domain for integrin or the MAP kinase as the case may be remains unaltered, the amino acid sequence of a region distant from the binding domain, or the amino acid sequence of either one or both regions immediately flanking the binding domain, is altered to achieve a change in the three dimensional conformation of the integrin subunit or MAP kinase such that the MAP kinase integrin binding interaction is inhibited.","The gene or nucleic acid sequence may be altered to achieve the desired outcome by the deletion, insertion or substitution of one or more nucleotides such that the corresponding amino acid sequence is modified to the extent that binding of the MAP kinase to the integrin is inhibited.","Inhibition in this context may be partial or total inhibition.","The gene or nucleic acid sequence can be introduced into a cell in an appropriate expression vector for expression of the gene or nucleic acid sequence extrachromosomally or more preferably, for facilitating integration of the gene or nucleic acid sequence into genomic DNA by heterologous or homologous recombination events.","In another strategy, down-regulation of the expression of an integrin subunit such as β6 and hence an integrin heterodimer such as αvβ6 or the down regulation of expression of a MAP kinase such as ERK2, is achieved using antisense nucleic acid sequences (e.g.","oligonucleotides) for inhibiting expression of the subunit or MAP kinase.","Typically, this will involve expression of a nucleic acid construct incorporating all or part of a coding region of the gene for the integrin subunit or MAP kinase inserted in the reverse orientation resulting in the synthesis of antisense RNA which inhibits translation of mRNA encoding the integrin subunit or MAP kinase by hybridisation of the antisense RNA to the mRNA.","More broadly, a method of down regulating an activity of a cell by inhibiting expression of an intact integrin subunit or a MAP kinase may comprise contacting the cell with a first nucleic acid molecule that is capable of interacting with a target nucleic acid sequence coding for the subunit or MAP kinase, or which first nucleic acid molecule is capable of being transcribed to a nucleic acid molecule capable of interacting with the target sequence whereby the interaction of the first nucleic acid molecule with the target sequence inhibits expression of the integrin subunit or MAP kinase.","Reference to the first nucleic acid molecule is to be understood as a reference to any nucleic acid molecule which directly or indirectly facilitates reduction, inhibition or other form of down regulation of the expression of the integrin molecule or MAP kinase.","Nucleic acid molecules which fall within the scope of this definition include antisense sequences administered to a cell and antisense sequences generated in situ which have sufficient complementarity with target sequence such as mRNA coding for the integrin subunit or MAP kinase or for instance, a transcription regulatory sequence controlling transcription of the gene coding for the integrin subunit or MAP kinase, to thereby be capable of hybridising with the target sequence and inhibit the expression of the integrin subunit or MAP kinase.","The first nucleic acid molecule may also be a ribozyme capable for instance, specifically binding to that region of nucleic acid encoding the binding domain of an integrin subunit or MAP kinase or the binding domain of a MAP kinase for an integrin, and cleaving the nucleic acid.","Typically, an antisense strategy in accordance with the present invention will be directed toward inhibition of an intact integrin subunit to thereby down regulate cell activity via down regulation of interaction of the integrin with a MAP kinase.","On a priori grounds, targeting the expression of the β6 subunit in malignant cells such as in colon cancer by means of adenoviral-mediated antisense therapy is preferred because down-regulating β6 by means of a non-adenoviral approach may increase cell surface expression of the β5 subunit.","The relevance to such therapy is that the vitronectin-binding integrin αvβ5 promotes adenovirus internalisation (Thomas et al, 1993).","Given that abundant αvβ5 is always present on the surface of colon cancer cells for example, a secondary benefit of inhibiting β6 expression during the course of therapy may be the concomitant rise of β5 in cells already transduced with antisense β6 nucleic acid.","This is likely to facilitate further virus uptake into such cells since the amount of integrin αvβ5 present has been shown to be closely related to levels of gene expression following adenovirus-mediated gene transfer (Takayama et al, 1998).","Typically, an antisense nucleic acid sequence will hybridise with all or part of that region of the target sense nucleic acid encoding the binding domain of an integrin subunit or the corresponding binding domain of a MAP kinase.","An antisense nucleic sequence may for instance be capable of hybridising to exon and/or intron sequences of pre-mRNA.","Preferably, an antisense nucleic acid sequence will be designed for specifically hybridising to mature mRNA in which intron sequences have been spliced out by normal cellular processing events.","It is not necessary that an antisense nucleic acid sequence have total complementarity with its target sequence only that substantial complementarity exists for specificity and to allow hybridisation under cellular conditions.","Preferably, an antisense nucleic sequence will have a complementarity of about 70% or greater, more preferably about 80% or greater and most preferably, about 90% or 95% or greater.","Preferred antisense sequences are oligonucleotides wherein the complementary sense nucleotides encode for about 50 amino acids or less, preferably about 35 or 30 amino acids or less, more preferably less than about 25 or 20 amino acids, most preferably about 15 amino acids or less and usually between about 5 to about 15 amino acids.","Antisense nucleic acid sequences may be generated in vivo by transcription of a suitable expression vector within a cell transformed with the vector, or ex vivo and then be introduced into a target cell to effect down regulation of the MAP kinase integrin interaction.","Antisense sequences will desirably be designed to be resistant to endogenous exonucleases and/or endonucleases to provide in vivo stability in target cells.","Modification to the phosphate backbone, sugar moieties or nucleic acid bases may also be made to enhance uptake by cells or for instance solubility, and all such modifications are expressly encompassed.","Such modifications include modification of the phosphodiester linkages between sugar moieties, the utilisation of synthetic nucleotides and substituted sugar moieties, linkage to liphophilic moieties and the such like as described in U.S. Pat.","No.","5,877,309.Methods for the construction of oligonucleotides for use in antisense therapy have previously been described (see Van der Krol et al, 1998 Biotechniques 6:958-976; and Stein et al, 1998 Cancer Res 48:2659-2668; Bachman et al, 1998, J. Mol.","Med.","76:126-132).","Any means able to achieve the introduction of a gene or a nucleic acid into a target cell may be used.","Gene transfer methods known in the art include viral and non-viral transfer methods.","Suitable virus into which appropriate viral expression vectors may be packaged for delivery to target cells include adenovirus (Berkner, 1992; Gorziglia and Kapikian, 1992); vaccinia virus (Moss, 1992); retroviruses of avian (Petropoulos et al, 1992); murine (Miller, 1992) and human origin (Shimada et al, 1991); herpes viruses including Herpes Simplex Virus (HSV) and EBV (Margolskee, 1992; Johnson et al, 1992; Fink et al, 1992; Breakfield and Geller, 1997; Freese et al, 1990); papovaviruses such as SV40 (Madzak et al, 1992), adeno-associated virus (Muzyczka, 1992); BCG and poliovirus.","Particularly preferred virus are replication deficient recombinant adenovirus (eg.","He et al, 1998).","Engineered virus may be administered locally or systemically to achieve delivery of the gene or nucleic acid sequence of interest into a target cell.","Gene transfer methods as described above may be used in the provision of transgenic animals for studying in vivo the effect of for instance, modifying the binding site of an integrin for a MAP kinase or the binding domain of the MAP kinase for the intergrin.","A transgenic animal is one with cells that contain heterologous nucleic acid as a result of the deliberate introduction of the nucleic acid.","The nucleic acid may be introduced indirectly by viral transfer as indicated above or directly by microinjection into a pronucleus of a fertilised egg prior to transfer of the egg to a surrogate mother for development to term.","Alternatively, a gene or nucleic acid sequence of interest may be introduced into an embryonal stem (ES) cell in culture and the transformed cell injected into a recipient blastocyst which is then transferred to a surrogate mother for development to term.","Techniques for generation of transgenic animals are for instance described in U.S. Pat.","No.","4,873,191; Van der Putten, 1985; Thompson et al, 1989 and Lo, 1983.A transgenic animal homozygous for a transferred gene for instance may be obtained by the mating of animals heterozygous for the gene as will be appreciated.","Both animal cells expressing a heterologous gene or nucleic acid sequence and transgenic animals in which a particular gene has been mutagenised by homologous recombination may be provided.","A transgenic animal may for instance be a mouse, rat, hamster or pig.","Typically, the transgenic animal will be a mouse.","Agents for modulating the functional activity of a cell arising from the interaction of a MAP kinase like ERK2 or JNK-1 with a integrin include antagonists and inhibitors capable of associating with the integrin or MAP kinase to thereby inhibit the MAP kinase and integrin interaction.","Antagonists and inhibitors include those agents that act by binding the integrin or MAP kinase adjacent to the relevant binding domain and stearically hindering the interaction of the MAP kinase with the integrin, as well as allostearic inhibitors that distort the binding domain upon associating with the integrin.","Additional agents include agonists of a MAP kinase or an integrin, that are capable of binding to the pertinent binding domain involved in the MAP kinase integrin interaction.","A binding domain involved in the MAP kinase integrin interaction may be identified and characterised using protocols and techniques described herein.","Specifically, a binding domain may be localised by assessing the capacity of respective overlapping peptide fragments of the cytoplasmic domain of an integrin subunit to bind with a MAP kinase or correspondingly, assessing the capacity of overlapping peptide fragments of the MAP kinase to bind with an integrin.","The specific amino acid sequence which constitute the binding domain may then be determined utilising progressively smaller peptide fragments of a fragment observed to interact with the MAP kinase or the integrin.","In particular, test peptides are readily synthesised to a desired length involving deletion of an amino acid or amino acids from either or both ends of the larger amino acid sequence, and tested for their ability to associate with the MAP kinase or integrin.","This process is repeated until the minimum length peptide capable of binding with the MAP kinase or integrin substantially without compromising the optimum observed level of binding is identified.","The amino acids that play an active role in the MAP kinase integrin interaction may be achieved with the use of further synthesised test peptides in which one or more amino acids of the sequence are deleted or substituted with a different amino acid or amino acids to determine the effect on the ability of the peptide of associate with the MAP kinase or integrin.","Typically, substitution mutagenesis will involve substitution of selected ones of the amino acid sequence with alanine or other relatively neutrally charged amino acid.","By deletion is meant deletion of one or more of the amino acids between the N-terminal and C-terminal amino acid residues of the identified amino acid sequence.","Nucleotide and amino acid sequence data for the β6 integrin subunit for instance is found in Sheppard et al, 1990.The amino acid sequence for β6 is also set out in SEQ ID NO: 1.The nucleotide and amino acid sequence for ERK2 may be found in Boulton et al, 1991 and is set out in FIG.","39.Reference to such published data allows the ready design of peptide fragments of a MAP kinase or an integrin subunit cytoplasmic domain for use in the identification and localisation of the binding domain for the MAP kinase or the integrin as is applicable, and the identification of the corresponding nucleic acid sequence encoding such peptide fragments as well as the amino acid sequence of the binding domain.","Localisation and characterisation of a binding domain for a MAP kinase or integrin enables the design of agents for binding to the binding domain for modulation of cell activity either for down regulating the functional activity of the integrin or the MAP kinase and more particularly the MAP kinase integrin binding interaction, or as agonists of the MAP kinase or integrin.","This will typically involve determining the physical properties of the binding domain such as size and charge distribution, and the tertiary structure of the binding domain.","Specifically, at least the region of the integrin containing the binding domain for the MAP kinase or the region of the MAP kinase containing the binding domain for the integrin is modelled taking into account the stereochemistry and physical properties of the binding domain such as size and charge distribution as well as its three dimensional structure as determined using x-ray chrstallography, nuclear magnetic resonance and/or commercially available computer modelling software.","Such modelling techniques are well known in the art.","In a variation of this approach, the modelling will take into account the interaction of the relevant binding domain with the MAP kinase or the integrin as the case may be such that any change in conformation arising from the interaction may be taken in to account in the design of an agent.","Modelling flanking regions adjacent the binding domain also allows the design of agents for associating with such flanking regions but which are nevertheless capable of inhibiting the MAP kinase integrin interaction either by stearic hindrance or by distorting the conformation of the binding domain of the MAP kinase or integrin (eg.","allostearic inhibitors).","The design of a mimetic of the binding domain of interest will usually involve selecting or deriving a template molecule onto which chemical groups are grafted to provide required physical and chemical characteristics.","The selection of template molecule and chemical groups is based on ease of synthesis, likely pharmacological acceptability, risk of or potential for degradation in vivo, stability and maintenance of biological activity upon administration.","Pharmacological acceptability and the like are also taken into consideration in the design of other agent types.","In order to constrain a polypeptide or other agent in a three dimensional conformation required for binding, it may be synthesised with side chain structures or otherwise be incorporated into a molecule with a known stable structure in vivo.","In particular, a polypeptide or the like may be incorporated into an amino acid sequence at least part of which folds into a β-pleated sheet or helical structure such as an α-helix (eg.","see Dedhar et al., 1997).","A polypeptide or other agent may also be cyclised to provide enhanced rigidity and thereby stability in vivo.","Various methods for cyclising peptides, fusion proteins or the like are known (eg.","Schiller et al., 1985).","For example, a synthetic peptide incorporating two cysteine residues distanced from each other along the peptide may be cyclised by the oxidation of the thiol groups of the residues to form a disulfide bridge between them.","Cyclisation may also be achieved by the formation of a peptide bond between the N-terminal and C-terminal amino acids of a synthetic peptide or for instance through the formation of a bond between the positively charged amino group on the side chain of a lysine residue and the negatively charged carboxyl group on the side chain of a glutamine acid residue.","As will be understood, the position of the various amino acid residues between which such bonds are formed will determine the size of the cycle.","Variation of cycle size for optimisation of binding affinity may be achieved by synthesising peptides in which the position of amino acids for achieving cyclisation has been altered.","The formation of direct chemical bonds between amino acids or the use of any suitable linker to achieve cyclisation is also well within the scope of the skilled addressee.","Further strategies for identifying possible agents include large scale screening techniques as are known to the skilled addressee.","For example, peptide library technology provides an efficient way of testing a vast number of potential agents.","Such libraries and their use are well known.","Prospective agents identified may be then further evaluated in suitable activity, competitive and other immunoassays.","A method of screening for an agent or evaluating whether an agent is capable of binding to a MAP kinase or an integrin, and in particular the binding domain of a MAP kinase for an integrin or the binding domain of an integrin for a MAP kinase, and thereby inhibiting the MAP kinase integrin interaction or acting as a MAP kinase or integrin agonist, may for instance involve utilising the agent in an assay whereby the agent has the opportunity of binding to the MAP kinase or the integrin in the presence of the integrin or the MAP kinase as the case may be or prior to the addition of the integrin or the MAP kinase, and determining whether inhibition of binding of the MAP kinase to the integrin results.","An alternate screening method may for instance involve selecting a test agent capable of binding with the integrin or MAP kinase, measuring cellular activity in the presence of the test agent, and comparing that activity with cellular activity in the absence of the test agent.","Cellular activity may be assessed by cell growth as indicated by [3H]-thymidine uptake or other measurement of cellular activity.","As will be understood, a difference in observed functional activity in the presence of the test agent is indicative of the modulating effect provided by the test agent.","It will be understood that the integrin in the context of such assays may be an integrin subunit or polypeptide or fragment incorporating the binding domain of the integrin for the MAP kinase, or a homolog, analog, variant or derivative of such a molecule to which the MAP kinase is capable of binding.","Similarly, a MAP kinase in this context may be an intact MAP kinase or a fragment thereof incorporating the binding domain for the integrin, or a homolog, analog, variant or derivative of such a molecule that is capable of binding with the binding domain of the integrin.","In addition, determination of whether an agent is capable of binding to the binding domain of an integrin or MAP kinase may be achieved using a polypeptide or fragment as described herein consisting of the binding domain or a core amino acid sequence of the binding domain that directly participates in the binding interaction, or analogs or the like of such molecules.","It is not necessary that an agent be proteinaceous in character and indeed, mimetics may be prepared which may not be a polypeptide at all but which nevertheless possess the capability of binding with the integrin or MAP kinase.","Polypeptides including fusion proteins and fragments of a MAP kinase comprising the binding domain for an integrin or fragments of an integrin subunit comprising the binding domain for a MAP kinase, or which incorporate sufficient core amino acid sequence of the binding domain of the integrin or MAP kinase for binding by the MAP kinase or integrin are contemplated herein.","Typically, a polypeptide will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less and generally, less than about 40 amino acids.","Preferably, the length will be from between about 5 to about 30 amino acids, and more preferably from between about 10 amino acids and about 25 amino acids.","Preferably, a polypeptide will comprise or incorporate the amino acid sequence RSKAKWQTGTNPLYR, more usually the amino acid sequence RSKAKNPLYR, or one or both of sequences RSKAK and NPLYR.","Alternatively, a polypeptide may have the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or PSNLLLNTTCDLKIC.","Polypeptides and fusion proteins or the like may be synthesised or produced using conventional recombinant techniques.","Nucleic acid encoding a fusion protein may for instance be provided via the joining together of separate DNA fragments encoding peptides or polypeptides having desired three dimensional conformations and/or other characteristics by employing blunt-ended termini and oligonucleotide linkers, digestion to provide staggered termini as appropriate, and ligation of cohesive ends prior to insertion of the resultant chimeric sequence into a suitable expression vector.","Alternatively, PCR amplification of DNA fragments can be utilised employing primers which give rise to amplicons with complementary termini which can be subsequently ligated together (eg.","see Current Protocols in Molecular Biology.","John Wiley & Sons, 1992).","Nucleic acid sequences encoding for the polypeptides of the invention, mutagenised integrin subunits and the like as described herein are also encompassed as are the respective complementary antisense nucleic acid sequences.","Sense oligonucleotides encoding for the binding site of an integrin subunit or MAP kinase or a partial amino acid sequence of a binding domain, and the complementary antisense oligonucleotides are particularly suitable for use as primers in polymerase chain reaction (PCR) amplification methods or as probes for detection of the presence the respective target nucleic acid sequences with which they hybridise such as in Southern blotting protocols, or in affinity chromatography purification of the target nucleic acid sequence.","Probes may be labelled with for instance commonly used isotopes such as P32, fluorophores, chemiluminescent agents and enzymes (see eg.","Essential Molecular Biology.","A Practical Approach Vol.","II, Oxford University Press, 1993; Current Protocols in Molecular Biology, Ausubel FM., John Wiley & Sons Inc., 1998).","The choice of a label will vary depending on the degree of sensitivity required, ease of conjugation with the probe, safety and other factors.","Oligonucleotides for use as probes or primers will usually have a length of less than about 60 nucleotides, usually less than about 50 or 40 nucleotides preferably, between about 14 and about 30 nucleotides, and more preferably, between about 14 and about 25 nucleotides.","While it is desirable that a primer or probe has 100% complementarity with its target sequence, oligonucleotides may be designed with less complementarity but which nevertheless hybridise with the target sequence.","Typically, a primer or probe will have a complementarity of about 70% or greater, more preferably about 80% or greater and most preferably about 90% or 95%, or greater.","A probe will generally be designed for being capable of hybridising with its target nucleic acid sequence under moderate or high stringency wash conditions.","Moderate stringency wash conditions are for example those that employ 0.2×SSC (0.015M NaCl/0.0015M sodium citrate)/0.1% SDS (sodium dodecylsulfate) wash buffer at 42° C. High stringency wash conditions employ for instance, 0.1×SSC wash buffer at 68° C. Generally, the content of purine relative to the content of pyrimidine nucleotides in the region of target nucleic acid of interest will be taken into account in the design of such primers and probes as will be their length in accordance with well accepted principles known in the art.","In addition, the present invention provides vectors incorporating nucleic acid sequences of the invention.","The term “vector” is to be taken to mean a nucleic acid molecule capable of facilitating the transport of a nucleic acid sequence inserted therein into a cell and includes expression vectors and cloning vectors.","Suitable expression vectors include plasmids and cosmids capable of expression of a DNA (eg.","genomic DNA or cDNA) insert.","An expression vector will typically include transcriptional regulatory control sequences to which the inserted nucleic acid sequence is operably linked.","By “operably linked” is meant the nucleic acid insert is linked to the transcriptional regulatory control sequences for permitting transcription of the inserted sequence without a shift in the reading frame of the insert.","Such transcriptional regulatory control sequences include promotors for facilitating binding of RNA polymerase to initiate transcription, expression control elements for enabling binding of ribosomes to transcribed mRNA, and enhancers for modulating promotor activity.","A promotor may be a tissue specific promotor which facilitates transcription of the nucleic acid insert only in specific cell lineages and not in other cell types or only to a relatively low level in such other cell types.","The design of an expression vector will depend on the host cell to be transfected, the mode of transfection and the desired level of transcription of the nucleic acid insert.","Numerous expression vectors suitable for transfection of prokaryotic (eg.","bacterial) or eukaryotic (eg yeast, insect or mammalian cells) are known in the art.","Expression vectors suitable for transfection of eukaryotic cells include pSV2neo, pEF.PGK.puro, pTk2, pRc/CNV, pcDNAI/neo, non-replicating adenoviral shuttle vectors incorporating the polyadenylation site and elongation factor 1-α promotor and pAdEasy based expression vectors most preferably incorporating a cytomegalovirus (CMV) promotor (eg.","see He et al, 1998).","For expression in insect cells, baculovirus expression vectors may be utilised examples of which include pVL based vectors such as pVL1392, and pVL941, and pAcUW based vectors such as pAcUW1.Viral expression vectors are particularly preferred.","Typical cloning vectors incorporate an origin of replication (ori) for permitting efficient replication of the vector, a reporter or marker gene for enabling selection of host cells transformed with the vector, and restriction enzyme cleavage sites for facilitating the insertion and subsequent excision of the nucleic acid sequence of interest.","Preferably, the cloning vector has a polylinker sequence incorporating an array of restriction sites.","The marker gene may be drug-resistance gene (eg.","Ampr for ampicillin resistance), a gene encoding an enzyme such as chloramphenicol acetyltransferase (CAT), β-lactamase, adenosine deaminase (ADA), aminoglycoside phosphotransferase (APH), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), or for instance β-galactosidase encoded by the E. coli lacZ gene (LacZ′).","Yeast reporter genes include imidazole glycerolphosphate dehydratase (HIS3), N-(5′-phosphoribosyl)-anthranilate isomerase (TRP1) and β-isopropylmalate dehydrogenase (LEU2).","As will be appreciated, expression vectors of the invention may also incorporate such marker genes.","Cloning vectors include cloning vectors for mammalian, yeast and insect cells.","Particular vectors that may find application include pBR322 based vectors and pUC vectors such as pUC118 and pUC119.Suitable expression and cloning vectors are for instance described in Molecular Cloning.","A Laboratory Manual., Sambrook et al., 2nd Ed.","Cold Spring Harbour Laboratory., 1989.Host cells suitable for being transformed by vectors of the invention include bacteria such as E. coli, Bacillus such as B. subtilis, Streptomyces and Pseudomonas bacterial strains, yeast such as Sacchromyces and Pichia, insect cells, avian cells and mammalian cells such as Chinese Hamster Ovary cells (CHO), COS, HeLa, HaRas, WI38, SW480, and NIH3T3 cells.","Host cells may be cultured in a suitable culture medium and under conditions for facilitating expression of nucleic acid sequences of the invention or replication of cloning vectors, prior to purification from the host cells, and/or supernatants as the case may be using standard purification techniques.","Rather than utilising viral mediated transfection of cells, nucleic acid sequences and other molecules of the invention may also be delivered to a cell in vitro or in vivo by liposome mediated transfection.","The liposomes may carry targeting molecules for maximising delivery of the agent or agents contained therein to specific cell types of interest.","Such targeting molecules may be for instance antibodies, ligands or cell surface receptors for facilitating fusion of liposomes to the specific cells of interest.","Agents may also be intracellularly delivered in vitro using conventional cold or heat shock techniques or for instance, calcium phosphate coprecipitation or electroporation protocols.","Yet another strategy is to design the agent to have the inherent ability to pass across the lipid bilayer of a cell.","A particularly preferred way of achieving intracellular delivery of an agent is to use “carrier peptides” which have the ability to deliver macro-molecules across cell membranes in an energy-independent manner (Prociantz, 1996).","Indeed, such peptides provide the possibility of both testing potential agents in cell culture without drastically altering cell membrane integrity and of delivering agents in vivo.","Carrier peptides that are known in the art include penetratins and variants thereof (Derossi et al, 1994, 1996), human immunodeficiency virus Tat derived peptide (Prociantz, 1996), and transportan derived peptide (Pooga et al.","1998).","Indeed, carrier peptides have been successfully used to facilitate internalisation of mimetics of Src homology 2 binding sites, and peptides which inhibit protein kinase C mediated axon development and CD44 (hyaluronate receptor) dependent migration (Theodore et al, 1995; Williams et al, 1997; Peck Isacke, 1998; Derossi et al, 1998).","Specific targetting to β6-expressing cancer cells may also be achieved by coupling humanised anti-β6 antibody to carrier molecules such as penetratin coupled to an agent capable of inhibiting binding of a MAP kinase with an integrin expressed by the cell or down regulation of the expression of the integrin.","Coupling may for instance be by a peptide bond or disulfide bridge.","Given that β6 expression enhances effective proteolysis at the cell surface by matrix metalloproteinase-9 (Agrez et al, 1999), such targetting approaches may include engineering an MMP-9 cleavage site between the antibody and the carrier peptide penetratin to facilitate internalisation of the pentratin-agent complex.","Another approach may employ coupling the penetratin-agent complex to β6 integrin receptor-targetted peptides, targetted for binding to the extracellular β6 domain by virtue of their DLXXL sequence.","For example, a ligand recognition motif for αVβ6 integrin, RTDLDSLRTYTL (Kraft et al, 1999) may be used in conjunction with or without an engineered MMP-9 cleavage site to release the penetratin-agent complex at the cell surface.","Further protocol for targetting nucleic acids to cells by targetting integrins is described in Bachmann et al, 1998.The toxicity profile of an agent of the invention may be tested on normal and malignant cells by evaluation of cell morphology, trypan-blue exclusion, assessment of apoptosis and cell proliferation studies (eg cell counts, 3H-thymidine uptake and MTT assay).","Agents of the invention may be co-administered with one or more other compounds or drugs.","For example, an agent or agents may be administered in combination with antisense therapy or chemotherapeutic drugs.","Alternatively, an agent may be administered in conjunction with antisense therapy and/or chemotherapeutic drugs.","By “co-administered” is meant simultaneous administration in the same formulation or in two different formulations by the same or different routes, or sequential administration by the same or different routes.","By “sequential” administration is meant a time difference of between the administration of the agents, drugs and other therapies which can be administered in any order.","The time difference may range from very short times up to hours or for instance days or weeks.","The agent or agents will typically be formulated into pharmaceutical composition incorporating pharmaceutically acceptable carriers, diluents and/or excipients for administration to the intended subject.","Pharmaceutical forms include sterile aqueous solutions suitable for injection, (where the agent or agents is water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions.","Such injectable compositions will be fluid to the extent that the syringability exists and typically, will be stable to allow for storage after manufacture.","The carrier may be a solvent or dispersion medium containing one or more of ethanol, polyol (eg glycerol, propylene glycol, liquid polyethylene glycol and the like), vegetable oils, and suitable mixtures thereof.","Fluidity may be maintained by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants.","Sterile injectable solutions will typically be prepared by incorporating the active agents in the desired amount in the appropriate solvent with various other components enumerated above, prior to sterilising the solution by filtration.","Generally, dispersions will be prepared by incorporating the sterile active agents into a sterile vehicle which contains the dispersion medium and other components.","In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active agent plus any additional desired ingredient from previously sterile filtered solutions thereof.","For oral administration, the active agents may be formulated into any orally acceptable carrier deemed suitable.","In particular, the active ingredient may be formulated with an inert diluent, an assimilable edible carrier or it may be enclosed in a hard or soft shell gelatin capsule.","Alternatively, it may be incorporated directly into food.","Moreover, an active agent may be incorporated with excipients and used in the form of ingestable tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and the like.","Such compositions will generally contain at least about 1% by weight of the active agent or agents.","The percentage may of course be varied and may conveniently be between about 5 to about 80% w/w of the composition or preparation.","As will be appreciated, the amount of active agent or agents in such compositions will be such that a suitable effective dosage will be delivered to the subject taking into account the proposed mode of administration.","Preferred oral compositions according to the invention will contain between about 0.1 μg and 2000 mg of each active agent, respectively.","Active agents may also be formulated into topically acceptable carriers conventionally used for forming creams, lotions, ointments and the like for internal or external application.","Topical formulations may be applied to a site to be treated by dressings and the like impregnated with the formulation.","Typically, a composition of the invention will incorporate one or more preservatives such as parabens, chlorobutanol, phenol, sorbic acid, and thimersal.","In many cases, a composition may furthermore include isotonic agents such as sugars or sodium chloride.","In addition, prolonged absorption of the composition may be brought about by the use in the compositions of agents for delaying absorption such as aluminium monosterate and gelatin.","Tablets, troches, pills, capsules and the like may also contain one or more of the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium sterate; a sweetening agent such as sucrose, lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, orange or cherry flavouring.","When the dosage unit form is a capsule, it may contain in addition to one or more of the above ingredients a liquid carrier.","Various other ingredients may be present as coatings or to otherwise modify the physical form of the dosage unit.","For instance, tablets, pills or capsules may be coated with shellac, sugars or both.","In addition, an active agent may be incorporated into any suitable sustained-release preparation or formulation.","Pharmaceutically acceptable carriers, diluents and/or excipients include any suitable conventionally known solvents, dispersion media and isotonic preparations or solutions.","Use of such ingredients and media for pharmaceutically active substances is well known.","Except insofar as any conventional media or agent is incompatible with the active agent, use thereof in therapeutic and prophylactic compositions is contemplated.","Supplementary active ingredients can also be incorporated into the compositions if desired.","It is particularly preferred to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.","Dosage unit form as used herein is to be taken to mean physically discrete units suited as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of active agent calculated to produce the desired therapeutic or prophylactic effect in association with the relevant carrier, diluent and/or excipient.","A unit dosage formed will generally contain each active agent in amounts ranging from about 0.5 μg to about 2000 mg/ml of carrier respectively.","A pharmaceutical composition may also comprise vectors capable of transfecting target cells where the vector carries a nucleic acid molecule for modulating functional activity or expression of an integrin or MAP kinase.","The vector may for instance, be packaged into a suitable virus for delivery of the vector into target cells as described above.","The dosage of an active agent will depend on a number of factors including whether the agent is to be administered for prophylactic or therapeutic use, the condition for which the agent is intended to be administered, the severity of the condition, the age of the subject, and related factors including weight and general health of the subject as may be determined by the physician or attendant in accordance with accepted principles.","Indeed, a low dosage may initially be given which is subsequently increased at each administration following evaluation of the subjects response.","Similarly, frequency of administration may be determined in the same way that is, by continuously monitoring the subjects response between each dosage and if necessary, increasing the frequency of administration or alternatively, reducing the frequency of administration.","The route of administration of a pharmaceutical composition will again depend on the nature of the condition for which the composition is to be administered.","Suitable routes of administration include but are not limited to respiritoraly, intratracheally, nasopharyngeally, intraveneously, intraperitonealy, subcutaneously, intracranialy, intradermially, intramuscularly, intraoccularly, intrathecally, intranasally, by infusion, orally, rectally, via IV group patch, topically and by implant.","With respect to intravenous routes, particularly suitable routes are via injection into blood vessels which supply a tumour or particular organs to be treated.","Agents may also be delivered into cavities such for example the pleural or peritoneal cavity, or be injected directly into tumour tissue.","The production of antibodies and monoclonal antibodies is well established in the art (eg.","see Antibodies, A Laboratory Manual.","Harlow & Lane Eds.","Cold Spring Harbour Press, 1988).","For polyclonal antibodies, a mammal such as a sheep or rat for instance is immunised with a polypeptide of the invention and antisera subsequently isolated prior to purification of the antibodies therefrom by standard affinity chromatography techniques such as Sepharose-Protein A chromatography.","Desirably, the mammal is periodically challenged with the relevant antigen to establish and/or maintain high antibody titre.","To produce monoclonal antibodies, B lymphocytes can be isolated from the immunised mammal and fused with immortalising cells (eg.","myeloma cells) by standard somatic cell fusion techniques (eg.","utilising polyethylene glycol) to produce hybridoma cells (Kohler and Milstein, 1975; see also Handbook of Experimental Immunology, Weir et al Eds.","Blackwell Scientific Publications.","4th Ed.","1986).","The resulting hybridoma cells may then be screened for production of antibodies specific for the peptide by an enzyme linked immunosorbant assay (ELISA) or other immunoassay.","Conventionally used methods for preparing monoclonal antibodies include those involving the use of Epstein-Barr virus (Cole et al.","Monoclonal Antibodies and Cancer Therapy, Allen R. Liss Inc. pp.","77-96, 1985).","The term “antibody” or “antibodies” as used herein is to be taken to include within its scope entire intact antibodies as well as binding fragments thereof such as Fab and (Fab′)2 fragments which may be obtained by papain or pepsin proteolytic cleavage, respectively.","An antibody of the invention may be labelled for enabling detection of antibody binding in immunoassays including competitive inhibition assays.","A “label” may be any molecule which by its nature is capable of providing or causing the production of an analytically identifiable signal which allows the detection of an antibody and antigen complex.","Such detection may be qualitative or quantitative.","An antibody can for instance be labelled with radioisotopes including 32p, 125I or 131I, an enzyme, a fluorescent label, chemiluminescent molecule or for instance an affinity label such as biotin, avidin, streptavidin and the like.","An enzyme can be conjugated with an antibody by means of coupling agents such as gluteraldehyde, carbodiimides, or for instance periodate although a wide variety of conjugation techniques exist.","Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase amongst others.","Detection utilising enzymes is achieved with use of a suitable substrate for the selected enzyme.","The substrate is generally chosen for the production upon hydrolysis of a detectable colour change.","However, fluorogenic substrates may also be used which yield a fluorescence product rather than a chromogen.","Suitable fluorescent labels are those capable of being conjugated to an antibody substantially without altering the binding capacity of the antibody and include fluorescein, phycoerythrin (PE) and rhodamine which emit light at a characteristic wavelength in the colour range following illumination with light at a different wavelength.","Methods for labelling of antibodies can be found in Current Protocols in Molecular Biology.","Ausubel FM., John Wiley & Sons Inc. Immunoassays in which antibodies of the invention may be utilised include radioimmunoassays (RIA) and ELISA (eg., see Handbook of Experimental Immunology, Weir et al., Vol.","1-4, Blackwell Scientific Publications 4th Edition, 1986).","Such assays include those in which a target antigen is detected by direct binding with a labelled antibody, and those in which the target antigen is bound by a first antibody, typically immobilised on a solid substrate (eg., a microtitre tissue culture plate formed from a suitable plastics material such as polystyrene or the like) and a labelled second antibody specific for the first antibody used to form an antigen-first antibody-second antibody complex that is detected by a signal emitted by the label.","Sandwich techniques in which the antigen is immobilised by an antibody for presentation to a labelled second antibody specific for the antigen are also well known.","An antibody can be bound to a solid substrate covalently utilising commonly used amide or ester linkers, or by adsorption.","Optimal concentrations of antibodies, temperatures, incubation times and other assay conditions can be determined by the skilled addressee with reference to conventional assay methodology and the application of routine experimentation.","Antibodies and other molecules of the invention including polypeptides and oligonucleotides as described herein when bound to a solid support can be used in affinity chromatography for the purification of a binding partner for which they are specific.","In particular, a polypeptide either alone or as a fusion protein comprising or consisting of the binding domain for a MAP kinase for instance can be utilised in the purification, isolation or concentration of an integrin or fragment thereof or other molecule capable of binding to the binding domain of the MAP kinase for the integrin.","It may also be used for assaying levels of the integrin for instance in cell extracts.","Similarly, an antibody or other such molecule as described herein that specifically binds to the binding domain of a MAP kinase has use in the purification of the MAP kinase or fragments thereof incorporating a binding domain for the integrin from a relatively crude preparation or mixture.","Suitable solid supports include agarose, sepharose and other commercially available supports such as beads formed from latex, polystyrene, polypropylene, dextran, glass or synthetic resins, typically packed in an affinity column through which the relevant sample containing the binding partner is passed at a pH and conditions (eg., low salt concentration) under which the binding partner becomes bound by the antibody, polypeptide or other such molecule.","The column is then washed utilising a suitable buffer whereby the binding partner is retained bound on the column, prior to being eluted therefrom utilising a suitable elution buffer (eg., with a higher salt concentration and at an altered pH, typically pH 2.5 or pH 11) that facilitates the release of the binding partner from the affinity column, and collected.","Protocols for affinity chromatography are described in Current Protocols in Molecular Biology-Ausubel FM., John Wiley & Sons Inc. Buffers and conditions utilised for the purification, isolation or concentration of a binding partner will vary depending on the affinity the antibody, polypeptide or the like for the binding partner.","A kit for use in assays as described herein may include one or more of an antibody, polypeptide, vector, nucleic acid or other molecule of the invention.","The kit may also comprise one or more other reagents such as washing solutions, dilution buffers and the like together with instructions for use.","The antibody or other molecule of the invention may or may not be labelled, bound to a solid support or be coupled with another molecule.","Particularly preferred kits are those provided for use in affinity chromotography or RIA, ELISA or other type of immunoassay.","The present invention will be described herein after with reference to a number of examples.","EXAMPLE 1 Anti-integrin antibodies and antibodies against the β1 subunit have been shown to inhibit proliferation of retinal pigment epithelial cells (Hergott et al, 1993).","In endothelial cells, inhibition of cell-matrix interactions by anti-integrin antibodies specifically against α2β1 converts the cells from a proliferative to a differentiated phenotype (Gamble et al, 1993).","In another study, synthetic peptides containing the integrin recognition sequence arginine-glycine-aspartate (RGD) have been shown to inhibit tumour cell invasion in vitro and tumour metastases from melanoma in an animal model (Humphries et; al, 1986; Gehlsen et al, 1988).","In colon cancer, the contribution made by the α5β1 receptor to regulation of growth appears to be ligand (fibronectin)-dependent.","For example, induced expression of α5β1 in human colon cancer cells constitutively lacking this integrin has been shown to result in decreased tumour cell proliferation in vitro (Varner et al, 1995).","Interestingly, when the appropriate ligand was present, cell proliferation was restored, indicating that the unoccupied receptor mediated a negative growth signal in these cells (Varner et al, 1995).","Moreover, induction of α5β1 expression was associated with a marked reduction of tumourigenicity in immune-deficient mice.","Failure to ligate all of the tumour cell α5β1 molecules with sufficient murine fibronectin most likely accounts for the in vivo tumour suppression in these studies (Varner et al, 1995).","In the present study the effect of down-regulating αvβ6 expression on colon cancer growth was examined.","1.1 Methods 1.1.1 Generation of sense and antisense β6 constructs in pEF.PGK.puro vector.","For β6 antisense constructs, β6 cDNA was excised from the vector pcDNA1neo β6 (Weinacker et al, 1994) using the restriction enzymes SnaB1 and Xba1.This produced a 5′ overhang (Xba1) and a 3′ blunt end (SnaB1).","The 5′ overhang was blunted with Klenow (Promega) prior to ligation.","pEF.PGK.puro vector (a gift from D. Huang, the Walter & Eliza Hall Institute, Melbourne Australia) was cut with EcoRV which produced blunt ends.","The pEF.puro vector was de-phosphorylated using calf intestinal alkaline phosphatase (CIAP, Promega) and the β6 cDNA ligated overnight into pEF.puro using T4 DNA ligase (Promega) at a ratio of vector:insert of 1:5.After ligation all the reaction mix was used for transformation into competent JM109 cells and the cells plated onto LB plates containing ampicillin.","After overnight incubation at 37° C., colonies were selected, the plasmid DNA extracted by microprepping, and the DNA cut with BstE11 to confirm the antisense orientation of β6.Digestion with BstE11 produced the expected two fragments, one of 6.0 basepairs and the other 3.5 basepairs.","To provide β6 sense constructs, β6 cDNA was also excised from the pcDNA1neo b6 vector using the restriction enzymes Snab1 and Xba1 as described above.","The pEF.puro vector was then cut with Xba1 and EcoRV and the insert was ligated in sense direction into the pEF.PGK.puro vector without having to blunt the insert.","The ligation reaction was prepared at a ratio of vector:insert of 1:1.Digestion with BstE11 produced two expected fragments, one of 7.5 basepairs and the other 2.0 basepairs.","1.1.2 Transfection of WiDr and HT29 Colon Cancer Cells The human colon cancer cell lines, WiDr and HT29 were obtained from the American Type Culture Collection (ATCC), Rockville, Md., USA and maintained as monolayers in standard medium comprising Dulbecco's Modified Eagle's medium (DMEM; 4.5 gm/litre of glucose) with 10% heat-inactivated foetal bovine serum (FBS) supplemented with HEPES and penicillin/streptomycin.","WiDr and HT29 cell lines constitutively express αvβ6.Stable transfectants of WiDr and HT29 cells expressing β6 in sense and antisense direction were generated using lipofectamine and puromycin as the selection antibiotic.","One stable antisense β6 transfectant from HT29 cells and three stable clones from WiDr cells were successfully established for use in all experiments.","Mock transfectants using vector alone were also generated as controls.","Initial killing curves performed using a range of concentrations of puromycin established that WiDr and HT29 cells could be stably transfected with puromycin concentrations of 1.0 μg and 2.5 μg/ml, respectively.","1.1.3 Assessment of β6 Expression in the Transfected Cell Lines β6 expression was assessed by means of FACScan analyses of parent cell lines and clones generated therefrom by means of limiting-dilution experiments.","Stability of the transfectants was confirmed regularly by repeated FACScan analyses, and surface biotinylation and immunoprecipitation as described below.","1.1.4 FACScan Analyses Monolayer cultures of cell lines were harvested with trypsin/EDTA and then blocked with goat serum at 4° C. for 10 min.","Cells were washed once with PBS, incubated with primary antibody against integrin subunits for 20 min at 4° C. and then washed twice with PBS.","Cells were then stained with secondary antibody conjugated with phycoerythrin for 20 min at 4° C., washed twice with PBS and resuspended in 0.5 ml PBS prior to FACScan analysis (Becton Dickinson, Rutherford, N.J., USA).","1.1.5 Integrin Immunoprecipitation Cells were harvested with trypsin/EDTA, the trypsin neutralised with standard culture medium, and the cell pellets washed once with cold PBS.","Cell pellets were then exposed to biotin-CNHS-ester (Sigma) in biotinylation buffer (10 mM sodium borate, 150 mM sodium chloride, pH 8.8) for 30 mins at 4° C. with continuous slow mixing.","Cell pellets were then centrifuged at 4° C., washed twice with cold PBS and exposed to lysis buffer (containing 100 mM Tris, 150 mM NaCl, 1 mM CaCl2, 1% Triton, 0.1% SDS and 0.1% NP-40 at pH 7.4 and containing 1 mM phenylmethylsulfonyl fluoride (PMSF)) for 30 min at 4° C. Lysates were then clarified by ultracentrifugation (10,000 g) for 30 min and the protein content measured using the BCA protein assay reagent kit.","Lysates containing equal protein amounts were pre-cleared with rabbit anti-mouse (RAM) immunoglobulin coupled to Sepharose-4B beads for 12 hrs.","Immunoprecipitations were carried out indirectly using RAM-Sepharose 4B and analysed by 7.5% SDS-PAGE under non-reducing conditions.","1.1.6 Reverse Transcriptase-PCR (RT-PCR) mRNA levels for β6 expression were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR).","Total RNA was extracted from cell cultures using the commercial TriPure isolation reagent based on the method of Chomozynski and Sacchi (1987).","0.4-2 μg of RNA was used to prepared cDNA by reverse transcription.","Briefly, a reaction mixture in a final volume of 40 μl containing 8 μl of 5×RT reaction buffer (250 mM Tris, 15 mM MgCl2, 375 mM KCL, pH 8.3), 8 μl of 2.5 mM of each dNTP, 4 μl of 100 mM DTT, 40U of an Rnase inhibitor, Rnasin (Promega, Madison, Wis., USA), 0.5 μg of random hexamers (Promega) and 200U of Moloney murine leukaemia virus (M-MLV) reverse transcriptase (Promega) were mixed and incubated at 38° C. for a minimum of 90 min.","The reaction was stopped by heating at 95° C. for 5 min and cDNA stored at 4° C. until PCR.","2-5 μl of this cDNA was combined with 5 μl of 10×PCR buffer (100 mM Tris, 500 mM KCl, 15 mM MgCl2, pH8.3) 8 μl of 1.25 mM dNTP each and 1.25 μl of 20 μM of both forward and reverse primers.","The forward primer sequence was 5′AGGATAGTTCTGTTTCCTGC3′ and the reverse primer sequence 5′ATCATAGGAATATTTGGAGG3′.","The reaction was initiated by 2.5U of Taq polymerase in a final volume of 50 μl.","After an initial 5 min incubation at 94° C., 30 cycles of amplification were performed under the following conditions: 94° C. 1 min, 54° C. 1 min and 72° C. for 1 min.","The reaction was stopped by incubating at 72° C. for 10 min.","To verify that equal amounts of RT product from cells were subjected to PCR amplification, the same amounts of cDNA were amplified for the “house-keeping” gene GAPDH using specific primers.","The same reaction conditions were used except that the annealing temperature was changed to 48° C. and PCR amplification performed for 35 cycles.","1.2 Results 1.2.1 αvβ6 Expression in HT29 and WiDr Transfected Cell Lines Transfection of the colon cancer cell lines HT29 and WiDr with the β6 gene construct in an antisense orientation resulted in a marked reduction of β6 expression at the transcript level and on the cell surface as shown in FIGS.","1 to 4.Transfection of cells with β6 in the sense orientation did not enhance β6 surface expression.","However, a consequence of down-regulation of the β6 subunit in antisense transfectants was a marked increase in surface expression of the β5 subunit.","The changes in surface expression of β6 and β5 subunits noted on FACScan analyses of antisense β6 transfectants was confirmed by surface-labelling cells with biotin and immunoprecipitating integrin subunits with either anti-β6 mAb (R6G9) or anti-β5 mAb (P1F6).","1.2.2 Effect of Suppression of αvβ6 Expression on Cell Binding to Fibronectin The major substrate for αvβ6 is fibronectin and to investigate the effect that reduction in β6 surface expression in antisense β6 transfectants might have on cell-matrix adhesion, WiDr and HT29 antisense β6 transfectants were seeded on fibronectin in adhesion assays.","Since both cell lines can adhere to fibronectin through members of the β1 integrin subfamily (either α3β1 or α5β1) αvβ6-mediated adhesion to fibronectin was assessed in the absence/presence of blocking anti-β1 antibody.","The cell-adhesion assays were performed in wells of non-tissue culture-treated polystyrene 96-well flat bottom microtitre plates (Nunc, Roskilde, Denmark).","Culture wells were coated with fibronectin, washed with phosphate-buffered saline (PBS) and then blocked with 0.5% bovine serum albumin (Sigma) in PBS for 1 hr at 37° C. Harvested cells were seeded at a density of 105 cells/well for HT29 and 1.5×105 cells/well for WiDr cells in 200 μl of standard DMEM which lacked FBS but contained 0.5% bovine serum albumin.","To block adhesion, cells were incubated with anti-β1 blocking antibodies for 15 min at 4° C. before plating.","The plates were centrifuged (top side up) at 10×g for 5 min, then incubated for 1 hr at 37° C. in humidified 5% carbon dioxide.","Non-adherent cells were removed by centrifugation top side down at 48×g for 5 min.","The attached cells were fixed and stained with 0.5% crystal violet (in 20% methanol and 1% formaldehyde) and the wells washed with phosphate-buffered saline.","The relative number of cells in each well was evaluated by measuring the absorbance at 595 nm in a Microplate Reader (Bio-Rad).","With reduction in cell surface expression of β6, β1-independent adhesion of cells to fibronectin was reduced compared with mock transfectants (containing vector alone) which express surface β6 at similar levels to wild-type cells.","The further addition of blocking anti-αv antibody completely prevented binding to fibronectin.","1.2.3 Effect of Suppression of αvβ6 Expression on Tumour Cell Proliferation and Tumour Growth In Vivo To investigate the effect of diminished αvβ6 surface expression on cell proliferation in vitro, WiDr and HT29 antisense β6 transfectants were seeded as monolayers in 96-well microtitre culture plates (5,000 viable cells per well) in standard culture medium containing the puromycin selection antibiotic.","Cells were pulsed with 1 μCi (3H)-thymidine (Amersham) per well for the last 24 hours of each experiment before automated harvesting and measurement of radioactivity.","WiDr wild-type, mock and antisense β6 transfectants, and HT29 wild-type, mock, sense β6 and antisense β6 transfectants were harvested second daily during a six-day culture period.","A marked increase in thymidine incorporation was observed for WiDR and HT29 cells expressing normal levels of αvβ6 compared with antisense β6 transfectants (see FIGS.","5 and 6).","1.2.4 Effect of Suppression of αvβ6 Expression on Tumour Formation The ability of HT29 antisense β6 transfactants to form tumours in immune-deficient mice was assessed.","BALB/C female athymic mice (8 weeks of age purchased from the Animal Resource Centre, Perth, Western Australia) were maintained under pathogen-free conditions and fed standard mouse chow and water ad lib.","The mice were divided into groups of ten each and all mice within each group inoculated with a single cell line.","Cells used were WiDr mock (transfected with vector alone) and antisense β6 transfected clones (clones 1-3) and HT29 mock, sense and antisense β6 cell lines.","Mice received subcutaneous flank injections of 106 viable tumour cells suspended in 0.2 ml of standard DMEM culture medium.","Animal weights and tumour sizes (breadth and length as measured with calipers) were recorded weekly.","Six weeks following the last injection, visible subcutaneous tumours were excised, weighed and fixed in 4% formalin.","At the time of euthanasia, all internal organs were routinely inspected for presence of metastases.","Tumour growth after six weeks following inoculation of HT29 mock and antisense β6 transfectants is shown in FIG.","7.Measurements of tumour growth for WiDr and HT29 cells are shown in FIGS.","8 and 9, respectively.","Similar tumour growth profiles for mock and antisense β6 WiDr clones 2 and 3 each inoculated into 10 mice are not shown.","Of a total of 40 mice injected with cells expressing antisense β6 (HT29-10 mice and WiDr cells lines, 3 clones-30 mice) tumours completely disappeared in 93% of animals.","In the remaining 3 animals tumour sizes diminished to 1 mm2 in size during the six week period compared with tumours at least 15 mm2 in size from cells expressing normal levels of β6.To confirm the presence of tumour xenografts histologically at one week following subcutaneous inoculation of mock and antisense β6 transfectants, tumour nodules were excised, fixed in formalin and stained with haematoxylin and eosin.","1.2.5 Effect of Suppression of αvβ6 Expression on Gelatinase B Secretion Serum-free tumour-conditioned medium was collected from each of the 3 WiDr mock and antisense β6 clones and concentrated ×44 for measurement of gelatinase B using the Biotrak MMP-9 activity assay system (Amersharn Pharmacia Biotech, Uppsala Sweden).","Down-regulation of αvβ6 expression resulted in a marked reduction in gelatinase B secretion.","1.3 Discussion of Results Induced expression of β6 in Chinese hamster ovary (CHO) cells has been shown to result in decreased surface expression of the β5 integrin subunit which also partners αv (Weinacker et al, 1994).","The concept of integrin switching depends on the availability of the promiscuous αv partner subunit.","In the present study, the reverse was observed.","As a consequence of down-regulation of β6 in colon cancer cells which constitutively express αvβ6, β5 surface expression increased, most likely secondary to increased availability of the αv subunit partner.","Heterologous expression of αvβ6 in colon cancer cells has previously been reported to enhance tumour growth in immune-deficient mice (Agrez et al, 1994).","Suppression of αvβ6 expression in the present study was shown to result in nearly complete disappearance of tumours in 93% of animals following subcutaneous inoculation of tumour cells.","Moreover, in the remaining 7% of animals, a 95% reduction in tumour size was observed over a six week period compared with large tumours seen in all animals injected with cells in which αvβ6 expression had not been perturbed.","Similar findings have been described with loss of the classical vitronectin receptor αvβ3 in melanoma.","For example, in experimental animal models, the loss of αvβ3 expression in melanoma cells has been shown to lead to reduced in vivo proliferation which is restored upon re-expression of the receptor (Felding-Habermann et al, 1992).","Although the mechanisms involved in αvβ6-mediated tumour growth remain to be elucidated, the present in vitro data show that loss of β6 expression is associated with decreased proliferative capacity of the cells.","Taken together with the marked reduction in gelatinase B secretion seen for colon cancer cells transfected with antisense β6, the findings reported in the present study suggest that intracellular signalling pathways activated via the integrin αvβ6 play a major role in promoting progression of this tumour type.","EXAMPLE 2 The association of αvβ6 expression and MAP kinase activity were evaluated using WiDr, HT29 and SW480 cell lines.","2.1 Methods 2.1.1 SW480 Colon Cancer β6 Transfectants Stable transfectants of SW480 colon cancer cells (ATCC) expressing gene constructs of either wild-type or mutant forms of the β6 integrin subunit or the expression plasmid only (pcDNA1neo) have been previously described (Agrez et al, 1994).","The transfected SW480 cell lines were maintained in standard medium supplemented with the neomycin analogue G418.Stable transfectants of WiDr and HT29 cells expressing wild-type β6 in antisense orientation were generated as described in Example 1.1.2 and maintained in standard medium supplemented with puromycin.","2.1.1 MAP Kinase Assay.","Cultures of WiDr and HT29 mock and antisense β6 transfectants were established by seeding 1×106 cells/5 ml of culture medium in 25 cm2 tissue culture flasks.","Cells were incubated at 37° C. in humidified CO2 for 24 hours before serum starvation in serum-free medium for the next 16 hours.","Foetal calf serum was then added to a final concentration of 10% for 30 mins before MAP kinase assays were performed.","Before each experiment the cells were washed twice with PBS, resuspended in extraction buffer (10 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 2 mM DTT, 1 mM orthovanadate, 1 mM PMSF, 4 μg/ml aprotonin, 2 μg/ml leupeptin and 1 μg/ml pepstatin, pH 7.4) and sonicated at a setting of 7, using a Soniprep 150 watt ultrasonic disintegrator for a total of 90 seconds in three 30 second pulses with an interval of 30 seconds between each pulse.","Cellular debris was removed by centrifugation at 900 g for 10 min at 4° C. The assay was performed on equal cell numbers using a MAP kinase assay system (Amersham Pharmacia Biotech, Uppsala Sweden).","The ability of cells to transfer phosphate from [γ32P]-ATP to a synthetic peptide that contains specifically a p42/p44 MAP kinase phosphorylation site was measured as described in the manufacturer's instructions.","[32P]-labelled peptides were spotted onto PEI-cellulose paper, unbound radioactivity was washed with 75 mM phosphoric acid and bound [32P]-labelled peptides were measured by liquid scintillation counting.","Protein estimation was performed on each cell lysate used and enzyme activity calculated as described in the manufacturer's instructions.","Where MEK inhibitors, PD98059 and U0126 were used, cells were cultured as described above and the inhibitors, at a final concentration of 40 μM, were added one hour before the addition of serum to the medium.","2.1.2 Western Blotting To detect the MAP kinases ERK1/2, cells were lysed in lysis buffer containing 100 mM Tris, 150 mM NaCl, 1 mM CaCl2, 1% Triton, 0.1% SDS and 0.5% NP-40 at pH7.4, supplemented with enzyme inhibitors (1 mM PMSF, 1 mM sodium orthovanadate, 1 μg/ml pepstatin A, 2.5 μg/ml aprotonin, 1 mM benzamidine, 1 μg/ml leupeptin).","Lysates were clarified by ultracentrifugation and equal protein loads electrophoresed in 8% or 10% SDS-PAGE under non-reducing conditions.","Electrophoresed proteins were transferred to nitrocellulose membranes (Biotrace NT, Gelman Sciences, Ann Arbor, Mich.) in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, 0.1% SDS) for 2 hours at a constant voltage of 40 volts in a Transfer Blot Cell (Bio-Rad).","Membranes were blocked with 5% casein for 1 hr at room temperature and probed with monoclonal anti-ERK antibodies (E10 which recognises only phosphorylated ERK1/2 (New England BioLabs) and SC-1647 which recognises total ERKs, non-phosphorylated and phosphorylated (Santa Cruz Biotechnology).","In some experiments, membranes were probed with polyclonal anti-ERK antibody (New England BioLabs) which also recognises total ERKs.","Membranes were then washed three times in Tris buffered saline, containing 0.1% Tween and incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit antibody.","Blots were visualised by the enhanced chemi-luminescence detection system according to the manufacturer's instructions (Du Pont).","2.1.3 Integrin β-Subunit Immunoprecipitations Tumour cells were harvested and divided into two equal aliquots based on cell counts.","One aliquot was surface biotinylated and the cells lysed for integrin immunoprecipitations as described in Example 1.1.5, with the exception that three sequential rounds of immuno-precipitation were performed to deplete the lysates of integrin subunits β5 and β6.Lysis buffer was the same as that used for Western blotting.","The other aliquot, intended for subsequent ERK2 immunoblotting to examine the effect of integrin immunodepletion on ERKs was not biotinylated but lysed immediately and divided into three samples each at a protein concentration of 1 mg/ml.","Three sequential rounds of immunoprecipitation were performed against anti-β6 monoclonal antibody, R6G9 (using isotype matched antibody, IgG2A, and anti-β5 monoclonal antibody, P1F6, in control immunoprecipitations).","The integrin-depleted lysates were electrophoresed in 8% or 10% SDS-PAGE under non-reducing conditions and transferred to nitrocellulose membranes.","Membranes were blocked with casein as for Western blotting and probed with anti-ERK monoclonal antibodies E10 and SC-1647.In parallel experiments, the sequentially immunoprecipitated β6 subunit bound to rabbit anti-mouse (RAM) coupled Sepharose B4 beads was also electrophoresed in 10% SDS-PAGE under non-reducing conditions, transferred to nitrocellulose membranes, and the membranes probed with anti-ERK monoclonal antibody E10 which recognises only phosphorylated forms of ERK1/2.2.3 Results 2.3.1 Effect of Serum on MAP Kinase Activity The effect of MEK (MAP kinase) inhibitors UO126 and PD98059 on MAP kinase activity in WiDr and HT29 wild-type cells was tested in the absence/presence of serum.","Adherent cell monolayers on plastic were grown for 24 hrs in standard culture medium, then washed three times in PBS followed by 16 hrs in culture under serum-free conditions.","Serum was then added for 30 min and MAP kinase activity assessed before and after addition of serum.","The addition of serum markedly stimulated MAP kinase activity for both cell lines and was inhibitable by both MEK inhibitors.","2.3.2 Effect of Altered β6 Expression on MAP Kinase Activity Following Serum Stimulation.","In these experiments, WiDr transfectants (3 mock and 3 antisense β6 clones), HT29 transfectants (mock and antisense β6) and SW480 β6 transfectants (mock and sense β6) were serum-starved for 16 hrs followed by 30 mins exposure to serum.","Increased expression of αvβ6 was associated with a marked increase in MAP kinase activity upon serum stimulation compared with cells lacking αvβ6 altogether (SW480 mock).","Cells in which β6 expression had been down-regulated (antisense β6 transfectants) exhibited suppressed MAP kinase activity.","Induced expression of β6 in the non-β6-expressing colon cancer cell line SW480 resulted in a three fold increase in serum-dependent MAP kinase activity.","2.3.3 αvβ6 Binds an Extracellular Signal-Related Kinase (ERK).","A highly surprising finding arising from phage display screening which underpins the present work is that the integrin β6 cytoplasmic domain binds a MAP kinase.","Use of phage display to screen a γgt11 cDNA colon cancer cell library with the β6 cytoplasmic domain as bait yielded a clone which coded for ERK2 at the 3′ end with 100% nucleotide sequence identity (across 393 bases) to the published sequence of ERK2 (Boulton et al, 1991).","This putative association was investigated further.","Integrin immunoprecipitations were performed on equal protein loads of tumour cell lysates and the transferred proteins blotted with antibodies recognising phosphorylated and non-phosphorylated forms of ERK1/2.The specificity of this interaction was examined in integrin immunoprecipitations against β1, β5, β6 and αv integrin subunits.","As shown in FIG.","10, the anti-ERK mAb′SC-1647 identified a β6-specific band migrating at the position of purified ERK2 protein.","The integrin-associated ERK band was identified only in immunoprecipitations of the β6 subunit and its partner αv and not in immunoprecipitations of the β1/β5 subunits or in immunoprecipitations using isotype-matched control antibodies.","2.3.4 Effect of Altered β6 Expression on Total Cellular ERK and β6-Bound ERK.","Equal protein loads from one representative clone each from WiDr mock and antisense β6 transfectants were electrophoresed, transferred to nitrocellulose and blotted with anti-ERK mAb (SC-1647) as shown in FIG.","11(A).","β6 immuno-precipitates from equal protein loads of the WiDr mock and antisense β6 clones were electrophoresed, transferred and probed with anti-ERK mAb (E10) as shown in FIG.","11(B).","As indicated, suppression of β6 expression resulted in a reduction of both total cellular and phosphorylated integrin-associated ERK compared with WiDr mock transfectants.","Similarly, β6 immunoprecipitations from SW480 β6 transfected clones expressing high and low levels of β6 (confirmed by FACScan and β6 immunoprecipitations) showed parallel changes in β6-bound phosphorylated ERK (FIG.","12).","2.3.5 Effect of β6 Immunodepletion on Total Cellular ERK and β6-Bound ERK WiDr wild-type cells were surface biotinylated and the cell lysates immunodepleted of β6 in three rounds of sequential immunoprecipitation using anti-β6 mAb (R6G9) resulting in a marked loss of β6 from the lysates as shown in FIG.","13(A).","β6-immunodepleted lysates were then transferred and blotted with anti-ERK1/2 antibody (SC-1647), which recognises both phosphorylated and non-phosphorylated forms of ERK1/2.As shown in FIG.","13(B), following three rounds of β6 immunodepletion, levels of ERK1/2 in β6-depleted lysates compared with non-immunodepleted lysates, were markedly reduced suggesting a significant contribution of β6-bound ERK to total cellular ERK.","In contrast, immunodepletion of β5 by three successive rounds of immunoprecipitation with mAb P1F6 or isotype-matched control antibody IgG2A did not result in any reduction of cellular ERK levels compared with non-immunodepleted cell lysates.","2.3.6 Effect of ERK Immunodepletion on β6-Bound ERK Cell lysates from WiDr wild-type cells were immunodepleted of ERK1/2 by means of sequential immunoprecipitations using the anti-ERK mAb, SC-1647.ERK-immunodepleted cell lysates probed with either E10 or SC-1647 mAbs contained markedly less ERKs.","To examine the effect of ERK-immunodepletion on β6-bound ERK, the ERK-immunodepleted cell lysates were immunoprecipitated with anti-β6 mAb (R6G9) and the β6-immunoprecipitates probed with anti-ERK mAb (E10 recognising phosphorylated ERK1/2).","ERK immunodepletion effectively reduced levels of β6-bound ERK.","2.3.7 Effect of Preventing Cell Attachment on αvβ6-Bound ERK SW480 mock and β6 expressing transfected cells were grown to 80-90% confluency.","Cells were washed twice with PBS and harvested after trypsinization.","Cells were divided into three equal portions (approximately 4×106/batch).","The first group was plated on a 75 cm2 plastic flask in normal 10% serum containing medium (attached cells) while the other two groups were plated on 0.3% agarose underlay in serum free medium (non-attached cells).","Cells were allowed to grow for 24 hours at 37° C., after which in one group of 0.3% agarose underlay, 15 ml of serum-free medium was added while in the other an equal volume of 10% serum-containing medium was added for 30 mins.","Cells were collected washed with PBS and lysed in cell lysis buffer (100 mM Tris-HCl pH-7.5, 150 mM NaCl, 1 mM CaCl2, 1% Triton, 0.1% SDS, 0.1% Np-40, 1 mM vanadate, 1 μg/ml pepstatin, 1 mM PMSF, 5 μg/ml aprotonin and 1 μg/ml of leupeptin).","10 μl (10 μg of protein) was used for the analyses of cell lysates after adding equal volumes of non-reducing Laemmli buffer.","For SW480-β6-transfected cells the rest of the cell lysate was used for immunoprecipitation of αvβ6 integrin.","Cell lysates and β6 immunoprecipitates were subjected to western blotting and probed with E10 monoclonal antibody (recognising phosphorylated ERK1/2).","Cell lysates from the non-attached cells were found to require serum factors to maintain phosphorylation of total cellular ERK.","In contrast, non-attached SW480 β6 colon cancer cells do not require serum factors to maintain the activation phosphorylation) state of β6-bound ERK.","2.3.8 Effect of PP2A Phosphatase on αvβ6-Bound ERK In experiments to examine the effect of protein phosphatase 2A (PP2A) on β6-bound ERK, SW480 β6-transfected cells were sonicated in buffer comprising 5 mM Tric-HCl (pH 8.0), 10 mM MgCl2 and 0.01 mM EGTA together with enzyme inhibitors, and cell lysates from both serum-starved and serum-induced cells treated with 0.5 units of PP2A (Promega) at 30° C. for 10 minutes.","The reaction mixture was stopped by addition of equal volumes of non-reducing Laemmli sample buffer.","In parallel, PP2A-treated cell lysates were immunoprecipitated with mAb R6G9 (anti-β6) followed by Western blot with anti-ERK mAb E10.MAP kinase activity assays were performed according to the manufacturer's instructions (Amersham Pharmacia Biotec) using γ32P-ATP.","Exposure of cell lysates prepared from serum-supplemented and serum-starved cells to the PP2A catalytic subunit resulted in dephosphorylation of total ERK.","In contrast, dephosphorylation of β6-bound ERK was not observed in β6 immunoprecipitates prepared from serum-supplemented or serum-starved cells β6 Bound ERK may therefore serve to maintain adjacent growth factor receptors in an activated state and thereby alter their sensitivity to exogenous co-factors.","2.3.9 Inhibition of MAP Kinase Activity Inhibits Secretion of Gelatinase B. SW480 β6 transfectants were cultured under serum-free conditions for 48 hours in the absence/presence of the MEK inhibitor PD98059 (40 μM) or DMSO (vehicle control).","Tumour-conditioned medium was assayed for gelatinase B by analysis of equal protein loads in a gelatin zymogram.","Inhibition of MAP kinase activity by the MEK inhibitor reduced gelatinase B secretion compared with controls.","2.3.10 β6-ERK2 Association in HaCaT and HaRas Cell Lines β6 immunoprecipitates were prepared from human kerotinocyte cell lines (HaCaT and HaRas were obtained from Prof N. Fusenig, The German Cancer Research Institute, Heidelberg, Germany) using mAb R6G9 (anti-β6) and the immunoprecipitates probed with mab E10 (against phosphorylated ERK 1/2).","FIG.","14 shows that ERK2 associates with β6 in both of HaCat and HaRas cells.","2.4 Discussion of Results The MAP kinase pathway has been shown to be important in experimental tumour metastases (Mansour et al, 1994) and recent data implicate MAP kinases in tumour growth and invasiveness of colon cancer cells (Sebolt-Leopold et al, 1999).","In the present study, up-/down-regulation of β6 expression in various colon cancer cell lines was shown to enhance/suppress respectively, MAP kinase activity.","The presence of serum induced a three-fold increase in MAP kinase activity above that observed for serum-starved β6-expressing cells.","In contrast, only a one-fold increase in serum-dependent MAP kinase activity was observed for cells in which β6 had been down-regulated consequent upon transfection with antisense β6.Moreover, induced expression of β6 in the non-β6-expressing colon cancer cell line SW480, was associated with a three-fold increase in serum-dependent MAP kinase activity.","Serum contains a mixture of growth factors raising the possibility that one role for αvβ6 in colon cancer cells is to lower the threshold for activation of MAP kinase signalling pathways at times when the supply of serum-containing growth factors is limited.","The ERK band co-immunoprecipitated with β6 migrated either at or 1-2 kD higher than the mobility of purified phosphorylated ERK2 depending on the acrylamide concentration used in SDS-PAGE showing that the kinase does indeed, associate with the β6 subunit.","Slight differences in mobility of the β6-associated ERK band compared with the pure ERK protein could arise if β6-bound ERK is hyper-phosphorylated and/or exists in an altered conformation consequent upon its association with β6.ERK bound to β6 may also be an alternatively spliced variant of ERK2 causing it to migrate differently.","Finally, the purified ERK2 protein is derived from mouse which differs slightly from human ERK2 by being two amino acid residues shorter and also containing a single amino acid substitution.","In the present study, increased/decreased β6 expression in colon cancer cell lines was associated with increased/decreased β6-bound ERK, respectively.","In addition, immunodepletion experiments suggest that β6-bound ERK makes a substantial contribution to total phosphorylated ERK within the cell and overall MAP kinase activity (see FIGS.","15(A) and 15(B)).","The observation that β6-mediated colon cancer growth in vitro is inhibitable by a matrix metalloproteinase inhibitor (Agrez et al, 1999) suggests that the increased gelatinase B secretion by β6-expressing colon cancer cells contributes to tumour progression.","Taken together with the finding that inhibition of MAP kinase activity by the MEK inhibitor PD98059 diminished gelatinase B secretion in β6-expressing cells, it seems that activation of MAP kinase signalling plays a role, at least in part, in β6-mediated induction of gelatinase B secretion.","EXAMPLE 3 3.1 Identification of the Binding Domain on the β6 Subunit Cytoplasmic Tail Domain for ERK2 Peptide fragments corresponding to regions of the cytoplasmic tail domain of the β6 subunit were screened in an enzyme-linked immunosorbent assay (ELISA) for binding with ERK2.The 52 amino acid long β6 cytoplasmic tail is shown in FIG.","16 as are the amino acid sequences for the cytoplasmic domains of the β1 to β3 subunits.","In particular, four synthetic peptides designated fragment 1 to fragment 4 were prepared and biotinylated at the N-terminal end of each, respectively (Auspep Pty Ltd, Melbourne Australia).","The region of the β6 tail to which each corresponds is indicated in FIG.","16 and set out below.","Fragment 1: HDRKEVAKFEAERSKAKWQTGT Fragment 2: RSKAKWQTGTNPLYRGSTST Fragment 3: NPLYRGSTSTFKNVTYKHRE Fragment 4: FKNVTYKHREKQKVDLSTDS The fragments overlap by 10 amino acids and are each 20 amino acids long with the exception of the fragment 1 with a length of 22 amino acids.","Fragment 4 was synthesised with a terminal serine rather than a cysteine as found in wild-type β6 to avoid formation of a disulfide bridge between peptides.","Overlapping biotinylated fragments 1 to 4 were coated onto streptavidin coated polystyrene plates (Pierce, Rockford Ill. USA, Cat No.","15125) and the ELISA performed substantially according to manufacturers instructions.","Briefly, wells are washed with 3×200 μl of wash buffer (TBS, 0.1% BSA, 0.05% Tween 20 or SuperBlock™ blocking buffer in TBS, Pierce, Prod.","No.","37535) prior to addition of biotinylated peptides (100 μl) and incubation for 1 hr at room temperature to allow for capture of the peptides on the plate.","Following another washing step, peptides were overlayed with GST-ERK, ERK (or JNK-1) alone at a volume of 100 μl per well, and the plates incubated for a further 1 hr at room temperature before removal of any unbound ERK by further washing.","Binding of ERK to the peptides is detected using 100 μl anti-ERK1/2 mAb SC1647 (Santa Cruz) as the primary antibody at a dilution of 1:700 (isotype matched antibody IgG2b is used as a control).","This is followed by another washing step and addition of 100 μl rabbit anti-mouse antibody (Biorad) conjugated to alkaline phosphatase at a concentration of 1:1000 for 30 minutes again at room temperature.","A 10 aliquot of detection reagent (alkaline phosphatase detection kit-Biorad) is then introduced into each well after a final washing step and allowed to react for 15-30 minutes at room temperature in the dark before absorbance is measured at 405 nm.","All dilutions of peptides, MAP kinase and antibodies were performed using the wash buffer.","GST-ERK is a fusion protein consisting of ERK coupled to glutathione-S-transferase and purified from host cells transfected with pGEX vector.","As shown in FIG.","17, significant binding of non-phosphorylated GST.ERK2 (0.25 μg/100 μl) to peptide fragment 2 was observed (1 μg/100 μl) while only negligible or low level binding for the other fragments was found.","Significant binding of non-phosphorylated ERK2 to both fragment 2 and β6 cytoplasmic tail peptide compared to fragments 1, 3 and 4 over a range of concentrations of ERK2 was also observed (see FIG.","18).","Similar results were observed using a range of concentrations of the peptide fragments as shown in FIG.","19.To further localise the binding domain on the cytoplasmic tail of the β6 subunit, progressively shorter peptides from the region of the β6 cytoplasmic tail corresponding to peptide fragment 2 were synthesised, biotinylated and the capacity to associate or otherwise bind to ERK2 assessed as described above.","The binding of GST.ERK2 to a 15 mer test peptide (seq.","4) having the amino acid sequence RSKAKWQTGTNPLYR and a 10 mer test peptide having the sequence RSKAKWQTGT is shown in FIG.","20 compared to fragment 2 over a range of concentrations of the peptides.","As can be seen, no reduction in binding to the seq.","4 peptide compared to fragment 2 was found.","Binding of ERK2 to the seq.","3 peptide was substantially less than that observed for seq.","4.A number of 10 mer biotinylated peptides corresponding to regions of fragment 2 or fragment 3 were then tested.","The amino acid sequence for each peptide is as follows and their location in the β6 cytoplasmic domain is indicated in FIG.","21.10(1): NPLYRGSTST 10(2): WQTGTNPLYR 10(3): KFEAERSKAK The results are set out in FIG.","22 and show that GST.ERK binding to the 10 mer peptides is substantially reduced compared to binding to the seq.","4 peptide suggesting that opposite end regions of seq.","4 participate in the binding of ERK2.Comparable binding of ERK2 to seq.","4 was found using a further 10 mer peptide identified as 10(4) in which amino acid sequence WQTGT of seq.","4 is omitted indicating that WQTGT is a linker sequence that does not participate directly in the binding of ERK to seq.","4.Negligible binding of ERK2 to the 5 mer peptide RSKAK was observed as shown in FIG.","23.ERK2 cleaved from GST-ERK2 by thrombin was used in this assay.","Results (not shown) indicate that greater than a 3 fold increase in assay sensitivity can be achieved using thrombin cleaved ERK2 rather than GST-ERK2.EXAMPLE 4 4.1 MAP Kinase JNK-1 Binds to the Cytoplasmic Tail Domain of β6 In view of the observation that ERK2 associates with the cytoplasmic tail of the β6 subunit, the MAP kinase JNK-1 was tested to evaluate whether it also could associate with the cytoplasmic tail of β6.Briefly, 0.05-1.5 μm/100 μl of JNK-1 (Santa Cruz) was aliquoted into wells of a 96 well culture place containing increasing concentrations of the β6 cytoplasmic domain tail peptide used in Example 3.For comparison purposes, non-phosphorylated GST-ERK2 (0.05-1.5 μm/100 μl) was aliquoted into wells containing β6 cytoplasmic tail peptide or peptide fragments 1 or 2, respectively.","Binding of JNK-1 was detected using mouse anti-JNK-1 mAb SC 474-G (Santa Cruz) and HRP-conjugated goat anti-mouse antibody.","Absorbance was read at 405 nm and the results are shown in FIG.","24.Significant binding of JNK-1 to the β6 cytoplasmic tail peptide was found.","EXAMPLE 5 5.1 Evaluation of Ability of ERK2 to Bind to β6 Δ746-764 Deletion Mutant.","To examine the role of the amino acid sequence RSKAKWQTGTNPLYR in the β6 cytoplasmic domain in situ, a β6 deletion construct lacking the coding sequence for AERSKAKWOTGTNPLYRG was transfected into colon cancer cell line SW480 which does not constitutively express the α0Vβ6 integrin using the calcium phosphate method previously described for transfections into this cell line (Agrez et al, 1994).","The location of the β6 Δ746-764 deletion is indicated in FIG.","25.Construction of the β6 Δ746-764 deletion mutant in the vector pcDNA1neo and failure of the expressed receptor to localise to focal adhesions in Chinese hamster ovary cells has been reported (Cone et al, 1994).","Facscan analysis revealed comparable levels of surface expression of mutant β6 to that seen for the full length wild-type receptor (see FIG.","26).","Equal protein loads of cell lysates prepared from SW480 cells were immunoprecipitated with either anti-β6 monoclonal antibody (mAb R6G9) or matched isotype control antibody.","Surface biotinylation prior to immunoprecipitation confirmed equal surface expression of mutant and wild-type β6 (see FIG.","27(A).","Aliquots of the immunoprecipitates were electrophoresed and transferred to nitrocellulose for Western blotting using monoclonal antibody E10 which recognises ERK1/2.As seen in FIG.","27(B), loss of the RSKAKWQTGTNPLYR sequence in the β6 cytoplasmic domain reduced levels of β6-bound ERK by greater than approximately 75% of that observed for the wild type receptor.","EXAMPLE 6 6.1 Growth Inhibition Study.","HT29 and SW480 β6-expressing colon cancer cell lines were seeded into wells of 96-well microtitre plates (Nunclon) in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% foetal bovine serum, glutamine, Hepes, and antibiotics.","Seeding cell densities were 3×103 cells per triplicate well for each condition tested and after 24 hours incubation of cell cultures in 5% CO2, 100% humidity at 37° C., the culture medium was exchanged for serum-free DMEM medium supplemented with insulin, transferrin, selenous acid, hydrocortisone, non-essential amino acids, glutamine, Hepes and antibiotics containing either peptide RSKAKWQTGTNPLYR alone or penetratin-peptide complex at a concentration of 10 μm for HT29 cells or 30 μm for SW480 β6-expressing cells.","Cell cultures were incubated for a further 24 hours following which cultures were photographed (Kodak Techpan Film at 100 ASA setting) and the experiments terminated by addition of the cell proliferation reagent WST-1 (Boehringer Mannheim) to monitor effects of the peptide on cell growth.","The cell proliferation reagent WST-1 is designed to be used for the non-radioactive, spectrophotometric quantification of cell growth and viability in proliferation and chemosensitivity assays.","The colourmetric assay is based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenase in viable cells.","Specifically, at the termination of experiments, 30 μl of WST-1 was added to 270 μl culture medium volume in each microtitre well and the colour change quantitated in an ELISA plate reader by measuring absorbance of the formazan product at 450 mn (using a reference wavelength of more than 600 nm).","The mean absorbance readings from triplicate wells (±standard error of the means) after subtraction of background control wells (culture medium without cells) was determined.","Only the carrier penetratin-peptide complex was effective in inhibiting cell proliferation in contrast to either peptide or penetratin alone as shown in FIGS.","28 and 29 indicating that the penetratin-peptide complex was internalised by both the HT29 and SW480 cells resulting in the observed suppression of colon cancer growth.","Photographs of the SW480 cells treated with penetratin alone or the penetratin-peptide complex are shown in FIG.","30(A) to (C).","EXAMPLE 7 7.1 Growth Inhibition in SW480 Mock and SW480 β6 Transfectants.","SW480 mock and SW480 β6 transfectants were cultured in DMEM medium supplemented with 1% foetal bovine serum in the presence of 20 μM seq.","4 coupled to penetratin.","Percentage inhibition was assessed by the WST-1 colorimetric dehydrogenase assay described in Example 6.The percentage inhibition of growth observed for the −β6 and +β6 expressing cells was 17% and 50%, respectively as indicated in FIG.","31A.","The −β6 and +β cultured cells are shown in FIG.","31B.","EXAMPLE 8 The proliferation of SW480 cells expressing a β6 Δ746-764 deletion mutant was compared with non-β6 expressing SW480 cells and SW480 cells expressing full length wild-type β6.Cells were cultured for 10 days within a 3-dimensional collagen type I matrix.","Collagen gels were prepared as bilayers (upper layer cell-containing and lower layer minus cells) in 24 well culture plates as previously described (Agrez, 1989; Agrez, 1994) except for the use of 5% foetal bovine serum as the supplement for DMEM.","Colonies were photographed in the gel FIG.","32(A) at 10 days (bar represents 200μ) and following dissolution of the collagen with collagenase FIG.","32(B) prior to visual colony counting of all colonies exceeding 200μ in diameter within each well FIG.","32(C).","As can be seen, significant proliferation of the SW480 cells expressing the full length wild-type β6 was observed compared to the non-β6 expressing cells and cells expressing the β6 Δ746-764 deletion mutant.","EXAMPLE 9 Growth inhibition of SW480 cells expressing full length wild-type β6 exposed to seq.4 coupled to penetratin or RSKAKWQTGTNPLYR peptide coupled to penetratin (5, 10, 20, 30 μM in DMEM minus foetal bovine serum) but which peptide contained alanine substitutions at the four positions indicated was assessed.","As shown in FIG.","33(A) and FIG.","33(B), progressive inhibition of proliferation in a dose-response manner was observed for the seq.4 penetratin complex compared with the alanine substituted peptide-penetratin complex which was without effect at all doses tested.","EXAMPLE 10 Binding of ERK2 to the seq.4 peptide (RSKAKWQTGTNPLYR) was compared with peptides corresponding to regions of the cytoplasmic domain of integrin subunits β1, β2, β3 and β5.The amino acid sequences for those peptides is shown below: β3 KFEKEKMNAKWDTGENPIYK β2 KEKLKSQWNNDNPLFK β3 RARAKWDTANNPLYK β5 RSRARYEMASNPLYR As shown in FIG.","34, significant binding of ERK2 to the seq.","4 peptide was observed.","Binding of ERK2 to the β5 and β3 peptides was also found.","The results have been corrected for non-specific binding and indicate a hierarchy of binding of ERK2 to integrin subunits.","EXAMPLE 11 Angiogenesis, the growth of new blood vessels, plays a role in diverse disease states including progression of cancer, diabetic retinopathy, rheumatoid arthritis, psoriasis, atherosclerosis, pregnancy and endometriosis.","For examples, in cancers, the ability of microscopic tumour deposits to enlarge beyond 1-2 mms in diameter is dependent upon tumour-induced angiogenesis or sprouting of new host-derived blood vessels to nourish enlarging tumour cell nodules.","The mitogen activated protein (MAP) kinase signalling pathway is critical for endothelial cell proliferation and new blood vessel formation.","Moreover, a relationship exists between extracellular signal-regulated (ERK) kinase 1/2 activation and vascular endothelial growth factor (VEGF) expression with each activating the other (Gupta et al, 1999; Yu & Sato, 1999: Pages et al, 2000).","Two growth factor-dependant pathways of angiogenisis have now been shown to exist defined by their dependency on the distinct vascular integrins αvβ3 and αvβ5 (Friedlander et al, 1995: Eliceiri and Cheresh, 1999).","It is noteworthy that the vascular system lacks αvβ6 expression (Breuss et al, 1993).","Antibodies against αvβ5 and inhibition of protein kinase C signalling have both been shown to block VEGF-induced angiogenesis (Freidlander et al, 1995).","The importance of αvβ5 in neovascularisation is further highlighted by the observation that β3 integrin-deficient mice exhibit normal blood vessel development in contrast to αv-deficient mice which implicates other beta integrin partners such as β5 (Eliceiri and Cheresh, 1999).","11.1 The β5 Integrin Subunit Binds ERK in Cells that Either Lack β6 or Express β6 Lacking the Binding Site for ERK2 The SW480 cell line expresses abundant αvβ5 but only minimal levels of αvβ3 and lacks αvβ1 and αvβ6.SW480 cells provide a model, therefore, with which to test whether the β5 integrin subunit can co-immunoprecipitate with ERK either in the absence of constitutive β6 expression or in the presence of β6 that lacks the binding site for ERK2 (RSKAKWQTGTNPLYR).","To evaluate this, cell lysates containing equal protein loads were prepared from SW480 wild-type cells and SW480 cells transfected with either vector alone (mock transfectants), wild type β6 or the β6 deletion mutant Δ746-764 (lacking the sequence EAERSKAKWQTGTNPLYRG, Cone et al, 1994).","The lysates were immunoprecipitated with mAbs R6G9 (anti-β6), P1F6 (anti-β5) or murine IgG control antibody (mlgG).","Western blotting of the immunoprecipitates with mAb E10 recognising phosphorylated ERK1 and 2 revealed no β5-bound ERK in the SW480 β6 transfectants expressing wild-type β6 in contrast to β5-associated ERK observed for the cell lines which either lack β6 or express the deletion mutant lacking the ERK2 binding sequence (FIGS.","35A and 35B).","To determine whether β5 can bind ERK2 in vitro, a 15-mer peptide fragment derived from the β5 cytoplasmic domain corresponding to the ERK binding domain on β6 was tested for its ability to bind ERK2 in an ELISA.","In vitro MAP kinase activity assays were performed using a non-radioactive assay kit (Cell Signalling Technology, Beverley, Mass.)","which measures phosphorylation of the ERK1 and 2 substrate Elk-1 presented as a fusion protein comprising the Elk-1 codons 307-428 coupled to GST.","In the assay, ERK kinase is first immunoprecipitated from complete or integrin-immunodepleted soluble cell lysates and relative enzyme activity determined by the in vitro phosphorylation of GST-Elk-1.Dose-dependant binding of the β5 fragment (761RSRARYEMASNPLYR775) to ERK2 was observed as shown in FIG.","35C although less avidly than the 15-mer β6 peptide.","In contrast, a synthetic peptide derived from the cytoplasmic domain of the β1 subunit (765KFEKEKMNAKWDTGENPIYK784) which corresponds to the ERK binding domain on β6 failed to bind ERK2 (FIG.","35C).","11.2 The Increase in Cytosolic MAP Kinase Activity Upon Growth-Factor Stimulation is All Accounted for by β5-Bound ERK in Human Umbilical Vein Endothelial Cells that Lack β6 To determine the amount of ERK 1/2 activity associated with β5 following the activation of the MAP kinase pathway, the activity of ERK1/2 was compared in non-β5 immunodepleted lysates with that in β5 immunodepleted lysates derived from human umbilical vein endothelial cells (HUVECs) before and after stimulation with epidermal growth factor (EGF).","Sequential immunodepletion of the β5 integrin subunit from cell lysates prepared from HUVECs results in total loss of all epidermal growth factor-induced MAP kinase (ERK 1/2) activity as determined by an Elk-1 phosphorylation assay (FIG.","36).","Briefly, HUVECs were cultured in the absence/presence of 50 ng/ml epidermal growth factor (EGF) for 10 minutes after a prior 24 hour culture period in serum-free and growth factor-free medium.","Bands show MAP kinase activity from immunodepleted cell lysates (anti-β5 or control IgG or anti-β6 immunodepletions, five rounds each) prepared from HUVECs before and after EGF stimulation.","Non-phosphorylated and phosphorylated Elk-1 controls are shown in the left hand two lanes.","These results demonstrate that in β5-expression endothelial cells, the rise in cytosolic MAP kinase activity above basal levels upon EGF stimulation is completely accounted for by β5-bound ERK.","The findings also indicate that a hierarchy of integrin-ERK2 interactions exists within cells specified by β6 over β5 in β6-expressing epithelial cells and β5 over other β integrins in endothelial cells.","Given the critical requirement of ERK1/2 activity in the process or angiogenesis and responsiveness of endothelial cells to various growth factors these findings show, for the first time, how the αvβ5 integrin may regulate angiogenesis through maintaining activated ERK at the plasma membrane.","Accordingly, targeting the binding site on β5 for ERK2 or the binding site on ERK2 for the integrin offers a means of inhibiting new blood vessel formation.","Moreover, the findings allow for anti-angiogenic therapies to benefit patients with a range of disorders including not only cancer, but also diabetic retinopathy, arthritis and other inflammatory conditions.","EXAMPLE 12 The β6 integrin immunodepletion/ERK immunoblotting experiments together with the antisense nucleic acid strategies reported herein show that the β6 integrin subunit contributes substantially to the total MAP kinase activity with for instance, colon cancer cells.","Moreover, the fact β6-bound phosphorylated ERK2 is reduced by more than 75% in colon cancer cells lacking constitutive β6 expression stably transfected with a β6 deletion mutant in which the precise ERK2 binding domain is lacking, and that tumour growth is also reduced significantly compared with β6-expressing cells, shows that ERK2 plays a significant role in promoting cancer growth.","Accordingly, interruption of the β6-MAP kinase pathway has important implications not only for control of advanced colon cancer, but epithelial cancers in general given the wide expression of this integrin in other cancer types such as oropharyngeal, breast and ovarian cancer.","Hence, identification of the ERK2 binding sequence to β6 facilitates the development of mimetics which interrupt the β6-MAP kinase growth-promoting axis in cancer.","12.1 ELISA Methodology for Determination of β6-Binding Domain in ERK2.To determine the β6-binding domain(s) of ERK2, a biotinylated β6 cytoplasmic tail peptide spanning the cytoplasmic tail of β6 was incubated with a soluble ERK2 peptide fragment in a total incubation volume of 20 μl phosphate buffered saline (PBS).","The mixture was stood for 45 minutes at room temperature (RT).","Soluble ERK2 in PBS was then added and the solution left for a further 30 minutes.","Following incubation the reaction volume was made to 400 μl with carbonate buffer (0.015 M Na2CO3 and 0.035M NaHCO3 at pH 9.6) and 100 μl of this mixture added to each of 4 wells of a polystyrene ELISA plate (Nunc, Maxicorp).","ERK2/peptides were allowed to attach to the plastic substrate for 18 to 20 hours at RT in the dark.","After immobilisation the overlying buffer was flicked from the wells and excess solid surface blocked with casein (0.5% skim milk) in PBS for 60 minutes at RT.","Wells were washed (×3) with was buffer (PBS plus 0.5% skim milk powder, 0.05% Tween-20) prior to incubation with the anti-β6 antibody SC-6632 (Santa Cruz) diluted 1:500 v/v in wash buffer (2 wells) or a control antibody (2 wells).","Incubation with the primary antibody was carried out for 30 minutes at RT.","The ELISA plate was flicked to remove the antibody mixture before washing 3 times with wash buffer.","Wells were then incubated with anti-species alkaline phosphatase (AP) conjugated antibody (1:2000 v/v in wash buffer) for 30 minutes at RT, flicked and washed as above.","Specific binding events were quantitatively determined using a colourimetric substrate system by measuring absorbency at 405 mm (Bio-Rad, AP substrate kit).","12.2 Identification of β6-Binding Domain of ERK2.In a preliminary study, ERK peptides were probed in small groups to identify a possible ERK2 competing peptide that binds with the β6 cytoplasmic tail peptide.","Overlapping ERK2 peptides 1 to 14 comprising the full length ERK2 sequence (360 amino acids) were utilised, and an inhibitory effect on β6-ERK2 binding was identified within the group comprising peptides 7, 8, and 9 (data not shown).","The amino acid sequences for the peptides and position within ERK2 are shown below.","1.MAAAAAAGAGPEMVRGQVFDVGPRYTNLSY 2.YTNLSYIGEGAYGMVCSAYDNVNKVRVAIK 3.VRVAIKKISPFEHQTYCQRTLREIKILLRF 4.KILLRFRHENIIGINDIIRAPTIEQMKDVY 5.QMKDVYIVQDLMETDLYKLLKTQHLSNDHI 6.LSNDHICYFLYQILRGLKYIHSANVLHRDLK 7.HRDLKPSNLLLNTTCDLKICDFGLAR 8.DFGLARVADPDHDHTGFLTEYVATRWYRAPEIMLNSKGY 9.NSKGYTKSIDIWSVGCILAEMLSNRPIFPG 10.PIFPGKHYLDQLNHILGILGSPSQEDLNCI 11.DLNGIINLKA RNYLLSLPHKNKVPWNRLFP 12.NRLFPNADSKALDLLDKMLTFNPHKRIEVE 13.RIEVEQALAHPYLEQYYDPSDEPIAEAPFK 14.EAPFKFDMELDDLPKEKLKELIFEETARFQPGYRS Individual overlapping peptides of the ERK2 sequence were then tested at equimolar concentrations for the ability to inhibit the β6-ERK interaction.","Only peptide 7 (HRDLKPSNLLLNTTCDLKICDFGLAR) was found to be effective (see FIG.","37).","Repeat testing using adjacent ERK2 overlapping peptides as controls yielded the same result (see FIG.","38).","Importantly, β6 bound to ERK2 does not interfere with ERK2 binding to the well, thereby confirming the validity of the ERK2 peptide competition assay.","To optimise the assay, β6 cytoplasmic tail peptide was incubated at increasing concentrations with ERK2.Maximal β6 detected when bound to ERK was found to be at a concentration of 0.25 μM (data not shown).","β6 cytoplasmic tail peptide (0.25 μM) was then either pre-incubated with ERK peptides 2, 7, 8 or 10 in the competition assay or directly with ERK2 (positive control).","An inhibition of 83% for β6-ERK2 binding was again demonstrated for sequence 7 (see FIG.","39).","To demonstrate that neither β6 alone or ERK2 peptide fragments 2, 7, 8 and 10 interfered with ERK2 binding to the plate, parallel samples of each condition identified in FIG.","38 were probed with anti-ERK2 antibody.","None of the peptide or β6 coincubations with ERK2 interfered with ERK binding to the ELISA plate.","EXAMPLE 13 13.1 Induction of Wound Healing Response Keratinocytes in vivo in unwounded skin do not express β6 integrin but do so after sub-culturing in vitro.","Hence, to avoid the conflicting presence of integrin αvβ6, the epithelial cell line SW480 which lacks αvβ6 expression was chosen in order to examine the effect of peptide β6 agonists on cell migration following artificial wounding in vitro.","The wound assay was performed by seeding 2×105 cells into 24 well (1.6 cm diameter) tissue culture plates in Dulbecco's modified Eagles medium (DMEM) supplemented by 10% foetal calf serum, HEPES, glutamine and antibotics.","The confluent cell monolayers were streaked with the tip of a plastic pipette in a cross-wire fashion, and loose non-adherent cells washed off gently with medium before replacing the culture medium with DMEM supplemented with 1% foetal calf serum.","In addition, RSKAKWQTGTNPLYR peptide ligated to penetratin (carrier peptide) by means of cysteine Cys (Npys) bonding was added to each well at a final concentration in the range of 1.25-10 μM.","Cell migration across the wounded (streaked) area was photographed (Techpan, Kodachrome, ASA100) after a further 48 hours in culture.","As shown in FIGS.","40A to 40D, a dose-dependent wound repair process (enhanced cell migration) was observed for the peptide-agonist complexed with the intracellular delivery vehicle penetratin.","The conditions utilised in the assay were as follows: A: no peptide-penetratin complex added, B: 2.5 μM peptide-penetratin complex added, C: 5 μM peptide-penetratin complex added, D: 10 μM peptide-penetratin complex added.","Cell migration was not stimulated by either the peptide-agonist uncoupled to penetratin or penetratin alone (not shown).","The bar length shown in FIG.","40D represents 80 μM in length.","13.2 Discussion The activity of ERK2 is tightly regulated.","Dual phosphorylation events, one on residue 185 (Y) and one on residue 183 (T) are required for ERK2 activation (Zhang et al, 1995).","In unphosphorylated ERK2, Y185 is found largely buried within the molecule whereas T183 is exposed.","In particular, the phosphorylation lip of ERK2 consists of 15 amino residues from residue 173 (D) to residue 187 (A) namely DPDHDHTGFLTEYVA.","This sequence is near the mouth of the active site (Zhang et al, 1995).","The 26 mer peptide fragment of ERK2 (peptide 7) found to inhibit binding of β6 cytoplasmic domain to ERK2 stretches from amino acid residue 145 (H) to residue 170 (R).","This inhibitory binding sequence lies adjacent to the phosphorylation lip of ERK2.The reason for low stability of the phosphorylation lip in ERK2 is uncertain, but may be due to the unusual cluster of three positively charged amino acids namely residue 146 (R), 170 (R) and 201 (K) (Zhang et al, 1995).","Residues 146(R) and 170(R) of ERK2, both of which are included within peptide 7, appear to be conserved among many serine/threonine specific kinases.","For conserved amino acid residue 146 (R) to bind to a phosphate ion, it must lose its interaction with the backbone of the loop defined by amino acid residues 199 to 205 of ERK2 adjacent to the phosphorylation lip (residues 173 (D) to 187 (A)).","Accordingly, β6 may bind to the 15 mer PSNLLLNTTCDLKIC within peptide 7 and break the interaction between residue 146 (R) and the backbone loop allowing ERK to be phosphorylated on residue 183 (T) and 185 (Y).","Although the present invention has been described hereinbefore with reference to a number of preferred embodiments, the skilled addressee will understand that numerous variations and modifications are possible without departing from the scope of the invention.","REFERENCES CITED Agrez, M. 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K., Integrin-dependent activation of MAP kinase: a link to shape-dependent cell proliferation.","Mol.","Biol.","Cell 6, 273-282 (1995)."]],"string":"[\n [\n \"Map kinase integrin-binding domain\",\n \"Dislosed is the integrin-binding domain of MAP kinase.\",\n \"Interaction between MAP kinase and integrin via this domain activates MAP kinase and initiates cellular activity.\",\n \"Methods of modulating cellular activity by inhibiting direct MAP kinase-integrin binding are provided.\",\n \"These methods have particular use in inhibiting growth of cancer cells.\"\n ],\n [\n \"1.A fragment of a MAP kinase wherein the fragment consists of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin.\",\n \"2.A fragment of a MAP kinase wherein the fragment comprises a binding domain of the MAP kinase for an integrin or a partial amino acid sequence of the binding domain that is capable of binding to the integrin and has a length of 100 amino acids or less, or an analog or derivative of the fragment that is capable of binding to the integrin.\",\n \"3.A fragment according to claim 2 wherein the fragment has a length of about 50 amino acids or less.\",\n \"4.A fragment according to claim 3 wherein the fragment has a length of about 40 amino acids or less.\",\n \"5.A fragment according to claim 4 wherein the fragment has a length of about 30 amino acids or less.\",\n \"6.A fragment according to claim 5 wherein the fragment has a length of about 20 amino acids or less.\",\n \"7.A fragment according to claim 2 comprising the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or a contiguous amino acid sequence thereof.\",\n \"8.A fragment according to claim 2 comprising the amino acid sequence PSNLLLNTTCDLKIC or a contiguous amino acid sequence thereof.\",\n \"9.A fragment according to claim 1 wherein the MAP kinase is an ERK or a JNK family member.\",\n \"10.A fragment according to claim 9 wherein the MAP kinase is ERK2.11.A fragment according to claim 1 wherein the integrin is selected from the group consisting of β3, β5 and β6.12.An isolated or purified polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase.\",\n \"13.A polypeptide according to claim 12 comprising a core amino acid sequence of a binding domain of the MAP kinase for the integrin.\",\n \"14.A polypeptide according to claim 12 wherein the MAP kinase is a member of the ERK family or the JNK family.\",\n \"15.A polypeptide according to claim 14 wherein the MAP kinase is ERK2.16.A polypeptide according to claim 12 wherein the integrin is selected from the group consisting of β3, β5 and β6.17.A fusion protein incorporating a binding moiety capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin.\",\n \"18.A fusion protein according to claim 17 adapted for passing across a cell membrane.\",\n \"19.A fusion protein according to claim 18 further incorporating a carrier moiety for facilitating passage across the cell membrane.\",\n \"20.A fusion protein according to claim 17 wherein the binding moiety comprises a fragment of a MAP kinase consisting of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin or a polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase.\",\n \"21.A fusion protein according to claim 18 adapted for releasing the binding moiety after passing across the cell membrane.\",\n \"22.An agent for inhibiting binding of a MAP kinase to an integrin, comprising: a targeting moiety for targeting a cell expressing the integrin; a binding moiety for binding to a binding domain of the integrin for the MAP kinase to thereby inhibit the binding of the MAP kinase to the integrin; and a carrier moiety for facilitating passage of the binding moiety across the cell membrane of the cell; wherein the binding moiety is other than an antibody or a binding fragment thereof.\",\n \"23.An agent according to claim 22 wherein the binding and carrier moieties are capable of being released from the targeting moiety at the cell for passage of the carrier moiety and the binding moiety across the cell membrane of the cell.\",\n \"24.An agent according to claim 23 wherein the agent incorporates an enzyme cleavage site for being cleaved to thereby release the carrier moiety and the inhibitor moiety at the cell.\",\n \"25.An agent according to claim 24 wherein the enzyme cleavage site is a cleavage site for matrix-metalloproteinase-9 (MMP-9).\",\n \"26.An agent according to claim 22 adapted for release of the inhibitor moiety from the carrier moiety after passage of the inhibitor moiety across the cell membrane of the cell.\",\n \"27.An agent according to claim 22 wherein the targeting moiety is an antibody or a binding fragment of an antibody.\",\n \"28.An agent according to claim 27 wherein the antibody or the binding fragment is specific for an extracellular region of the integrin.\",\n \"29.An agent according to claim 22 wherein the targeting moiety is an integrin receptor targeted peptide for binding to the integrin.\",\n \"30.An agent according to claim 22 wherein the binding moiety is capable of binding to a binding domain of the integrin for the MAP kinase.\",\n \"31.An agent according to claim 30 wherein the binding moiety comprises a fragment consisting of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin or a polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase, or an analog or derivative of the fragment or polypeptide that is capable of binding to the binding domain of the integrin for the MAP kinase.\",\n \"32.An agent according to claim 22 wherein the binding moiety is an analog of a binding domain of a MAP kinase for an integrin.\",\n \"33.An agent capable of inhibiting binding of a MAP kinase to an integrin, wherein the agent comprises an analog of a binding domain of the MAP kinase for the integrin.\",\n \"34.An agent according to claim 33 wherein the analog comprises a mimetic of the binding domain of the MAP kinase.\",\n \"35.An isolated nucleic acid sequence encoding a fragment as defined in claim 1 or an analog or derivative thereof capable of binding to the binding domain of the integrin for the MAP kinase.\",\n \"36.An isolated nucleic acid sequence encoding a polypeptide as defined in claim 12 or an analog or derivative thereof capable of binding to the binding domain of the integrin for the MAP kinase.\",\n \"37.An isolated nucleic acid sequence encoding a fusion protein as defined in claim 17.38.An isolated antisense nucleic acid sequence complementary to a nucleic acid sequence as defined in claim 35.39.An isolated antisense nucleic acid sequence according to claim 38 wherein the antisense nucleic acid sequence is labelled.\",\n \"40.An oligonucleotide comprising from 14 to 45 contiguous nucleotides of a nucleic acid sequence as defined in claim 35.41.An oligonucleotide comprising from 14 to 45 nucleotides and which is capable of hybridising with a nucleic acid sequence as defined in claim 35.42.A vector incorporating a nucleic acid sequence as defined in claim 35.43.An expression vector incorporating a nucleic acid sequence as defined in claim 35.44.A host cell transformed with a vector as defined in claim 42.45.A host cell transformed according to claim 44 wherein the host cell is selected from the group consisting of a mammalian cell, an epithelial cell, a neoplastic cell and a cancer cell.\",\n \"46.A host cell according to claim 45 wherein the host cell is a colon cancer cell.\",\n \"47.A pharmaceutical composition comprising a fragment as defined in claim 1 or an analog or derivative thereof capable of binding to the binding domain of the integrin for the MAP kinase, together with a pharmaceutically acceptable carrier or diluent.\",\n \"48.A pharmaceutical composition comprising a polypeptide as defined in claim 12 or an analog or derivative thereof capable of binding to the binding domain of the integrin of the MAP kinase, together with a pharmaceutically acceptable carrier or diluent.\",\n \"49.A pharmaceutical composition comprising fusion protein as defined in claim 17 together with a pharmaceutically acceptable carrier or diluent.\",\n \"50.A pharmaceutical composition comprising an agent for inhibiting binding of a MAP kinase with an integrin as defined in claim 22, together with a pharmaceutically acceptable carrier or diluent.\",\n \"51.A pharmaceutical composition comprising a vector as defined in claim 43 together with a pharmaceutically acceptable carrier or diluent.\",\n \"52.A pharmaceutical composition comprising an antisense nucleic acid sequence as defined in claim 38 together with a pharmaceutically acceptable carrier or diluent.\",\n \"53.A pharmaceutical composition comprising an oligonucleotide as defined in claim 40 together with a pharmaceutically acceptable carrier or diluent.\",\n \"54.An antibody specific for a binding domain of a MAP kinase for an integrin, or a binding fragment of the antibody.\",\n \"55.An antibody specific for a fragment as defined in claim 1 or an analog or derivative of the fragment that is capable of binding to the binding domain of the integrin for the MAP kinase.\",\n \"56.An antibody specific for a polypeptide as defined in claim 12 or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin in the MAP kinase.\",\n \"57.An antibody according to claim 54 wherein the antibody is a monoclonal antibody.\",\n \"58.A method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin comprising: (a) testing a number of agents for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if any said agent is capable of binding to the binding domain of the MAP kinase on the basis of the testing.\",\n \"59.A method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing a number of agents for ability to bind to the MAP kinase; (b) selecting an agent or agents identified as being able to bind to the MAP kinase on the basis of the testing; and (c) utilising the selected said agent or agents in an assay for indicating whether the or any of the selected said agents is capable of binding to the binding domain of the MAP kinase for the integrin.\",\n \"60.A method of evaluating whether an agent is capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing the agent for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if the agent is capable of binding to the binding domain of the MAP kinase on the basis of the testing.\",\n \"61.A method according to claim 60 wherein the integrin is selected from the group consisting of β3, β5 and β6.62.A method according to claim 61 wherein the MAP kinase is an ERK family member or a JNK family member.\",\n \"63.A method according to claim 62 wherein the MAP kinase is ERK2.64.An agent identified to be capable of binding to a binding domain of a MAP kinase for an integrin by a method as defined in claim 61.65.A method of isolating an agent from a sample utilizing a molecule immobilised on a solid support and which is capable of binding to a binding domain of an integrin for a MAP kinase, comprising: (a) contacting the molecule immobilised on the solid support with the sample under conditions suitable for binding of the agent to the molecule; (b) eluting the agent from the solid support; and (c) collecting the eluted agent; wherein the molecule is other than an antibody or binding fragment of an antibody.\",\n \"66.A method according to claim 65 wherein the molecule is a MAP kinase or fragment thereof, or an analog or derivative of a fragment of a MAP kinase.\",\n \"67.A method according to claim 65 wherein the molecule is a fragment consisting of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin, or an analog or derivative thereof.\",\n \"68.A method according to claim 65 wherein the molecule is a polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase, or an analog or derivative thereof.\",\n \"69.A method according to claim 65 wherein the integrin is selected from the group consisting of β3, β5 and β6.70.A method according to claim 65 wherein the MAP kinase is an ERK family member or a JNK family member.\",\n \"71.A method according to claim 70 wherein the MAP kinase is ERK2.72.An agent isolated from a sample by a method as defined in claim 61.73.A method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an antisense oligonucleotide for hybridising with at least part of a nucleic acid sequence coding for the binding domain of the MAP kinase for the integrin to thereby cause expression of the MAP kinase to be down-regulated.\",\n \"74.A method according to claim 73 wherein the oligonucleotide is about 50 nucleotides or less in length.\",\n \"75.A method according to claim 74 wherein the oligonucleotide is 45 nucleotides in length or less.\",\n \"76.A method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising treating the cell with an antisense oligonucleotide for hybridising with at least part of a nucleic acid sequence coding for the binding domain of the MAP kinase for the integrin to thereby cause expression of the MAP kinase to be down-regulated, wherein the antisense oligonucleotide is an oligonucleotide as defined in claim 38.77.A method according to claim 73 wherein the cell is transfected with the antisense oligonucleotide or the antisense oligonucleotide is transcribed from a vector introduced into the cell.\",\n \"78.A method according to claim 73 wherein the cell is a cancer cell.\",\n \"79.A method according to claim 73 wherein the MAP kinase is an ERK family member or a JNK family member.\",\n \"80.A method according to claim 79 wherein the MAP kinase is ERK2.81.A method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: transfecting the cell with an expression vector incorporating a nucleic acid sequence encoding a MAP kinase of a fragment thereof, or a homolog, analog or variant of the MAP kinase or fragment, with a mutagenised binding domain for the integrin or in which the binding domain has been deleted whereby binding to the integrin is inhibited or prevented.\",\n \"82.A method according to claim 81 wherein the binding domain is mutagenised.\",\n \"83.A method according to claim 81 wherein the MAP kinase is an ERK family member of a JNK family member.\",\n \"84.A method according to claim 81 wherein the MAP kinase is ERK2.85.A method according to claim 81 wherein the integrin subunit is selected from the group consisting of β3, β5 and β6.86.A method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the integrin for the MAP kinase, wherein the agent is other than an antibody or a binding fragment thereof.\",\n \"87.A method according to claim 86 wherein the agent is a fragment consisting of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin, or an analog or derivative thereof capable of binding to the binding domain of the integrin.\",\n \"88.A method according to claim 86 wherein the agent is a polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase, or an analog or derivative thereof capable of binding to the binding domain of the integrin.\",\n \"89.A method according to claim 86 wherein the agent is a fusion protein incorporating a binding moiety capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin.\",\n \"90.A method according to claim 86 wherein the agent is an agent comprising: a targeting moiety for targeting a cell expressing the integrin; a binding moiety for binding to a binding domain of the integrin for the MAP kinase to thereby inhibit the binding of the MAP kinase to the integrin; and a carrier moiety for facilitating passage of the binding moiety across the cell membrane of the cell; wherein the binding moiety is other than an antibody or a binding fragment thereof.\",\n \"91.A method according to claim 86 wherein the activity of the cell is growth, migration, spreading, invasion, matrix-degrading enzyme secretion, differentiation and/or apoptosis of the cell.\",\n \"92.A method according to claim 86 wherein the cell is a cancer cell.\",\n \"93.A method according to claim 92 wherein the cancer cell is a colon cancer cell.\",\n \"94.A method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the MAP kinase, wherein the agent is other than an agent comprising a binding domain of the integrin for the MAP kinase or an analog or derivative thereof.\",\n \"95.A method according to claim 94 wherein the agent comprises an antibody or binding fragment thereof.\",\n \"96.A method according to claim 94 wherein the activity of the cell is growth, migration, spreading, invasion, matrix-degrading enzyme secretion, differentiation and/or apoptosis.\",\n \"97.A method according to claim 94 wherein the cell is a cancer cell.\",\n \"98.A method according to claim 94 wherein the cancer cell is a colon cancer cell.\",\n \"99.A method according to claim 73 comprising treating a mammal in need of such treatment.\",\n \"100.A method according to claim 73 wherein the method comprises treatment or prophylaxis of cancer or a condition associated with a predisposition to cancer.\",\n \"101.A method according to claim 100 wherein the cancer is selected from the group consisting of cancer of the lip, tongue, salivary glands, gums, floor and other areas of the mouth, oropharynx, nasopharynx, hypopharynx and other oral cavities, oesophagus, stomach, small intestine, duodenum colon, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung, breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid and skin.\",\n \"102.A method according to claim 101 wherein the cancer is colon cancer.\",\n \"103.A method of prophylaxis or treatment of cancer in a mammal, comprising: treating the mammal with an effective amount of an antisense nucleic acid for hybridising with a nucleic acid encoding a MAP kinase to cause expression of the MAP kinase to be down-regulated wherein the MAP kinase has a binding domain for an integrin.\",\n \"104.A method according to claim 103 comprising administering an effective amount of the antisense nucleic acid to the mammal.\",\n \"105.A method according to claim 103 comprising administering to the mammal an effective amount of a vector incorporating an nucleic acid sequence complementary to the antisense nucleic acid for generation of the antisense nucleic sequence in vivo.\",\n \"106.A method according to claim 103 wherein the antisense nucleic acid is an antisense oligonucleotide for hybridising with at least part of the sequence of the nucleic acid encoding the binding domain of the MAP kinase.\",\n \"107.A method according to claim 106 wherein the oligonucleotide has a length of 150 nucleotides or less.\",\n \"108.A method according to claim 107 wherein the oligonucleotide has a length of 90 nucleotides or less.\",\n \"109.A method according to claim 108 wherein the oligonucleotide has a length of 50 nucleotides or less.\",\n \"110.A method according to claim 109 wherein the oligonucleotide has a length of from 15 to 30 nucleotides.\",\n \"111.A method according to claim 103 wherein the integrin subunit is selected from the group consisting of β3, β5 and β6.112.A method according to claim 103 wherein the cancer is colon cancer.\",\n \"113.A fragment according to claim 9 wherein the integrin is selected from the group consisting of β3, β5 and β6.114.A polypeptide according to claim 15 wherein the integrin is selected from the group consisting of β3, β5 and β6.115.A vector incorporating an oligonucleotide as defined in claim 40.116.An expression vector incorporating an oligonucleotide as defined in claim 41 for being expressed in a cell.\",\n \"117.A host cell transformed with a vector as defined in claim 43.118.A pharmaceutical composition comprising an antisense nucleic acid sequence as defined in claim 39 together with a pharmaceutically acceptable carrier or diluent.\",\n \"119.A pharmaceutical composition comprising an oligonucleotide as defined in claim 41 together with a pharmaceutically acceptable carrier or diluent.\",\n \"120.An antibody according to claim 55 wherein the antibody is a monoclonal antibody.\",\n \"121.An antibody according to claim 56 wherein the antibody is a monoclonal antibody.\",\n \"122.A method according to claim 69 wherein the MAP kinase is an ERK family member or a JNK family member.\",\n \"123.A method according to claim 86 comprising treating a mammal in need of such treatment.\",\n \"124.A method according to claim 86 wherein the method comprises treatment or prophylaxis of cancer or a condition associated with a predisposition to cancer.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>Colorectal cancer is the commonest internal malignancy affecting men and women in Australia.\",\n \"About 4% of individuals develop this disease during the course of their lifetime and it was responsible for 14% of cancer deaths in that country in 1990.In 1995 there were 10,615 cases of colorectal cancer and 4508 deaths in Australia.\",\n \"Worldwide, an estimated 875,000 cases of colorectal cancer occurred in 1996, accounting for 8.5% of all new cases of cancer.\",\n \"Incidence rates vary approximately 20-fold around the world, with the highest rates seen in the developed world and lowest in India.\",\n \"Australian incidence rates are towards the higher end of the scale internationally alongside those for North America and New Zealand.\",\n \"Five-year survival following diagnosis of colon cancer is around 55% in the developed world and has altered little during the past few decades despite advances in chemo-, immuno- and radiotherapy.\",\n \"Colorectal cancer is a malignant tumour that starts in the bowel wall and is confined locally for a relatively long period before spreading through the bowel wall and metastasising to lymph nodes and other parts of the body.\",\n \"Survival rates are significantly improved where the disease is detected and treated early.\",\n \"The aetiology of colorectal cancer is complex and appears to involve interactions between inherited susceptibility and environmental factors.\",\n \"Recognition of the genetic component of colorectal cancer is growing.\",\n \"Mutations are present as inherited germline defects or arise in somatic cells secondary to environmental insult.\",\n \"There are two main inherited predisposition syndromes: Familial Adenomatous Polyposis (FAP) and Hereditary Non-Polyposis Colorectal Cancer (HNPCC); the remaining cases are attributed to so-called sporadic colorectal cancer.\",\n \"FAP and HNPCC contribute to approximately 1% and 4%, respectively, of all colorectal cancers and a strong family history of bowel cancer in first-degree relatives is obtained in another 10-15% of patients.\",\n \"However, in the vast majority of patients the aetiology of large bowel cancer remains unknown.\",\n \"Most colon cancer arises within pre-existing benign precursor lesions or adenomas.\",\n \"Adenomas are classified by histological architecture as tubular, tubulovillous or villous.\",\n \"Villous change is associated with a higher malignant potential, as are large and high-grade epithelial dysplasia.\",\n \"Environmental risk factors for development of colorectal cancer include diets low in fibre and vegetables and high in fat, red meat and alcohol and cigarette smoking which may induce mutations in somatic cells.\",\n \"Studies have shown that persistent genetic instability and accumulation of mutations in several genes that are mainly concerned with cell growth or DNA repair, may be critical for the development of all colorectal cancers.\",\n \"For example, a normal mucosal cell with inactivation of tumour suppressor genes such as the Adenomatous Polyposis Coli (APC) gene or Mutated in Colon Cancer (MCC) gene can proliferate and become a small adenomatous polyp.\",\n \"Mutations in oncogenes such as k ras and in tumour suppressor genes such as p53 and the Deleted in Colon Cancer gene (DCC) may then occur and lead to the transformation of the polyp into a large adenoma, from which a carcinoma can eventually arise.\",\n \"The uncontrolled cell growth that leads to the development of neoplasia is believed to result, therefore, from a series of inherited and acquired accumulated genetic changes.\",\n \"This multistep process confers on cells the capacity to survive and proliferate in a manner freed from the constraints imposed on normal cell growth.\",\n \"Spread of cancer cells involves tumour cell migration through the extracellular matrix scaffold, invasion of basement membranes, arrest of circulating tumour cells, and tumour cell extravasation and proliferation at metastatic sites.\",\n \"Detachment of cells from the primary tumour mass and modification of the peri-cellular environment aid penetration of tumour cells into blood and lymphatic vessels.\",\n \"It is the invasive and metastatic potential of tumour cells that ultimately dictates the fate of most patients suffering from malignant diseases.\",\n \"Hence, tumourigenesis can be viewed as a tissue remodelling process that reflects the ability of cancer cells to proliferate and digest surrounding matrix barriers.\",\n \"These events are thought to be regulated, at least in part, by cell adhesion molecules and matrix-degrading enzymes.\",\n \"Cell adhesion receptors on the surface of colon cancer cells are involved in complex cell signalling which may regulate cell proliferation, migration, invasion and metastasis and several families of adhesion molecules have now been identified including integrins, cadherins, the immunoglobulin superfamily, hyaluronate receptors, and mucins (Agrez, 1996.)\",\n \"In general, these cell surface molecules mediate both cell-cell and cell-matrix binding, the latter involving attachment of tumour cells to extracellular scaffolding molecules such as collagen, fibronectin and laminin.\",\n \"It is now clear that multiple and varied cell adhesion receptors exist on colon cancer cells at any one time and an understanding of the role of individual receptors in promoting growth and spread of colon cancer is only just beginning to be elucidated.\",\n \"Of all the families of cell adhesion molecules, the best-characterised at the present time is the family known as integrins.\",\n \"Integrins are involved in several fundamental processes including leucocyte recruitment, immune activation, thrombosis, wound healing, embryogenesis, virus internalisation and tumourigenesis.\",\n \"Integrins are transmembrane glycoproteins consisting of an alpha (α) and beta (β) chain in close association that provide a structural and functional bridge between extracellular matrix molecules and cytoskeletal components with the cell.\",\n \"The integrin family comprises 17 different α and 8β subunits and the αβ combinations are subsumed under 3 subfamilies.\",\n \"Excluding the leucocyte integrin subfamily that is designated by the β2 nomenclature, the remaining integrins are arranged into two major subgroups, designated β1 and αv based on sharing common chains.\",\n \"In the β1 subfamily, the β1 chain combines with any one of nine a chain members (α1-9), and the a chain which associates with β1 determines the matrix-binding specificity of that receptor.\",\n \"For example, α2β1 binds collagen and laminin, α3β1 binds collagen, laminin and fibronectin, and α5β1 binds fibronectin.\",\n \"In the αv subfamily of receptors, the abundant and promiscuous αv chain combines with any one of five β chains, and a distinguishing feature of αv integrins is that they all recognise and bind with high affinity to arginine-glycine-aspartate (RGD) sequences present in the matrix molecules to which they adhere (Hynes, 1992).\",\n \"The current picture of integrins is that the N-terminal domains of α and β subunits combine to form a ligand-binding head on each integrin.\",\n \"This head, containing the cation binding domains, is connected by two stalks representing both subunits, to the membrane-spanning segments and thus to the two cytoplasmic domains.\",\n \"The β subunits all show considerable similarity at the amino acid level (Loftus et al, 1994).\",\n \"All have a molecular mass between 90 and 110 kDa, with the exception of β4 which is larger at 210 kDa.\",\n \"Similarly, they all contain 56 conserved cysteine residues, except for β4 which has 48.These cysteines are arranged in four repeating patterns which are thought to be linked internally by disulphide bonds.\",\n \"The α-subunits have a molecular mass ranging from 150-200 kDa.\",\n \"They exhibit a lower degree of similarity than the β chains, although all contain seven repeating amino acid sequences interspaced with non-repeating domains.\",\n \"The β subunit cytoplasmic domain is required for linking integrins to the cytoskeleton (Hynes, 1992).\",\n \"In many cases, this linkage is reflected in localisation to focal contacts, which is believed to lead to the assembly of signalling complexes that include α-actinin, talin, and focal adhesion kinase (FAK) (Otey et al, 1990; Guan and Shalloway, 1992; Kornberg et al, 1992).\",\n \"At least three different regions that are required for focal contact localisation of β1 integrins have been delineated (Reszka et al, 1992).\",\n \"These regions contain conserved sequences that are also found in the cytoplasmic domains of the β2, β3, β5, β6 and β7 integrin subunits.\",\n \"The functional differences between these cytoplasmic domains with regard to their signalling capacity have not yet been established.\",\n \"Ligation of integrins by their extracellular matrix protein ligands induces a cascade of intracellular signals that include tyrosine phosphorylation of focal adhesion kinase, increases in intracellular Ca 2+ levels, inositol lipid synthesis, synthesis of cyclins and expression of immediate early genes.\",\n \"In contrast, prevention of integrin-ligand interactions suppresses cellular growth or induces apoptotic cell death (Meredith et al, 1993; Montgomery et al, 1994; Brooks et al, 1994; Varner et al, 1995; Boudreau et al, 1995).\",\n \"Thus, integrins play roles in a number of cellular processes that impact on the development of tumours, including the regulation of proliferation and apoptosis.\",\n \"The integrin β6 subunit was first identified in cultured epithelial cells as part of the αvβ6 heterodimer, and the αvβ6 complex was shown to bind fibronectin in an arginine-glycine-aspartate (RGD)-dependent manner in human pancreatic carcinoma cells (Sheppard et al, 1990; Busk et al, 1992).\",\n \"The β6 subunit is composed of 788 amino acids and shares 34-51% sequence homology with other β integrin subunits β1-β5.The β6 subunit also contains 9 potential glycosylation sites on the extracellular domain (Sheppard et al, 1990).\",\n \"The cytoplasmic domain differs from other β subunits in that it is composed of a 41 amino acid region that is highly conserved among integrin β subunits, and a unique 11 amino acid carboxy-terminal extension.\",\n \"The 11 amino acid extension has been shown not to be necessary for localisation of β6 to focal contacts; in fact, its removal appears to increase receptor localisation.\",\n \"However, removal of any of the three conserved regions previously identified as important for the localisation of β1 integrins to focal contacts (Reszka et al, 1992) has been shown to eliminate recruitment of β6 to focal contacts (Cone et al, 1994).\",\n \"The integrin αvβ6 has previously been shown to enhance growth of colon cancer cells in vitro and in vivo, and this growth-enhancing effect is due, at least in part, to αvβ6-mediated gelatinase B secretion (Agrez et al, 1994; Niu et al, 1998 Agrez et al, 1999).\",\n \"What makes this epithelial-restricted integrin of particular interest in cancer is that it is not expressed on normal epithelial cells but is highly expressed during would healing and tumourigenesis, particularly at the invading edge of tumour cell islands (Breuss et al, 1995; Agrez et al, 1996).\",\n \"Further more, αvβ6 has been found to induce its own expression in an autocrine manner with cell crowding and a self-perpetuating model of colon cancer progression regulated by αvβ6-mediated gelatinase B secretion has been proposed (Niu et al, 2000).\",\n \"Invasion of the extracellular matrix and metastatic spread of colon cancer is also likely to reflect the ability of tumour cells to digest their surrounding matrix scaffold through secretion of matrix-degrading enzymes such as matrix metalloproteinases (MMPs).\",\n \"The mechanisms whereby human colon cancer cells escape the constraints on growth imposed on normal cells by cell crowding and dense pericellular matrices is unclear.\",\n \"However, even colon cancer cells are subject to relative growth inhibition in vitro in a dense extracellular matrix environment (Agrez, 1989).\",\n \"Integrins can signal through the cell membrane in either direction.\",\n \"The extracellular binding activity of integrins can be regulated from the cell interior as, for example, by phosphorylation of integrin cytolasmic domains (inside-out signalling), while the binding of the extracellular matrix (ECM) elicits signals that are transmitted into the cell (outside-in signalling) (Giancotti and Ruoslahti, 1999).\",\n \"Outside-in signalling can be roughly divided into two descriptive categories.\",\n \"The first is ‘direct signalling’ in which ligation and clustering of integrins is the only extracellular stimulus.\",\n \"Thus, adhesion to ECM proteins can activate cytoplasmic tyrosine kinases (e.g.\",\n \"focal adhesion kinase FAK) and serine/threonine kinases (such as those in the mitogen-activated protein kinase (MAPK) cascade) and stimulate lipid metabolism (eg phosphatidylinositol-4,5-biphosphate (P 1 P 2 ) synthesis.\",\n \"The second category of integrin signalling is ‘collaborative signalling’, in which integrin-mediated cell adhesion modulates signalling events initiated through other types of receptors, particularly receptor tyrosine kinases that are activated by polypeptide growth factors (Howe et al, 1998).\",\n \"In all cases, however, integrin-mediated adhesion seems to be required for efficient transduction of signals into the cytosol or nucleus.\",\n \"MAP kinases behave as a convergence point for diverse receptor-initiated signalling events at the plasma membrane.\",\n \"The core unit of MAP kinase pathways is a three-member protein kinase cascade in which MAP kinases are phosphorylated by MAP kinase kinases (MEKs) which are in turn phosphorylated by MAP kinase kinases (e.g.\",\n \"Raf-1) (Garrington and Johnson, 1999).\",\n \"Amongst the 12 member proteins of the MAP kinase family are the extracellular signal-regulated kinases (ERKs) (Boulton et al, 1991) activated by phosphorylation of tyrosine and threonine residues (Payne et al, 1991) which is the type of activation common to all known MAP kinase isoforms.\",\n \"ERK 1/2 (44 kD and 42 kD MAPks, respectively) share 90% amino acid identity and are ubiquitous components of signal transduction pathways (Boulton et al, 1991).\",\n \"These serine/threonine kinases phosphorylate and modulate the function of many proteins with regulatory functions including other protein kinases (such as p90 rsk ) cytoskeletal proteins (such as microtubule-associated phospholipase A 2 ), upstream regulators (such as the epidermal growth factor receptor and Ras exchange factor) and transcription factors (such as C-Myc and Elk-1).\",\n \"ERKs play a major role in growth-promoting events, especially when the concentration of growth factors available to a cell is limited (Giancotti and Ruoslahti, 1999).\",\n \"MAP kinases can be activated through non-receptor tyrosine kinases such as focal adhesion kinase (FAK), cytoplasmic tyrosine kinase (pp60 c-srk) (Schlaepher and Hunter, 1998), and growth factors acting through membrane-associated receptor tyrosine kinases.\",\n \"The FAK pathway is activated by most integrins.\",\n \"In addition to activating FAK, some β1 and αv integrins also activate the tyrosine kinase Fynu and through it, the adaptor protein Shc (Wang et al, 1996).\",\n \"It is likely that both FAK and Shc contribute to the activation of Ras and thence to the downstream kinase cascade of Raf-1, MEK, and MAP kinases (Schlaepfer et al, 1994; 1997).\",\n \"It is now generally accepted that the activation of ERK in response to integrin ligation requires Ras signalling (Wary et al, 1996; Schlaepfer and Hunter, 1997).\",\n \"The laminin receptor α6β4, the laminin/collagen receptor α1β1, the fibronectin receptor α5β1 and the RGD binding receptor αvβ3 are linked to the Ras-Raf-MEK-ERK signalling pathway and control of immediate early gene expression (Wary et al, 1996; Maniero et al, 1995; 1997).\",\n \"The ability of integrins to activate ERK may be especially important when the concentration of growth factors available to the cell is limited.\",\n \"In this setting, proliferation is likely to require co-stimulation of ERK through integrins and growth factor receptors (Giancotti and Ruoslahti, 1999).\",\n \"Moreover, activation of ERK in response to integrin ligation may play a role in regulating cell migration (Klemke et al, 1997) possibly by initiating matrix-degrading enzyme secretion.\",\n \"While there is a good deal of evidence in support of a key role for FAK and the phosphotyrosine-domain-containing adaptor protein Shc (Howe et al, 1998; Giancotti & Ruoslahti, 1999) in the Ras-Raf MEK-MAP kinase activation pathway there are also data implicating alternate pathways independent of MEKs.\",\n \"For example, MEK-independent regulation of MAP kinase activation in NIH3T3 fibroblasts has been shown to be mediated by phosphatidylinositol-3-kinases and the conventional protein kinase C (PKC) isoforms and is thought to be due to inactivation of a MAP kinase inhibitor (Grammer and Blenis, 1997).\",\n \"Although the mechanism by which PKC regulates integrin function is not known, PKC has been shown to regulate integrin-induced activation of the MAP kinase pathway upstream of Shc.\",\n \"For example, PKC inhibition has been shown to inhibit ERK2 activation by fibronectin receptors without any effect on integrin-induced FAK or paxillin tyrosine phosphorylation (Miranti et al, 1999).\",\n \"Hence, MAP kinase activation is more complicated than a simple linear pathway, and the mechanistic basis for the commonly observed integrin-mediated activation of MAP kinases remains controversial.\",\n \"The importance of the MAP kinase pathway in promoting colon cancer growth in vivo is now no longer in question.\",\n \"Although relatively little is known about the role of MAP kinase activation in for instance, colon tumour progression, MAP kinase pathways have been shown to be highly activated during the late progression of colorectal cancer (Licato et al, 1997).\",\n \"In a recent breakthrough in this field, a highly potent inhibitor of MAP kinase activation has been identified which is capable of inhibiting human colon cancer growth in immune-deficient mice raising hops for the clinical application of MAP kinase inhibitors in the treatment of colon cancer (Sebolt-Leopold et at, 1999).\",\n \"Various intracellular proteins may be linked directly or spatially to integrin cytoplasmic domains.\",\n \"Direct interactions have been identified between cytoskeletal proteins such as α-actinin and talin and β1 and β3 integrin tails (Horwitz et al, 1986; Otey et al, 1990; Knezevic et al, 1996; Pfaff et al, 1998).\",\n \"A direct association between FAK and the β1 integrin tail has been suggested based on in vitro β1 peptide studies, but this remains to be confirmed (Schaller et al, 1995).\",\n \"More recently, the cytoplasmic domain of the α4 subunit has been found to be physically associated with the signalling adaptor protein paxillin in Jurkat T cells, and this binding event regulates the kinetics of FAK tyrosine phosphorylation (Liu et al, 1999).\",\n \"Direct integrin links with the intracellular calcium-binding protein, calreticulin, and integrin-linked kinase (ILK) (Hannigan et al, 1996) have been shown to regulate “inside-out” integrin signalling.\",\n \"For example, calreticulin has been shown to bind to a chain cytoplasmic domains (Rojiani et al, 1991) and modify α2β1 integrin activation by phorbol esters and anti-integrin antibodies (Coppolino et al, 1995).\",\n \"Newly identified integrin-binding molecules include the serine/threonine integrin-linked kinase, ILK, which can associate with the β1, β2 and β3 subunits.\",\n \"When over-expressed, ILK has been shown to reduce anchorage-independent growth and tumourigenicity in nude mice (Hannigan et al, 1996).\",\n \"Co-immunoprecipitation strategies have also demonstrated interactions between integrins and the integral plasma membrane protein IAP, and members of the four transmembrane domain protein family (tetraspans).\",\n \"The extracellular Ig region of the IAP molecule mediates association with αvβ3 and is required for cell binding to vitronectin-coated particles (Lindberg et al, 1996).\",\n \"An emerging model for tetraspans is that they recruit signalling enzymes such as phosphatidylinositol-4-kinase and PKC into complexes with integrins (Hemler, 1998).\",\n \"Integrins have also been shown to be physically linked with matrix-degrading enzymes and growth factors.\",\n \"For example, the integrin αvβ6 has been shown to bind and activate latent TGFβ1 in keratinocytes (Munger et al, 1999) which is thought to be important in modulating the inflammatory process following epithelial injury.\",\n \"In melanoma cells, αvβ3 binds activated gelatinase A (Brooks et al, 1996), and both insulin and platelet-derived growth factor (PDGF) co-immunoprecipitate with this integrin in NIH3T3 mouse fibroblasts (Schneller et al, 1997).\",\n \"Synergism between integrin-mediated signalling processes and growth factor responses is now well-recognised and Schneller et al (1997) showed that a small subset of each of the insulin receptor and PDGF β-receptor is tyrosine phosphorylated upon growth factor stimulation.\",\n \"Interestingly, this subset can associate with the αvβ3 integrin, and PDGF activity is enhanced in association with increased MAP kinase activity in cells plated on the αvβ3 ligand, vitronectin.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>Broadly stated, the present invention relates to the surprising finding that members of the mitogen activated protein (MAP) kinase family can associate with the cytoplasmic domain of an integrin molecule.\",\n \"It is believed that no member of the MAP kinase family has previously been found to directly associate with any integrin.\",\n \"The identification of this functional relationship permits the rational design of agents for therapeutically or prophylactically modulating cellular activity mediated by the MAP kinase and integrin interaction.\",\n \"In an aspect of the present invention there is provided an agent capable of binding to a binding domain of an integrin for a MAP kinase.\",\n \"In another aspect of the present invention there is provided an isolated or purified polypeptide capable of binding to a binding domain of an integrin for a MAP kinase or a homolog, analog, variant or derivative of the polypeptide.\",\n \"The polypeptide may consist of or comprise the binding domain of the MAP kinase to which the integrin binds or sufficient core amino acid sequence of the binding domain of the MAP kinase to enable binding of the polypeptide to the integrin.\",\n \"Preferably, the polypeptide will be a fragment of a MAP kinase.\",\n \"Most preferably, the polypeptide will comprise the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or PSNLLLNTTCDLKIC or a region or regions of such sequences.\",\n \"Accordingly, in a further aspect of the present invention there is provided a fragment of a MAP kinase wherein the fragment is capable of binding with an integrin, or a homolog, analog, variant or derivative of the fragment.\",\n \"In another aspect of the present invention there is provided a fragment of a MAP kinase wherein the fragment consists of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin.\",\n \"In another aspect of the present invention there is provided an isolated or purified polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase.\",\n \"Preferably, a polypeptide or fragment of the invention will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less and more usually about 40 amino acids or less.\",\n \"Typically, the length will be between about 5 to about 30 amino acids.\",\n \"In yet another aspect of the present invention there is provided a fusion protein a fusion protein incorporating a binding moiety capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin.\",\n \"Preferably, the binding moiety will comprise a fragment or polypeptide of the invention.\",\n \"Most preferably, the fusion protein will incorporate a carrier moiety for facilitating passage across the cell membrane.\",\n \"The carrier moiety may be penetratin.\",\n \"In still another aspect of the present invention there is provided an agent for inhibiting binding of a MAP kinase to an integrin, comprising: a targeting moiety for targeting a cell expressing the integrin; a binding moiety for binding to a binding domain of the integrin for the MAP kinase to thereby inhibit the binding of the MAP kinase to the integrin; and a carrier moiety for facilitating passage of the binding moiety across the cell membrane of the cell; wherein the binding moiety is other than an antibody or an binding fragment thereof.\",\n \"In another aspect of the present invention there is provided a MAP kinase or a fragment thereof with a mutagenised binding domain for an integrin or in which the binding domain is deleted, or a homolog, analog or variant of the MAP kinase or fragment, wherein capability to bind with the integrin is thereby reduced.\",\n \"In another aspect of the present invention there is provided an isolated nucleic acid sequence encoding a fragment or polypeptide of the invention or a homolog, analog, variant or derivative of the fragment or polypeptide.\",\n \"In another aspect of the invention there is provided a nucleic acid sequence coding for a MAP kinase or fragment thereof having a mutagenised binding domain for an integrin or in which the binding domain is deleted or a homolog, analog or variant of the MAP kinase or fragment, wherein capability to bind with the integrin is thereby reduced.\",\n \"In a still further aspect there is provided an isolated nucleic acid sequence encoding a fusion protein or agent of the invention.\",\n \"There are also provided antisense nucleic acid sequences complimentary to the sense nucleic sequences of the invention.\",\n \"Such antisense sequences find application in antisense therapy of cells in which down regulation of cellular activity is desired, and include oligonucleotides.\",\n \"Sense oligonucleotides coding for all or part of the binding domain of a MAP kinase for an integrin, or that of a homolog, analog or variant thereof, and complimentary antisense oligonucleotides find particular application as primers or probes.\",\n \"A nucleic acid primer or probe of the invention may be labelled with a suitable reporter molecule for enabling detection of hybridisation of the primer or probe to a target nucleic acid sequence.\",\n \"In yet another aspect of the present invention there is provided a vector incorporating a nucleic acid sequence of the invention.\",\n \"Typically, the vector will be an expression vector and the nucleic acid sequence will be capable of being transcribed.\",\n \"In a further aspect of the present invention there is provided a host cell transformed with a vector of the invention.\",\n \"Preferably, the host cell will be selected from the group consisting of a mammalian cell, an epithelial cell, a neoplastic cell, and a cancer cell.\",\n \"Preferably, the host cell will be a mammalian cell and most preferably, a colon cancer cell.\",\n \"In yet another aspect of the present invention there is provided an antibody generated with the use of a polypeptide, fragment, agent or fusion protein of the invention.\",\n \"In a still further aspect of the invention there is provided an antibody and binding fragment thereof capable of binding to a binding domain of a MAP kinase for an integrin.\",\n \"The antibody may be a polyclonal or monoclonal antibody.\",\n \"Preferably, the antibody is a monoclonal antibody.\",\n \"In still another aspect of the present invention there is provided a method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing a number of agents for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if any said agent is capable of binding to the binding domain of the MAP kinase on the basis of the testing.\",\n \"In yet another aspect of the invention there is provided a method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing a number of agents for ability to bind to the MAP kinase; (b) selecting an agent or agents identified as being able to bind to the MAP kinase on the basis of the testing; and (c) utilising the selected said agent or agents in an assay for indicating whether the or any of the selected said agents is capable of binding to the binding domain of the MAP kinase for the integrin.\",\n \"In another aspect of the present invention there is provided a method of evaluating whether an agent is capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing the agent for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if the agent is capable of binding to the binding domain on the basis of the testing.\",\n \"Preferably, a polypeptide or fragment of the invention comprising or consisting of the binding domain of the MAP kinase or core amino acid sequence of the binding domain or a homolog, analog, variant or derivative of the polypeptide or fragment or core amino acid sequence is used in the testing or assaying for whether an agent is capable of binding to the binding domain of the MAP kinase.\",\n \"Most preferably, the polypeptide or fragment will comprise or consist of the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or PSNLLLNTTCDLKIC, core amino acid sequence of such sequences capable of binding to an integrin, or a homolog variant, analog or derivative of such sequences.\",\n \"Testing or assaying of an agent for ability to bind to the binding domain of the MAP kinase for the integrin and thereby inhibit binding of the MAP kinase to the integrin, may comprise exposing the MAP kinase to the agent(s) to enable binding of the agent(s) to the MAP kinase to occur either in the presence of the integrin or prior to the addition of the integrin.\",\n \"Rather than using the MAP kinase, a polypeptide, fragment of a MAP kinase or other molecule capable of binding to the binding site on the integrin for the MAP kinase may be used in such testing or assaying.\",\n \"Similarly, rather than utilising an intact integrin, a polypeptide or integrin fragment comprising or incorporating the binding domain of the integrin for the MAP kinase, or other molecule capable of binding with the binding domain on the MAP kinase for the integrin, may be utilised.\",\n \"In still another aspect of the present invention there is provided a method of isolating an agent from a sample utilising a molecule immobilised on a solid support and which is capable of binding to a binding domain of an integrin for a MAP kinase, comprising: (a) contacting the molecule immobilised on the solid support with the sample under conditions suitable for binding of the agent to the molecule; (b) eluting the agent from the solid support; and (c) collecting the eluted agent; wherein the molecule is other than an antibody or binding fragment of an antibody.\",\n \"The molecule may be the MAP kinase, or a fusion protein, a polypeptide, or a fragment of the invention to which the integrin is capable of binding, or for instance a homolog, analog, variant or derivative of such a polypeptide or fragment.\",\n \"In yet another aspect of the present invention there is provided the agent so isolated.\",\n \"An agent of the invention will usually be provided in the form of a pharmaceutical composition.\",\n \"Accordingly, in another aspect of the present invention there is provided a pharmaceutical composition comprising an agent of the invention capable of binding to a binding domain of an integrin for a MAP kinase, and a pharmaceutically acceptable carrier or diluent.\",\n \"The agent may or not be proteinaceous in nature.\",\n \"Preferably, the agent will comprise a fusion protein, or polypeptide.\",\n \"Most preferably, the agent will be coupled to a carrier molecule for facilitating entry of the agent into a cell.\",\n \"In a further aspect of the invention there is provided a pharmaceutical composition comprising a nucleic acid sequence of the invention and a pharmaceutically acceptable carrier or diluent.\",\n \"Preferably, the nucleic acid sequence is incorporated into a vector as described herein.\",\n \"Alternatively, the nucleic acid sequence may be joined to a carrier molecule such as penetratin for facilitating entry of the nucleic acid sequence into a target cell.\",\n \"In another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: transfecting the cell with an expression vector incorporating a nucleic acid sequence encoding a MAP kinase of a fragment thereof, or a homolog, analog or variant of the MAP kinase or fragment, for being expressed by the cell and having a mutagenised binding domain for the integrin or in which the binding domain has been deleted whereby binding to the integrin is inhibited or prevented.\",\n \"In still another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: transfecting the cell with a nucleic acid sequence encoding a fragment of a MAP kinase, or a homolog, analog or variant of the fragment, for being expressed by the cell and capable of binding to the binding domain of an integrin for the MAP kinase.\",\n \"In yet another aspect of the present invention there is provided a method of modulating activity of a cell, comprising: transfecting the cell with a nucleic acid encoding a polypeptide for being expressed by the cell wherein the polypeptide is capable of binding to a binding domain of an integrin for a MAP kinase.\",\n \"In still another aspect of the invention there is provided a method of modulating activity of a cell, comprising causing the expression of a MAP kinase to which an integrin is able to bind to be down-regulated.\",\n \"Preferably, down-regulation of the expression of the MAP kinase is achieved using an antisense nucleic acid sequence that inhibits expression of gene coding for the MAP kinase.\",\n \"The antisense nucleic acid sequence may be administered to the cell or generated within the cell.\",\n \"Preferably, the cell will be transformed with a vector of the invention for generation of the antisense nucleic acid sequence.\",\n \"Preferably, the antisense nucleic acid sequence will specifically hybridise with sense nucleic acid sequence coding for at least part of the binding domain of the MAP kinase for the integrin and/or intron sequence between such coding sequence.\",\n \"Accordingly, in another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an antisense oligonucleotide for hybridising with at least part of a nucleic acid sequence coding for the binding domain of a MAP kinase for an integrin to thereby cause expression of the MAP kinase to be down-regulated.\",\n \"In another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the integrin for the MAP kinase, wherein the agent is other than an antibody or a binding fragment thereof.\",\n \"In a further aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the MAP kinase, wherein the agent is other than an agent comprising a binding domain of the integrin for the MAP kinase or an analog or derivative thereof.\",\n \"In yet another aspect of the invention there is provided a method of modulating the activity of a cell, comprising: contacting the cell with an effective amount of a fusion protein, antibody or other molecule of the invention capable of binding to the binding domain of an integrin for a MAP kinase.\",\n \"Preferably, modulation of the activity of a cell by a method as described herein will comprise treating a mammal in need of such treatment.\",\n \"Accordingly, in a further aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid sequence encoding a MAP kinase or a fragment thereof or a homolog, analog or variant of the MAP kinase or fragment, with a mutagenised binding domain for an integrin or in which the binding domain has been deleted and wherein the nucleic acid sequence is capable of being expressed.\",\n \"In another aspect of the invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid capable of causing the expression of a MAP kinase to which an integrin is able to bind to be down regulated.\",\n \"In a further aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal a nucleic acid sequence encoding a fragment of a MAP kinase, or a homolog, analog or variant of the fragment, for being expressed by the cell and capable of binding to the binding domain of an integrin for the MAP kinase.\",\n \"In still another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid sequence for expression of a polypeptide or a fragment of a MAP kinase within the cell, wherein the polypeptide or fragment is capable of binding to a binding domain of an integrin for the MAP kinase to thereby modulate the cellular activity.\",\n \"In another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of an agent capable of binding to the binding domain of an integrin for a MAP kinase, wherein the agent is other than an antibody or a binding fragment thereof.\",\n \"In another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of an agent capable of binding to the binding domain of a MAP kinase for an integrin wherein the agent is other than an agent comprising the binding domain of the integrin for the MAP kinase or an analog or derivative thereof.\",\n \"In another aspect of there is provided a method of treatment or prophylaxis of a disease or condition in a mammal, wherein the disease or condition is responsive to an agent capable of binding to a binding domain of an integrin for a MAP kinase and wherein the agent comprises a polypeptide or fragment of the invention or a homolog, variant, analog or derivative of such a polypeptide or fragment, and the method comprises administering an effective amount of the agent to the mammal.\",\n \"In yet another aspect of the present invention there is provided a method of treatment or prophylaxis of a disease or condition in a mammal, wherein said condition is responsive to an agent capable of binding to a binding domain of an integrin for a MAP kinase wherein the agent is a fusion protein of the invention.\",\n \"In another aspect there is provided use of a nucleic acid of the invention in the manufacture of a medicament for administration to a mammal in the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable.\",\n \"In a still further aspect of the invention there is provided the use of an agent capable of binding to a binding domain of an integrin for a MAP kinase in the manufacture of a medicament for administration to a mammal for the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable, wherein the agent is other than an antibody or binding fragment thereof.\",\n \"The agent may for instance be a polypeptide, fragment or the like as described herein.\",\n \"In another aspect of the present invention there is provided the use of an agent capable of binding to the binding domain of a MAP kinase for an integrin wherein the agent is other than an agent comprising the binding domain of the integrin for the MAP kinase or an analog or derivative thereof.\",\n \"In another aspect there is provided the use of fusion protein of the invention in the manufacture of a medicament for administration to a mammal for the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable.\",\n \"Methods as described above will typically comprise treatment or prophylaxis of cancer or a condition associated with a predisposition to cancer.\",\n \"The cancer may for instance be selected from the group consisting of cancer of the lip, tongue, salivary glands, gums, floor and other areas of the mouth, oropharynx, nasopharynx, hypopharynx and other oral cavities, oesophagus, stomach, small intestine, duodenum, colon, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung, breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid and skin.\",\n \"In still another aspect of the present invention there is provided a method of causing an activity of a cell to be upregulated, comprising: contacting the cell with an effective amount of a nucleic acid capable of being expressed within the cell and coding for an agent capable of binding to a binding domain of a MAP kinase for an integrin or a binding domain of the integrin for the MAP kinase.\",\n \"In a still further aspect of the invention there is provided a method of causing an activity of a cell to be upregulated, comprising contacting the cell with an effective amount of an agent capable of binding to a binding domain of a MAP kinase for an integrin or a binding domain of the integrin for the MAP kinase.\",\n \"Typically, the agent will be a polypeptide or fragment of an integrin comprising or consisting of the binding domain of an integrin for a MAP kinase or comprising or consisting of the core amino acid sequence of the binding domain directly involved in the binding to the MAP kinase, or a homolog, variant, analog or derivative of such polypeptide or fragment.\",\n \"Preferably, the polypeptide or fragment will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less and more usually about 40 amino acids or less.\",\n \"Typically, the length will be between about 5 to about 30 amino acids.\",\n \"Most preferably, the polypeptide will comprise or consist of the amino acid sequence RSKAKWQTGTNPLYR or RSKAKNPLYR.\",\n \"Up regulation of cellular activity is particularly desirable for instance in wound healing, re-endothelialisation within natural or artificial blood vessels, nerve growth and for instance induction of labour.\",\n \"The cellular activity desired to be modulated will typically but not exclusively, be cell growth or proliferation.\",\n \"Indeed, any activity mediated by MAP kinase signalling is expressly included within the scope of the invention.\",\n \"The cell may be any cell type in which functional activity arising from signalling mediated by a MAP kinase may occur.\",\n \"Preferably, the cell will be an epithelial cell and usually, a neoplastic cell.\",\n \"Usually, the MAP kinase will be selected from the group consisting of an extracellular signal-regulated kinase (ERK) and a JNK MAP kinase.\",\n \"Preferably, the MAP kinase is ERK2 or JNK-1.Most preferably, the MAP kinase is ERK2.The mammal may be any mammal treatable with a method of the invention.\",\n \"For instance, the mammal may be a member of the bovine, porcine, ovine or equine families, a laboratory test animal such as a mouse, rabbit, guinea pig, a cat or dog, a primate or a human being.\",\n \"Preferably, the mammal will be a human being.\",\n \"As will be understood, modulation of cellular activity within the context of the invention may be up regulation or down regulation of cellular activity.\",\n \"Reference to “down modulation” or “down-regulating” or the like should be understood to include preventing, reducing or otherwise inhibiting one or more aspects of the activity of the cell.\",\n \"Conversely, reference to “up regulation” or like terms should be understood to include enhancing or increasing one or more aspects of the activity of the cell.\",\n \"In the broadest sense, the term “integrin” unless otherwise specified, is to be taken to encompass an integrin family member or a homolog, derivative, variant or analog of an integrin subunit, or an integrin family member incorporating at least one such homolog, derivative, variant or analog of an integrin subunit.\",\n \"Usually, the integrin will be a member of the αv subfamily.\",\n \"Preferably, the integrin is or incorporates an integrin subunit selected from the group consisting of β3, β5 and β6.Most preferably, the integrin comprises β6.By “binding domain” is meant the minimum length of contiguous amino acid sequence required for binding.\",\n \"By “core amino acid sequence” is meant regions or amino acids of the binding domain that directly participate in the binding as distinct from any amino acids that do not directly participate in the binding interaction.\",\n \"Typically, the core amino acid sequence of the binding domain will comprise regions of the binding domain linked together by a number of intervening amino acids which do not directly participate in the binding.\",\n \"The term “homolog” is to be taken to mean a molecule that has amino acid sequence similarity.\",\n \"The homology between amino acid sequences can be determined by comparing amino acids at each position in the sequences when optimally aligned for the purpose of comparison.\",\n \"The sequences are considered homologous at a position if the amino acids at that position are the same.\",\n \"Typically, a homolog will have an overall amino acid sequence homology of at least about 30% more preferably at least about 50% or 70% and most preferably, greater than about 80%, 90% or 98% sequence homology.\",\n \"Homology with a binding domain may be greater than the overall amino acid sequence homology of the homolog, and will usually be greater than about 60% or 80%, and more usually greater than about 90%, 95% or 98%.\",\n \"The term “analog” is to be taken to mean a molecule that has one or more aspects of biological function characteristic of the molecule on which at least part of the analog is based or which was otherwise utilised in the design or preparation of the analog.\",\n \"An analog may have substantial overall structural similarity with the molecule or only structural similarity with one or more regions or domains thereof responsible for the desired characteristic biological function.\",\n \"By “structural” similarity is meant similarity in shape, conformation and/or other structural features responsible for the provision of the biological function or which otherwise have involvement in the provision of the biological function.\",\n \"Alternatively, it will be understood that with knowledge of the region(s) or domain(s) of a molecule that provide(s) the characteristic biological function, analogs may be designed that while differing in structure nevertheless possess such biological function.\",\n \"Indeed, it is not necessary that an analog have amino acid sequence homology, and an analog may not be proteinaceous at all.\",\n \"An analog may for instance be a mimetic of a molecule.\",\n \"By the term “variant” is meant an isoform, an allelic variant of a gene or region thereof, a naturally occurring mutant form of a gene or region thereof, or a polypeptide or fragment having an amino acid sequence that differs in one or more amino acids but which retains one or more aspects of desired characteristic biological function.\",\n \"This may be achieved by the addition of one or more amino acids to an amino acid sequence, deletion of one or more amino acids from an amino acid sequence and/or the substitution of one or more amino acids with another amino acid or amino acids.\",\n \"Inversion of amino acids and any other mutational change that results in alteration of an amino acid sequence are also encompassed.\",\n \"A variant may be prepared by introducing nucleotide changes in a nucleic acid sequence such that the desired amino acid changes are achieved upon expression of the mutagenised nucleic acid sequence, or for instance by synthesising an amino acid sequence incorporating the desired amino acid changes which possibility is well within the capability of the skilled addressee.\",\n \"Substitution of an amino acid may involve a conservative or non-conservative amino acid substitution.\",\n \"By conservative amino acid substitution is meant replacing an amino acid residue with another amino acid having similar stereochemical properties (eg.\",\n \"structure, charge, acidity or basicity characteristics) and which does not substantially effect conformation or the desired aspect or aspects of characteristic biological function.\",\n \"Preferred variants include ones having amino acid sequences in which one or more amino acids have been substituted with alanine or other neutrally charged amino acid residue(s), or to which one or more such residues have been added.\",\n \"A variant may also incorporate an amino acid or amino acids that are not encoded by the genetic code.\",\n \"By the term “derivative” is meant a molecule that is derived or obtained from another molecule and which retains one or more aspects of characteristic biological function of that molecule.\",\n \"A derivative may for instance arise as a result of the cleavage of the parent molecule, cyclisation and/or coupling with one or more additional moieties that improve solubility, lipophilic characteristics to enhance uptake by cells, stability or biological half-life, decreased cellular toxicity, or for instance to act as a label for subsequent detection or the like.\",\n \"A derivative may also result from post-translational or post-synthesis modification such as the attachment of carbohydrate moieties or chemical reaction(s) resulting in structural modification(s) such as the alkylation or acetylation of amino acid residues or other changes involving the formation of chemical bonds.\",\n \"The term “polypeptide” is used interchangeably herein with “peptide” and encompasses amino acid sequences incorporating only a few amino acid residues or many amino acid residues coupled by peptide bonds.\",\n \"The term “neoplastic cell” is to be taken to mean a cell exhibiting abnormal growth and may or may not be a malignant cell.\",\n \"“Growth” is to be taken in its broadest sense and includes proliferation of the cell.\",\n \"In this regard, an example of abnormal cell growth is the uncontrolled proliferation of a cell.\",\n \"Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.\",\n \"The features and advantages of the present invention will become further apparent from the following detailed description of preferred embodiments and the accompanying drawings.\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention relates to a method of modulating cell activity mediated by a mitogen activated protein kinase (MAP) and more particularly, modulation of the growth and/or the proliferation of a cell such as a neoplastic cell.\",\n \"In particular, the invention finds use inter alia in applications requiring modulation of cell activity.\",\n \"The present invention also provides agents for use in the above.\",\n \"BACKGROUND OF THE INVENTION Colorectal cancer is the commonest internal malignancy affecting men and women in Australia.\",\n \"About 4% of individuals develop this disease during the course of their lifetime and it was responsible for 14% of cancer deaths in that country in 1990.In 1995 there were 10,615 cases of colorectal cancer and 4508 deaths in Australia.\",\n \"Worldwide, an estimated 875,000 cases of colorectal cancer occurred in 1996, accounting for 8.5% of all new cases of cancer.\",\n \"Incidence rates vary approximately 20-fold around the world, with the highest rates seen in the developed world and lowest in India.\",\n \"Australian incidence rates are towards the higher end of the scale internationally alongside those for North America and New Zealand.\",\n \"Five-year survival following diagnosis of colon cancer is around 55% in the developed world and has altered little during the past few decades despite advances in chemo-, immuno- and radiotherapy.\",\n \"Colorectal cancer is a malignant tumour that starts in the bowel wall and is confined locally for a relatively long period before spreading through the bowel wall and metastasising to lymph nodes and other parts of the body.\",\n \"Survival rates are significantly improved where the disease is detected and treated early.\",\n \"The aetiology of colorectal cancer is complex and appears to involve interactions between inherited susceptibility and environmental factors.\",\n \"Recognition of the genetic component of colorectal cancer is growing.\",\n \"Mutations are present as inherited germline defects or arise in somatic cells secondary to environmental insult.\",\n \"There are two main inherited predisposition syndromes: Familial Adenomatous Polyposis (FAP) and Hereditary Non-Polyposis Colorectal Cancer (HNPCC); the remaining cases are attributed to so-called sporadic colorectal cancer.\",\n \"FAP and HNPCC contribute to approximately 1% and 4%, respectively, of all colorectal cancers and a strong family history of bowel cancer in first-degree relatives is obtained in another 10-15% of patients.\",\n \"However, in the vast majority of patients the aetiology of large bowel cancer remains unknown.\",\n \"Most colon cancer arises within pre-existing benign precursor lesions or adenomas.\",\n \"Adenomas are classified by histological architecture as tubular, tubulovillous or villous.\",\n \"Villous change is associated with a higher malignant potential, as are large and high-grade epithelial dysplasia.\",\n \"Environmental risk factors for development of colorectal cancer include diets low in fibre and vegetables and high in fat, red meat and alcohol and cigarette smoking which may induce mutations in somatic cells.\",\n \"Studies have shown that persistent genetic instability and accumulation of mutations in several genes that are mainly concerned with cell growth or DNA repair, may be critical for the development of all colorectal cancers.\",\n \"For example, a normal mucosal cell with inactivation of tumour suppressor genes such as the Adenomatous Polyposis Coli (APC) gene or Mutated in Colon Cancer (MCC) gene can proliferate and become a small adenomatous polyp.\",\n \"Mutations in oncogenes such as k ras and in tumour suppressor genes such as p53 and the Deleted in Colon Cancer gene (DCC) may then occur and lead to the transformation of the polyp into a large adenoma, from which a carcinoma can eventually arise.\",\n \"The uncontrolled cell growth that leads to the development of neoplasia is believed to result, therefore, from a series of inherited and acquired accumulated genetic changes.\",\n \"This multistep process confers on cells the capacity to survive and proliferate in a manner freed from the constraints imposed on normal cell growth.\",\n \"Spread of cancer cells involves tumour cell migration through the extracellular matrix scaffold, invasion of basement membranes, arrest of circulating tumour cells, and tumour cell extravasation and proliferation at metastatic sites.\",\n \"Detachment of cells from the primary tumour mass and modification of the peri-cellular environment aid penetration of tumour cells into blood and lymphatic vessels.\",\n \"It is the invasive and metastatic potential of tumour cells that ultimately dictates the fate of most patients suffering from malignant diseases.\",\n \"Hence, tumourigenesis can be viewed as a tissue remodelling process that reflects the ability of cancer cells to proliferate and digest surrounding matrix barriers.\",\n \"These events are thought to be regulated, at least in part, by cell adhesion molecules and matrix-degrading enzymes.\",\n \"Cell adhesion receptors on the surface of colon cancer cells are involved in complex cell signalling which may regulate cell proliferation, migration, invasion and metastasis and several families of adhesion molecules have now been identified including integrins, cadherins, the immunoglobulin superfamily, hyaluronate receptors, and mucins (Agrez, 1996.)\",\n \"In general, these cell surface molecules mediate both cell-cell and cell-matrix binding, the latter involving attachment of tumour cells to extracellular scaffolding molecules such as collagen, fibronectin and laminin.\",\n \"It is now clear that multiple and varied cell adhesion receptors exist on colon cancer cells at any one time and an understanding of the role of individual receptors in promoting growth and spread of colon cancer is only just beginning to be elucidated.\",\n \"Of all the families of cell adhesion molecules, the best-characterised at the present time is the family known as integrins.\",\n \"Integrins are involved in several fundamental processes including leucocyte recruitment, immune activation, thrombosis, wound healing, embryogenesis, virus internalisation and tumourigenesis.\",\n \"Integrins are transmembrane glycoproteins consisting of an alpha (α) and beta (β) chain in close association that provide a structural and functional bridge between extracellular matrix molecules and cytoskeletal components with the cell.\",\n \"The integrin family comprises 17 different α and 8β subunits and the αβ combinations are subsumed under 3 subfamilies.\",\n \"Excluding the leucocyte integrin subfamily that is designated by the β2 nomenclature, the remaining integrins are arranged into two major subgroups, designated β1 and αv based on sharing common chains.\",\n \"In the β1 subfamily, the β1 chain combines with any one of nine a chain members (α1-9), and the a chain which associates with β1 determines the matrix-binding specificity of that receptor.\",\n \"For example, α2β1 binds collagen and laminin, α3β1 binds collagen, laminin and fibronectin, and α5β1 binds fibronectin.\",\n \"In the αv subfamily of receptors, the abundant and promiscuous αv chain combines with any one of five β chains, and a distinguishing feature of αv integrins is that they all recognise and bind with high affinity to arginine-glycine-aspartate (RGD) sequences present in the matrix molecules to which they adhere (Hynes, 1992).\",\n \"The current picture of integrins is that the N-terminal domains of α and β subunits combine to form a ligand-binding head on each integrin.\",\n \"This head, containing the cation binding domains, is connected by two stalks representing both subunits, to the membrane-spanning segments and thus to the two cytoplasmic domains.\",\n \"The β subunits all show considerable similarity at the amino acid level (Loftus et al, 1994).\",\n \"All have a molecular mass between 90 and 110 kDa, with the exception of β4 which is larger at 210 kDa.\",\n \"Similarly, they all contain 56 conserved cysteine residues, except for β4 which has 48.These cysteines are arranged in four repeating patterns which are thought to be linked internally by disulphide bonds.\",\n \"The α-subunits have a molecular mass ranging from 150-200 kDa.\",\n \"They exhibit a lower degree of similarity than the β chains, although all contain seven repeating amino acid sequences interspaced with non-repeating domains.\",\n \"The β subunit cytoplasmic domain is required for linking integrins to the cytoskeleton (Hynes, 1992).\",\n \"In many cases, this linkage is reflected in localisation to focal contacts, which is believed to lead to the assembly of signalling complexes that include α-actinin, talin, and focal adhesion kinase (FAK) (Otey et al, 1990; Guan and Shalloway, 1992; Kornberg et al, 1992).\",\n \"At least three different regions that are required for focal contact localisation of β1 integrins have been delineated (Reszka et al, 1992).\",\n \"These regions contain conserved sequences that are also found in the cytoplasmic domains of the β2, β3, β5, β6 and β7 integrin subunits.\",\n \"The functional differences between these cytoplasmic domains with regard to their signalling capacity have not yet been established.\",\n \"Ligation of integrins by their extracellular matrix protein ligands induces a cascade of intracellular signals that include tyrosine phosphorylation of focal adhesion kinase, increases in intracellular Ca2+ levels, inositol lipid synthesis, synthesis of cyclins and expression of immediate early genes.\",\n \"In contrast, prevention of integrin-ligand interactions suppresses cellular growth or induces apoptotic cell death (Meredith et al, 1993; Montgomery et al, 1994; Brooks et al, 1994; Varner et al, 1995; Boudreau et al, 1995).\",\n \"Thus, integrins play roles in a number of cellular processes that impact on the development of tumours, including the regulation of proliferation and apoptosis.\",\n \"The integrin β6 subunit was first identified in cultured epithelial cells as part of the αvβ6 heterodimer, and the αvβ6 complex was shown to bind fibronectin in an arginine-glycine-aspartate (RGD)-dependent manner in human pancreatic carcinoma cells (Sheppard et al, 1990; Busk et al, 1992).\",\n \"The β6 subunit is composed of 788 amino acids and shares 34-51% sequence homology with other β integrin subunits β1-β5.The β6 subunit also contains 9 potential glycosylation sites on the extracellular domain (Sheppard et al, 1990).\",\n \"The cytoplasmic domain differs from other β subunits in that it is composed of a 41 amino acid region that is highly conserved among integrin β subunits, and a unique 11 amino acid carboxy-terminal extension.\",\n \"The 11 amino acid extension has been shown not to be necessary for localisation of β6 to focal contacts; in fact, its removal appears to increase receptor localisation.\",\n \"However, removal of any of the three conserved regions previously identified as important for the localisation of β1 integrins to focal contacts (Reszka et al, 1992) has been shown to eliminate recruitment of β6 to focal contacts (Cone et al, 1994).\",\n \"The integrin αvβ6 has previously been shown to enhance growth of colon cancer cells in vitro and in vivo, and this growth-enhancing effect is due, at least in part, to αvβ6-mediated gelatinase B secretion (Agrez et al, 1994; Niu et al, 1998 Agrez et al, 1999).\",\n \"What makes this epithelial-restricted integrin of particular interest in cancer is that it is not expressed on normal epithelial cells but is highly expressed during would healing and tumourigenesis, particularly at the invading edge of tumour cell islands (Breuss et al, 1995; Agrez et al, 1996).\",\n \"Further more, αvβ6 has been found to induce its own expression in an autocrine manner with cell crowding and a self-perpetuating model of colon cancer progression regulated by αvβ6-mediated gelatinase B secretion has been proposed (Niu et al, 2000).\",\n \"Invasion of the extracellular matrix and metastatic spread of colon cancer is also likely to reflect the ability of tumour cells to digest their surrounding matrix scaffold through secretion of matrix-degrading enzymes such as matrix metalloproteinases (MMPs).\",\n \"The mechanisms whereby human colon cancer cells escape the constraints on growth imposed on normal cells by cell crowding and dense pericellular matrices is unclear.\",\n \"However, even colon cancer cells are subject to relative growth inhibition in vitro in a dense extracellular matrix environment (Agrez, 1989).\",\n \"Integrins can signal through the cell membrane in either direction.\",\n \"The extracellular binding activity of integrins can be regulated from the cell interior as, for example, by phosphorylation of integrin cytolasmic domains (inside-out signalling), while the binding of the extracellular matrix (ECM) elicits signals that are transmitted into the cell (outside-in signalling) (Giancotti and Ruoslahti, 1999).\",\n \"Outside-in signalling can be roughly divided into two descriptive categories.\",\n \"The first is ‘direct signalling’ in which ligation and clustering of integrins is the only extracellular stimulus.\",\n \"Thus, adhesion to ECM proteins can activate cytoplasmic tyrosine kinases (e.g.\",\n \"focal adhesion kinase FAK) and serine/threonine kinases (such as those in the mitogen-activated protein kinase (MAPK) cascade) and stimulate lipid metabolism (eg phosphatidylinositol-4,5-biphosphate (P1P2) synthesis.\",\n \"The second category of integrin signalling is ‘collaborative signalling’, in which integrin-mediated cell adhesion modulates signalling events initiated through other types of receptors, particularly receptor tyrosine kinases that are activated by polypeptide growth factors (Howe et al, 1998).\",\n \"In all cases, however, integrin-mediated adhesion seems to be required for efficient transduction of signals into the cytosol or nucleus.\",\n \"MAP kinases behave as a convergence point for diverse receptor-initiated signalling events at the plasma membrane.\",\n \"The core unit of MAP kinase pathways is a three-member protein kinase cascade in which MAP kinases are phosphorylated by MAP kinase kinases (MEKs) which are in turn phosphorylated by MAP kinase kinases (e.g.\",\n \"Raf-1) (Garrington and Johnson, 1999).\",\n \"Amongst the 12 member proteins of the MAP kinase family are the extracellular signal-regulated kinases (ERKs) (Boulton et al, 1991) activated by phosphorylation of tyrosine and threonine residues (Payne et al, 1991) which is the type of activation common to all known MAP kinase isoforms.\",\n \"ERK 1/2 (44 kD and 42 kD MAPks, respectively) share 90% amino acid identity and are ubiquitous components of signal transduction pathways (Boulton et al, 1991).\",\n \"These serine/threonine kinases phosphorylate and modulate the function of many proteins with regulatory functions including other protein kinases (such as p90rsk) cytoskeletal proteins (such as microtubule-associated phospholipase A2), upstream regulators (such as the epidermal growth factor receptor and Ras exchange factor) and transcription factors (such as C-Myc and Elk-1).\",\n \"ERKs play a major role in growth-promoting events, especially when the concentration of growth factors available to a cell is limited (Giancotti and Ruoslahti, 1999).\",\n \"MAP kinases can be activated through non-receptor tyrosine kinases such as focal adhesion kinase (FAK), cytoplasmic tyrosine kinase (pp60 c-srk) (Schlaepher and Hunter, 1998), and growth factors acting through membrane-associated receptor tyrosine kinases.\",\n \"The FAK pathway is activated by most integrins.\",\n \"In addition to activating FAK, some β1 and αv integrins also activate the tyrosine kinase Fynu and through it, the adaptor protein Shc (Wang et al, 1996).\",\n \"It is likely that both FAK and Shc contribute to the activation of Ras and thence to the downstream kinase cascade of Raf-1, MEK, and MAP kinases (Schlaepfer et al, 1994; 1997).\",\n \"It is now generally accepted that the activation of ERK in response to integrin ligation requires Ras signalling (Wary et al, 1996; Schlaepfer and Hunter, 1997).\",\n \"The laminin receptor α6β4, the laminin/collagen receptor α1β1, the fibronectin receptor α5β1 and the RGD binding receptor αvβ3 are linked to the Ras-Raf-MEK-ERK signalling pathway and control of immediate early gene expression (Wary et al, 1996; Maniero et al, 1995; 1997).\",\n \"The ability of integrins to activate ERK may be especially important when the concentration of growth factors available to the cell is limited.\",\n \"In this setting, proliferation is likely to require co-stimulation of ERK through integrins and growth factor receptors (Giancotti and Ruoslahti, 1999).\",\n \"Moreover, activation of ERK in response to integrin ligation may play a role in regulating cell migration (Klemke et al, 1997) possibly by initiating matrix-degrading enzyme secretion.\",\n \"While there is a good deal of evidence in support of a key role for FAK and the phosphotyrosine-domain-containing adaptor protein Shc (Howe et al, 1998; Giancotti & Ruoslahti, 1999) in the Ras-Raf MEK-MAP kinase activation pathway there are also data implicating alternate pathways independent of MEKs.\",\n \"For example, MEK-independent regulation of MAP kinase activation in NIH3T3 fibroblasts has been shown to be mediated by phosphatidylinositol-3-kinases and the conventional protein kinase C (PKC) isoforms and is thought to be due to inactivation of a MAP kinase inhibitor (Grammer and Blenis, 1997).\",\n \"Although the mechanism by which PKC regulates integrin function is not known, PKC has been shown to regulate integrin-induced activation of the MAP kinase pathway upstream of Shc.\",\n \"For example, PKC inhibition has been shown to inhibit ERK2 activation by fibronectin receptors without any effect on integrin-induced FAK or paxillin tyrosine phosphorylation (Miranti et al, 1999).\",\n \"Hence, MAP kinase activation is more complicated than a simple linear pathway, and the mechanistic basis for the commonly observed integrin-mediated activation of MAP kinases remains controversial.\",\n \"The importance of the MAP kinase pathway in promoting colon cancer growth in vivo is now no longer in question.\",\n \"Although relatively little is known about the role of MAP kinase activation in for instance, colon tumour progression, MAP kinase pathways have been shown to be highly activated during the late progression of colorectal cancer (Licato et al, 1997).\",\n \"In a recent breakthrough in this field, a highly potent inhibitor of MAP kinase activation has been identified which is capable of inhibiting human colon cancer growth in immune-deficient mice raising hops for the clinical application of MAP kinase inhibitors in the treatment of colon cancer (Sebolt-Leopold et at, 1999).\",\n \"Various intracellular proteins may be linked directly or spatially to integrin cytoplasmic domains.\",\n \"Direct interactions have been identified between cytoskeletal proteins such as α-actinin and talin and β1 and β3 integrin tails (Horwitz et al, 1986; Otey et al, 1990; Knezevic et al, 1996; Pfaff et al, 1998).\",\n \"A direct association between FAK and the β1 integrin tail has been suggested based on in vitro β1 peptide studies, but this remains to be confirmed (Schaller et al, 1995).\",\n \"More recently, the cytoplasmic domain of the α4 subunit has been found to be physically associated with the signalling adaptor protein paxillin in Jurkat T cells, and this binding event regulates the kinetics of FAK tyrosine phosphorylation (Liu et al, 1999).\",\n \"Direct integrin links with the intracellular calcium-binding protein, calreticulin, and integrin-linked kinase (ILK) (Hannigan et al, 1996) have been shown to regulate “inside-out” integrin signalling.\",\n \"For example, calreticulin has been shown to bind to a chain cytoplasmic domains (Rojiani et al, 1991) and modify α2β1 integrin activation by phorbol esters and anti-integrin antibodies (Coppolino et al, 1995).\",\n \"Newly identified integrin-binding molecules include the serine/threonine integrin-linked kinase, ILK, which can associate with the β1, β2 and β3 subunits.\",\n \"When over-expressed, ILK has been shown to reduce anchorage-independent growth and tumourigenicity in nude mice (Hannigan et al, 1996).\",\n \"Co-immunoprecipitation strategies have also demonstrated interactions between integrins and the integral plasma membrane protein IAP, and members of the four transmembrane domain protein family (tetraspans).\",\n \"The extracellular Ig region of the IAP molecule mediates association with αvβ3 and is required for cell binding to vitronectin-coated particles (Lindberg et al, 1996).\",\n \"An emerging model for tetraspans is that they recruit signalling enzymes such as phosphatidylinositol-4-kinase and PKC into complexes with integrins (Hemler, 1998).\",\n \"Integrins have also been shown to be physically linked with matrix-degrading enzymes and growth factors.\",\n \"For example, the integrin αvβ6 has been shown to bind and activate latent TGFβ1 in keratinocytes (Munger et al, 1999) which is thought to be important in modulating the inflammatory process following epithelial injury.\",\n \"In melanoma cells, αvβ3 binds activated gelatinase A (Brooks et al, 1996), and both insulin and platelet-derived growth factor (PDGF) co-immunoprecipitate with this integrin in NIH3T3 mouse fibroblasts (Schneller et al, 1997).\",\n \"Synergism between integrin-mediated signalling processes and growth factor responses is now well-recognised and Schneller et al (1997) showed that a small subset of each of the insulin receptor and PDGF β-receptor is tyrosine phosphorylated upon growth factor stimulation.\",\n \"Interestingly, this subset can associate with the αvβ3 integrin, and PDGF activity is enhanced in association with increased MAP kinase activity in cells plated on the αvβ3 ligand, vitronectin.\",\n \"SUMMARY OF THE INVENTION Broadly stated, the present invention relates to the surprising finding that members of the mitogen activated protein (MAP) kinase family can associate with the cytoplasmic domain of an integrin molecule.\",\n \"It is believed that no member of the MAP kinase family has previously been found to directly associate with any integrin.\",\n \"The identification of this functional relationship permits the rational design of agents for therapeutically or prophylactically modulating cellular activity mediated by the MAP kinase and integrin interaction.\",\n \"In an aspect of the present invention there is provided an agent capable of binding to a binding domain of an integrin for a MAP kinase.\",\n \"In another aspect of the present invention there is provided an isolated or purified polypeptide capable of binding to a binding domain of an integrin for a MAP kinase or a homolog, analog, variant or derivative of the polypeptide.\",\n \"The polypeptide may consist of or comprise the binding domain of the MAP kinase to which the integrin binds or sufficient core amino acid sequence of the binding domain of the MAP kinase to enable binding of the polypeptide to the integrin.\",\n \"Preferably, the polypeptide will be a fragment of a MAP kinase.\",\n \"Most preferably, the polypeptide will comprise the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or PSNLLLNTTCDLKIC or a region or regions of such sequences.\",\n \"Accordingly, in a further aspect of the present invention there is provided a fragment of a MAP kinase wherein the fragment is capable of binding with an integrin, or a homolog, analog, variant or derivative of the fragment.\",\n \"In another aspect of the present invention there is provided a fragment of a MAP kinase wherein the fragment consists of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin.\",\n \"In another aspect of the present invention there is provided an isolated or purified polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase.\",\n \"Preferably, a polypeptide or fragment of the invention will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less and more usually about 40 amino acids or less.\",\n \"Typically, the length will be between about 5 to about 30 amino acids.\",\n \"In yet another aspect of the present invention there is provided a fusion protein a fusion protein incorporating a binding moiety capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin.\",\n \"Preferably, the binding moiety will comprise a fragment or polypeptide of the invention.\",\n \"Most preferably, the fusion protein will incorporate a carrier moiety for facilitating passage across the cell membrane.\",\n \"The carrier moiety may be penetratin.\",\n \"In still another aspect of the present invention there is provided an agent for inhibiting binding of a MAP kinase to an integrin, comprising: a targeting moiety for targeting a cell expressing the integrin; a binding moiety for binding to a binding domain of the integrin for the MAP kinase to thereby inhibit the binding of the MAP kinase to the integrin; and a carrier moiety for facilitating passage of the binding moiety across the cell membrane of the cell; wherein the binding moiety is other than an antibody or an binding fragment thereof.\",\n \"In another aspect of the present invention there is provided a MAP kinase or a fragment thereof with a mutagenised binding domain for an integrin or in which the binding domain is deleted, or a homolog, analog or variant of the MAP kinase or fragment, wherein capability to bind with the integrin is thereby reduced.\",\n \"In another aspect of the present invention there is provided an isolated nucleic acid sequence encoding a fragment or polypeptide of the invention or a homolog, analog, variant or derivative of the fragment or polypeptide.\",\n \"In another aspect of the invention there is provided a nucleic acid sequence coding for a MAP kinase or fragment thereof having a mutagenised binding domain for an integrin or in which the binding domain is deleted or a homolog, analog or variant of the MAP kinase or fragment, wherein capability to bind with the integrin is thereby reduced.\",\n \"In a still further aspect there is provided an isolated nucleic acid sequence encoding a fusion protein or agent of the invention.\",\n \"There are also provided antisense nucleic acid sequences complimentary to the sense nucleic sequences of the invention.\",\n \"Such antisense sequences find application in antisense therapy of cells in which down regulation of cellular activity is desired, and include oligonucleotides.\",\n \"Sense oligonucleotides coding for all or part of the binding domain of a MAP kinase for an integrin, or that of a homolog, analog or variant thereof, and complimentary antisense oligonucleotides find particular application as primers or probes.\",\n \"A nucleic acid primer or probe of the invention may be labelled with a suitable reporter molecule for enabling detection of hybridisation of the primer or probe to a target nucleic acid sequence.\",\n \"In yet another aspect of the present invention there is provided a vector incorporating a nucleic acid sequence of the invention.\",\n \"Typically, the vector will be an expression vector and the nucleic acid sequence will be capable of being transcribed.\",\n \"In a further aspect of the present invention there is provided a host cell transformed with a vector of the invention.\",\n \"Preferably, the host cell will be selected from the group consisting of a mammalian cell, an epithelial cell, a neoplastic cell, and a cancer cell.\",\n \"Preferably, the host cell will be a mammalian cell and most preferably, a colon cancer cell.\",\n \"In yet another aspect of the present invention there is provided an antibody generated with the use of a polypeptide, fragment, agent or fusion protein of the invention.\",\n \"In a still further aspect of the invention there is provided an antibody and binding fragment thereof capable of binding to a binding domain of a MAP kinase for an integrin.\",\n \"The antibody may be a polyclonal or monoclonal antibody.\",\n \"Preferably, the antibody is a monoclonal antibody.\",\n \"In still another aspect of the present invention there is provided a method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing a number of agents for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if any said agent is capable of binding to the binding domain of the MAP kinase on the basis of the testing.\",\n \"In yet another aspect of the invention there is provided a method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing a number of agents for ability to bind to the MAP kinase; (b) selecting an agent or agents identified as being able to bind to the MAP kinase on the basis of the testing; and (c) utilising the selected said agent or agents in an assay for indicating whether the or any of the selected said agents is capable of binding to the binding domain of the MAP kinase for the integrin.\",\n \"In another aspect of the present invention there is provided a method of evaluating whether an agent is capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing the agent for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if the agent is capable of binding to the binding domain on the basis of the testing.\",\n \"Preferably, a polypeptide or fragment of the invention comprising or consisting of the binding domain of the MAP kinase or core amino acid sequence of the binding domain or a homolog, analog, variant or derivative of the polypeptide or fragment or core amino acid sequence is used in the testing or assaying for whether an agent is capable of binding to the binding domain of the MAP kinase.\",\n \"Most preferably, the polypeptide or fragment will comprise or consist of the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or PSNLLLNTTCDLKIC, core amino acid sequence of such sequences capable of binding to an integrin, or a homolog variant, analog or derivative of such sequences.\",\n \"Testing or assaying of an agent for ability to bind to the binding domain of the MAP kinase for the integrin and thereby inhibit binding of the MAP kinase to the integrin, may comprise exposing the MAP kinase to the agent(s) to enable binding of the agent(s) to the MAP kinase to occur either in the presence of the integrin or prior to the addition of the integrin.\",\n \"Rather than using the MAP kinase, a polypeptide, fragment of a MAP kinase or other molecule capable of binding to the binding site on the integrin for the MAP kinase may be used in such testing or assaying.\",\n \"Similarly, rather than utilising an intact integrin, a polypeptide or integrin fragment comprising or incorporating the binding domain of the integrin for the MAP kinase, or other molecule capable of binding with the binding domain on the MAP kinase for the integrin, may be utilised.\",\n \"In still another aspect of the present invention there is provided a method of isolating an agent from a sample utilising a molecule immobilised on a solid support and which is capable of binding to a binding domain of an integrin for a MAP kinase, comprising: (a) contacting the molecule immobilised on the solid support with the sample under conditions suitable for binding of the agent to the molecule; (b) eluting the agent from the solid support; and (c) collecting the eluted agent; wherein the molecule is other than an antibody or binding fragment of an antibody.\",\n \"The molecule may be the MAP kinase, or a fusion protein, a polypeptide, or a fragment of the invention to which the integrin is capable of binding, or for instance a homolog, analog, variant or derivative of such a polypeptide or fragment.\",\n \"In yet another aspect of the present invention there is provided the agent so isolated.\",\n \"An agent of the invention will usually be provided in the form of a pharmaceutical composition.\",\n \"Accordingly, in another aspect of the present invention there is provided a pharmaceutical composition comprising an agent of the invention capable of binding to a binding domain of an integrin for a MAP kinase, and a pharmaceutically acceptable carrier or diluent.\",\n \"The agent may or not be proteinaceous in nature.\",\n \"Preferably, the agent will comprise a fusion protein, or polypeptide.\",\n \"Most preferably, the agent will be coupled to a carrier molecule for facilitating entry of the agent into a cell.\",\n \"In a further aspect of the invention there is provided a pharmaceutical composition comprising a nucleic acid sequence of the invention and a pharmaceutically acceptable carrier or diluent.\",\n \"Preferably, the nucleic acid sequence is incorporated into a vector as described herein.\",\n \"Alternatively, the nucleic acid sequence may be joined to a carrier molecule such as penetratin for facilitating entry of the nucleic acid sequence into a target cell.\",\n \"In another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: transfecting the cell with an expression vector incorporating a nucleic acid sequence encoding a MAP kinase of a fragment thereof, or a homolog, analog or variant of the MAP kinase or fragment, for being expressed by the cell and having a mutagenised binding domain for the integrin or in which the binding domain has been deleted whereby binding to the integrin is inhibited or prevented.\",\n \"In still another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: transfecting the cell with a nucleic acid sequence encoding a fragment of a MAP kinase, or a homolog, analog or variant of the fragment, for being expressed by the cell and capable of binding to the binding domain of an integrin for the MAP kinase.\",\n \"In yet another aspect of the present invention there is provided a method of modulating activity of a cell, comprising: transfecting the cell with a nucleic acid encoding a polypeptide for being expressed by the cell wherein the polypeptide is capable of binding to a binding domain of an integrin for a MAP kinase.\",\n \"In still another aspect of the invention there is provided a method of modulating activity of a cell, comprising causing the expression of a MAP kinase to which an integrin is able to bind to be down-regulated.\",\n \"Preferably, down-regulation of the expression of the MAP kinase is achieved using an antisense nucleic acid sequence that inhibits expression of gene coding for the MAP kinase.\",\n \"The antisense nucleic acid sequence may be administered to the cell or generated within the cell.\",\n \"Preferably, the cell will be transformed with a vector of the invention for generation of the antisense nucleic acid sequence.\",\n \"Preferably, the antisense nucleic acid sequence will specifically hybridise with sense nucleic acid sequence coding for at least part of the binding domain of the MAP kinase for the integrin and/or intron sequence between such coding sequence.\",\n \"Accordingly, in another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an antisense oligonucleotide for hybridising with at least part of a nucleic acid sequence coding for the binding domain of a MAP kinase for an integrin to thereby cause expression of the MAP kinase to be down-regulated.\",\n \"In another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the integrin for the MAP kinase, wherein the agent is other than an antibody or a binding fragment thereof.\",\n \"In a further aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the MAP kinase, wherein the agent is other than an agent comprising a binding domain of the integrin for the MAP kinase or an analog or derivative thereof.\",\n \"In yet another aspect of the invention there is provided a method of modulating the activity of a cell, comprising: contacting the cell with an effective amount of a fusion protein, antibody or other molecule of the invention capable of binding to the binding domain of an integrin for a MAP kinase.\",\n \"Preferably, modulation of the activity of a cell by a method as described herein will comprise treating a mammal in need of such treatment.\",\n \"Accordingly, in a further aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid sequence encoding a MAP kinase or a fragment thereof or a homolog, analog or variant of the MAP kinase or fragment, with a mutagenised binding domain for an integrin or in which the binding domain has been deleted and wherein the nucleic acid sequence is capable of being expressed.\",\n \"In another aspect of the invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid capable of causing the expression of a MAP kinase to which an integrin is able to bind to be down regulated.\",\n \"In a further aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal a nucleic acid sequence encoding a fragment of a MAP kinase, or a homolog, analog or variant of the fragment, for being expressed by the cell and capable of binding to the binding domain of an integrin for the MAP kinase.\",\n \"In still another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid sequence for expression of a polypeptide or a fragment of a MAP kinase within the cell, wherein the polypeptide or fragment is capable of binding to a binding domain of an integrin for the MAP kinase to thereby modulate the cellular activity.\",\n \"In another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of an agent capable of binding to the binding domain of an integrin for a MAP kinase, wherein the agent is other than an antibody or a binding fragment thereof.\",\n \"In another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of an agent capable of binding to the binding domain of a MAP kinase for an integrin wherein the agent is other than an agent comprising the binding domain of the integrin for the MAP kinase or an analog or derivative thereof.\",\n \"In another aspect of there is provided a method of treatment or prophylaxis of a disease or condition in a mammal, wherein the disease or condition is responsive to an agent capable of binding to a binding domain of an integrin for a MAP kinase and wherein the agent comprises a polypeptide or fragment of the invention or a homolog, variant, analog or derivative of such a polypeptide or fragment, and the method comprises administering an effective amount of the agent to the mammal.\",\n \"In yet another aspect of the present invention there is provided a method of treatment or prophylaxis of a disease or condition in a mammal, wherein said condition is responsive to an agent capable of binding to a binding domain of an integrin for a MAP kinase wherein the agent is a fusion protein of the invention.\",\n \"In another aspect there is provided use of a nucleic acid of the invention in the manufacture of a medicament for administration to a mammal in the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable.\",\n \"In a still further aspect of the invention there is provided the use of an agent capable of binding to a binding domain of an integrin for a MAP kinase in the manufacture of a medicament for administration to a mammal for the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable, wherein the agent is other than an antibody or binding fragment thereof.\",\n \"The agent may for instance be a polypeptide, fragment or the like as described herein.\",\n \"In another aspect of the present invention there is provided the use of an agent capable of binding to the binding domain of a MAP kinase for an integrin wherein the agent is other than an agent comprising the binding domain of the integrin for the MAP kinase or an analog or derivative thereof.\",\n \"In another aspect there is provided the use of fusion protein of the invention in the manufacture of a medicament for administration to a mammal for the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable.\",\n \"Methods as described above will typically comprise treatment or prophylaxis of cancer or a condition associated with a predisposition to cancer.\",\n \"The cancer may for instance be selected from the group consisting of cancer of the lip, tongue, salivary glands, gums, floor and other areas of the mouth, oropharynx, nasopharynx, hypopharynx and other oral cavities, oesophagus, stomach, small intestine, duodenum, colon, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung, breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid and skin.\",\n \"In still another aspect of the present invention there is provided a method of causing an activity of a cell to be upregulated, comprising: contacting the cell with an effective amount of a nucleic acid capable of being expressed within the cell and coding for an agent capable of binding to a binding domain of a MAP kinase for an integrin or a binding domain of the integrin for the MAP kinase.\",\n \"In a still further aspect of the invention there is provided a method of causing an activity of a cell to be upregulated, comprising contacting the cell with an effective amount of an agent capable of binding to a binding domain of a MAP kinase for an integrin or a binding domain of the integrin for the MAP kinase.\",\n \"Typically, the agent will be a polypeptide or fragment of an integrin comprising or consisting of the binding domain of an integrin for a MAP kinase or comprising or consisting of the core amino acid sequence of the binding domain directly involved in the binding to the MAP kinase, or a homolog, variant, analog or derivative of such polypeptide or fragment.\",\n \"Preferably, the polypeptide or fragment will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less and more usually about 40 amino acids or less.\",\n \"Typically, the length will be between about 5 to about 30 amino acids.\",\n \"Most preferably, the polypeptide will comprise or consist of the amino acid sequence RSKAKWQTGTNPLYR or RSKAKNPLYR.\",\n \"Up regulation of cellular activity is particularly desirable for instance in wound healing, re-endothelialisation within natural or artificial blood vessels, nerve growth and for instance induction of labour.\",\n \"The cellular activity desired to be modulated will typically but not exclusively, be cell growth or proliferation.\",\n \"Indeed, any activity mediated by MAP kinase signalling is expressly included within the scope of the invention.\",\n \"The cell may be any cell type in which functional activity arising from signalling mediated by a MAP kinase may occur.\",\n \"Preferably, the cell will be an epithelial cell and usually, a neoplastic cell.\",\n \"Usually, the MAP kinase will be selected from the group consisting of an extracellular signal-regulated kinase (ERK) and a JNK MAP kinase.\",\n \"Preferably, the MAP kinase is ERK2 or JNK-1.Most preferably, the MAP kinase is ERK2.The mammal may be any mammal treatable with a method of the invention.\",\n \"For instance, the mammal may be a member of the bovine, porcine, ovine or equine families, a laboratory test animal such as a mouse, rabbit, guinea pig, a cat or dog, a primate or a human being.\",\n \"Preferably, the mammal will be a human being.\",\n \"As will be understood, modulation of cellular activity within the context of the invention may be up regulation or down regulation of cellular activity.\",\n \"Reference to “down modulation” or “down-regulating” or the like should be understood to include preventing, reducing or otherwise inhibiting one or more aspects of the activity of the cell.\",\n \"Conversely, reference to “up regulation” or like terms should be understood to include enhancing or increasing one or more aspects of the activity of the cell.\",\n \"In the broadest sense, the term “integrin” unless otherwise specified, is to be taken to encompass an integrin family member or a homolog, derivative, variant or analog of an integrin subunit, or an integrin family member incorporating at least one such homolog, derivative, variant or analog of an integrin subunit.\",\n \"Usually, the integrin will be a member of the αv subfamily.\",\n \"Preferably, the integrin is or incorporates an integrin subunit selected from the group consisting of β3, β5 and β6.Most preferably, the integrin comprises β6.By “binding domain” is meant the minimum length of contiguous amino acid sequence required for binding.\",\n \"By “core amino acid sequence” is meant regions or amino acids of the binding domain that directly participate in the binding as distinct from any amino acids that do not directly participate in the binding interaction.\",\n \"Typically, the core amino acid sequence of the binding domain will comprise regions of the binding domain linked together by a number of intervening amino acids which do not directly participate in the binding.\",\n \"The term “homolog” is to be taken to mean a molecule that has amino acid sequence similarity.\",\n \"The homology between amino acid sequences can be determined by comparing amino acids at each position in the sequences when optimally aligned for the purpose of comparison.\",\n \"The sequences are considered homologous at a position if the amino acids at that position are the same.\",\n \"Typically, a homolog will have an overall amino acid sequence homology of at least about 30% more preferably at least about 50% or 70% and most preferably, greater than about 80%, 90% or 98% sequence homology.\",\n \"Homology with a binding domain may be greater than the overall amino acid sequence homology of the homolog, and will usually be greater than about 60% or 80%, and more usually greater than about 90%, 95% or 98%.\",\n \"The term “analog” is to be taken to mean a molecule that has one or more aspects of biological function characteristic of the molecule on which at least part of the analog is based or which was otherwise utilised in the design or preparation of the analog.\",\n \"An analog may have substantial overall structural similarity with the molecule or only structural similarity with one or more regions or domains thereof responsible for the desired characteristic biological function.\",\n \"By “structural” similarity is meant similarity in shape, conformation and/or other structural features responsible for the provision of the biological function or which otherwise have involvement in the provision of the biological function.\",\n \"Alternatively, it will be understood that with knowledge of the region(s) or domain(s) of a molecule that provide(s) the characteristic biological function, analogs may be designed that while differing in structure nevertheless possess such biological function.\",\n \"Indeed, it is not necessary that an analog have amino acid sequence homology, and an analog may not be proteinaceous at all.\",\n \"An analog may for instance be a mimetic of a molecule.\",\n \"By the term “variant” is meant an isoform, an allelic variant of a gene or region thereof, a naturally occurring mutant form of a gene or region thereof, or a polypeptide or fragment having an amino acid sequence that differs in one or more amino acids but which retains one or more aspects of desired characteristic biological function.\",\n \"This may be achieved by the addition of one or more amino acids to an amino acid sequence, deletion of one or more amino acids from an amino acid sequence and/or the substitution of one or more amino acids with another amino acid or amino acids.\",\n \"Inversion of amino acids and any other mutational change that results in alteration of an amino acid sequence are also encompassed.\",\n \"A variant may be prepared by introducing nucleotide changes in a nucleic acid sequence such that the desired amino acid changes are achieved upon expression of the mutagenised nucleic acid sequence, or for instance by synthesising an amino acid sequence incorporating the desired amino acid changes which possibility is well within the capability of the skilled addressee.\",\n \"Substitution of an amino acid may involve a conservative or non-conservative amino acid substitution.\",\n \"By conservative amino acid substitution is meant replacing an amino acid residue with another amino acid having similar stereochemical properties (eg.\",\n \"structure, charge, acidity or basicity characteristics) and which does not substantially effect conformation or the desired aspect or aspects of characteristic biological function.\",\n \"Preferred variants include ones having amino acid sequences in which one or more amino acids have been substituted with alanine or other neutrally charged amino acid residue(s), or to which one or more such residues have been added.\",\n \"A variant may also incorporate an amino acid or amino acids that are not encoded by the genetic code.\",\n \"By the term “derivative” is meant a molecule that is derived or obtained from another molecule and which retains one or more aspects of characteristic biological function of that molecule.\",\n \"A derivative may for instance arise as a result of the cleavage of the parent molecule, cyclisation and/or coupling with one or more additional moieties that improve solubility, lipophilic characteristics to enhance uptake by cells, stability or biological half-life, decreased cellular toxicity, or for instance to act as a label for subsequent detection or the like.\",\n \"A derivative may also result from post-translational or post-synthesis modification such as the attachment of carbohydrate moieties or chemical reaction(s) resulting in structural modification(s) such as the alkylation or acetylation of amino acid residues or other changes involving the formation of chemical bonds.\",\n \"The term “polypeptide” is used interchangeably herein with “peptide” and encompasses amino acid sequences incorporating only a few amino acid residues or many amino acid residues coupled by peptide bonds.\",\n \"The term “neoplastic cell” is to be taken to mean a cell exhibiting abnormal growth and may or may not be a malignant cell.\",\n \"“Growth” is to be taken in its broadest sense and includes proliferation of the cell.\",\n \"In this regard, an example of abnormal cell growth is the uncontrolled proliferation of a cell.\",\n \"Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.\",\n \"The features and advantages of the present invention will become further apparent from the following detailed description of preferred embodiments and the accompanying drawings.\",\n \"BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS FIG.\",\n \"1: Surface biotinylation and immunoprecipitation of integrin subunits β5 and β6 in HT29 colon cancer cells stably transfected with either vector alone (mock transfectants) or antisense β6 construct; FIG.\",\n \"2: Amplification of β6 and glyceraldehyde dehydrogenase (GAPDHu) mRNA: Ethidium-stained agarose gels with amplification products following RT-PCR from total RNA extracted from transfected HT29 and WiDr cell lines.\",\n \"Equal amounts of PCR product obtained from RT-PCR reactions were loaded on each lane and the β6 (141 basepairs) and GAPDH (216 basepairs) bands are indicated (WT, wild-type; S, sense β6; A/S, antisense β6; mock, vector alone); FIG.\",\n \"3: Non-transfected HT29 cells (wild) and cells transfected with vector alone (mock), sense β6 and antisense β6 analysed by FACScan for expression of the β6 subunit.\",\n \"White and black histograms represent cells stained in the absence and presence, respectively, of mAb E7P6 (anti-β6); FIG.\",\n \"4: WiDr cells transfected with vector alone (mock) or antisense β6 and analysed by FACScan for expression of the β6 subunit.\",\n \"White and black histograms represent cells stained in the absence and presence, respectively, of mAb E7P6 (anti-β6); FIG.\",\n \"5: Tumor cell proliferation in vitro assessed by (3H)-thymidine uptake for WiDr wild-type cells and transfectants (mock and antisense β6) cultured on plastic for the times indicated.\",\n \"FIG.\",\n \"6: Tumor cell proliferation in vitro assessed by (3H)-thymidine uptake for HT29 wild-type cells and transfectants (mock, sense β6 and antisense β6) cultured on plastic for the times indicated.\",\n \"FIG.\",\n \"7: Tumour growth after 6 weeks following subcutaneous inoculation of 106 viable cells of HT29 mock (vector alone) and antisense β6 transfectants; FIG.\",\n \"8: Graph showing average tumour size at weekly intervals for each subgroup of 10 animals following subcutaneous inoculation of clone 1 from WiDr mock (vector alone) and antisense β6 transfectants; FIG.\",\n \"9: Graph showing average tumour size at weekly intervals for each subgroup of 10 animals following subcutaneous inoculation of HT29 mock (vector alone) and antisense β6 transfectants; FIG.\",\n \"10: β6-associated ERK identified by immunoprecipitations of integrin subunits from SW480 β6 transfectants.\",\n \"(Top) Cell lysates were immunoprecipitated with mAbs QE2E5 (anti-β1), L230 (anti-αv), P1F6 (anti-β5), R6G9 (anti-β6), or matched isotype control Abs (IgG1 and IgG2A), and (bottom) blotted with anti-ERK1/2 mAb, SC-1647, which recognises total ERK (phosphorylated and non-phosphorylated).\",\n \"Purified, non-phosphorylated ERK2 is shown in the left hand lane; FIGS.\",\n \"11(A) and 11(B): (A) Western blotting: equal protein loads of cell lysates from one representative clone each from WiDr mock and antisense β6 blotted with anti-ERK mAb (SC-1647) against total ERK.\",\n \"Purified non-phosphorylated ERK2 is shown in the left hand lane.\",\n \"(B) β6 immunoprecipitates (mAb R6G9) from equal protein loads of the cell lysates in (A) probed with anti-ERK mAb (E10).\",\n \"Purified phosphorylated ERK2 is shown in the left hand lane; FIG.\",\n \"12: β6-bound ERK shown for the high and low SW480 β6-expressing clones by probing β6 immunoprecipitates with anti-ERK mAb (E10) against phosphorylated forms of ERK1/2.Purified, phosphorylated ERK2 is shown in the left hand lane; FIGS.\",\n \"13(A) and 13(B): (A) Surface biotinylation of WiDr wild-type cells and β6 immunodepletion of the cell lysates by three successive rounds of β6 and β5 immunoprecipitations using mAb R6G9 and P1F6, respectively or control mAb (IgG2A).\",\n \"The β6 and partner αv bands are arrowed.\",\n \"(B) β6-immunodepleted WiDr cell lysates after 3 successive rounds of β6-immunoprecipitations probed with anti-ERK mAb SC-1647 recognising both phosphorylated and non-phosphorylated forms of ERK1/2 and compared with non-β6 immunodepleted lysates and control lysates sequentially immunoprecipitated 3 times with either isotype matched control mAb (IgG2A) or mAb P1F6 (anti-β5); FIG.\",\n \"14: Non-transformed (HaCaT) and Ras-transformed (HaRas) human keratinocytes: β6 immunoprecipitation and ERK western blots probed with monoclonal antibody E10 (against Phosphorylated ERK 1/2) and monoclonal antibody SC1647 (against total ERK 1/2), respectively.\",\n \"FIGS.\",\n \"15(A) and 15(B): (A) Depletion of β6 with sequential rounds of ERK immunodepletion of cell lysates using anti-ERK mAb (SC-1647).\",\n \"(B) MAP kinase activity in cell lysates before and after 5 rounds of sequential β6 immunodepletion from SW480 β6 and SW480 mock transfectants (full grey and hatched bars, respectively).\",\n \"MAP kinase activity is shown as the mean of three independent experiments.\",\n \"The reduction in MAP kinase activity following β6 immunodepletion was highly significant (P≦0.005, students T test).\",\n \"FIG.\",\n \"16: Shows the amino acid sequence of the cytoplasmic domain of the β6 subunit as well as the amino acid sequences for the β1 to β3 subunits, respectively; FIG.\",\n \"17: Graph showing binding of non-phosphorylated ERK2 (GST.ERK2) to overlapping fragments corresponding to different regions of the cytoplasmic domain of the β6 subunit; FIG.\",\n \"18: Graph showing binding of ERK2 (GST.ERK2) to the β6 cytoplasmic domain and the peptide fragments indicated in FIG.\",\n \"16 over a range of concentrations of ERK2; FIG.\",\n \"19: Graph showing binding of ERK2 (GST.ERK2) to the β6 cytoplasmic domain and fragments thereof; FIG.\",\n \"20: Graph showing binding of ERK2 (GST.ERK2) to a 15 mer fragment of the β6 cytoplasmic domain and which has the amino acid sequence RSKAKWQTGTNPLYR; and FIG.\",\n \"21: Shows regions of the cytoplasmic domain of the β6 subunit corresponding to synthesised fragments thereof evaluated for capacity to be bound by ERK2.FIG.\",\n \"22: Graph showing assay results for ERK2 (GST.ERK) binding to synthesised 10 mer fragments identified in FIG.\",\n \"21.FIG.\",\n \"23: Graph showing binding of ERK2 (thrombin cleaved) to synthesised peptide having the amino acid sequence RSKAKNPLYR compared to the 15 mer RSKAKWQTGTNPLYR fragment of the cytoplasmic domain of the β6 subunit.\",\n \"FIG.\",\n \"24: Graph showing binding of JNK-1 to the β6 cytoplasmic domain.\",\n \"FIG.\",\n \"25: Location of β6 Δ746-764, β6(770t) and β6(777t) deletions in the cytoplasmic domain of the β6 subunit.\",\n \"FIG.\",\n \"26: SW480 cells transfected with wild-type full length coding sequence for β6 or β6 Δ746-764 deletion mutant analysed by FACScan for expression of wild-type or mutant β6.White and black histograms represent cells stained in the absence and presence of the integrin subunit, respectively.\",\n \"FIGS.\",\n \"27(A) and 27(B): (A) Western blotting: equal protein loads of cell lyates from SW480 cells expressing wild-type β6 or β6 Δ746-764 deletion mutant.\",\n \"(B) β6 immunoprecipitates (mAb R6G9) from equal protein loads of cell lysates (A) probed with anti-ERK mAb (E10).\",\n \"FIG.\",\n \"28: Proliferation of HT29 colon cancer cells cultured for 48 hours and treated with penetratin, the fragment of β6 cytoplasmic domain having amino acid sequence RSKAKWQTGTNPLYR alone or the fragment coupled to penetratin for the final 24 hours of the incubation period.\",\n \"FIG.\",\n \"29: Proliferation of SW480 cells expressing wild-type β6 cultured on plastic for 48 hours and treated with penetratin, the RSKAKWQTGTNPLYR peptide alone or the peptide coupled to penetratin for the final 24 hours of the incubation period.\",\n \"FIGS.\",\n \"30(A) to 29 (C): (A) SW480 cells cultured with control additive for 24 hours; (B) SW480 cells cultured with penetratin for 24 hours; (C) SW480 cells cultured with RSKAKWQTGTNPLYR bound to penetratin for 24 hours.\",\n \"FIGS.\",\n \"31(A) and 31 (B): (A) SW480 mock (−β6) and SW480 β6 transfectants (+β6) cultured in presence of the RSKAKWQTGTNPLYR peptide coupled to penetratin.\",\n \"(B) Photomicrographs of cells shown in (A) cultured in the presence/absence of the peptide penetratin complex.\",\n \"FIGS.\",\n \"32(A) to 32(C): (A) SW480 cells cultured in a 3-dimensional collagen type I matrix photographed in the gel at the end of 10 days.\",\n \"(B) The SW480 cells following dissolution of the collagen with collagenase.\",\n \"(C) Graph showing numbers of colonies of the SW480 cells having a diameter exceeding 200μ.\",\n \"FIGS.\",\n \"33(A) and 33(B): Graphs showing inhibition of proliferation of SW480 cells expressing full length wild-type β6 in the presence of RSKAKWQTGTNPLYR peptide bound to penetratin.\",\n \"FIG.\",\n \"34: Graph showing binding of ERK2 to RSKAKWQTGTNPLYR peptide and peptides corresponding to regions of the cytoplasmic domain of β3 and β5.FIG.\",\n \"35(A) to 35(C): (A) Immunoprecipitations of β5 and β6 subunits followed by immunoblotting with anti-biotin antibody.\",\n \"αv/β5/β6 bands are shown for the SW480 cell line: wild-type cells, mock transfectants (vector alone) or cells expressing either full length β6 or a Δ746-764 deletion mutant.\",\n \"(B) Indentical amounts of precipitated proteins shown in (A) were immunoblotted with anti-ERK mAb (E10) against phosphorylated ERK1/2.Purified phosphorylated ERK2 is shown in the left hand lane.\",\n \"(C) Comparative binding of ERK2 (1 μm) to synthetic peptides comprising either RSKAKWQTGTNPLYR or peptide sequences derived from β5 and β1 cytoplasmic domains that correspond to the binding domain on β6 for ERK.\",\n \"FIG.\",\n \"36: In vitro phosphorylation of GST-Elk-1 by immunoprecipitated ERK from cell lysates prepared from human umbilical vein endothelial cells (HUVECs) cultured in the absence/presence of epidermal growth factor (EGF) after a 24 hour culture period in serum-free and growth factor free medium.\",\n \"FIGS.\",\n \"37 and 38: Overlapping peptides derived from ERK2 sequence tested for ability to inhibit the β6-ERK2 interaction.\",\n \"FIG.\",\n \"39: Inhibition of β6-ERK2 interaction using ERK2 peptide HRDLKPSNLLLNTTCDLK (0.25 μm) and β6 cytoplasmic tail peptide.\",\n \"FIGS.\",\n \"40(A) to 40(D): Illustrate a dose-dependent wound repair process for peptide-agonist complexed with penetratin as a delivery vehicle.\",\n \"FIG.\",\n \"41: Nucleotide and amino acid sequence for ERK2.DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE PRESENT INVENTION The distribution of β6 integrin subunit within various tissues has been assessed by both in situ hybridisation and immunostaining and reported in the art.\",\n \"For instance, β6 mRNA in adult primate tissues was detected only in epithelial cells and at very low or undetectable levels in most normal tissues (Breuss et al, 1993).\",\n \"High-level expression of β6 has been observed in secretory endometrial glands while low-level expression was detected in the ductal epithelia of salivary gland, mammary gland and epididymis, in gall and urinary bladder, and in the digestive tract.\",\n \"Immunostaining data has also shown that β6 expression is restricted to epithelia and is up-regulated in parallel with morphogenetic events, tumourigenesis and epithelial repair (Breuss et al, 1993; 1995).\",\n \"During development of the kidney, lung and skin, β6 is expressed by specific types of epithelial cells, whereas it is mostly undetectable in normal adult kidney, lung and skin.\",\n \"In contrast, high level expression of β6 has been observed in several types of carcinoma.\",\n \"For example, β6 is almost invariably neo-expressed in squamous cell carcinomas derived from the oral mucosa, and often focally localised at the infiltrating edges of tumour cell islands (Breuss et al, 1995; Thomas et al, 1997).\",\n \"Moreover, expression of the β6 subunit has been observed in renal cell carcinoma and testicular tumour cell lines (Takiuchi et al, 1994) and 50% of lung cancers have been shown to express this subunit (Smythe et al, 1995).\",\n \"Recent studies have shown that αvβ6 is a major fibronectin-binding receptor in colorectal cancer (Agrez et al, 1996).\",\n \"Moreover, normal colonic epithelium from cancer patients does not express αvβ6 in immunostaining studies, and as with squamous cell carcinomas from the oral mucosa (Thomas et al, 1997) maximal β6 expression in colon cancer has been observed at the invading edges of tumour cell islands (Agrez et al, 1996).\",\n \"Importantly, heterologous expression of the β6 subunit in colon cancer cells lacking constitutive expression of this receptor has been shown to stimulate tumour cell proliferation in vitro and tumour growth in athymic immune-deficient mice, and this growth-promoting effect is mediated through the 11 amino acid C-terminal cytoplasmic extension unique to the β6 subunit (Agrez et al, 1994).\",\n \"These findings suggest that one mechanism of the enhanced growth effect involves induced secretion of matrix-degrading enzymes as has been previously suggested for invasive melanoma cells.\",\n \"Support for this arises from findings that the proliferative capacity of colon cancer cells within 3-dimensional collagen matrices is inversely related to the density of the extracellular matrix (Agrez, 1989), and the observation that induced expression of αVβ6 in colon cancer cells markedly enhances gelatinase B secretion (Agrez et al, 1999).\",\n \"Indeed, the β6 subunit is widely observed in cancers of various origins (Breuss et al, 1995).\",\n \"As described above, β6 is detected in at least 50% of bowel cancer tumours.\",\n \"Others have reported its presence in oropharyngeal cancers where it is also present and strongly expressed in the invading margins of the cancer cell islands as is commonly found in bowel cancer.\",\n \"In the oropharyngeal mucosa, no β6 is observed in the normal lining cells of the mouth but in both primary and metastatic tumours from the oropharyngeal mucosa, strong β6 expression is seen which does not correlate with degree of differentiation and in particular, is restricted to the basal layer of epithelial cells.\",\n \"In colon cancer, β6 expression is similarly maximal at the advancing edges of tumour cell islands (Agrez et al, 1996).\",\n \"Hence, modulation of MAP kinase interaction with the β6 subunit in epithelial cells is of interest in the prophylaxis or treatment of cancer of the lip, tongue, salivary glands, gums, floor and other areas of the mouth, oropharynx, nasopharynx, hypopharynx and other oral cavities, oesophagus, stomach, small intestine, duodenum, colon, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung, female and male breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid and skin.\",\n \"In terms of prophylactic use in relation to cancer, modulation of the MAP kinase β6 interaction and specifically down regulation of the interaction may find application in protecting against ultraviolet-induced skin cancer, lung cancer in smokers, cancer of the gut where polyps are present as in polyposis coli or other inheritable disease where a pre-disposition to the development of polyps exists, breast cancer in high risk patients with a familial history of breast cancer or otherwise identified as carrying known mutations of the breast cancer susceptibility gene BRCA1 or BRCA2 associated with breast cancer, transitional cell cancers arising from bladder papillomas, and cancer of the cervix in individuals deemed to be at high risk.\",\n \"Expression of β6 is also up-regulated in migrating keratinocytes at the wound edge during experimental epidermal wound healing.\",\n \"αvβ6 is not expressed in normal epithelium (Jones et al, 1997).\",\n \"However, following experimental wounding, αv appears to switch its heterodimeric association from β5 to β6 subunit during re-epithelialisation.\",\n \"At day 3 after wounding, β6 is absent but then appears around the perimeter of the basal cells of the migrating epidermis (Clark et al, 1996).\",\n \"By day 14 after wounding, when re-epithelialisation is complete, all suprabasalar cells overlying the wound express β6 but not β5.In human mucosal wounds, maximal expression of β6 has been observed relatively late when epithelial sheets are fused and granulation tissue is present (Haapasalmi et al, 1996).\",\n \"Furthermore, those investigators observed maximal expression of tenascin with αvβ6 expression.\",\n \"Interestingly, freshly isolated keratinocytes have not been found to express β6 but begin to express this after subculturing (Haapasalmi et al, 1996).\",\n \"Moreover, TGF-β1 has been shown to induce the de novo expression of αvβ6 at the cell surface on keratinocytes (Zambruno et al, 1995).\",\n \"This is particularly relevant in view of the recent observation that αvβ6 binds and activates latent TGF-β1 which may be a means of locally regulating TGF-β1 function in vivo during tissue response to injury (Munger et al, 1999).\",\n \"In contrast to persistent αvβ6 expression observed in colon cancer cells, new expression of αvβ6 in migrating keratinocytes is down-regulated to undetectable levels once re-epithelialisation is complete (Happasalami et al, 1996; Cass et al, 1998).\",\n \"While in the normal unwounded skin, just as in other normal epithelia, αvβ6 expression is absent indicating that this MAP kinase activation pathway is normally suppressed.\",\n \"However, alterations in cell shape trigger biochemical signalling by MAP kinases in human keratinocytes and it is likely that alterations of skin mechanics in vivo during wound formation and healing trigger physiological responses via the MAP kinase pathway (Krippenberg et al, 2000).\",\n \"In addition, gelatinase B secretion by keratinocytes which facilitates the process of cell migration during normal re-epithelialisation is MAP kinase-dependent (McCawley et al, 1999).\",\n \"As outlined above, MAP kinases behave as a convergence point for diverse receptor-initiated signalling events at the plasma membrane which regulate cell proliferation, differentiation, migration and invasion.\",\n \"In particular, extracellular signal-regulated kinases (ERKs) become activated upon integrin ligation and, thereby, regulate cell migration possibly by initiating matrix degrading enzyme secretion (Klemke et al, 1997) such as gelatinase B. Gelatinase B production has been shown to be dependent on endogenous ERK signalling in growth factor-stimulated human kertinocytes (McCawley et al, 1999; Zeigler et all, 1999).\",\n \"Aside from integrins, growth factors such as foetal calf serum or epidermal growth factor (EGF) have been shown to stimulate ERK activity via the Ras-Raf-Mek-MAPk cascade resulting in directed cell migration through a porous membrane (Giehl et al, 2000).\",\n \"There is also abundant data indicating that the MAP kinase cascade plays a major role in alterations of cell shape and migration for a range of normal cell types.\",\n \"For example, the MAP kinase signalling pathway has been shown to mediate neurite outgrowth by cerebellar neurons (Schmid et al, 2000) implying a role for MAP kinase in nerve repair, while growth factor-induced migration of endothelial cells has been shown to be dependant on MAP kinase signalling and this dependency for new blood vessel formation (angiogenesis) on sustained ERK activity is regulated by the ligation state of both growth factor receptors and integrins (Eliceiri et al, 1998).\",\n \"In addition, ERK activation has been demonstrated to be essential for up-regulation of vascular endothelial growth factor (VEGF) in other cell types (Jung et al, 1999), and induction of endothelial tube formation by human umbilical vein endothelial cells (HUVECs) dispersed within a 3-dimensional collagen gel has been shown to be dependent on MAP kinase activation (Ilan et al, 1998).\",\n \"Growth factors and their receptors are also known to play important roles in tissue repair and ulcer healing.\",\n \"For example, EGF accelerates gastroduodenal ulcer healing by stimulating cell proliferation, and this event is regulated through the Ras/Raf/MAP kinase pathway (Tarnawski and Jones, 1998).\",\n \"Moreover, in the healing process of gastric ulcers, ERKs have been shown to translocate to cell nuclei with activation of the c-fos gene which triggers cell proliferation (Tarnawski et al, 1998).\",\n \"Cutaneous wound re-epithelialisation and keratinocyte migration requires ordered gene expression and the fibroblast growth factor (FGF)-inducible response element (FiRE) required for migrating (but no proliferating) keratinocytes has been shown to be induced by treatment of cells with okadaic acid, the inhibitor of protein phosphatases which inactivate ERKs (Jaakkola et al, 1998).\",\n \"The MAP kinase cascade is also be implicated in events relating to parturition.\",\n \"For example, induction of labour is commonly achieved with oxytocin and oxytocin induction of prostaglandin synthesis has been shown to be MAP kinase-dependent (Strakova et al, 1998).\",\n \"In fact, oxytocin causes rapid activation of MAP kinases in both human and rat puerperal uterine myometrial cultured cells (Nohara et al, 1996).\",\n \"Moreover, MAP kinase inhibition has been shown to inhibit prostaglandin-induced uterine contraction in animal models (Ohmichi et al, 1997).\",\n \"In addition, gelatinase B has been identified in amniotic fluid association with normal labour (Vadillo-Ortega et al, 1996) and gelatinase B production has been shown to be dependent on endogenous ERK signalling in other cell types such as keratinocytes (Zeigler et al, 1999; McCawley et al, 1999).\",\n \"Taken together, these date suggest that MAP kinase signalling may be an important regulator of parturition.\",\n \"αvβ6 expression is also upregulated in type II alveolar epithelial cells during lung injury caused by injection of live bacteria and αvβ6 mRNA is induced within 5 hours of acute injury (Breuss et al, 1995).\",\n \"Interestingly, αvβ6 has been shown to be expressed on proximal airway epithelial cells in 50% of smokers undergoing lung resection (Weinacker et al, 1995).\",\n \"Just as in human keratinocytes, in primary cultures of human airway epithelial cells, TGF-β1 has been shown to dramatically increase expression of αvβ6 without affecting surface expression of any other integrin.\",\n \"Moreover, epidermal growth factor (EGF) also induces αvβ6 in these cells, and the effect of both growth factors on β6 expression has been shown to be additive (Wang et al, 1996).\",\n \"αvβ6 expression has also been observed in adult lungs and kidneys at focal sites of sub-clinical inflammation as well as in a variety of clinical specimens from patients with chronic or acute inflammation of the lungs or kidneys (Breuss et al, 1995).\",\n \"Taken together, this data and studies outlined above indicate that αvβ6 has a role in inflammation and affects for instance cell spreading, migration and growth during re-organisation of epithelial in development, tissue repair and neoplasia.\",\n \"Hence, the application of agents, on either unwounded epithelial tissues (such as before surgery) or upon wounded epithelium, which up-relate MAP kinase activity may enhance cell migration and wound re-epithelialisation and thereby, hasten the healing process.\",\n \"Such agents may be based upon the binding domain on β6 for ERK2 and act therefore as β6 agonists (in the absence of this integrin) leading to MAP kinase activation.\",\n \"Examples of the application of such agents may include cell migration in wound healing, re-endothelialisation within natural or artificial blood vessels, or renewed nerve outgrowth following injury or disease.\",\n \"In addition, use of such agonists may for instance be useful in promoting induction of labour.\",\n \"The epithelial restricted integrin subunit β6 is shown herein to interact with at least MAP kinases ERK2 and JNK-1, and its down-regulation dramatically suppresses growth of colon cancer.\",\n \"Hence, therapeutic strategies to inhibit growth and proliferation of colon cancer and growth of other malignant cancer cells by switching off either the permissive β6 integrin and/or inhibiting the β6 MAP kinase interaction are of particular interest.\",\n \"In particular, the fact that β6 expression may be significantly upregulated on tumour cells compared to normal cells offers the potential for tumour cell specificity substantially without impairment of normal cellular function.\",\n \"Down-regulation of the functional activity of an integrin or MAP kinase may be achieved by preventing or down-regulating the expression of the integrin molecule or MAP kinase or by inhibiting the signalling function of the integrin or MAP kinase.\",\n \"Gene therapy is one strategy for treating cancers of different types.\",\n \"The use of recombinant adenoviruses has for instance been utilised for restoring expression of polypeptide encoded by a wild-type p53 tumour suppressor gene (Bookstein et al, 1996), and adenoviral vectors for expression of wild-type p53 have been shown to suppress growth of human colon cancers by as much as 60% in animal models following intra-tumoural injection of recombinant virus (Spitz et al, 1996).\",\n \"Rather than replacing a defective gene with one encoding a polypeptide the expression of which restores normal function of the cell as in the above examples, the possibility exists to achieve down regulation of cancer cells such as colon cells by introducing a gene that codes for an integrin subunit in which the binding domain for a MAP kinase has been rendered defective by mutagenesis, or in which the binding domain has been wholly or partially deleted, to thereby achieve down regulation through the inhibition of the MAP kinase integrin interaction.\",\n \"The defective integrin subunit will nevertheless usually be able to associate with its normal partner integrin subunit and be expressed on the cell membrane.\",\n \"Preferably, the defective integrin subunit will be expressed at a higher level than the corresponding wild-type integrin subunit such that down regulation is achieved by a dominant negative effect.\",\n \"Alternatively, such therapy may involve the introduction and expression of a nucleic acid sequence that encodes a fragment or truncated form of an integrin subunit that excludes the binding domain for the MAP kinase or in which the binding domain has been partially or wholly deleted or otherwise mutagenised so as to be defective.\",\n \"Similarly, a gene coding for a MAP kinase or a nucleic acid sequence coding for a fragment of a MAP kinase comprising the binding domain for an integrin may also be modified such that the binding domain of the MAP kinase is mutagenised or deleted.\",\n \"Another option is to introduce a nucleic acid sequence encoding a polypeptide capable of binding to the binding domain on the integrin for the MAP kinase or to the binding site on the MAP kinase for the integrin upon being expressed within the cell to thereby inhibit intracellular binding of the MAP kinase to the integrin and thereby achieve down regulation of cellular activity.\",\n \"A gene or nucleic acid sequence encoding an integrin subunit or MAP kinase may also be modified such that although the encoded binding domain for integrin or the MAP kinase as the case may be remains unaltered, the amino acid sequence of a region distant from the binding domain, or the amino acid sequence of either one or both regions immediately flanking the binding domain, is altered to achieve a change in the three dimensional conformation of the integrin subunit or MAP kinase such that the MAP kinase integrin binding interaction is inhibited.\",\n \"The gene or nucleic acid sequence may be altered to achieve the desired outcome by the deletion, insertion or substitution of one or more nucleotides such that the corresponding amino acid sequence is modified to the extent that binding of the MAP kinase to the integrin is inhibited.\",\n \"Inhibition in this context may be partial or total inhibition.\",\n \"The gene or nucleic acid sequence can be introduced into a cell in an appropriate expression vector for expression of the gene or nucleic acid sequence extrachromosomally or more preferably, for facilitating integration of the gene or nucleic acid sequence into genomic DNA by heterologous or homologous recombination events.\",\n \"In another strategy, down-regulation of the expression of an integrin subunit such as β6 and hence an integrin heterodimer such as αvβ6 or the down regulation of expression of a MAP kinase such as ERK2, is achieved using antisense nucleic acid sequences (e.g.\",\n \"oligonucleotides) for inhibiting expression of the subunit or MAP kinase.\",\n \"Typically, this will involve expression of a nucleic acid construct incorporating all or part of a coding region of the gene for the integrin subunit or MAP kinase inserted in the reverse orientation resulting in the synthesis of antisense RNA which inhibits translation of mRNA encoding the integrin subunit or MAP kinase by hybridisation of the antisense RNA to the mRNA.\",\n \"More broadly, a method of down regulating an activity of a cell by inhibiting expression of an intact integrin subunit or a MAP kinase may comprise contacting the cell with a first nucleic acid molecule that is capable of interacting with a target nucleic acid sequence coding for the subunit or MAP kinase, or which first nucleic acid molecule is capable of being transcribed to a nucleic acid molecule capable of interacting with the target sequence whereby the interaction of the first nucleic acid molecule with the target sequence inhibits expression of the integrin subunit or MAP kinase.\",\n \"Reference to the first nucleic acid molecule is to be understood as a reference to any nucleic acid molecule which directly or indirectly facilitates reduction, inhibition or other form of down regulation of the expression of the integrin molecule or MAP kinase.\",\n \"Nucleic acid molecules which fall within the scope of this definition include antisense sequences administered to a cell and antisense sequences generated in situ which have sufficient complementarity with target sequence such as mRNA coding for the integrin subunit or MAP kinase or for instance, a transcription regulatory sequence controlling transcription of the gene coding for the integrin subunit or MAP kinase, to thereby be capable of hybridising with the target sequence and inhibit the expression of the integrin subunit or MAP kinase.\",\n \"The first nucleic acid molecule may also be a ribozyme capable for instance, specifically binding to that region of nucleic acid encoding the binding domain of an integrin subunit or MAP kinase or the binding domain of a MAP kinase for an integrin, and cleaving the nucleic acid.\",\n \"Typically, an antisense strategy in accordance with the present invention will be directed toward inhibition of an intact integrin subunit to thereby down regulate cell activity via down regulation of interaction of the integrin with a MAP kinase.\",\n \"On a priori grounds, targeting the expression of the β6 subunit in malignant cells such as in colon cancer by means of adenoviral-mediated antisense therapy is preferred because down-regulating β6 by means of a non-adenoviral approach may increase cell surface expression of the β5 subunit.\",\n \"The relevance to such therapy is that the vitronectin-binding integrin αvβ5 promotes adenovirus internalisation (Thomas et al, 1993).\",\n \"Given that abundant αvβ5 is always present on the surface of colon cancer cells for example, a secondary benefit of inhibiting β6 expression during the course of therapy may be the concomitant rise of β5 in cells already transduced with antisense β6 nucleic acid.\",\n \"This is likely to facilitate further virus uptake into such cells since the amount of integrin αvβ5 present has been shown to be closely related to levels of gene expression following adenovirus-mediated gene transfer (Takayama et al, 1998).\",\n \"Typically, an antisense nucleic acid sequence will hybridise with all or part of that region of the target sense nucleic acid encoding the binding domain of an integrin subunit or the corresponding binding domain of a MAP kinase.\",\n \"An antisense nucleic sequence may for instance be capable of hybridising to exon and/or intron sequences of pre-mRNA.\",\n \"Preferably, an antisense nucleic acid sequence will be designed for specifically hybridising to mature mRNA in which intron sequences have been spliced out by normal cellular processing events.\",\n \"It is not necessary that an antisense nucleic acid sequence have total complementarity with its target sequence only that substantial complementarity exists for specificity and to allow hybridisation under cellular conditions.\",\n \"Preferably, an antisense nucleic sequence will have a complementarity of about 70% or greater, more preferably about 80% or greater and most preferably, about 90% or 95% or greater.\",\n \"Preferred antisense sequences are oligonucleotides wherein the complementary sense nucleotides encode for about 50 amino acids or less, preferably about 35 or 30 amino acids or less, more preferably less than about 25 or 20 amino acids, most preferably about 15 amino acids or less and usually between about 5 to about 15 amino acids.\",\n \"Antisense nucleic acid sequences may be generated in vivo by transcription of a suitable expression vector within a cell transformed with the vector, or ex vivo and then be introduced into a target cell to effect down regulation of the MAP kinase integrin interaction.\",\n \"Antisense sequences will desirably be designed to be resistant to endogenous exonucleases and/or endonucleases to provide in vivo stability in target cells.\",\n \"Modification to the phosphate backbone, sugar moieties or nucleic acid bases may also be made to enhance uptake by cells or for instance solubility, and all such modifications are expressly encompassed.\",\n \"Such modifications include modification of the phosphodiester linkages between sugar moieties, the utilisation of synthetic nucleotides and substituted sugar moieties, linkage to liphophilic moieties and the such like as described in U.S. Pat.\",\n \"No.\",\n \"5,877,309.Methods for the construction of oligonucleotides for use in antisense therapy have previously been described (see Van der Krol et al, 1998 Biotechniques 6:958-976; and Stein et al, 1998 Cancer Res 48:2659-2668; Bachman et al, 1998, J. Mol.\",\n \"Med.\",\n \"76:126-132).\",\n \"Any means able to achieve the introduction of a gene or a nucleic acid into a target cell may be used.\",\n \"Gene transfer methods known in the art include viral and non-viral transfer methods.\",\n \"Suitable virus into which appropriate viral expression vectors may be packaged for delivery to target cells include adenovirus (Berkner, 1992; Gorziglia and Kapikian, 1992); vaccinia virus (Moss, 1992); retroviruses of avian (Petropoulos et al, 1992); murine (Miller, 1992) and human origin (Shimada et al, 1991); herpes viruses including Herpes Simplex Virus (HSV) and EBV (Margolskee, 1992; Johnson et al, 1992; Fink et al, 1992; Breakfield and Geller, 1997; Freese et al, 1990); papovaviruses such as SV40 (Madzak et al, 1992), adeno-associated virus (Muzyczka, 1992); BCG and poliovirus.\",\n \"Particularly preferred virus are replication deficient recombinant adenovirus (eg.\",\n \"He et al, 1998).\",\n \"Engineered virus may be administered locally or systemically to achieve delivery of the gene or nucleic acid sequence of interest into a target cell.\",\n \"Gene transfer methods as described above may be used in the provision of transgenic animals for studying in vivo the effect of for instance, modifying the binding site of an integrin for a MAP kinase or the binding domain of the MAP kinase for the intergrin.\",\n \"A transgenic animal is one with cells that contain heterologous nucleic acid as a result of the deliberate introduction of the nucleic acid.\",\n \"The nucleic acid may be introduced indirectly by viral transfer as indicated above or directly by microinjection into a pronucleus of a fertilised egg prior to transfer of the egg to a surrogate mother for development to term.\",\n \"Alternatively, a gene or nucleic acid sequence of interest may be introduced into an embryonal stem (ES) cell in culture and the transformed cell injected into a recipient blastocyst which is then transferred to a surrogate mother for development to term.\",\n \"Techniques for generation of transgenic animals are for instance described in U.S. Pat.\",\n \"No.\",\n \"4,873,191; Van der Putten, 1985; Thompson et al, 1989 and Lo, 1983.A transgenic animal homozygous for a transferred gene for instance may be obtained by the mating of animals heterozygous for the gene as will be appreciated.\",\n \"Both animal cells expressing a heterologous gene or nucleic acid sequence and transgenic animals in which a particular gene has been mutagenised by homologous recombination may be provided.\",\n \"A transgenic animal may for instance be a mouse, rat, hamster or pig.\",\n \"Typically, the transgenic animal will be a mouse.\",\n \"Agents for modulating the functional activity of a cell arising from the interaction of a MAP kinase like ERK2 or JNK-1 with a integrin include antagonists and inhibitors capable of associating with the integrin or MAP kinase to thereby inhibit the MAP kinase and integrin interaction.\",\n \"Antagonists and inhibitors include those agents that act by binding the integrin or MAP kinase adjacent to the relevant binding domain and stearically hindering the interaction of the MAP kinase with the integrin, as well as allostearic inhibitors that distort the binding domain upon associating with the integrin.\",\n \"Additional agents include agonists of a MAP kinase or an integrin, that are capable of binding to the pertinent binding domain involved in the MAP kinase integrin interaction.\",\n \"A binding domain involved in the MAP kinase integrin interaction may be identified and characterised using protocols and techniques described herein.\",\n \"Specifically, a binding domain may be localised by assessing the capacity of respective overlapping peptide fragments of the cytoplasmic domain of an integrin subunit to bind with a MAP kinase or correspondingly, assessing the capacity of overlapping peptide fragments of the MAP kinase to bind with an integrin.\",\n \"The specific amino acid sequence which constitute the binding domain may then be determined utilising progressively smaller peptide fragments of a fragment observed to interact with the MAP kinase or the integrin.\",\n \"In particular, test peptides are readily synthesised to a desired length involving deletion of an amino acid or amino acids from either or both ends of the larger amino acid sequence, and tested for their ability to associate with the MAP kinase or integrin.\",\n \"This process is repeated until the minimum length peptide capable of binding with the MAP kinase or integrin substantially without compromising the optimum observed level of binding is identified.\",\n \"The amino acids that play an active role in the MAP kinase integrin interaction may be achieved with the use of further synthesised test peptides in which one or more amino acids of the sequence are deleted or substituted with a different amino acid or amino acids to determine the effect on the ability of the peptide of associate with the MAP kinase or integrin.\",\n \"Typically, substitution mutagenesis will involve substitution of selected ones of the amino acid sequence with alanine or other relatively neutrally charged amino acid.\",\n \"By deletion is meant deletion of one or more of the amino acids between the N-terminal and C-terminal amino acid residues of the identified amino acid sequence.\",\n \"Nucleotide and amino acid sequence data for the β6 integrin subunit for instance is found in Sheppard et al, 1990.The amino acid sequence for β6 is also set out in SEQ ID NO: 1.The nucleotide and amino acid sequence for ERK2 may be found in Boulton et al, 1991 and is set out in FIG.\",\n \"39.Reference to such published data allows the ready design of peptide fragments of a MAP kinase or an integrin subunit cytoplasmic domain for use in the identification and localisation of the binding domain for the MAP kinase or the integrin as is applicable, and the identification of the corresponding nucleic acid sequence encoding such peptide fragments as well as the amino acid sequence of the binding domain.\",\n \"Localisation and characterisation of a binding domain for a MAP kinase or integrin enables the design of agents for binding to the binding domain for modulation of cell activity either for down regulating the functional activity of the integrin or the MAP kinase and more particularly the MAP kinase integrin binding interaction, or as agonists of the MAP kinase or integrin.\",\n \"This will typically involve determining the physical properties of the binding domain such as size and charge distribution, and the tertiary structure of the binding domain.\",\n \"Specifically, at least the region of the integrin containing the binding domain for the MAP kinase or the region of the MAP kinase containing the binding domain for the integrin is modelled taking into account the stereochemistry and physical properties of the binding domain such as size and charge distribution as well as its three dimensional structure as determined using x-ray chrstallography, nuclear magnetic resonance and/or commercially available computer modelling software.\",\n \"Such modelling techniques are well known in the art.\",\n \"In a variation of this approach, the modelling will take into account the interaction of the relevant binding domain with the MAP kinase or the integrin as the case may be such that any change in conformation arising from the interaction may be taken in to account in the design of an agent.\",\n \"Modelling flanking regions adjacent the binding domain also allows the design of agents for associating with such flanking regions but which are nevertheless capable of inhibiting the MAP kinase integrin interaction either by stearic hindrance or by distorting the conformation of the binding domain of the MAP kinase or integrin (eg.\",\n \"allostearic inhibitors).\",\n \"The design of a mimetic of the binding domain of interest will usually involve selecting or deriving a template molecule onto which chemical groups are grafted to provide required physical and chemical characteristics.\",\n \"The selection of template molecule and chemical groups is based on ease of synthesis, likely pharmacological acceptability, risk of or potential for degradation in vivo, stability and maintenance of biological activity upon administration.\",\n \"Pharmacological acceptability and the like are also taken into consideration in the design of other agent types.\",\n \"In order to constrain a polypeptide or other agent in a three dimensional conformation required for binding, it may be synthesised with side chain structures or otherwise be incorporated into a molecule with a known stable structure in vivo.\",\n \"In particular, a polypeptide or the like may be incorporated into an amino acid sequence at least part of which folds into a β-pleated sheet or helical structure such as an α-helix (eg.\",\n \"see Dedhar et al., 1997).\",\n \"A polypeptide or other agent may also be cyclised to provide enhanced rigidity and thereby stability in vivo.\",\n \"Various methods for cyclising peptides, fusion proteins or the like are known (eg.\",\n \"Schiller et al., 1985).\",\n \"For example, a synthetic peptide incorporating two cysteine residues distanced from each other along the peptide may be cyclised by the oxidation of the thiol groups of the residues to form a disulfide bridge between them.\",\n \"Cyclisation may also be achieved by the formation of a peptide bond between the N-terminal and C-terminal amino acids of a synthetic peptide or for instance through the formation of a bond between the positively charged amino group on the side chain of a lysine residue and the negatively charged carboxyl group on the side chain of a glutamine acid residue.\",\n \"As will be understood, the position of the various amino acid residues between which such bonds are formed will determine the size of the cycle.\",\n \"Variation of cycle size for optimisation of binding affinity may be achieved by synthesising peptides in which the position of amino acids for achieving cyclisation has been altered.\",\n \"The formation of direct chemical bonds between amino acids or the use of any suitable linker to achieve cyclisation is also well within the scope of the skilled addressee.\",\n \"Further strategies for identifying possible agents include large scale screening techniques as are known to the skilled addressee.\",\n \"For example, peptide library technology provides an efficient way of testing a vast number of potential agents.\",\n \"Such libraries and their use are well known.\",\n \"Prospective agents identified may be then further evaluated in suitable activity, competitive and other immunoassays.\",\n \"A method of screening for an agent or evaluating whether an agent is capable of binding to a MAP kinase or an integrin, and in particular the binding domain of a MAP kinase for an integrin or the binding domain of an integrin for a MAP kinase, and thereby inhibiting the MAP kinase integrin interaction or acting as a MAP kinase or integrin agonist, may for instance involve utilising the agent in an assay whereby the agent has the opportunity of binding to the MAP kinase or the integrin in the presence of the integrin or the MAP kinase as the case may be or prior to the addition of the integrin or the MAP kinase, and determining whether inhibition of binding of the MAP kinase to the integrin results.\",\n \"An alternate screening method may for instance involve selecting a test agent capable of binding with the integrin or MAP kinase, measuring cellular activity in the presence of the test agent, and comparing that activity with cellular activity in the absence of the test agent.\",\n \"Cellular activity may be assessed by cell growth as indicated by [3H]-thymidine uptake or other measurement of cellular activity.\",\n \"As will be understood, a difference in observed functional activity in the presence of the test agent is indicative of the modulating effect provided by the test agent.\",\n \"It will be understood that the integrin in the context of such assays may be an integrin subunit or polypeptide or fragment incorporating the binding domain of the integrin for the MAP kinase, or a homolog, analog, variant or derivative of such a molecule to which the MAP kinase is capable of binding.\",\n \"Similarly, a MAP kinase in this context may be an intact MAP kinase or a fragment thereof incorporating the binding domain for the integrin, or a homolog, analog, variant or derivative of such a molecule that is capable of binding with the binding domain of the integrin.\",\n \"In addition, determination of whether an agent is capable of binding to the binding domain of an integrin or MAP kinase may be achieved using a polypeptide or fragment as described herein consisting of the binding domain or a core amino acid sequence of the binding domain that directly participates in the binding interaction, or analogs or the like of such molecules.\",\n \"It is not necessary that an agent be proteinaceous in character and indeed, mimetics may be prepared which may not be a polypeptide at all but which nevertheless possess the capability of binding with the integrin or MAP kinase.\",\n \"Polypeptides including fusion proteins and fragments of a MAP kinase comprising the binding domain for an integrin or fragments of an integrin subunit comprising the binding domain for a MAP kinase, or which incorporate sufficient core amino acid sequence of the binding domain of the integrin or MAP kinase for binding by the MAP kinase or integrin are contemplated herein.\",\n \"Typically, a polypeptide will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less and generally, less than about 40 amino acids.\",\n \"Preferably, the length will be from between about 5 to about 30 amino acids, and more preferably from between about 10 amino acids and about 25 amino acids.\",\n \"Preferably, a polypeptide will comprise or incorporate the amino acid sequence RSKAKWQTGTNPLYR, more usually the amino acid sequence RSKAKNPLYR, or one or both of sequences RSKAK and NPLYR.\",\n \"Alternatively, a polypeptide may have the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or PSNLLLNTTCDLKIC.\",\n \"Polypeptides and fusion proteins or the like may be synthesised or produced using conventional recombinant techniques.\",\n \"Nucleic acid encoding a fusion protein may for instance be provided via the joining together of separate DNA fragments encoding peptides or polypeptides having desired three dimensional conformations and/or other characteristics by employing blunt-ended termini and oligonucleotide linkers, digestion to provide staggered termini as appropriate, and ligation of cohesive ends prior to insertion of the resultant chimeric sequence into a suitable expression vector.\",\n \"Alternatively, PCR amplification of DNA fragments can be utilised employing primers which give rise to amplicons with complementary termini which can be subsequently ligated together (eg.\",\n \"see Current Protocols in Molecular Biology.\",\n \"John Wiley & Sons, 1992).\",\n \"Nucleic acid sequences encoding for the polypeptides of the invention, mutagenised integrin subunits and the like as described herein are also encompassed as are the respective complementary antisense nucleic acid sequences.\",\n \"Sense oligonucleotides encoding for the binding site of an integrin subunit or MAP kinase or a partial amino acid sequence of a binding domain, and the complementary antisense oligonucleotides are particularly suitable for use as primers in polymerase chain reaction (PCR) amplification methods or as probes for detection of the presence the respective target nucleic acid sequences with which they hybridise such as in Southern blotting protocols, or in affinity chromatography purification of the target nucleic acid sequence.\",\n \"Probes may be labelled with for instance commonly used isotopes such as P32, fluorophores, chemiluminescent agents and enzymes (see eg.\",\n \"Essential Molecular Biology.\",\n \"A Practical Approach Vol.\",\n \"II, Oxford University Press, 1993; Current Protocols in Molecular Biology, Ausubel FM., John Wiley & Sons Inc., 1998).\",\n \"The choice of a label will vary depending on the degree of sensitivity required, ease of conjugation with the probe, safety and other factors.\",\n \"Oligonucleotides for use as probes or primers will usually have a length of less than about 60 nucleotides, usually less than about 50 or 40 nucleotides preferably, between about 14 and about 30 nucleotides, and more preferably, between about 14 and about 25 nucleotides.\",\n \"While it is desirable that a primer or probe has 100% complementarity with its target sequence, oligonucleotides may be designed with less complementarity but which nevertheless hybridise with the target sequence.\",\n \"Typically, a primer or probe will have a complementarity of about 70% or greater, more preferably about 80% or greater and most preferably about 90% or 95%, or greater.\",\n \"A probe will generally be designed for being capable of hybridising with its target nucleic acid sequence under moderate or high stringency wash conditions.\",\n \"Moderate stringency wash conditions are for example those that employ 0.2×SSC (0.015M NaCl/0.0015M sodium citrate)/0.1% SDS (sodium dodecylsulfate) wash buffer at 42° C. High stringency wash conditions employ for instance, 0.1×SSC wash buffer at 68° C. Generally, the content of purine relative to the content of pyrimidine nucleotides in the region of target nucleic acid of interest will be taken into account in the design of such primers and probes as will be their length in accordance with well accepted principles known in the art.\",\n \"In addition, the present invention provides vectors incorporating nucleic acid sequences of the invention.\",\n \"The term “vector” is to be taken to mean a nucleic acid molecule capable of facilitating the transport of a nucleic acid sequence inserted therein into a cell and includes expression vectors and cloning vectors.\",\n \"Suitable expression vectors include plasmids and cosmids capable of expression of a DNA (eg.\",\n \"genomic DNA or cDNA) insert.\",\n \"An expression vector will typically include transcriptional regulatory control sequences to which the inserted nucleic acid sequence is operably linked.\",\n \"By “operably linked” is meant the nucleic acid insert is linked to the transcriptional regulatory control sequences for permitting transcription of the inserted sequence without a shift in the reading frame of the insert.\",\n \"Such transcriptional regulatory control sequences include promotors for facilitating binding of RNA polymerase to initiate transcription, expression control elements for enabling binding of ribosomes to transcribed mRNA, and enhancers for modulating promotor activity.\",\n \"A promotor may be a tissue specific promotor which facilitates transcription of the nucleic acid insert only in specific cell lineages and not in other cell types or only to a relatively low level in such other cell types.\",\n \"The design of an expression vector will depend on the host cell to be transfected, the mode of transfection and the desired level of transcription of the nucleic acid insert.\",\n \"Numerous expression vectors suitable for transfection of prokaryotic (eg.\",\n \"bacterial) or eukaryotic (eg yeast, insect or mammalian cells) are known in the art.\",\n \"Expression vectors suitable for transfection of eukaryotic cells include pSV2neo, pEF.PGK.puro, pTk2, pRc/CNV, pcDNAI/neo, non-replicating adenoviral shuttle vectors incorporating the polyadenylation site and elongation factor 1-α promotor and pAdEasy based expression vectors most preferably incorporating a cytomegalovirus (CMV) promotor (eg.\",\n \"see He et al, 1998).\",\n \"For expression in insect cells, baculovirus expression vectors may be utilised examples of which include pVL based vectors such as pVL1392, and pVL941, and pAcUW based vectors such as pAcUW1.Viral expression vectors are particularly preferred.\",\n \"Typical cloning vectors incorporate an origin of replication (ori) for permitting efficient replication of the vector, a reporter or marker gene for enabling selection of host cells transformed with the vector, and restriction enzyme cleavage sites for facilitating the insertion and subsequent excision of the nucleic acid sequence of interest.\",\n \"Preferably, the cloning vector has a polylinker sequence incorporating an array of restriction sites.\",\n \"The marker gene may be drug-resistance gene (eg.\",\n \"Ampr for ampicillin resistance), a gene encoding an enzyme such as chloramphenicol acetyltransferase (CAT), β-lactamase, adenosine deaminase (ADA), aminoglycoside phosphotransferase (APH), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), or for instance β-galactosidase encoded by the E. coli lacZ gene (LacZ′).\",\n \"Yeast reporter genes include imidazole glycerolphosphate dehydratase (HIS3), N-(5′-phosphoribosyl)-anthranilate isomerase (TRP1) and β-isopropylmalate dehydrogenase (LEU2).\",\n \"As will be appreciated, expression vectors of the invention may also incorporate such marker genes.\",\n \"Cloning vectors include cloning vectors for mammalian, yeast and insect cells.\",\n \"Particular vectors that may find application include pBR322 based vectors and pUC vectors such as pUC118 and pUC119.Suitable expression and cloning vectors are for instance described in Molecular Cloning.\",\n \"A Laboratory Manual., Sambrook et al., 2nd Ed.\",\n \"Cold Spring Harbour Laboratory., 1989.Host cells suitable for being transformed by vectors of the invention include bacteria such as E. coli, Bacillus such as B. subtilis, Streptomyces and Pseudomonas bacterial strains, yeast such as Sacchromyces and Pichia, insect cells, avian cells and mammalian cells such as Chinese Hamster Ovary cells (CHO), COS, HeLa, HaRas, WI38, SW480, and NIH3T3 cells.\",\n \"Host cells may be cultured in a suitable culture medium and under conditions for facilitating expression of nucleic acid sequences of the invention or replication of cloning vectors, prior to purification from the host cells, and/or supernatants as the case may be using standard purification techniques.\",\n \"Rather than utilising viral mediated transfection of cells, nucleic acid sequences and other molecules of the invention may also be delivered to a cell in vitro or in vivo by liposome mediated transfection.\",\n \"The liposomes may carry targeting molecules for maximising delivery of the agent or agents contained therein to specific cell types of interest.\",\n \"Such targeting molecules may be for instance antibodies, ligands or cell surface receptors for facilitating fusion of liposomes to the specific cells of interest.\",\n \"Agents may also be intracellularly delivered in vitro using conventional cold or heat shock techniques or for instance, calcium phosphate coprecipitation or electroporation protocols.\",\n \"Yet another strategy is to design the agent to have the inherent ability to pass across the lipid bilayer of a cell.\",\n \"A particularly preferred way of achieving intracellular delivery of an agent is to use “carrier peptides” which have the ability to deliver macro-molecules across cell membranes in an energy-independent manner (Prociantz, 1996).\",\n \"Indeed, such peptides provide the possibility of both testing potential agents in cell culture without drastically altering cell membrane integrity and of delivering agents in vivo.\",\n \"Carrier peptides that are known in the art include penetratins and variants thereof (Derossi et al, 1994, 1996), human immunodeficiency virus Tat derived peptide (Prociantz, 1996), and transportan derived peptide (Pooga et al.\",\n \"1998).\",\n \"Indeed, carrier peptides have been successfully used to facilitate internalisation of mimetics of Src homology 2 binding sites, and peptides which inhibit protein kinase C mediated axon development and CD44 (hyaluronate receptor) dependent migration (Theodore et al, 1995; Williams et al, 1997; Peck Isacke, 1998; Derossi et al, 1998).\",\n \"Specific targetting to β6-expressing cancer cells may also be achieved by coupling humanised anti-β6 antibody to carrier molecules such as penetratin coupled to an agent capable of inhibiting binding of a MAP kinase with an integrin expressed by the cell or down regulation of the expression of the integrin.\",\n \"Coupling may for instance be by a peptide bond or disulfide bridge.\",\n \"Given that β6 expression enhances effective proteolysis at the cell surface by matrix metalloproteinase-9 (Agrez et al, 1999), such targetting approaches may include engineering an MMP-9 cleavage site between the antibody and the carrier peptide penetratin to facilitate internalisation of the pentratin-agent complex.\",\n \"Another approach may employ coupling the penetratin-agent complex to β6 integrin receptor-targetted peptides, targetted for binding to the extracellular β6 domain by virtue of their DLXXL sequence.\",\n \"For example, a ligand recognition motif for αVβ6 integrin, RTDLDSLRTYTL (Kraft et al, 1999) may be used in conjunction with or without an engineered MMP-9 cleavage site to release the penetratin-agent complex at the cell surface.\",\n \"Further protocol for targetting nucleic acids to cells by targetting integrins is described in Bachmann et al, 1998.The toxicity profile of an agent of the invention may be tested on normal and malignant cells by evaluation of cell morphology, trypan-blue exclusion, assessment of apoptosis and cell proliferation studies (eg cell counts, 3H-thymidine uptake and MTT assay).\",\n \"Agents of the invention may be co-administered with one or more other compounds or drugs.\",\n \"For example, an agent or agents may be administered in combination with antisense therapy or chemotherapeutic drugs.\",\n \"Alternatively, an agent may be administered in conjunction with antisense therapy and/or chemotherapeutic drugs.\",\n \"By “co-administered” is meant simultaneous administration in the same formulation or in two different formulations by the same or different routes, or sequential administration by the same or different routes.\",\n \"By “sequential” administration is meant a time difference of between the administration of the agents, drugs and other therapies which can be administered in any order.\",\n \"The time difference may range from very short times up to hours or for instance days or weeks.\",\n \"The agent or agents will typically be formulated into pharmaceutical composition incorporating pharmaceutically acceptable carriers, diluents and/or excipients for administration to the intended subject.\",\n \"Pharmaceutical forms include sterile aqueous solutions suitable for injection, (where the agent or agents is water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions.\",\n \"Such injectable compositions will be fluid to the extent that the syringability exists and typically, will be stable to allow for storage after manufacture.\",\n \"The carrier may be a solvent or dispersion medium containing one or more of ethanol, polyol (eg glycerol, propylene glycol, liquid polyethylene glycol and the like), vegetable oils, and suitable mixtures thereof.\",\n \"Fluidity may be maintained by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants.\",\n \"Sterile injectable solutions will typically be prepared by incorporating the active agents in the desired amount in the appropriate solvent with various other components enumerated above, prior to sterilising the solution by filtration.\",\n \"Generally, dispersions will be prepared by incorporating the sterile active agents into a sterile vehicle which contains the dispersion medium and other components.\",\n \"In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active agent plus any additional desired ingredient from previously sterile filtered solutions thereof.\",\n \"For oral administration, the active agents may be formulated into any orally acceptable carrier deemed suitable.\",\n \"In particular, the active ingredient may be formulated with an inert diluent, an assimilable edible carrier or it may be enclosed in a hard or soft shell gelatin capsule.\",\n \"Alternatively, it may be incorporated directly into food.\",\n \"Moreover, an active agent may be incorporated with excipients and used in the form of ingestable tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and the like.\",\n \"Such compositions will generally contain at least about 1% by weight of the active agent or agents.\",\n \"The percentage may of course be varied and may conveniently be between about 5 to about 80% w/w of the composition or preparation.\",\n \"As will be appreciated, the amount of active agent or agents in such compositions will be such that a suitable effective dosage will be delivered to the subject taking into account the proposed mode of administration.\",\n \"Preferred oral compositions according to the invention will contain between about 0.1 μg and 2000 mg of each active agent, respectively.\",\n \"Active agents may also be formulated into topically acceptable carriers conventionally used for forming creams, lotions, ointments and the like for internal or external application.\",\n \"Topical formulations may be applied to a site to be treated by dressings and the like impregnated with the formulation.\",\n \"Typically, a composition of the invention will incorporate one or more preservatives such as parabens, chlorobutanol, phenol, sorbic acid, and thimersal.\",\n \"In many cases, a composition may furthermore include isotonic agents such as sugars or sodium chloride.\",\n \"In addition, prolonged absorption of the composition may be brought about by the use in the compositions of agents for delaying absorption such as aluminium monosterate and gelatin.\",\n \"Tablets, troches, pills, capsules and the like may also contain one or more of the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium sterate; a sweetening agent such as sucrose, lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, orange or cherry flavouring.\",\n \"When the dosage unit form is a capsule, it may contain in addition to one or more of the above ingredients a liquid carrier.\",\n \"Various other ingredients may be present as coatings or to otherwise modify the physical form of the dosage unit.\",\n \"For instance, tablets, pills or capsules may be coated with shellac, sugars or both.\",\n \"In addition, an active agent may be incorporated into any suitable sustained-release preparation or formulation.\",\n \"Pharmaceutically acceptable carriers, diluents and/or excipients include any suitable conventionally known solvents, dispersion media and isotonic preparations or solutions.\",\n \"Use of such ingredients and media for pharmaceutically active substances is well known.\",\n \"Except insofar as any conventional media or agent is incompatible with the active agent, use thereof in therapeutic and prophylactic compositions is contemplated.\",\n \"Supplementary active ingredients can also be incorporated into the compositions if desired.\",\n \"It is particularly preferred to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.\",\n \"Dosage unit form as used herein is to be taken to mean physically discrete units suited as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of active agent calculated to produce the desired therapeutic or prophylactic effect in association with the relevant carrier, diluent and/or excipient.\",\n \"A unit dosage formed will generally contain each active agent in amounts ranging from about 0.5 μg to about 2000 mg/ml of carrier respectively.\",\n \"A pharmaceutical composition may also comprise vectors capable of transfecting target cells where the vector carries a nucleic acid molecule for modulating functional activity or expression of an integrin or MAP kinase.\",\n \"The vector may for instance, be packaged into a suitable virus for delivery of the vector into target cells as described above.\",\n \"The dosage of an active agent will depend on a number of factors including whether the agent is to be administered for prophylactic or therapeutic use, the condition for which the agent is intended to be administered, the severity of the condition, the age of the subject, and related factors including weight and general health of the subject as may be determined by the physician or attendant in accordance with accepted principles.\",\n \"Indeed, a low dosage may initially be given which is subsequently increased at each administration following evaluation of the subjects response.\",\n \"Similarly, frequency of administration may be determined in the same way that is, by continuously monitoring the subjects response between each dosage and if necessary, increasing the frequency of administration or alternatively, reducing the frequency of administration.\",\n \"The route of administration of a pharmaceutical composition will again depend on the nature of the condition for which the composition is to be administered.\",\n \"Suitable routes of administration include but are not limited to respiritoraly, intratracheally, nasopharyngeally, intraveneously, intraperitonealy, subcutaneously, intracranialy, intradermially, intramuscularly, intraoccularly, intrathecally, intranasally, by infusion, orally, rectally, via IV group patch, topically and by implant.\",\n \"With respect to intravenous routes, particularly suitable routes are via injection into blood vessels which supply a tumour or particular organs to be treated.\",\n \"Agents may also be delivered into cavities such for example the pleural or peritoneal cavity, or be injected directly into tumour tissue.\",\n \"The production of antibodies and monoclonal antibodies is well established in the art (eg.\",\n \"see Antibodies, A Laboratory Manual.\",\n \"Harlow & Lane Eds.\",\n \"Cold Spring Harbour Press, 1988).\",\n \"For polyclonal antibodies, a mammal such as a sheep or rat for instance is immunised with a polypeptide of the invention and antisera subsequently isolated prior to purification of the antibodies therefrom by standard affinity chromatography techniques such as Sepharose-Protein A chromatography.\",\n \"Desirably, the mammal is periodically challenged with the relevant antigen to establish and/or maintain high antibody titre.\",\n \"To produce monoclonal antibodies, B lymphocytes can be isolated from the immunised mammal and fused with immortalising cells (eg.\",\n \"myeloma cells) by standard somatic cell fusion techniques (eg.\",\n \"utilising polyethylene glycol) to produce hybridoma cells (Kohler and Milstein, 1975; see also Handbook of Experimental Immunology, Weir et al Eds.\",\n \"Blackwell Scientific Publications.\",\n \"4th Ed.\",\n \"1986).\",\n \"The resulting hybridoma cells may then be screened for production of antibodies specific for the peptide by an enzyme linked immunosorbant assay (ELISA) or other immunoassay.\",\n \"Conventionally used methods for preparing monoclonal antibodies include those involving the use of Epstein-Barr virus (Cole et al.\",\n \"Monoclonal Antibodies and Cancer Therapy, Allen R. Liss Inc. pp.\",\n \"77-96, 1985).\",\n \"The term “antibody” or “antibodies” as used herein is to be taken to include within its scope entire intact antibodies as well as binding fragments thereof such as Fab and (Fab′)2 fragments which may be obtained by papain or pepsin proteolytic cleavage, respectively.\",\n \"An antibody of the invention may be labelled for enabling detection of antibody binding in immunoassays including competitive inhibition assays.\",\n \"A “label” may be any molecule which by its nature is capable of providing or causing the production of an analytically identifiable signal which allows the detection of an antibody and antigen complex.\",\n \"Such detection may be qualitative or quantitative.\",\n \"An antibody can for instance be labelled with radioisotopes including 32p, 125I or 131I, an enzyme, a fluorescent label, chemiluminescent molecule or for instance an affinity label such as biotin, avidin, streptavidin and the like.\",\n \"An enzyme can be conjugated with an antibody by means of coupling agents such as gluteraldehyde, carbodiimides, or for instance periodate although a wide variety of conjugation techniques exist.\",\n \"Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase amongst others.\",\n \"Detection utilising enzymes is achieved with use of a suitable substrate for the selected enzyme.\",\n \"The substrate is generally chosen for the production upon hydrolysis of a detectable colour change.\",\n \"However, fluorogenic substrates may also be used which yield a fluorescence product rather than a chromogen.\",\n \"Suitable fluorescent labels are those capable of being conjugated to an antibody substantially without altering the binding capacity of the antibody and include fluorescein, phycoerythrin (PE) and rhodamine which emit light at a characteristic wavelength in the colour range following illumination with light at a different wavelength.\",\n \"Methods for labelling of antibodies can be found in Current Protocols in Molecular Biology.\",\n \"Ausubel FM., John Wiley & Sons Inc. Immunoassays in which antibodies of the invention may be utilised include radioimmunoassays (RIA) and ELISA (eg., see Handbook of Experimental Immunology, Weir et al., Vol.\",\n \"1-4, Blackwell Scientific Publications 4th Edition, 1986).\",\n \"Such assays include those in which a target antigen is detected by direct binding with a labelled antibody, and those in which the target antigen is bound by a first antibody, typically immobilised on a solid substrate (eg., a microtitre tissue culture plate formed from a suitable plastics material such as polystyrene or the like) and a labelled second antibody specific for the first antibody used to form an antigen-first antibody-second antibody complex that is detected by a signal emitted by the label.\",\n \"Sandwich techniques in which the antigen is immobilised by an antibody for presentation to a labelled second antibody specific for the antigen are also well known.\",\n \"An antibody can be bound to a solid substrate covalently utilising commonly used amide or ester linkers, or by adsorption.\",\n \"Optimal concentrations of antibodies, temperatures, incubation times and other assay conditions can be determined by the skilled addressee with reference to conventional assay methodology and the application of routine experimentation.\",\n \"Antibodies and other molecules of the invention including polypeptides and oligonucleotides as described herein when bound to a solid support can be used in affinity chromatography for the purification of a binding partner for which they are specific.\",\n \"In particular, a polypeptide either alone or as a fusion protein comprising or consisting of the binding domain for a MAP kinase for instance can be utilised in the purification, isolation or concentration of an integrin or fragment thereof or other molecule capable of binding to the binding domain of the MAP kinase for the integrin.\",\n \"It may also be used for assaying levels of the integrin for instance in cell extracts.\",\n \"Similarly, an antibody or other such molecule as described herein that specifically binds to the binding domain of a MAP kinase has use in the purification of the MAP kinase or fragments thereof incorporating a binding domain for the integrin from a relatively crude preparation or mixture.\",\n \"Suitable solid supports include agarose, sepharose and other commercially available supports such as beads formed from latex, polystyrene, polypropylene, dextran, glass or synthetic resins, typically packed in an affinity column through which the relevant sample containing the binding partner is passed at a pH and conditions (eg., low salt concentration) under which the binding partner becomes bound by the antibody, polypeptide or other such molecule.\",\n \"The column is then washed utilising a suitable buffer whereby the binding partner is retained bound on the column, prior to being eluted therefrom utilising a suitable elution buffer (eg., with a higher salt concentration and at an altered pH, typically pH 2.5 or pH 11) that facilitates the release of the binding partner from the affinity column, and collected.\",\n \"Protocols for affinity chromatography are described in Current Protocols in Molecular Biology-Ausubel FM., John Wiley & Sons Inc. Buffers and conditions utilised for the purification, isolation or concentration of a binding partner will vary depending on the affinity the antibody, polypeptide or the like for the binding partner.\",\n \"A kit for use in assays as described herein may include one or more of an antibody, polypeptide, vector, nucleic acid or other molecule of the invention.\",\n \"The kit may also comprise one or more other reagents such as washing solutions, dilution buffers and the like together with instructions for use.\",\n \"The antibody or other molecule of the invention may or may not be labelled, bound to a solid support or be coupled with another molecule.\",\n \"Particularly preferred kits are those provided for use in affinity chromotography or RIA, ELISA or other type of immunoassay.\",\n \"The present invention will be described herein after with reference to a number of examples.\",\n \"EXAMPLE 1 Anti-integrin antibodies and antibodies against the β1 subunit have been shown to inhibit proliferation of retinal pigment epithelial cells (Hergott et al, 1993).\",\n \"In endothelial cells, inhibition of cell-matrix interactions by anti-integrin antibodies specifically against α2β1 converts the cells from a proliferative to a differentiated phenotype (Gamble et al, 1993).\",\n \"In another study, synthetic peptides containing the integrin recognition sequence arginine-glycine-aspartate (RGD) have been shown to inhibit tumour cell invasion in vitro and tumour metastases from melanoma in an animal model (Humphries et; al, 1986; Gehlsen et al, 1988).\",\n \"In colon cancer, the contribution made by the α5β1 receptor to regulation of growth appears to be ligand (fibronectin)-dependent.\",\n \"For example, induced expression of α5β1 in human colon cancer cells constitutively lacking this integrin has been shown to result in decreased tumour cell proliferation in vitro (Varner et al, 1995).\",\n \"Interestingly, when the appropriate ligand was present, cell proliferation was restored, indicating that the unoccupied receptor mediated a negative growth signal in these cells (Varner et al, 1995).\",\n \"Moreover, induction of α5β1 expression was associated with a marked reduction of tumourigenicity in immune-deficient mice.\",\n \"Failure to ligate all of the tumour cell α5β1 molecules with sufficient murine fibronectin most likely accounts for the in vivo tumour suppression in these studies (Varner et al, 1995).\",\n \"In the present study the effect of down-regulating αvβ6 expression on colon cancer growth was examined.\",\n \"1.1 Methods 1.1.1 Generation of sense and antisense β6 constructs in pEF.PGK.puro vector.\",\n \"For β6 antisense constructs, β6 cDNA was excised from the vector pcDNA1neo β6 (Weinacker et al, 1994) using the restriction enzymes SnaB1 and Xba1.This produced a 5′ overhang (Xba1) and a 3′ blunt end (SnaB1).\",\n \"The 5′ overhang was blunted with Klenow (Promega) prior to ligation.\",\n \"pEF.PGK.puro vector (a gift from D. Huang, the Walter & Eliza Hall Institute, Melbourne Australia) was cut with EcoRV which produced blunt ends.\",\n \"The pEF.puro vector was de-phosphorylated using calf intestinal alkaline phosphatase (CIAP, Promega) and the β6 cDNA ligated overnight into pEF.puro using T4 DNA ligase (Promega) at a ratio of vector:insert of 1:5.After ligation all the reaction mix was used for transformation into competent JM109 cells and the cells plated onto LB plates containing ampicillin.\",\n \"After overnight incubation at 37° C., colonies were selected, the plasmid DNA extracted by microprepping, and the DNA cut with BstE11 to confirm the antisense orientation of β6.Digestion with BstE11 produced the expected two fragments, one of 6.0 basepairs and the other 3.5 basepairs.\",\n \"To provide β6 sense constructs, β6 cDNA was also excised from the pcDNA1neo b6 vector using the restriction enzymes Snab1 and Xba1 as described above.\",\n \"The pEF.puro vector was then cut with Xba1 and EcoRV and the insert was ligated in sense direction into the pEF.PGK.puro vector without having to blunt the insert.\",\n \"The ligation reaction was prepared at a ratio of vector:insert of 1:1.Digestion with BstE11 produced two expected fragments, one of 7.5 basepairs and the other 2.0 basepairs.\",\n \"1.1.2 Transfection of WiDr and HT29 Colon Cancer Cells The human colon cancer cell lines, WiDr and HT29 were obtained from the American Type Culture Collection (ATCC), Rockville, Md., USA and maintained as monolayers in standard medium comprising Dulbecco's Modified Eagle's medium (DMEM; 4.5 gm/litre of glucose) with 10% heat-inactivated foetal bovine serum (FBS) supplemented with HEPES and penicillin/streptomycin.\",\n \"WiDr and HT29 cell lines constitutively express αvβ6.Stable transfectants of WiDr and HT29 cells expressing β6 in sense and antisense direction were generated using lipofectamine and puromycin as the selection antibiotic.\",\n \"One stable antisense β6 transfectant from HT29 cells and three stable clones from WiDr cells were successfully established for use in all experiments.\",\n \"Mock transfectants using vector alone were also generated as controls.\",\n \"Initial killing curves performed using a range of concentrations of puromycin established that WiDr and HT29 cells could be stably transfected with puromycin concentrations of 1.0 μg and 2.5 μg/ml, respectively.\",\n \"1.1.3 Assessment of β6 Expression in the Transfected Cell Lines β6 expression was assessed by means of FACScan analyses of parent cell lines and clones generated therefrom by means of limiting-dilution experiments.\",\n \"Stability of the transfectants was confirmed regularly by repeated FACScan analyses, and surface biotinylation and immunoprecipitation as described below.\",\n \"1.1.4 FACScan Analyses Monolayer cultures of cell lines were harvested with trypsin/EDTA and then blocked with goat serum at 4° C. for 10 min.\",\n \"Cells were washed once with PBS, incubated with primary antibody against integrin subunits for 20 min at 4° C. and then washed twice with PBS.\",\n \"Cells were then stained with secondary antibody conjugated with phycoerythrin for 20 min at 4° C., washed twice with PBS and resuspended in 0.5 ml PBS prior to FACScan analysis (Becton Dickinson, Rutherford, N.J., USA).\",\n \"1.1.5 Integrin Immunoprecipitation Cells were harvested with trypsin/EDTA, the trypsin neutralised with standard culture medium, and the cell pellets washed once with cold PBS.\",\n \"Cell pellets were then exposed to biotin-CNHS-ester (Sigma) in biotinylation buffer (10 mM sodium borate, 150 mM sodium chloride, pH 8.8) for 30 mins at 4° C. with continuous slow mixing.\",\n \"Cell pellets were then centrifuged at 4° C., washed twice with cold PBS and exposed to lysis buffer (containing 100 mM Tris, 150 mM NaCl, 1 mM CaCl2, 1% Triton, 0.1% SDS and 0.1% NP-40 at pH 7.4 and containing 1 mM phenylmethylsulfonyl fluoride (PMSF)) for 30 min at 4° C. Lysates were then clarified by ultracentrifugation (10,000 g) for 30 min and the protein content measured using the BCA protein assay reagent kit.\",\n \"Lysates containing equal protein amounts were pre-cleared with rabbit anti-mouse (RAM) immunoglobulin coupled to Sepharose-4B beads for 12 hrs.\",\n \"Immunoprecipitations were carried out indirectly using RAM-Sepharose 4B and analysed by 7.5% SDS-PAGE under non-reducing conditions.\",\n \"1.1.6 Reverse Transcriptase-PCR (RT-PCR) mRNA levels for β6 expression were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR).\",\n \"Total RNA was extracted from cell cultures using the commercial TriPure isolation reagent based on the method of Chomozynski and Sacchi (1987).\",\n \"0.4-2 μg of RNA was used to prepared cDNA by reverse transcription.\",\n \"Briefly, a reaction mixture in a final volume of 40 μl containing 8 μl of 5×RT reaction buffer (250 mM Tris, 15 mM MgCl2, 375 mM KCL, pH 8.3), 8 μl of 2.5 mM of each dNTP, 4 μl of 100 mM DTT, 40U of an Rnase inhibitor, Rnasin (Promega, Madison, Wis., USA), 0.5 μg of random hexamers (Promega) and 200U of Moloney murine leukaemia virus (M-MLV) reverse transcriptase (Promega) were mixed and incubated at 38° C. for a minimum of 90 min.\",\n \"The reaction was stopped by heating at 95° C. for 5 min and cDNA stored at 4° C. until PCR.\",\n \"2-5 μl of this cDNA was combined with 5 μl of 10×PCR buffer (100 mM Tris, 500 mM KCl, 15 mM MgCl2, pH8.3) 8 μl of 1.25 mM dNTP each and 1.25 μl of 20 μM of both forward and reverse primers.\",\n \"The forward primer sequence was 5′AGGATAGTTCTGTTTCCTGC3′ and the reverse primer sequence 5′ATCATAGGAATATTTGGAGG3′.\",\n \"The reaction was initiated by 2.5U of Taq polymerase in a final volume of 50 μl.\",\n \"After an initial 5 min incubation at 94° C., 30 cycles of amplification were performed under the following conditions: 94° C. 1 min, 54° C. 1 min and 72° C. for 1 min.\",\n \"The reaction was stopped by incubating at 72° C. for 10 min.\",\n \"To verify that equal amounts of RT product from cells were subjected to PCR amplification, the same amounts of cDNA were amplified for the “house-keeping” gene GAPDH using specific primers.\",\n \"The same reaction conditions were used except that the annealing temperature was changed to 48° C. and PCR amplification performed for 35 cycles.\",\n \"1.2 Results 1.2.1 αvβ6 Expression in HT29 and WiDr Transfected Cell Lines Transfection of the colon cancer cell lines HT29 and WiDr with the β6 gene construct in an antisense orientation resulted in a marked reduction of β6 expression at the transcript level and on the cell surface as shown in FIGS.\",\n \"1 to 4.Transfection of cells with β6 in the sense orientation did not enhance β6 surface expression.\",\n \"However, a consequence of down-regulation of the β6 subunit in antisense transfectants was a marked increase in surface expression of the β5 subunit.\",\n \"The changes in surface expression of β6 and β5 subunits noted on FACScan analyses of antisense β6 transfectants was confirmed by surface-labelling cells with biotin and immunoprecipitating integrin subunits with either anti-β6 mAb (R6G9) or anti-β5 mAb (P1F6).\",\n \"1.2.2 Effect of Suppression of αvβ6 Expression on Cell Binding to Fibronectin The major substrate for αvβ6 is fibronectin and to investigate the effect that reduction in β6 surface expression in antisense β6 transfectants might have on cell-matrix adhesion, WiDr and HT29 antisense β6 transfectants were seeded on fibronectin in adhesion assays.\",\n \"Since both cell lines can adhere to fibronectin through members of the β1 integrin subfamily (either α3β1 or α5β1) αvβ6-mediated adhesion to fibronectin was assessed in the absence/presence of blocking anti-β1 antibody.\",\n \"The cell-adhesion assays were performed in wells of non-tissue culture-treated polystyrene 96-well flat bottom microtitre plates (Nunc, Roskilde, Denmark).\",\n \"Culture wells were coated with fibronectin, washed with phosphate-buffered saline (PBS) and then blocked with 0.5% bovine serum albumin (Sigma) in PBS for 1 hr at 37° C. Harvested cells were seeded at a density of 105 cells/well for HT29 and 1.5×105 cells/well for WiDr cells in 200 μl of standard DMEM which lacked FBS but contained 0.5% bovine serum albumin.\",\n \"To block adhesion, cells were incubated with anti-β1 blocking antibodies for 15 min at 4° C. before plating.\",\n \"The plates were centrifuged (top side up) at 10×g for 5 min, then incubated for 1 hr at 37° C. in humidified 5% carbon dioxide.\",\n \"Non-adherent cells were removed by centrifugation top side down at 48×g for 5 min.\",\n \"The attached cells were fixed and stained with 0.5% crystal violet (in 20% methanol and 1% formaldehyde) and the wells washed with phosphate-buffered saline.\",\n \"The relative number of cells in each well was evaluated by measuring the absorbance at 595 nm in a Microplate Reader (Bio-Rad).\",\n \"With reduction in cell surface expression of β6, β1-independent adhesion of cells to fibronectin was reduced compared with mock transfectants (containing vector alone) which express surface β6 at similar levels to wild-type cells.\",\n \"The further addition of blocking anti-αv antibody completely prevented binding to fibronectin.\",\n \"1.2.3 Effect of Suppression of αvβ6 Expression on Tumour Cell Proliferation and Tumour Growth In Vivo To investigate the effect of diminished αvβ6 surface expression on cell proliferation in vitro, WiDr and HT29 antisense β6 transfectants were seeded as monolayers in 96-well microtitre culture plates (5,000 viable cells per well) in standard culture medium containing the puromycin selection antibiotic.\",\n \"Cells were pulsed with 1 μCi (3H)-thymidine (Amersham) per well for the last 24 hours of each experiment before automated harvesting and measurement of radioactivity.\",\n \"WiDr wild-type, mock and antisense β6 transfectants, and HT29 wild-type, mock, sense β6 and antisense β6 transfectants were harvested second daily during a six-day culture period.\",\n \"A marked increase in thymidine incorporation was observed for WiDR and HT29 cells expressing normal levels of αvβ6 compared with antisense β6 transfectants (see FIGS.\",\n \"5 and 6).\",\n \"1.2.4 Effect of Suppression of αvβ6 Expression on Tumour Formation The ability of HT29 antisense β6 transfactants to form tumours in immune-deficient mice was assessed.\",\n \"BALB/C female athymic mice (8 weeks of age purchased from the Animal Resource Centre, Perth, Western Australia) were maintained under pathogen-free conditions and fed standard mouse chow and water ad lib.\",\n \"The mice were divided into groups of ten each and all mice within each group inoculated with a single cell line.\",\n \"Cells used were WiDr mock (transfected with vector alone) and antisense β6 transfected clones (clones 1-3) and HT29 mock, sense and antisense β6 cell lines.\",\n \"Mice received subcutaneous flank injections of 106 viable tumour cells suspended in 0.2 ml of standard DMEM culture medium.\",\n \"Animal weights and tumour sizes (breadth and length as measured with calipers) were recorded weekly.\",\n \"Six weeks following the last injection, visible subcutaneous tumours were excised, weighed and fixed in 4% formalin.\",\n \"At the time of euthanasia, all internal organs were routinely inspected for presence of metastases.\",\n \"Tumour growth after six weeks following inoculation of HT29 mock and antisense β6 transfectants is shown in FIG.\",\n \"7.Measurements of tumour growth for WiDr and HT29 cells are shown in FIGS.\",\n \"8 and 9, respectively.\",\n \"Similar tumour growth profiles for mock and antisense β6 WiDr clones 2 and 3 each inoculated into 10 mice are not shown.\",\n \"Of a total of 40 mice injected with cells expressing antisense β6 (HT29-10 mice and WiDr cells lines, 3 clones-30 mice) tumours completely disappeared in 93% of animals.\",\n \"In the remaining 3 animals tumour sizes diminished to 1 mm2 in size during the six week period compared with tumours at least 15 mm2 in size from cells expressing normal levels of β6.To confirm the presence of tumour xenografts histologically at one week following subcutaneous inoculation of mock and antisense β6 transfectants, tumour nodules were excised, fixed in formalin and stained with haematoxylin and eosin.\",\n \"1.2.5 Effect of Suppression of αvβ6 Expression on Gelatinase B Secretion Serum-free tumour-conditioned medium was collected from each of the 3 WiDr mock and antisense β6 clones and concentrated ×44 for measurement of gelatinase B using the Biotrak MMP-9 activity assay system (Amersharn Pharmacia Biotech, Uppsala Sweden).\",\n \"Down-regulation of αvβ6 expression resulted in a marked reduction in gelatinase B secretion.\",\n \"1.3 Discussion of Results Induced expression of β6 in Chinese hamster ovary (CHO) cells has been shown to result in decreased surface expression of the β5 integrin subunit which also partners αv (Weinacker et al, 1994).\",\n \"The concept of integrin switching depends on the availability of the promiscuous αv partner subunit.\",\n \"In the present study, the reverse was observed.\",\n \"As a consequence of down-regulation of β6 in colon cancer cells which constitutively express αvβ6, β5 surface expression increased, most likely secondary to increased availability of the αv subunit partner.\",\n \"Heterologous expression of αvβ6 in colon cancer cells has previously been reported to enhance tumour growth in immune-deficient mice (Agrez et al, 1994).\",\n \"Suppression of αvβ6 expression in the present study was shown to result in nearly complete disappearance of tumours in 93% of animals following subcutaneous inoculation of tumour cells.\",\n \"Moreover, in the remaining 7% of animals, a 95% reduction in tumour size was observed over a six week period compared with large tumours seen in all animals injected with cells in which αvβ6 expression had not been perturbed.\",\n \"Similar findings have been described with loss of the classical vitronectin receptor αvβ3 in melanoma.\",\n \"For example, in experimental animal models, the loss of αvβ3 expression in melanoma cells has been shown to lead to reduced in vivo proliferation which is restored upon re-expression of the receptor (Felding-Habermann et al, 1992).\",\n \"Although the mechanisms involved in αvβ6-mediated tumour growth remain to be elucidated, the present in vitro data show that loss of β6 expression is associated with decreased proliferative capacity of the cells.\",\n \"Taken together with the marked reduction in gelatinase B secretion seen for colon cancer cells transfected with antisense β6, the findings reported in the present study suggest that intracellular signalling pathways activated via the integrin αvβ6 play a major role in promoting progression of this tumour type.\",\n \"EXAMPLE 2 The association of αvβ6 expression and MAP kinase activity were evaluated using WiDr, HT29 and SW480 cell lines.\",\n \"2.1 Methods 2.1.1 SW480 Colon Cancer β6 Transfectants Stable transfectants of SW480 colon cancer cells (ATCC) expressing gene constructs of either wild-type or mutant forms of the β6 integrin subunit or the expression plasmid only (pcDNA1neo) have been previously described (Agrez et al, 1994).\",\n \"The transfected SW480 cell lines were maintained in standard medium supplemented with the neomycin analogue G418.Stable transfectants of WiDr and HT29 cells expressing wild-type β6 in antisense orientation were generated as described in Example 1.1.2 and maintained in standard medium supplemented with puromycin.\",\n \"2.1.1 MAP Kinase Assay.\",\n \"Cultures of WiDr and HT29 mock and antisense β6 transfectants were established by seeding 1×106 cells/5 ml of culture medium in 25 cm2 tissue culture flasks.\",\n \"Cells were incubated at 37° C. in humidified CO2 for 24 hours before serum starvation in serum-free medium for the next 16 hours.\",\n \"Foetal calf serum was then added to a final concentration of 10% for 30 mins before MAP kinase assays were performed.\",\n \"Before each experiment the cells were washed twice with PBS, resuspended in extraction buffer (10 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 2 mM DTT, 1 mM orthovanadate, 1 mM PMSF, 4 μg/ml aprotonin, 2 μg/ml leupeptin and 1 μg/ml pepstatin, pH 7.4) and sonicated at a setting of 7, using a Soniprep 150 watt ultrasonic disintegrator for a total of 90 seconds in three 30 second pulses with an interval of 30 seconds between each pulse.\",\n \"Cellular debris was removed by centrifugation at 900 g for 10 min at 4° C. The assay was performed on equal cell numbers using a MAP kinase assay system (Amersham Pharmacia Biotech, Uppsala Sweden).\",\n \"The ability of cells to transfer phosphate from [γ32P]-ATP to a synthetic peptide that contains specifically a p42/p44 MAP kinase phosphorylation site was measured as described in the manufacturer's instructions.\",\n \"[32P]-labelled peptides were spotted onto PEI-cellulose paper, unbound radioactivity was washed with 75 mM phosphoric acid and bound [32P]-labelled peptides were measured by liquid scintillation counting.\",\n \"Protein estimation was performed on each cell lysate used and enzyme activity calculated as described in the manufacturer's instructions.\",\n \"Where MEK inhibitors, PD98059 and U0126 were used, cells were cultured as described above and the inhibitors, at a final concentration of 40 μM, were added one hour before the addition of serum to the medium.\",\n \"2.1.2 Western Blotting To detect the MAP kinases ERK1/2, cells were lysed in lysis buffer containing 100 mM Tris, 150 mM NaCl, 1 mM CaCl2, 1% Triton, 0.1% SDS and 0.5% NP-40 at pH7.4, supplemented with enzyme inhibitors (1 mM PMSF, 1 mM sodium orthovanadate, 1 μg/ml pepstatin A, 2.5 μg/ml aprotonin, 1 mM benzamidine, 1 μg/ml leupeptin).\",\n \"Lysates were clarified by ultracentrifugation and equal protein loads electrophoresed in 8% or 10% SDS-PAGE under non-reducing conditions.\",\n \"Electrophoresed proteins were transferred to nitrocellulose membranes (Biotrace NT, Gelman Sciences, Ann Arbor, Mich.) in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, 0.1% SDS) for 2 hours at a constant voltage of 40 volts in a Transfer Blot Cell (Bio-Rad).\",\n \"Membranes were blocked with 5% casein for 1 hr at room temperature and probed with monoclonal anti-ERK antibodies (E10 which recognises only phosphorylated ERK1/2 (New England BioLabs) and SC-1647 which recognises total ERKs, non-phosphorylated and phosphorylated (Santa Cruz Biotechnology).\",\n \"In some experiments, membranes were probed with polyclonal anti-ERK antibody (New England BioLabs) which also recognises total ERKs.\",\n \"Membranes were then washed three times in Tris buffered saline, containing 0.1% Tween and incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit antibody.\",\n \"Blots were visualised by the enhanced chemi-luminescence detection system according to the manufacturer's instructions (Du Pont).\",\n \"2.1.3 Integrin β-Subunit Immunoprecipitations Tumour cells were harvested and divided into two equal aliquots based on cell counts.\",\n \"One aliquot was surface biotinylated and the cells lysed for integrin immunoprecipitations as described in Example 1.1.5, with the exception that three sequential rounds of immuno-precipitation were performed to deplete the lysates of integrin subunits β5 and β6.Lysis buffer was the same as that used for Western blotting.\",\n \"The other aliquot, intended for subsequent ERK2 immunoblotting to examine the effect of integrin immunodepletion on ERKs was not biotinylated but lysed immediately and divided into three samples each at a protein concentration of 1 mg/ml.\",\n \"Three sequential rounds of immunoprecipitation were performed against anti-β6 monoclonal antibody, R6G9 (using isotype matched antibody, IgG2A, and anti-β5 monoclonal antibody, P1F6, in control immunoprecipitations).\",\n \"The integrin-depleted lysates were electrophoresed in 8% or 10% SDS-PAGE under non-reducing conditions and transferred to nitrocellulose membranes.\",\n \"Membranes were blocked with casein as for Western blotting and probed with anti-ERK monoclonal antibodies E10 and SC-1647.In parallel experiments, the sequentially immunoprecipitated β6 subunit bound to rabbit anti-mouse (RAM) coupled Sepharose B4 beads was also electrophoresed in 10% SDS-PAGE under non-reducing conditions, transferred to nitrocellulose membranes, and the membranes probed with anti-ERK monoclonal antibody E10 which recognises only phosphorylated forms of ERK1/2.2.3 Results 2.3.1 Effect of Serum on MAP Kinase Activity The effect of MEK (MAP kinase) inhibitors UO126 and PD98059 on MAP kinase activity in WiDr and HT29 wild-type cells was tested in the absence/presence of serum.\",\n \"Adherent cell monolayers on plastic were grown for 24 hrs in standard culture medium, then washed three times in PBS followed by 16 hrs in culture under serum-free conditions.\",\n \"Serum was then added for 30 min and MAP kinase activity assessed before and after addition of serum.\",\n \"The addition of serum markedly stimulated MAP kinase activity for both cell lines and was inhibitable by both MEK inhibitors.\",\n \"2.3.2 Effect of Altered β6 Expression on MAP Kinase Activity Following Serum Stimulation.\",\n \"In these experiments, WiDr transfectants (3 mock and 3 antisense β6 clones), HT29 transfectants (mock and antisense β6) and SW480 β6 transfectants (mock and sense β6) were serum-starved for 16 hrs followed by 30 mins exposure to serum.\",\n \"Increased expression of αvβ6 was associated with a marked increase in MAP kinase activity upon serum stimulation compared with cells lacking αvβ6 altogether (SW480 mock).\",\n \"Cells in which β6 expression had been down-regulated (antisense β6 transfectants) exhibited suppressed MAP kinase activity.\",\n \"Induced expression of β6 in the non-β6-expressing colon cancer cell line SW480 resulted in a three fold increase in serum-dependent MAP kinase activity.\",\n \"2.3.3 αvβ6 Binds an Extracellular Signal-Related Kinase (ERK).\",\n \"A highly surprising finding arising from phage display screening which underpins the present work is that the integrin β6 cytoplasmic domain binds a MAP kinase.\",\n \"Use of phage display to screen a γgt11 cDNA colon cancer cell library with the β6 cytoplasmic domain as bait yielded a clone which coded for ERK2 at the 3′ end with 100% nucleotide sequence identity (across 393 bases) to the published sequence of ERK2 (Boulton et al, 1991).\",\n \"This putative association was investigated further.\",\n \"Integrin immunoprecipitations were performed on equal protein loads of tumour cell lysates and the transferred proteins blotted with antibodies recognising phosphorylated and non-phosphorylated forms of ERK1/2.The specificity of this interaction was examined in integrin immunoprecipitations against β1, β5, β6 and αv integrin subunits.\",\n \"As shown in FIG.\",\n \"10, the anti-ERK mAb′SC-1647 identified a β6-specific band migrating at the position of purified ERK2 protein.\",\n \"The integrin-associated ERK band was identified only in immunoprecipitations of the β6 subunit and its partner αv and not in immunoprecipitations of the β1/β5 subunits or in immunoprecipitations using isotype-matched control antibodies.\",\n \"2.3.4 Effect of Altered β6 Expression on Total Cellular ERK and β6-Bound ERK.\",\n \"Equal protein loads from one representative clone each from WiDr mock and antisense β6 transfectants were electrophoresed, transferred to nitrocellulose and blotted with anti-ERK mAb (SC-1647) as shown in FIG.\",\n \"11(A).\",\n \"β6 immuno-precipitates from equal protein loads of the WiDr mock and antisense β6 clones were electrophoresed, transferred and probed with anti-ERK mAb (E10) as shown in FIG.\",\n \"11(B).\",\n \"As indicated, suppression of β6 expression resulted in a reduction of both total cellular and phosphorylated integrin-associated ERK compared with WiDr mock transfectants.\",\n \"Similarly, β6 immunoprecipitations from SW480 β6 transfected clones expressing high and low levels of β6 (confirmed by FACScan and β6 immunoprecipitations) showed parallel changes in β6-bound phosphorylated ERK (FIG.\",\n \"12).\",\n \"2.3.5 Effect of β6 Immunodepletion on Total Cellular ERK and β6-Bound ERK WiDr wild-type cells were surface biotinylated and the cell lysates immunodepleted of β6 in three rounds of sequential immunoprecipitation using anti-β6 mAb (R6G9) resulting in a marked loss of β6 from the lysates as shown in FIG.\",\n \"13(A).\",\n \"β6-immunodepleted lysates were then transferred and blotted with anti-ERK1/2 antibody (SC-1647), which recognises both phosphorylated and non-phosphorylated forms of ERK1/2.As shown in FIG.\",\n \"13(B), following three rounds of β6 immunodepletion, levels of ERK1/2 in β6-depleted lysates compared with non-immunodepleted lysates, were markedly reduced suggesting a significant contribution of β6-bound ERK to total cellular ERK.\",\n \"In contrast, immunodepletion of β5 by three successive rounds of immunoprecipitation with mAb P1F6 or isotype-matched control antibody IgG2A did not result in any reduction of cellular ERK levels compared with non-immunodepleted cell lysates.\",\n \"2.3.6 Effect of ERK Immunodepletion on β6-Bound ERK Cell lysates from WiDr wild-type cells were immunodepleted of ERK1/2 by means of sequential immunoprecipitations using the anti-ERK mAb, SC-1647.ERK-immunodepleted cell lysates probed with either E10 or SC-1647 mAbs contained markedly less ERKs.\",\n \"To examine the effect of ERK-immunodepletion on β6-bound ERK, the ERK-immunodepleted cell lysates were immunoprecipitated with anti-β6 mAb (R6G9) and the β6-immunoprecipitates probed with anti-ERK mAb (E10 recognising phosphorylated ERK1/2).\",\n \"ERK immunodepletion effectively reduced levels of β6-bound ERK.\",\n \"2.3.7 Effect of Preventing Cell Attachment on αvβ6-Bound ERK SW480 mock and β6 expressing transfected cells were grown to 80-90% confluency.\",\n \"Cells were washed twice with PBS and harvested after trypsinization.\",\n \"Cells were divided into three equal portions (approximately 4×106/batch).\",\n \"The first group was plated on a 75 cm2 plastic flask in normal 10% serum containing medium (attached cells) while the other two groups were plated on 0.3% agarose underlay in serum free medium (non-attached cells).\",\n \"Cells were allowed to grow for 24 hours at 37° C., after which in one group of 0.3% agarose underlay, 15 ml of serum-free medium was added while in the other an equal volume of 10% serum-containing medium was added for 30 mins.\",\n \"Cells were collected washed with PBS and lysed in cell lysis buffer (100 mM Tris-HCl pH-7.5, 150 mM NaCl, 1 mM CaCl2, 1% Triton, 0.1% SDS, 0.1% Np-40, 1 mM vanadate, 1 μg/ml pepstatin, 1 mM PMSF, 5 μg/ml aprotonin and 1 μg/ml of leupeptin).\",\n \"10 μl (10 μg of protein) was used for the analyses of cell lysates after adding equal volumes of non-reducing Laemmli buffer.\",\n \"For SW480-β6-transfected cells the rest of the cell lysate was used for immunoprecipitation of αvβ6 integrin.\",\n \"Cell lysates and β6 immunoprecipitates were subjected to western blotting and probed with E10 monoclonal antibody (recognising phosphorylated ERK1/2).\",\n \"Cell lysates from the non-attached cells were found to require serum factors to maintain phosphorylation of total cellular ERK.\",\n \"In contrast, non-attached SW480 β6 colon cancer cells do not require serum factors to maintain the activation phosphorylation) state of β6-bound ERK.\",\n \"2.3.8 Effect of PP2A Phosphatase on αvβ6-Bound ERK In experiments to examine the effect of protein phosphatase 2A (PP2A) on β6-bound ERK, SW480 β6-transfected cells were sonicated in buffer comprising 5 mM Tric-HCl (pH 8.0), 10 mM MgCl2 and 0.01 mM EGTA together with enzyme inhibitors, and cell lysates from both serum-starved and serum-induced cells treated with 0.5 units of PP2A (Promega) at 30° C. for 10 minutes.\",\n \"The reaction mixture was stopped by addition of equal volumes of non-reducing Laemmli sample buffer.\",\n \"In parallel, PP2A-treated cell lysates were immunoprecipitated with mAb R6G9 (anti-β6) followed by Western blot with anti-ERK mAb E10.MAP kinase activity assays were performed according to the manufacturer's instructions (Amersham Pharmacia Biotec) using γ32P-ATP.\",\n \"Exposure of cell lysates prepared from serum-supplemented and serum-starved cells to the PP2A catalytic subunit resulted in dephosphorylation of total ERK.\",\n \"In contrast, dephosphorylation of β6-bound ERK was not observed in β6 immunoprecipitates prepared from serum-supplemented or serum-starved cells β6 Bound ERK may therefore serve to maintain adjacent growth factor receptors in an activated state and thereby alter their sensitivity to exogenous co-factors.\",\n \"2.3.9 Inhibition of MAP Kinase Activity Inhibits Secretion of Gelatinase B. SW480 β6 transfectants were cultured under serum-free conditions for 48 hours in the absence/presence of the MEK inhibitor PD98059 (40 μM) or DMSO (vehicle control).\",\n \"Tumour-conditioned medium was assayed for gelatinase B by analysis of equal protein loads in a gelatin zymogram.\",\n \"Inhibition of MAP kinase activity by the MEK inhibitor reduced gelatinase B secretion compared with controls.\",\n \"2.3.10 β6-ERK2 Association in HaCaT and HaRas Cell Lines β6 immunoprecipitates were prepared from human kerotinocyte cell lines (HaCaT and HaRas were obtained from Prof N. Fusenig, The German Cancer Research Institute, Heidelberg, Germany) using mAb R6G9 (anti-β6) and the immunoprecipitates probed with mab E10 (against phosphorylated ERK 1/2).\",\n \"FIG.\",\n \"14 shows that ERK2 associates with β6 in both of HaCat and HaRas cells.\",\n \"2.4 Discussion of Results The MAP kinase pathway has been shown to be important in experimental tumour metastases (Mansour et al, 1994) and recent data implicate MAP kinases in tumour growth and invasiveness of colon cancer cells (Sebolt-Leopold et al, 1999).\",\n \"In the present study, up-/down-regulation of β6 expression in various colon cancer cell lines was shown to enhance/suppress respectively, MAP kinase activity.\",\n \"The presence of serum induced a three-fold increase in MAP kinase activity above that observed for serum-starved β6-expressing cells.\",\n \"In contrast, only a one-fold increase in serum-dependent MAP kinase activity was observed for cells in which β6 had been down-regulated consequent upon transfection with antisense β6.Moreover, induced expression of β6 in the non-β6-expressing colon cancer cell line SW480, was associated with a three-fold increase in serum-dependent MAP kinase activity.\",\n \"Serum contains a mixture of growth factors raising the possibility that one role for αvβ6 in colon cancer cells is to lower the threshold for activation of MAP kinase signalling pathways at times when the supply of serum-containing growth factors is limited.\",\n \"The ERK band co-immunoprecipitated with β6 migrated either at or 1-2 kD higher than the mobility of purified phosphorylated ERK2 depending on the acrylamide concentration used in SDS-PAGE showing that the kinase does indeed, associate with the β6 subunit.\",\n \"Slight differences in mobility of the β6-associated ERK band compared with the pure ERK protein could arise if β6-bound ERK is hyper-phosphorylated and/or exists in an altered conformation consequent upon its association with β6.ERK bound to β6 may also be an alternatively spliced variant of ERK2 causing it to migrate differently.\",\n \"Finally, the purified ERK2 protein is derived from mouse which differs slightly from human ERK2 by being two amino acid residues shorter and also containing a single amino acid substitution.\",\n \"In the present study, increased/decreased β6 expression in colon cancer cell lines was associated with increased/decreased β6-bound ERK, respectively.\",\n \"In addition, immunodepletion experiments suggest that β6-bound ERK makes a substantial contribution to total phosphorylated ERK within the cell and overall MAP kinase activity (see FIGS.\",\n \"15(A) and 15(B)).\",\n \"The observation that β6-mediated colon cancer growth in vitro is inhibitable by a matrix metalloproteinase inhibitor (Agrez et al, 1999) suggests that the increased gelatinase B secretion by β6-expressing colon cancer cells contributes to tumour progression.\",\n \"Taken together with the finding that inhibition of MAP kinase activity by the MEK inhibitor PD98059 diminished gelatinase B secretion in β6-expressing cells, it seems that activation of MAP kinase signalling plays a role, at least in part, in β6-mediated induction of gelatinase B secretion.\",\n \"EXAMPLE 3 3.1 Identification of the Binding Domain on the β6 Subunit Cytoplasmic Tail Domain for ERK2 Peptide fragments corresponding to regions of the cytoplasmic tail domain of the β6 subunit were screened in an enzyme-linked immunosorbent assay (ELISA) for binding with ERK2.The 52 amino acid long β6 cytoplasmic tail is shown in FIG.\",\n \"16 as are the amino acid sequences for the cytoplasmic domains of the β1 to β3 subunits.\",\n \"In particular, four synthetic peptides designated fragment 1 to fragment 4 were prepared and biotinylated at the N-terminal end of each, respectively (Auspep Pty Ltd, Melbourne Australia).\",\n \"The region of the β6 tail to which each corresponds is indicated in FIG.\",\n \"16 and set out below.\",\n \"Fragment 1: HDRKEVAKFEAERSKAKWQTGT Fragment 2: RSKAKWQTGTNPLYRGSTST Fragment 3: NPLYRGSTSTFKNVTYKHRE Fragment 4: FKNVTYKHREKQKVDLSTDS The fragments overlap by 10 amino acids and are each 20 amino acids long with the exception of the fragment 1 with a length of 22 amino acids.\",\n \"Fragment 4 was synthesised with a terminal serine rather than a cysteine as found in wild-type β6 to avoid formation of a disulfide bridge between peptides.\",\n \"Overlapping biotinylated fragments 1 to 4 were coated onto streptavidin coated polystyrene plates (Pierce, Rockford Ill. USA, Cat No.\",\n \"15125) and the ELISA performed substantially according to manufacturers instructions.\",\n \"Briefly, wells are washed with 3×200 μl of wash buffer (TBS, 0.1% BSA, 0.05% Tween 20 or SuperBlock™ blocking buffer in TBS, Pierce, Prod.\",\n \"No.\",\n \"37535) prior to addition of biotinylated peptides (100 μl) and incubation for 1 hr at room temperature to allow for capture of the peptides on the plate.\",\n \"Following another washing step, peptides were overlayed with GST-ERK, ERK (or JNK-1) alone at a volume of 100 μl per well, and the plates incubated for a further 1 hr at room temperature before removal of any unbound ERK by further washing.\",\n \"Binding of ERK to the peptides is detected using 100 μl anti-ERK1/2 mAb SC1647 (Santa Cruz) as the primary antibody at a dilution of 1:700 (isotype matched antibody IgG2b is used as a control).\",\n \"This is followed by another washing step and addition of 100 μl rabbit anti-mouse antibody (Biorad) conjugated to alkaline phosphatase at a concentration of 1:1000 for 30 minutes again at room temperature.\",\n \"A 10 aliquot of detection reagent (alkaline phosphatase detection kit-Biorad) is then introduced into each well after a final washing step and allowed to react for 15-30 minutes at room temperature in the dark before absorbance is measured at 405 nm.\",\n \"All dilutions of peptides, MAP kinase and antibodies were performed using the wash buffer.\",\n \"GST-ERK is a fusion protein consisting of ERK coupled to glutathione-S-transferase and purified from host cells transfected with pGEX vector.\",\n \"As shown in FIG.\",\n \"17, significant binding of non-phosphorylated GST.ERK2 (0.25 μg/100 μl) to peptide fragment 2 was observed (1 μg/100 μl) while only negligible or low level binding for the other fragments was found.\",\n \"Significant binding of non-phosphorylated ERK2 to both fragment 2 and β6 cytoplasmic tail peptide compared to fragments 1, 3 and 4 over a range of concentrations of ERK2 was also observed (see FIG.\",\n \"18).\",\n \"Similar results were observed using a range of concentrations of the peptide fragments as shown in FIG.\",\n \"19.To further localise the binding domain on the cytoplasmic tail of the β6 subunit, progressively shorter peptides from the region of the β6 cytoplasmic tail corresponding to peptide fragment 2 were synthesised, biotinylated and the capacity to associate or otherwise bind to ERK2 assessed as described above.\",\n \"The binding of GST.ERK2 to a 15 mer test peptide (seq.\",\n \"4) having the amino acid sequence RSKAKWQTGTNPLYR and a 10 mer test peptide having the sequence RSKAKWQTGT is shown in FIG.\",\n \"20 compared to fragment 2 over a range of concentrations of the peptides.\",\n \"As can be seen, no reduction in binding to the seq.\",\n \"4 peptide compared to fragment 2 was found.\",\n \"Binding of ERK2 to the seq.\",\n \"3 peptide was substantially less than that observed for seq.\",\n \"4.A number of 10 mer biotinylated peptides corresponding to regions of fragment 2 or fragment 3 were then tested.\",\n \"The amino acid sequence for each peptide is as follows and their location in the β6 cytoplasmic domain is indicated in FIG.\",\n \"21.10(1): NPLYRGSTST 10(2): WQTGTNPLYR 10(3): KFEAERSKAK The results are set out in FIG.\",\n \"22 and show that GST.ERK binding to the 10 mer peptides is substantially reduced compared to binding to the seq.\",\n \"4 peptide suggesting that opposite end regions of seq.\",\n \"4 participate in the binding of ERK2.Comparable binding of ERK2 to seq.\",\n \"4 was found using a further 10 mer peptide identified as 10(4) in which amino acid sequence WQTGT of seq.\",\n \"4 is omitted indicating that WQTGT is a linker sequence that does not participate directly in the binding of ERK to seq.\",\n \"4.Negligible binding of ERK2 to the 5 mer peptide RSKAK was observed as shown in FIG.\",\n \"23.ERK2 cleaved from GST-ERK2 by thrombin was used in this assay.\",\n \"Results (not shown) indicate that greater than a 3 fold increase in assay sensitivity can be achieved using thrombin cleaved ERK2 rather than GST-ERK2.EXAMPLE 4 4.1 MAP Kinase JNK-1 Binds to the Cytoplasmic Tail Domain of β6 In view of the observation that ERK2 associates with the cytoplasmic tail of the β6 subunit, the MAP kinase JNK-1 was tested to evaluate whether it also could associate with the cytoplasmic tail of β6.Briefly, 0.05-1.5 μm/100 μl of JNK-1 (Santa Cruz) was aliquoted into wells of a 96 well culture place containing increasing concentrations of the β6 cytoplasmic domain tail peptide used in Example 3.For comparison purposes, non-phosphorylated GST-ERK2 (0.05-1.5 μm/100 μl) was aliquoted into wells containing β6 cytoplasmic tail peptide or peptide fragments 1 or 2, respectively.\",\n \"Binding of JNK-1 was detected using mouse anti-JNK-1 mAb SC 474-G (Santa Cruz) and HRP-conjugated goat anti-mouse antibody.\",\n \"Absorbance was read at 405 nm and the results are shown in FIG.\",\n \"24.Significant binding of JNK-1 to the β6 cytoplasmic tail peptide was found.\",\n \"EXAMPLE 5 5.1 Evaluation of Ability of ERK2 to Bind to β6 Δ746-764 Deletion Mutant.\",\n \"To examine the role of the amino acid sequence RSKAKWQTGTNPLYR in the β6 cytoplasmic domain in situ, a β6 deletion construct lacking the coding sequence for AERSKAKWOTGTNPLYRG was transfected into colon cancer cell line SW480 which does not constitutively express the α0Vβ6 integrin using the calcium phosphate method previously described for transfections into this cell line (Agrez et al, 1994).\",\n \"The location of the β6 Δ746-764 deletion is indicated in FIG.\",\n \"25.Construction of the β6 Δ746-764 deletion mutant in the vector pcDNA1neo and failure of the expressed receptor to localise to focal adhesions in Chinese hamster ovary cells has been reported (Cone et al, 1994).\",\n \"Facscan analysis revealed comparable levels of surface expression of mutant β6 to that seen for the full length wild-type receptor (see FIG.\",\n \"26).\",\n \"Equal protein loads of cell lysates prepared from SW480 cells were immunoprecipitated with either anti-β6 monoclonal antibody (mAb R6G9) or matched isotype control antibody.\",\n \"Surface biotinylation prior to immunoprecipitation confirmed equal surface expression of mutant and wild-type β6 (see FIG.\",\n \"27(A).\",\n \"Aliquots of the immunoprecipitates were electrophoresed and transferred to nitrocellulose for Western blotting using monoclonal antibody E10 which recognises ERK1/2.As seen in FIG.\",\n \"27(B), loss of the RSKAKWQTGTNPLYR sequence in the β6 cytoplasmic domain reduced levels of β6-bound ERK by greater than approximately 75% of that observed for the wild type receptor.\",\n \"EXAMPLE 6 6.1 Growth Inhibition Study.\",\n \"HT29 and SW480 β6-expressing colon cancer cell lines were seeded into wells of 96-well microtitre plates (Nunclon) in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% foetal bovine serum, glutamine, Hepes, and antibiotics.\",\n \"Seeding cell densities were 3×103 cells per triplicate well for each condition tested and after 24 hours incubation of cell cultures in 5% CO2, 100% humidity at 37° C., the culture medium was exchanged for serum-free DMEM medium supplemented with insulin, transferrin, selenous acid, hydrocortisone, non-essential amino acids, glutamine, Hepes and antibiotics containing either peptide RSKAKWQTGTNPLYR alone or penetratin-peptide complex at a concentration of 10 μm for HT29 cells or 30 μm for SW480 β6-expressing cells.\",\n \"Cell cultures were incubated for a further 24 hours following which cultures were photographed (Kodak Techpan Film at 100 ASA setting) and the experiments terminated by addition of the cell proliferation reagent WST-1 (Boehringer Mannheim) to monitor effects of the peptide on cell growth.\",\n \"The cell proliferation reagent WST-1 is designed to be used for the non-radioactive, spectrophotometric quantification of cell growth and viability in proliferation and chemosensitivity assays.\",\n \"The colourmetric assay is based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenase in viable cells.\",\n \"Specifically, at the termination of experiments, 30 μl of WST-1 was added to 270 μl culture medium volume in each microtitre well and the colour change quantitated in an ELISA plate reader by measuring absorbance of the formazan product at 450 mn (using a reference wavelength of more than 600 nm).\",\n \"The mean absorbance readings from triplicate wells (±standard error of the means) after subtraction of background control wells (culture medium without cells) was determined.\",\n \"Only the carrier penetratin-peptide complex was effective in inhibiting cell proliferation in contrast to either peptide or penetratin alone as shown in FIGS.\",\n \"28 and 29 indicating that the penetratin-peptide complex was internalised by both the HT29 and SW480 cells resulting in the observed suppression of colon cancer growth.\",\n \"Photographs of the SW480 cells treated with penetratin alone or the penetratin-peptide complex are shown in FIG.\",\n \"30(A) to (C).\",\n \"EXAMPLE 7 7.1 Growth Inhibition in SW480 Mock and SW480 β6 Transfectants.\",\n \"SW480 mock and SW480 β6 transfectants were cultured in DMEM medium supplemented with 1% foetal bovine serum in the presence of 20 μM seq.\",\n \"4 coupled to penetratin.\",\n \"Percentage inhibition was assessed by the WST-1 colorimetric dehydrogenase assay described in Example 6.The percentage inhibition of growth observed for the −β6 and +β6 expressing cells was 17% and 50%, respectively as indicated in FIG.\",\n \"31A.\",\n \"The −β6 and +β cultured cells are shown in FIG.\",\n \"31B.\",\n \"EXAMPLE 8 The proliferation of SW480 cells expressing a β6 Δ746-764 deletion mutant was compared with non-β6 expressing SW480 cells and SW480 cells expressing full length wild-type β6.Cells were cultured for 10 days within a 3-dimensional collagen type I matrix.\",\n \"Collagen gels were prepared as bilayers (upper layer cell-containing and lower layer minus cells) in 24 well culture plates as previously described (Agrez, 1989; Agrez, 1994) except for the use of 5% foetal bovine serum as the supplement for DMEM.\",\n \"Colonies were photographed in the gel FIG.\",\n \"32(A) at 10 days (bar represents 200μ) and following dissolution of the collagen with collagenase FIG.\",\n \"32(B) prior to visual colony counting of all colonies exceeding 200μ in diameter within each well FIG.\",\n \"32(C).\",\n \"As can be seen, significant proliferation of the SW480 cells expressing the full length wild-type β6 was observed compared to the non-β6 expressing cells and cells expressing the β6 Δ746-764 deletion mutant.\",\n \"EXAMPLE 9 Growth inhibition of SW480 cells expressing full length wild-type β6 exposed to seq.4 coupled to penetratin or RSKAKWQTGTNPLYR peptide coupled to penetratin (5, 10, 20, 30 μM in DMEM minus foetal bovine serum) but which peptide contained alanine substitutions at the four positions indicated was assessed.\",\n \"As shown in FIG.\",\n \"33(A) and FIG.\",\n \"33(B), progressive inhibition of proliferation in a dose-response manner was observed for the seq.4 penetratin complex compared with the alanine substituted peptide-penetratin complex which was without effect at all doses tested.\",\n \"EXAMPLE 10 Binding of ERK2 to the seq.4 peptide (RSKAKWQTGTNPLYR) was compared with peptides corresponding to regions of the cytoplasmic domain of integrin subunits β1, β2, β3 and β5.The amino acid sequences for those peptides is shown below: β3 KFEKEKMNAKWDTGENPIYK β2 KEKLKSQWNNDNPLFK β3 RARAKWDTANNPLYK β5 RSRARYEMASNPLYR As shown in FIG.\",\n \"34, significant binding of ERK2 to the seq.\",\n \"4 peptide was observed.\",\n \"Binding of ERK2 to the β5 and β3 peptides was also found.\",\n \"The results have been corrected for non-specific binding and indicate a hierarchy of binding of ERK2 to integrin subunits.\",\n \"EXAMPLE 11 Angiogenesis, the growth of new blood vessels, plays a role in diverse disease states including progression of cancer, diabetic retinopathy, rheumatoid arthritis, psoriasis, atherosclerosis, pregnancy and endometriosis.\",\n \"For examples, in cancers, the ability of microscopic tumour deposits to enlarge beyond 1-2 mms in diameter is dependent upon tumour-induced angiogenesis or sprouting of new host-derived blood vessels to nourish enlarging tumour cell nodules.\",\n \"The mitogen activated protein (MAP) kinase signalling pathway is critical for endothelial cell proliferation and new blood vessel formation.\",\n \"Moreover, a relationship exists between extracellular signal-regulated (ERK) kinase 1/2 activation and vascular endothelial growth factor (VEGF) expression with each activating the other (Gupta et al, 1999; Yu & Sato, 1999: Pages et al, 2000).\",\n \"Two growth factor-dependant pathways of angiogenisis have now been shown to exist defined by their dependency on the distinct vascular integrins αvβ3 and αvβ5 (Friedlander et al, 1995: Eliceiri and Cheresh, 1999).\",\n \"It is noteworthy that the vascular system lacks αvβ6 expression (Breuss et al, 1993).\",\n \"Antibodies against αvβ5 and inhibition of protein kinase C signalling have both been shown to block VEGF-induced angiogenesis (Freidlander et al, 1995).\",\n \"The importance of αvβ5 in neovascularisation is further highlighted by the observation that β3 integrin-deficient mice exhibit normal blood vessel development in contrast to αv-deficient mice which implicates other beta integrin partners such as β5 (Eliceiri and Cheresh, 1999).\",\n \"11.1 The β5 Integrin Subunit Binds ERK in Cells that Either Lack β6 or Express β6 Lacking the Binding Site for ERK2 The SW480 cell line expresses abundant αvβ5 but only minimal levels of αvβ3 and lacks αvβ1 and αvβ6.SW480 cells provide a model, therefore, with which to test whether the β5 integrin subunit can co-immunoprecipitate with ERK either in the absence of constitutive β6 expression or in the presence of β6 that lacks the binding site for ERK2 (RSKAKWQTGTNPLYR).\",\n \"To evaluate this, cell lysates containing equal protein loads were prepared from SW480 wild-type cells and SW480 cells transfected with either vector alone (mock transfectants), wild type β6 or the β6 deletion mutant Δ746-764 (lacking the sequence EAERSKAKWQTGTNPLYRG, Cone et al, 1994).\",\n \"The lysates were immunoprecipitated with mAbs R6G9 (anti-β6), P1F6 (anti-β5) or murine IgG control antibody (mlgG).\",\n \"Western blotting of the immunoprecipitates with mAb E10 recognising phosphorylated ERK1 and 2 revealed no β5-bound ERK in the SW480 β6 transfectants expressing wild-type β6 in contrast to β5-associated ERK observed for the cell lines which either lack β6 or express the deletion mutant lacking the ERK2 binding sequence (FIGS.\",\n \"35A and 35B).\",\n \"To determine whether β5 can bind ERK2 in vitro, a 15-mer peptide fragment derived from the β5 cytoplasmic domain corresponding to the ERK binding domain on β6 was tested for its ability to bind ERK2 in an ELISA.\",\n \"In vitro MAP kinase activity assays were performed using a non-radioactive assay kit (Cell Signalling Technology, Beverley, Mass.)\",\n \"which measures phosphorylation of the ERK1 and 2 substrate Elk-1 presented as a fusion protein comprising the Elk-1 codons 307-428 coupled to GST.\",\n \"In the assay, ERK kinase is first immunoprecipitated from complete or integrin-immunodepleted soluble cell lysates and relative enzyme activity determined by the in vitro phosphorylation of GST-Elk-1.Dose-dependant binding of the β5 fragment (761RSRARYEMASNPLYR775) to ERK2 was observed as shown in FIG.\",\n \"35C although less avidly than the 15-mer β6 peptide.\",\n \"In contrast, a synthetic peptide derived from the cytoplasmic domain of the β1 subunit (765KFEKEKMNAKWDTGENPIYK784) which corresponds to the ERK binding domain on β6 failed to bind ERK2 (FIG.\",\n \"35C).\",\n \"11.2 The Increase in Cytosolic MAP Kinase Activity Upon Growth-Factor Stimulation is All Accounted for by β5-Bound ERK in Human Umbilical Vein Endothelial Cells that Lack β6 To determine the amount of ERK 1/2 activity associated with β5 following the activation of the MAP kinase pathway, the activity of ERK1/2 was compared in non-β5 immunodepleted lysates with that in β5 immunodepleted lysates derived from human umbilical vein endothelial cells (HUVECs) before and after stimulation with epidermal growth factor (EGF).\",\n \"Sequential immunodepletion of the β5 integrin subunit from cell lysates prepared from HUVECs results in total loss of all epidermal growth factor-induced MAP kinase (ERK 1/2) activity as determined by an Elk-1 phosphorylation assay (FIG.\",\n \"36).\",\n \"Briefly, HUVECs were cultured in the absence/presence of 50 ng/ml epidermal growth factor (EGF) for 10 minutes after a prior 24 hour culture period in serum-free and growth factor-free medium.\",\n \"Bands show MAP kinase activity from immunodepleted cell lysates (anti-β5 or control IgG or anti-β6 immunodepletions, five rounds each) prepared from HUVECs before and after EGF stimulation.\",\n \"Non-phosphorylated and phosphorylated Elk-1 controls are shown in the left hand two lanes.\",\n \"These results demonstrate that in β5-expression endothelial cells, the rise in cytosolic MAP kinase activity above basal levels upon EGF stimulation is completely accounted for by β5-bound ERK.\",\n \"The findings also indicate that a hierarchy of integrin-ERK2 interactions exists within cells specified by β6 over β5 in β6-expressing epithelial cells and β5 over other β integrins in endothelial cells.\",\n \"Given the critical requirement of ERK1/2 activity in the process or angiogenesis and responsiveness of endothelial cells to various growth factors these findings show, for the first time, how the αvβ5 integrin may regulate angiogenesis through maintaining activated ERK at the plasma membrane.\",\n \"Accordingly, targeting the binding site on β5 for ERK2 or the binding site on ERK2 for the integrin offers a means of inhibiting new blood vessel formation.\",\n \"Moreover, the findings allow for anti-angiogenic therapies to benefit patients with a range of disorders including not only cancer, but also diabetic retinopathy, arthritis and other inflammatory conditions.\",\n \"EXAMPLE 12 The β6 integrin immunodepletion/ERK immunoblotting experiments together with the antisense nucleic acid strategies reported herein show that the β6 integrin subunit contributes substantially to the total MAP kinase activity with for instance, colon cancer cells.\",\n \"Moreover, the fact β6-bound phosphorylated ERK2 is reduced by more than 75% in colon cancer cells lacking constitutive β6 expression stably transfected with a β6 deletion mutant in which the precise ERK2 binding domain is lacking, and that tumour growth is also reduced significantly compared with β6-expressing cells, shows that ERK2 plays a significant role in promoting cancer growth.\",\n \"Accordingly, interruption of the β6-MAP kinase pathway has important implications not only for control of advanced colon cancer, but epithelial cancers in general given the wide expression of this integrin in other cancer types such as oropharyngeal, breast and ovarian cancer.\",\n \"Hence, identification of the ERK2 binding sequence to β6 facilitates the development of mimetics which interrupt the β6-MAP kinase growth-promoting axis in cancer.\",\n \"12.1 ELISA Methodology for Determination of β6-Binding Domain in ERK2.To determine the β6-binding domain(s) of ERK2, a biotinylated β6 cytoplasmic tail peptide spanning the cytoplasmic tail of β6 was incubated with a soluble ERK2 peptide fragment in a total incubation volume of 20 μl phosphate buffered saline (PBS).\",\n \"The mixture was stood for 45 minutes at room temperature (RT).\",\n \"Soluble ERK2 in PBS was then added and the solution left for a further 30 minutes.\",\n \"Following incubation the reaction volume was made to 400 μl with carbonate buffer (0.015 M Na2CO3 and 0.035M NaHCO3 at pH 9.6) and 100 μl of this mixture added to each of 4 wells of a polystyrene ELISA plate (Nunc, Maxicorp).\",\n \"ERK2/peptides were allowed to attach to the plastic substrate for 18 to 20 hours at RT in the dark.\",\n \"After immobilisation the overlying buffer was flicked from the wells and excess solid surface blocked with casein (0.5% skim milk) in PBS for 60 minutes at RT.\",\n \"Wells were washed (×3) with was buffer (PBS plus 0.5% skim milk powder, 0.05% Tween-20) prior to incubation with the anti-β6 antibody SC-6632 (Santa Cruz) diluted 1:500 v/v in wash buffer (2 wells) or a control antibody (2 wells).\",\n \"Incubation with the primary antibody was carried out for 30 minutes at RT.\",\n \"The ELISA plate was flicked to remove the antibody mixture before washing 3 times with wash buffer.\",\n \"Wells were then incubated with anti-species alkaline phosphatase (AP) conjugated antibody (1:2000 v/v in wash buffer) for 30 minutes at RT, flicked and washed as above.\",\n \"Specific binding events were quantitatively determined using a colourimetric substrate system by measuring absorbency at 405 mm (Bio-Rad, AP substrate kit).\",\n \"12.2 Identification of β6-Binding Domain of ERK2.In a preliminary study, ERK peptides were probed in small groups to identify a possible ERK2 competing peptide that binds with the β6 cytoplasmic tail peptide.\",\n \"Overlapping ERK2 peptides 1 to 14 comprising the full length ERK2 sequence (360 amino acids) were utilised, and an inhibitory effect on β6-ERK2 binding was identified within the group comprising peptides 7, 8, and 9 (data not shown).\",\n \"The amino acid sequences for the peptides and position within ERK2 are shown below.\",\n \"1.MAAAAAAGAGPEMVRGQVFDVGPRYTNLSY 2.YTNLSYIGEGAYGMVCSAYDNVNKVRVAIK 3.VRVAIKKISPFEHQTYCQRTLREIKILLRF 4.KILLRFRHENIIGINDIIRAPTIEQMKDVY 5.QMKDVYIVQDLMETDLYKLLKTQHLSNDHI 6.LSNDHICYFLYQILRGLKYIHSANVLHRDLK 7.HRDLKPSNLLLNTTCDLKICDFGLAR 8.DFGLARVADPDHDHTGFLTEYVATRWYRAPEIMLNSKGY 9.NSKGYTKSIDIWSVGCILAEMLSNRPIFPG 10.PIFPGKHYLDQLNHILGILGSPSQEDLNCI 11.DLNGIINLKA RNYLLSLPHKNKVPWNRLFP 12.NRLFPNADSKALDLLDKMLTFNPHKRIEVE 13.RIEVEQALAHPYLEQYYDPSDEPIAEAPFK 14.EAPFKFDMELDDLPKEKLKELIFEETARFQPGYRS Individual overlapping peptides of the ERK2 sequence were then tested at equimolar concentrations for the ability to inhibit the β6-ERK interaction.\",\n \"Only peptide 7 (HRDLKPSNLLLNTTCDLKICDFGLAR) was found to be effective (see FIG.\",\n \"37).\",\n \"Repeat testing using adjacent ERK2 overlapping peptides as controls yielded the same result (see FIG.\",\n \"38).\",\n \"Importantly, β6 bound to ERK2 does not interfere with ERK2 binding to the well, thereby confirming the validity of the ERK2 peptide competition assay.\",\n \"To optimise the assay, β6 cytoplasmic tail peptide was incubated at increasing concentrations with ERK2.Maximal β6 detected when bound to ERK was found to be at a concentration of 0.25 μM (data not shown).\",\n \"β6 cytoplasmic tail peptide (0.25 μM) was then either pre-incubated with ERK peptides 2, 7, 8 or 10 in the competition assay or directly with ERK2 (positive control).\",\n \"An inhibition of 83% for β6-ERK2 binding was again demonstrated for sequence 7 (see FIG.\",\n \"39).\",\n \"To demonstrate that neither β6 alone or ERK2 peptide fragments 2, 7, 8 and 10 interfered with ERK2 binding to the plate, parallel samples of each condition identified in FIG.\",\n \"38 were probed with anti-ERK2 antibody.\",\n \"None of the peptide or β6 coincubations with ERK2 interfered with ERK binding to the ELISA plate.\",\n \"EXAMPLE 13 13.1 Induction of Wound Healing Response Keratinocytes in vivo in unwounded skin do not express β6 integrin but do so after sub-culturing in vitro.\",\n \"Hence, to avoid the conflicting presence of integrin αvβ6, the epithelial cell line SW480 which lacks αvβ6 expression was chosen in order to examine the effect of peptide β6 agonists on cell migration following artificial wounding in vitro.\",\n \"The wound assay was performed by seeding 2×105 cells into 24 well (1.6 cm diameter) tissue culture plates in Dulbecco's modified Eagles medium (DMEM) supplemented by 10% foetal calf serum, HEPES, glutamine and antibotics.\",\n \"The confluent cell monolayers were streaked with the tip of a plastic pipette in a cross-wire fashion, and loose non-adherent cells washed off gently with medium before replacing the culture medium with DMEM supplemented with 1% foetal calf serum.\",\n \"In addition, RSKAKWQTGTNPLYR peptide ligated to penetratin (carrier peptide) by means of cysteine Cys (Npys) bonding was added to each well at a final concentration in the range of 1.25-10 μM.\",\n \"Cell migration across the wounded (streaked) area was photographed (Techpan, Kodachrome, ASA100) after a further 48 hours in culture.\",\n \"As shown in FIGS.\",\n \"40A to 40D, a dose-dependent wound repair process (enhanced cell migration) was observed for the peptide-agonist complexed with the intracellular delivery vehicle penetratin.\",\n \"The conditions utilised in the assay were as follows: A: no peptide-penetratin complex added, B: 2.5 μM peptide-penetratin complex added, C: 5 μM peptide-penetratin complex added, D: 10 μM peptide-penetratin complex added.\",\n \"Cell migration was not stimulated by either the peptide-agonist uncoupled to penetratin or penetratin alone (not shown).\",\n \"The bar length shown in FIG.\",\n \"40D represents 80 μM in length.\",\n \"13.2 Discussion The activity of ERK2 is tightly regulated.\",\n \"Dual phosphorylation events, one on residue 185 (Y) and one on residue 183 (T) are required for ERK2 activation (Zhang et al, 1995).\",\n \"In unphosphorylated ERK2, Y185 is found largely buried within the molecule whereas T183 is exposed.\",\n \"In particular, the phosphorylation lip of ERK2 consists of 15 amino residues from residue 173 (D) to residue 187 (A) namely DPDHDHTGFLTEYVA.\",\n \"This sequence is near the mouth of the active site (Zhang et al, 1995).\",\n \"The 26 mer peptide fragment of ERK2 (peptide 7) found to inhibit binding of β6 cytoplasmic domain to ERK2 stretches from amino acid residue 145 (H) to residue 170 (R).\",\n \"This inhibitory binding sequence lies adjacent to the phosphorylation lip of ERK2.The reason for low stability of the phosphorylation lip in ERK2 is uncertain, but may be due to the unusual cluster of three positively charged amino acids namely residue 146 (R), 170 (R) and 201 (K) (Zhang et al, 1995).\",\n \"Residues 146(R) and 170(R) of ERK2, both of which are included within peptide 7, appear to be conserved among many serine/threonine specific kinases.\",\n \"For conserved amino acid residue 146 (R) to bind to a phosphate ion, it must lose its interaction with the backbone of the loop defined by amino acid residues 199 to 205 of ERK2 adjacent to the phosphorylation lip (residues 173 (D) to 187 (A)).\",\n \"Accordingly, β6 may bind to the 15 mer PSNLLLNTTCDLKIC within peptide 7 and break the interaction between residue 146 (R) and the backbone loop allowing ERK to be phosphorylated on residue 183 (T) and 185 (Y).\",\n \"Although the present invention has been described hereinbefore with reference to a number of preferred embodiments, the skilled addressee will understand that numerous variations and modifications are possible without departing from the scope of the invention.\",\n \"REFERENCES CITED Agrez, M. 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D., MAP kinases, phosphatidylinositol 3-kinase, and p70 S6 kinase mediate the mitogenic response of human endothelial cells to vascular endothelial growth factor.\",\n \"J Cell Physiol.\",\n \"178(2): 235-46 (1999).\",\n \"Zambruno, G., Marchisio, P. C., Marconi, A., Vaschieri, C., Melchiori, A., Giannetti, A. and De Luca, M., Transforming growth factor—β1 modulates β1 and β5 integrin receptors and induces the de novo expression of the αvβ6 heterodimer in normal human keratinocytes: implications for wound healing.\",\n \"J.\",\n \"Cell Biol.\",\n \"129, 853-865 (1995).\",\n \"Zhu, X. and Assoian, R. K., Integrin-dependent activation of MAP kinase: a link to shape-dependent cell proliferation.\",\n \"Mol.\",\n \"Biol.\",\n \"Cell 6, 273-282 (1995).\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10451291"}}},{"rowIdx":299,"cells":{"text":{"kind":"list like","value":[["Modified tachykinin receptors","The invention provides a modified tachykinin receptor in which the three amino acids of the DRY sequence that occurs adjacent to the junction of the TM3 domain with intracellular loop 2 are replaced with amino acids whose side chains are neither lipophilic nor contain charged groups.","The receptor exhibits similar ligand binding characteristics to the wild type receptor but is incapable or substantially incapable of initiating an endogenous signal.","Thus the ligand exhibits no or substantially no intra-cellular coupling of the receptor to the G protein, whereby there is substantially no transduction of ligand binding signals to the cell, The invention also includes fragments of the receptor containing the modified DRY sequence, and polynucleotides that encode the modified receptor as aforesaid.","Therapeutic and diagnostic uses for the receptor are disclosed."],["1.A mutant tachykinin receptor in which the three amino acids of the DRY sequence that occurs adjacent to the junction of the TM3 domain with 5 intracellular loop 2 are replaced with amino acids whose side chains are neither lipophilic nor contain charged groups, said receptor exhibiting similar ligand binding characteristics to the wild type receptor but exhibiting substantially no intra-cellular coupling of the receptor to the G-protein, whereby there is substantially no transduction of ligand binding 10 signals to the cell; or a fragment of said receptor containing said modified DRY sequence; or an isolated protein or polypeptide containing an amino acid sequence at least 80% identical to the above sequence; or a variant thereof with sequential amino acid deletions from either the C terminus or the N-terminus; or an allelic variant, heterospecific homologue or biologically active proteolytic or other fragment thereof containing said modified DRY sequence.","2.A non-human mammalian receptor according to claim 1.3.A rat or mouse receptor according to claim 1.4.A human receptor according to claim 1.5.A mutant NIL-1 receptor according to claim 1.6.A mutant NK-2 receptor according to claim 1.7.A mutant NK-3 receptor according to claim 1.8.A receptor according to claim 1, wherein the replacement amino acids are selected from G and A.","9.The receptor of claim 8, wherein DRY is replaced by GGA.","10.A tachykinin receptor of SEQ ID No5, or a receptor having at least 80% amino acid identity with the receptor of SEQ ID No5, and that is capable of binding to substance P but is substantially incapable of initiating its endogenous signal, or a fragment of said receptor.","11.An isolated cell membrane incorporating a tachykinin receptor as defined in claim 1.12.Any of the following: (a) an isolated nucleic acid molecule comprising a polynucleotide that encodes a tachykinin receptor as claimed in claim 1; (b) an isolated nucleic acid molecule comprising a sequence that is hybridizable to the above sequence; (c) a gene which is the result of extending the above sequence or any sequence that is hybridizable to the above sequence; (d) a sequence or gene that is functionally equivalent to the above sequence or to a gene that is an extension of the above sequence, i.e.","that is not identical to the sequence or gene referred to but functions biologically as equivalent to the sequence or gene referred to, including any allelic variants and heterospecific mammalian homologues, including artificial or recombinant sequences created from cDNA or genomic DNA; (e) a recombinant vector comprising the above gene sequence; and (f) a host cell transformed with the vector.","13.Any of the following: (a) an isolated nucleic acid molecule having the nucleotide sequence of SEQ ID No6; (b) an isolated nucleic acid molecule comprising a sequence that is hybridizable to the above sequence; (c) a gene which is the result of extending the above sequence or any sequence that is hybridizable to the above sequence; (d) a sequence or gene that is functionally equivalent to the above sequence or to a gene that is an extension of the above sequence, i.e.","that is not identical to the sequence or gene referred to but functions biologically as equivalent to the sequence or gene referred to, including any allelic variants and heterospecific mammalian homologues, including artificial or recombinant sequences created from cDNA or genomic DNA; (e) a recombinant vector comprising the above gene sequence; and (f) a host cell transformed with the vector.","14.A method for producing a receptor protein having an amino acid sequenceas defined in claim 1, which method comprises the steps of: (a) inserting said nucleic acid sequence into an appropriate vector; (b) culturing, in an a culture medium, a host cell previously transformed or transfected with the recombinant vector of step (a); (c) harvesting cells containing the receptor protein obtained from step (b); and (d) separating or purifying, from said culture medium or from said host cell, the thus-produced receptor protein.","15.A pharmaceutical composition comprising an effective amount of a modified tachykinin ligand as defined in claim 1 or a nucleic acid sequence encoding said ligand and a pharmaceutically and pharmacologically acceptable carrier.","16.Use of a modified tachykinin receptor as defined in claim 1 in the preparation of a medicament for the treatment or prophylaxis of a condition associated with substance P or other tachykinin (neurokinin) receptor-binding ligand; 17.A method for the treatment or prevention of a condition associated with over-expression or inappropriate expression of an endogenous tachykinin ligand, which method comprises administration to a patient in need thereof of a non-toxic, effective amount of such a modified tachykinin ligand as defined in claim 1.18.A method for screening for therapeutically active compounds, said method comprising the following steps: (a) providing a cell line expressing a modified tachykinin receptor as defined in claim 1; (b) adding test sample to a solution containing labeled tachykinin ligand and the cell line from step (a); (c) incubating the cell line, test sample and labeled ligand mixture from step (b) to allow binding of said ligand and test sample to the modified tachykinin receptor; (d) optionally, separating the non-bound labeled ligand from the labeled ligand bound to the modified tachykinin receptor; and, if desired, (e) measuring the amount of labeled ligand that is bound to the modified tachykinin receptor.","19.Use of a modified tachykinin receptor as defined in claim 1 as a substitute in an assay to identify and/or evaluate entities that bind to the wild type tachykinin receptor.","20.Use of a modified tachykinin receptor as defined in claim 1 as a substitute in an assay in order to determine the concentration of ligand in body fluids in patients with arthritis, pain, migraine, anxiety, schizophrenia, asthma, rheumatoid arthritis, and in gastrointestinal disorders and diseases of the GI tract.","21.An assay procedure comprising the following steps: (a) providing a cell line is provided that expresses a modified tachykinin receptor as defined in claim 1; (b) labeling the cell line; (c) adding the test sample and labeled cells to a matrix binding SP or other ligand; (d) incubating the labeled cells, test sample and matrix-bound SP or other ligand to allow binding of SP or other ligand and test sample to the expressed modified tachykinin receptor; (e) separating the labelled non-bound cells from the SP or other ligand bound cells; and, if desired, (f) measuring the amount of labelled cells containing the modified tachykinin receptor that has bound to SF or other ligand.","22.Use of a modified tachykinin receptor as defined in claim 1 in protein therapy to reduce the effects of an excess of or inappropriately produced endogenous ligand.","23.A method for treatment of a patient in need thereof, which comprises administering to said patient a composition in the form of an aerosol that comprises a modified tachykinin receptor as defined in claim 1.24.A method for gene therapy treatment of a patient in need thereof, which comprises administering to said patient a nucleic acid sequence, virus or plasmid encoding a modified tachykinin receptor as defined in claim 1."],["<SOH> BACKGROUND TO THE INVENTION <EOH>Tachykinins are important in the mediation of many physiological and pathological processes including inflammation, pain, migraine, headache and allergy induced asthma.","They belong to an evolutionary conserved family of peptide neurotransmitters that have an established role in neurotransmission.","They share the C-terminal sequence Phe-Xaa-Gly-Leu-Met-NH 2 (SEQ ID NO 12) in which Xaa represents a hydrophobic residue.","That sequence is characteristic of tachykinins and believed to be mainly responsible for their biological activity at neurokinin receptors.","Mammalian tachykinins include substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) which exert their effects by binding to specific receptors.","SP is the most prominent member of the tachykinergic system and is released from sensory nerve endings throughout the body.","Its amino acid sequence is: in-line-formulae description=\"In-line Formulae\" end=\"lead\"?","H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH 2 (SEQ ID N o 1) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?","The amino acid sequence for NKA is: in-line-formulae description=\"In-line Formulae\" end=\"lead\"?","H-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH 2 (SEQ ID N o 2) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?","The amino acid sequence for NKB is: in-line-formulae description=\"In-line Formulae\" end=\"lead\"?","H-Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH 2 (SEQ ID N o 3) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?","SP has been implicated in the pathology of numerous diseases.","For example, it has been shown to be involved in the transmission of pain, in conditions associated with vasodilation, smooth muscle contraction, bronchoconstriction, activation of the immune system and neurogenic inflammation.","It has been implicated in migraine, as well as in disorders of the central nervous system, such as anxiety and schizophrenia; in respiratory and inflammatory diseases, such as asthma and rheumatoid arthritis; and in gastrointestinal (GI) disorders and diseases of the GI tract, such as ulcerative colitis and Crohn's disease.","Tachykinin receptors include NK-1, NK-2 and NK-3 and are membrane proteins of the super-family of guanine nucleotide-binding protein (G-protein)-coupled receptors (GPCR).","They have extra-cellular binding sites that have preferential affinities for the ligands SP, NKA and NKB respectively.","Like other G-protein coupled receptors, they possess an extra-cellular N-terminus, an intra-cellular C-terminus and seven trans-membrane (TM) α-helices of 20-30 amino acids connected by first, second and third extra-cellular loops and by first, second and third intra-cellular or cytoplasmic loops.","The greatest homology is found in the membrane-spanning α-helices of trans-membrane domains TM1-TM7 while the N- and C-termini show greater diversity between the three types of neurokinin receptor.","Cloned NK-1 receptors have been reported, including those for the Rana catesbeina , (Simmons et al., Neuroscience, 79: 1219-1229 (1997)), Mus musculus , (Sundelin et al, Eur.","J. Biochem.","203: 625-631 (1992)), Rattus norvegicus , (Hershey et al, J. Biol Chem., 266: 4366-4374 (1991) and Yokota et al, J. Biol.","Chem., 264: 17649-17652 (1989)), Cavia porcellus , (Gorbulev et al., Biochem.","Biophys.","Acta 1131: 99-102 (1992)), and Homo sapiens (human), (Takeda et al, Biochem.","Biophys.","Res.","Comm.","179: 1232-1240 (1991) and Fong et al U.S. Pat.","No.","5,525,712 and U.S. Pat.","No.","5,584,886).","Cloned rat and bovine neurokinin-2 receptors have been reported (Y. Sasi et al., Biochem.","Biophys.","Res.","Comm., 165: 695 (1989), and Y. Masu, et al., Nature 329: 836 (1987)).","Cloned rat neurokinin-3 receptor has also been reported (R. Shigemoto, et al., J. Biol.","Chem., 265:623 (1990)).","All three receptors share the signal transduction mechanisms of a G-protein coupled receptor.","The receptor is in an OFF state when no ligand is present, but, is triggered into an ON state when an agonist ligand binds to the receptor.","The G-protein when in its ON state triggers a downstream signaling pathway or signal cascade.","The G-protein comprises α-, β- and γ-units that are bound together in the OFF state, the α-subunit then having GDP bound to it.","In the ON state GTP replaces the GDP bound to the α-subunit.","The α-subunit becomes dissociated from the β- and γ-subunits and becomes available for activating the signal cascade.","After a short period, the GTP becomes hydrolyzed to GDP and the G-protein returns to its non activated OFF state.","Hydrolysis provides a negative feedback mechanism that ensures that the G protein is only in its activated ON state for a short period.","Various studies have been undertaken, involving different G-protein receptors, to determine how the various regions of the protein structure affect intra-cellular coupling of the receptor to the G-protein and consequential transduction of ligand binding signals to the cell.","G-protein receptors have a well-conserved sequence in the second intracellular loop where the loop joins the third trans-membrane domain that is known as the DRY sequence.","It has the residues in-line-formulae description=\"In-line Formulae\" end=\"lead\"?","5′-DRYXXV(P)XXPL-3′ (SEQ ID N o 4) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?","in which L represents Leu, Ile, Val, Met or Phe and X represents any amino acid.","It has been suggested that the DRY sequence contributes to the efficient binding and activation of G-proteins.","Fraser et al., Proc.","Natl Acad Sci.","USA, 85: 5478-5482 (1988) report a change of Asp to Asn at position 130 of the human β-adrenergic receptor (i.e.","the D of the DRY sequence) resulting in human β-adrenergic receptor that exhibits high affinity binding of agonist whilst being unable to interact effectively with G-protein.","However, a second paper from the same laboratory reports that the previous very high agonist binding efficiency in human β-adrenergic receptor mutated at position 130, upon which the above mentioned conclusion had been based, had not been reproduced (Wang et al., Mol Pharmacol 40(2): 168-79 (1991)).","In a review article Savarese and Fraser said that this locus may be important for coupling to some, but not all, G-proteins ( Biochem J., 283: 1-19 (1992)).","Moro et al made mutants of the Hm1 muscarinic cholinergic receptor with changes towards the 5′-end of the DRY sequence and found that replacing L at position 131 with A gave the strongest reduction in coupling efficiency ( J. Biol.","Chem.","268: 22273-22278 (1993)).","Subsequently, Shibata et al made a mutant of the angiotensin II receptor type I in which DRY at positions 125-127 is replaced by GGA and M at position 134 is replaced by A, resulting in uncoupling of the mutant A receptor from G-proteins ( Biochem.","Biophys.","Res.","Com.","218: 383-389 (1996)).","The authors concluded that DRY sequence as a whole including the final lipophilic amino acid L serves as a general site for G-protein coupling but they did not go on to consider what effects might be obtained by change confined to the DRY portion of the sequence.","Comparing the binding affinities of the two known isoforms of the human NK-1 receptor shows the importance of the cytoplasmic tail.","The long form (407 amino acids) and short form (311 amino acids) differ in the length of the C-terminus.","The long form has similar substance P binding characteristics to the rat NK-1 receptor, while the short form of the receptor has an apparent substance P binding affinity 10-fold less than the rat NK-1 receptor.","Furthermore, studies on these and other receptors have shown that the effect of mutations is unpredictable and specific to certain families of receptors.","Hence, a change in one G-protein coupling family will not necessarily have the same effect on another.","Other attempts to distinguish protein binding and signaling effects bear this out."],["<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides a mutant tachykinin receptor in which the three amino acids of the DRY sequence that occurs adjacent to the junction of the TM3 domain with intracellular loop 2 are replaced with amino acids whose side chains are neither lipophilic nor contain charged groups, said receptor exhibiting similar ligand binding characteristics to the wild type receptor but being incapable or substantially incapable of initiating an endogenous signal.","Thus the ligand exhibits no or substantially no intra-cellular coupling of the receptor to the G-protein, whereby there is substantially no transduction of ligand binding signals to the cell.","The way in which tachykinin receptors attach to cell membranes, the trans-membrane domains extra-cellular and cytoplasmic loops and the place where the DRY sequence referred to above occurs are apparent by inspection of FIG.","5 of the accompanying drawings.","The invention further provides any of the following: a fragment of said mutant tachykinin receptor containing said modified DRY sequence; an isolated protein or polypeptide containing an amino acid sequence at least 95% identical to the above sequence; a variant thereof with sequential amino acid deletions from either the C terminus or the N-terminus; and an allelic variant, heterospecific homologue or biologically active proteolytic or other fragment thereof containing said modified DRY sequence.","The invention also comprises an isolated cell membrane in which a modified tachykinin receptor as aforesaid is incorporated as membrane protein.","Such cell membrane material finds utility for research and in particular for screening for therapeutically useful compounds as described below.","The invention yet further provides any of the following: (a) an isolated nucleic acid molecule comprising a polynucleotide that encodes a modified tachykinin receptor as aforesaid; (b) an isolated nucleic acid molecule comprising a sequence that is hybridizable to the above sequence; (c) a gene which is the result of extending the above sequence or any sequence that is hybridizable to the above sequence; (d) a sequence or gene that is functionally equivalent to the above sequence or to a gene that is an extension of the above sequence, i.e.","that is not identical to the sequence or gene referred to but functions biologically as equivalent to the sequence or gene referred to, including any allelic variants and heterospecific mammalian homologues, including artificial or recombinant sequences created from cDNA or genomic DNA; (e) a recombinant vector comprising the above gene sequence; and (f) a host cell transformed with the vector.","The invention also provides a method for producing one of the amino acid sequences described herein and especially the receptor protein defined by SEQ ID N o 5, which method comprises the steps of: (a) inserting said nucleic acid sequence into an appropriate vector; (b) culturing in a culture medium a host cell previously transformed or transfected with the recombinant vector of step (a); (c) harvesting cells containing the receptor protein obtained from step (b); and (d) separating or purifying from said culture medium or from said host cells the thus-produced receptor protein.","In step (d) of the above method, the receptor protein may be obtained either from the culture medium and/or by lysing the host cell, for example by sonication or osmotic shock.","The invention yet further provides a method for screening for therapeutically active compounds, said method comprising the following steps: (a) providing a cell line expressing a modified tachykinin receptor as aforesaid; (b) adding test sample to a solution containing labeled SP or other tachykinin ligand and the cell line from step (a); (c) incubating the cell line, test sample and labeled SP or other ligand mixture from step (b) to allow binding of SP or other ligand and test sample to the modified tachykinin receptor; (d) optionally separating the non-bound labeled SP or other ligand from the labeled SP or other ligand bound to the modified tachykinin receptor and, if desired, (e) measuring the amount of labeled SP or other ligand that is bound to the modified tachykinin receptor.","The invention further provides: (a) the use of a modified tachykinin receptor according to the invention in the preparation of a medicament for the treatment or prophylaxis of a condition associated with substance P or other tachykinin (neurokinin) receptor-binding ligand; (b) the use of such a modified tachykinin receptor in therapy; (c) a method for the treatment or prevention of a condition associated with over-expression of an endogenous tachykinin ligand, which method comprises administration to a patient in need thereof of a non-toxic, effective amount of such a modified tackykinin ligand as described above; (d) a composition comprising a modified tachykinin ligand as described above in association with a pharmaceutically and pharmacologically acceptable carrier therefor; and (e) a use, method or composition according to any one of (a) to (d) above, in which the modified tachykinin ligand becomes generated in vivo from a nucleic acid sequence encoding such a ligand."],["FIELD OF THE INVENTION The present invention relates to a nucleic acid sequence encoding a tachykinin receptor, the tachykinin receptor encoded by said sequence, methods for its preparation and its use in therapy and screening.","In particular, the invention relates to a modification to tachykinin receptor proteins that gives rise to unexpected and useful properties.","BACKGROUND TO THE INVENTION Tachykinins are important in the mediation of many physiological and pathological processes including inflammation, pain, migraine, headache and allergy induced asthma.","They belong to an evolutionary conserved family of peptide neurotransmitters that have an established role in neurotransmission.","They share the C-terminal sequence Phe-Xaa-Gly-Leu-Met-NH2 (SEQ ID NO 12) in which Xaa represents a hydrophobic residue.","That sequence is characteristic of tachykinins and believed to be mainly responsible for their biological activity at neurokinin receptors.","Mammalian tachykinins include substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) which exert their effects by binding to specific receptors.","SP is the most prominent member of the tachykinergic system and is released from sensory nerve endings throughout the body.","Its amino acid sequence is: H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (SEQ ID No1) The amino acid sequence for NKA is: H-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 (SEQ ID No2) The amino acid sequence for NKB is: H-Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2 (SEQ ID No3) SP has been implicated in the pathology of numerous diseases.","For example, it has been shown to be involved in the transmission of pain, in conditions associated with vasodilation, smooth muscle contraction, bronchoconstriction, activation of the immune system and neurogenic inflammation.","It has been implicated in migraine, as well as in disorders of the central nervous system, such as anxiety and schizophrenia; in respiratory and inflammatory diseases, such as asthma and rheumatoid arthritis; and in gastrointestinal (GI) disorders and diseases of the GI tract, such as ulcerative colitis and Crohn's disease.","Tachykinin receptors include NK-1, NK-2 and NK-3 and are membrane proteins of the super-family of guanine nucleotide-binding protein (G-protein)-coupled receptors (GPCR).","They have extra-cellular binding sites that have preferential affinities for the ligands SP, NKA and NKB respectively.","Like other G-protein coupled receptors, they possess an extra-cellular N-terminus, an intra-cellular C-terminus and seven trans-membrane (TM) α-helices of 20-30 amino acids connected by first, second and third extra-cellular loops and by first, second and third intra-cellular or cytoplasmic loops.","The greatest homology is found in the membrane-spanning α-helices of trans-membrane domains TM1-TM7 while the N- and C-termini show greater diversity between the three types of neurokinin receptor.","Cloned NK-1 receptors have been reported, including those for the Rana catesbeina, (Simmons et al., Neuroscience, 79: 1219-1229 (1997)), Mus musculus, (Sundelin et al, Eur.","J. Biochem.","203: 625-631 (1992)), Rattus norvegicus, (Hershey et al, J. Biol Chem., 266: 4366-4374 (1991) and Yokota et al, J. Biol.","Chem., 264: 17649-17652 (1989)), Cavia porcellus, (Gorbulev et al., Biochem.","Biophys.","Acta 1131: 99-102 (1992)), and Homo sapiens (human), (Takeda et al, Biochem.","Biophys.","Res.","Comm.","179: 1232-1240 (1991) and Fong et al U.S. Pat.","No.","5,525,712 and U.S. Pat.","No.","5,584,886).","Cloned rat and bovine neurokinin-2 receptors have been reported (Y. Sasi et al., Biochem.","Biophys.","Res.","Comm., 165: 695 (1989), and Y. Masu, et al., Nature 329: 836 (1987)).","Cloned rat neurokinin-3 receptor has also been reported (R. Shigemoto, et al., J. Biol.","Chem., 265:623 (1990)).","All three receptors share the signal transduction mechanisms of a G-protein coupled receptor.","The receptor is in an OFF state when no ligand is present, but, is triggered into an ON state when an agonist ligand binds to the receptor.","The G-protein when in its ON state triggers a downstream signaling pathway or signal cascade.","The G-protein comprises α-, β- and γ-units that are bound together in the OFF state, the α-subunit then having GDP bound to it.","In the ON state GTP replaces the GDP bound to the α-subunit.","The α-subunit becomes dissociated from the β- and γ-subunits and becomes available for activating the signal cascade.","After a short period, the GTP becomes hydrolyzed to GDP and the G-protein returns to its non activated OFF state.","Hydrolysis provides a negative feedback mechanism that ensures that the G protein is only in its activated ON state for a short period.","Various studies have been undertaken, involving different G-protein receptors, to determine how the various regions of the protein structure affect intra-cellular coupling of the receptor to the G-protein and consequential transduction of ligand binding signals to the cell.","G-protein receptors have a well-conserved sequence in the second intracellular loop where the loop joins the third trans-membrane domain that is known as the DRY sequence.","It has the residues 5′-DRYXXV(P)XXPL-3′ (SEQ ID No4) in which L represents Leu, Ile, Val, Met or Phe and X represents any amino acid.","It has been suggested that the DRY sequence contributes to the efficient binding and activation of G-proteins.","Fraser et al., Proc.","Natl Acad Sci.","USA, 85: 5478-5482 (1988) report a change of Asp to Asn at position 130 of the human β-adrenergic receptor (i.e.","the D of the DRY sequence) resulting in human β-adrenergic receptor that exhibits high affinity binding of agonist whilst being unable to interact effectively with G-protein.","However, a second paper from the same laboratory reports that the previous very high agonist binding efficiency in human β-adrenergic receptor mutated at position 130, upon which the above mentioned conclusion had been based, had not been reproduced (Wang et al., Mol Pharmacol 40(2): 168-79 (1991)).","In a review article Savarese and Fraser said that this locus may be important for coupling to some, but not all, G-proteins (Biochem J., 283: 1-19 (1992)).","Moro et al made mutants of the Hm1 muscarinic cholinergic receptor with changes towards the 5′-end of the DRY sequence and found that replacing L at position 131 with A gave the strongest reduction in coupling efficiency (J. Biol.","Chem.","268: 22273-22278 (1993)).","Subsequently, Shibata et al made a mutant of the angiotensin II receptor type I in which DRY at positions 125-127 is replaced by GGA and M at position 134 is replaced by A, resulting in uncoupling of the mutant A receptor from G-proteins (Biochem.","Biophys.","Res.","Com.","218: 383-389 (1996)).","The authors concluded that DRY sequence as a whole including the final lipophilic amino acid L serves as a general site for G-protein coupling but they did not go on to consider what effects might be obtained by change confined to the DRY portion of the sequence.","Comparing the binding affinities of the two known isoforms of the human NK-1 receptor shows the importance of the cytoplasmic tail.","The long form (407 amino acids) and short form (311 amino acids) differ in the length of the C-terminus.","The long form has similar substance P binding characteristics to the rat NK-1 receptor, while the short form of the receptor has an apparent substance P binding affinity 10-fold less than the rat NK-1 receptor.","Furthermore, studies on these and other receptors have shown that the effect of mutations is unpredictable and specific to certain families of receptors.","Hence, a change in one G-protein coupling family will not necessarily have the same effect on another.","Other attempts to distinguish protein binding and signaling effects bear this out.","SUMMARY OF THE INVENTION The invention provides a mutant tachykinin receptor in which the three amino acids of the DRY sequence that occurs adjacent to the junction of the TM3 domain with intracellular loop 2 are replaced with amino acids whose side chains are neither lipophilic nor contain charged groups, said receptor exhibiting similar ligand binding characteristics to the wild type receptor but being incapable or substantially incapable of initiating an endogenous signal.","Thus the ligand exhibits no or substantially no intra-cellular coupling of the receptor to the G-protein, whereby there is substantially no transduction of ligand binding signals to the cell.","The way in which tachykinin receptors attach to cell membranes, the trans-membrane domains extra-cellular and cytoplasmic loops and the place where the DRY sequence referred to above occurs are apparent by inspection of FIG.","5 of the accompanying drawings.","The invention further provides any of the following: a fragment of said mutant tachykinin receptor containing said modified DRY sequence; an isolated protein or polypeptide containing an amino acid sequence at least 95% identical to the above sequence; a variant thereof with sequential amino acid deletions from either the C terminus or the N-terminus; and an allelic variant, heterospecific homologue or biologically active proteolytic or other fragment thereof containing said modified DRY sequence.","The invention also comprises an isolated cell membrane in which a modified tachykinin receptor as aforesaid is incorporated as membrane protein.","Such cell membrane material finds utility for research and in particular for screening for therapeutically useful compounds as described below.","The invention yet further provides any of the following: (a) an isolated nucleic acid molecule comprising a polynucleotide that encodes a modified tachykinin receptor as aforesaid; (b) an isolated nucleic acid molecule comprising a sequence that is hybridizable to the above sequence; (c) a gene which is the result of extending the above sequence or any sequence that is hybridizable to the above sequence; (d) a sequence or gene that is functionally equivalent to the above sequence or to a gene that is an extension of the above sequence, i.e.","that is not identical to the sequence or gene referred to but functions biologically as equivalent to the sequence or gene referred to, including any allelic variants and heterospecific mammalian homologues, including artificial or recombinant sequences created from cDNA or genomic DNA; (e) a recombinant vector comprising the above gene sequence; and (f) a host cell transformed with the vector.","The invention also provides a method for producing one of the amino acid sequences described herein and especially the receptor protein defined by SEQ ID No5, which method comprises the steps of: (a) inserting said nucleic acid sequence into an appropriate vector; (b) culturing in a culture medium a host cell previously transformed or transfected with the recombinant vector of step (a); (c) harvesting cells containing the receptor protein obtained from step (b); and (d) separating or purifying from said culture medium or from said host cells the thus-produced receptor protein.","In step (d) of the above method, the receptor protein may be obtained either from the culture medium and/or by lysing the host cell, for example by sonication or osmotic shock.","The invention yet further provides a method for screening for therapeutically active compounds, said method comprising the following steps: (a) providing a cell line expressing a modified tachykinin receptor as aforesaid; (b) adding test sample to a solution containing labeled SP or other tachykinin ligand and the cell line from step (a); (c) incubating the cell line, test sample and labeled SP or other ligand mixture from step (b) to allow binding of SP or other ligand and test sample to the modified tachykinin receptor; (d) optionally separating the non-bound labeled SP or other ligand from the labeled SP or other ligand bound to the modified tachykinin receptor and, if desired, (e) measuring the amount of labeled SP or other ligand that is bound to the modified tachykinin receptor.","The invention further provides: (a) the use of a modified tachykinin receptor according to the invention in the preparation of a medicament for the treatment or prophylaxis of a condition associated with substance P or other tachykinin (neurokinin) receptor-binding ligand; (b) the use of such a modified tachykinin receptor in therapy; (c) a method for the treatment or prevention of a condition associated with over-expression of an endogenous tachykinin ligand, which method comprises administration to a patient in need thereof of a non-toxic, effective amount of such a modified tackykinin ligand as described above; (d) a composition comprising a modified tachykinin ligand as described above in association with a pharmaceutically and pharmacologically acceptable carrier therefor; and (e) a use, method or composition according to any one of (a) to (d) above, in which the modified tachykinin ligand becomes generated in vivo from a nucleic acid sequence encoding such a ligand.","BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be further described with reference to the accompanying drawings, in which: FIG.","1: describes a protein sequence, being a translation of the sequence shown in FIG.","1 and being the sequence of a modified human NK-1 receptor according to the invention (hNK-1Rv1).","FIG.","2: is a consensus cDNA sequence encoding a modified human NK-1 receptor according to the invention.","FIG.","3: is a bar chart showing the results of Calcium imaging experiments on NK-1 wild type receptor and the modified receptor of FIG.","1 (hNK-1Rv1), and wild type receptor antisense transfected COS cells.","FIG.","4: shows the sequence alignments between NK-1 and other NK receptors in various species in the region of the DRY motif in TM3, demonstrating high conservancy (In the Figure, NK-1 sequences 1-4 provide SEQ ID NO 13, NK-1 sequence 5 provides SEQ ID NO 14, NK-2 sequences 1-2 and 4-7 provide SEQ ID NO 15, NK-2 sequence 3 provides SEQ ID NO 16 and NK-3 sequences 1-3 provide SEQ ID NO 17).","FIG.","5: is a diagram showing the nNK-1Rv1 receptor and portions of the cell membrane into which it is incorporated (SEQ ID NO 18; in addition to the DRY motif, L223 is changed to I; this change is believed to be inconsequential).","DESCRIPTION OF PREFERRED EMBODIMENTS Definitions Definitions for a number of terms used in this specification are given below: Alleles or allelic sequences mean alternative forms of the receptor genes referred to above resulting from one or more variations in the nucleic acid sequence, resulting in altered mRNAs and proteins or polypeptides Functional equivalence when used in relation to gene sequences or amino acid sequences covers sequences that are not identical to the sequence referred to but function biologically or chemically as equivalents of the disclosed sequence.","Incapable of initiating an endogenous signal in the present context means that, within experimental error (±5% of control), the modified NKR does not result in conversion of GTP to GDP in the cell or a recombinant construct; and/or does not evoke a calcium flux therein; and/or does not evoke other components of the signal transduction pathway.","Isolated, when used in relation to a polynucleotide sequence, means such a sequence that has been removed from its natural environment, i.e.","from the organism in which it occurs in nature and/or from genes that are immediately contiguous (one at the 5′ end and the other at the 3′ end) in the naturally occurring genome of the organism from which it is derived.","Isolated when used in relation to a cell membrane refers to the membrane as a discrete entity substantially separate from cytoplasmic material.","Operably linked refers to a linkage of polynucleotide elements in a functional relationship.","For instance, a promoter or an enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.","More specifically, two DNA molecules (such as a polynucleotide containing a promoter region and a polynucleotide encoding a desired polypeptide or polynucleotide) are said to be “operably linked” if the nature of the linkage between the two polynucleotides neither results in the introduction of a frame-shift mutation nor interferes with the ability of the polynucleotide containing the promoter to direct the transcription of the coding polynucleotide.","Stringent hybridisation conditions is a recognized term in the art and for a given nucleic acid sequence refers to those conditions which permit hybridisation of that sequence to its complementary sequence and not to a substantially different sequence.","It generally implies at least about 97% identity between the sequences.","Protein and Nucleic Acid Sequences In the above mentioned mutant receptors, amino acids for modifying the DRY sequence that are non-polar and have the correct hydrophilic/lipophilic balance include glycine and alanine which are preferred.","However, the inventors also envisage the use of other non polar amino acids.","In a preferred aspect, the invention provides a nucleic acid sequence as shown in FIG.","2 [SEQ ID No6], which encodes the consensus amino acid sequence for the modified hNK-1v or a sequence that hybridizes thereto under stringent hybridization conditions.","The invention also provides a preferred hNK-1v receptor sequence shown in FIG.","1 [SEQ ID No5].","The invention further provides a nucleic acid sequence encoding a receptor protein having at least 80%, preferably 90%, more preferably 95%, and most preferably 98% amino acid identity with the hNK-1Rv1 encoded by the nucleic acid sequence of SEQ ID No6, and which encodes protein that can bind to SP, but cannot initiate its endogenous signal, or encodes a peptide fragment thereof according to sequence (d), or encodes a sequence complementary thereto according to sequence (e), as defined hereinabove.","The invention also provides a protein that: (a) has at least 80%, preferably 90%, more preferably 95%, and most preferably 98% amino acid identity with the hNK-1Rv1 protein having the amino acid sequence of SEQ ID No5; and (b) can bind to SP or to a peptide fragment thereof, or to a sequence complementary thereto, but cannot initiate its endogenous signal to G protein.","The hNK-1Rv1 variant of the NK-1 receptor shown in SEQ ID No5 has similar ligand binding characteristics to the wild type receptor, but is deficient in cell signaling capabilities, as demonstrated by the results of tests described in Examples 4 and 5.Some mammalian receptors may exhibit overall homology with the hNK-1 receptor of less than 80% (for example, rat NK-2 receptor has about 48% homology to rat NK-1 receptor), but are nevertheless included within the scope of this invention in view of their conservancy in the intracellular DRY region of TM3.Accordingly, the present invention provides a polypeptide having as low as 40% overall amino acid identity with the hNK-1Rv1 protein, but having at least 75%, such as at least 80%, preferably 90%, more preferably 95%, and most preferably 98% amino acid identity with the TM3 intracellular loop of the bNK-1Rv1 protein in the vicinity of the DRY motif, and having the properties previously specified.","In view of the high level of conservancy demonstrated by other NK receptors in the DRY region of TM3 intracellular loop, and also the high level of conservancy observed in that region between human and other species, the present invention also provides a nucleotide sequence that encodes a modified neurokinin receptor, wherein the modification is or includes substitution of the DRY motif within the intracellular loop of TM3, namely, Asp129, Arg130 and Tyr131, by Gly, Gly and Ala, respectively, for example wherein the receptor is a modified NK-2 or modified NK-3 mammalian receptor.","Vectors The invention further provides a vector (e.g.","a plasmid or virus) comprising a nucleic acid encoding the modified tachykinin receptor, especially the hNK-1Rv1 receptor or any other modified hNK-1R of this invention.","A recombinant vector of the invention comprises an expression vector comprising a nucleic acid sequence encoding the modified NK-R, especially the hNK-1Rv1, polypeptide.","One suitable vector for the expression of a human variant NK-1 receptor of the invention is a baculovirus vector that can be propagated in insect cells and in insect cell-lines.","Expression requires that appropriate signals are provided in the vector, said signals including various regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells.","The regulatory sequences of the expression vectors are operably linked to the nucleic acid encoding the modified tachykinin receptor, especially the human NK-1 variant receptor.","Generally, recombinant expression vectors include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence.","The heterologous structural sequence is assembled in an appropriate frame with the translation, initiation and termination sequences, and preferably a leader sequence capable of directing sequences of the translated protein into the periplasmic space or the extra-cellular medium.","Where the vector is adapted for transfecting and expressing desired sequences in eukaryotic host cells, preferred vectors comprise an origin of replication from the desired host, a suitable promoter and an enhancer, and also any necessary ribosome binding sites, polyadenylation site, transcriptional termination sequences, and optionally 5′-flanking non-transcribed sequences.","Suitable promoter regions used in the expression vectors according to the invention are chosen taking into account the host cell in which the heterologous nucleic acids have to be expressed.","A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression, or may be endogenous to the native polynucleotide containing the coding sequence to be expressed.","Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.","A recombinant vector of the invention may be used to amplify a polynucleotide derived from the nucleic acid sequence encoding the modified NKR, especially the hNK-1Rv1 polypeptide that has been inserted in a suitable host cell, this polynucleotide being amplified every time the recombinant vector replicates.","DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, and polyadenylation sites may be used to provide the require non-transcribed genetic elements.","The suitable promoter regions used in the expression vectors according to the invention are chosen taking into account the host cell in which the heterologous nucleic acids are to be expressed.","A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively it can be endogenous to the native polynucleotide containing the coding sequence to be expressed.","Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.","Preferred bacterial promoters are the LacI, LacZ, T3 or T7 bacteriophage RNA polymerase promoters, the lambda PR, PL and trp promoters (EP 0 036 776), the polyhedrin promotor, or the p10 protein promoter from baculovirus (Kit Novagen; Smith et al., 1983); O'Reilly et al., 1992, Baculovirus expression vectors: A Laboratory Manual.","W.H.","Freeman and Co., New York).","Preferred selectable marker genes contained in the expression recombinant vectors of the invention for selection of transformed host cells are preferably dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, TRP1 for S. cerevisiae or tetracyclin, rifampicin or ampicillin resistance in E. coli, or Levan saccharase for mycobacteria, this latter marker being a negative selection marker.","Preferred bacterial vectors of the invention are listed hereafter as illustrative but not limitative examples: pQE70, pQE60, pQE-9 (Qiagen), pD10, fephagescript, psiX174, p.Bluescript SK, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (Qiagen).","Preferred bacteriophage recombinant vectors of the invention are P1 bacteriophage vectors such as described by Sternberg N. L. (1992;1994).","A suitable vector for the expression of hNK-1vRa polypeptide of the invention or a fragment thereof, is a baculovirus vector that can be propagated in insect cells and in insect cell-lines.","A specific suitable host vector system is the pVL 1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC NoCRL 1711) which is derived from spodoptera frugiperda.","The recombinant expression vectors of the invention may also be derived from an adenovirus such as those described by Feldman and Steg.","(1996) or Ohno et al.","(1994).","Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type two or five (Ad 2 or Ad 5) or an adenovirus of animal origin (French Patent Application noFR 93 05 954).","Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vivo gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, murine sarcoma virus, and Ross Sarcoma Virus.","Other preferred retroviral 5 vectors are those described in Roth et al.","(1996), in PCT Application WO 93/25 234, in PCT Application WO 94/06920, and also in Roux et al.","(1989), Julan et al.","(1992) and Nada et al.","(1991).","Yet, another viral vector system that is contemplated by the invention consists in the adeno associated virus (AAV) such as those described by Flotte et al.","(1992), Samulski et al.","(1989) and McLaughlin et al.","(1996).","Expression Systems For expression of the modified neurokinin receptors, the invention provides host cells transformed (prokaryotic cells) or transfected (eukaryotic cells) with such a vector; and any cell, or live organism, including a non-human mammal, that has been genetically engineered to produce such a polypeptide, said cell or live organism incorporating expressibly therein a nucleic acid sequence according to this invention.","Heterologous expression systems may be used to express cloned NK-1 receptor, NK-2 receptor and NK-3 receptor, including human and non-human mammalian variants thereof.","The choice of expression system depends on a number of factors including stability of protein expression, post translational modification and required yield.","However, as a general rule, the more complex the organism the lower the yield of expressed receptor, but the greater the likelihood that the receptor will be in its native conformation.","Several expression hosts are commonly available Prokaryotic host cells, e.g.","Escherichia coli DH5-α (see Sambrook et al., for a comprehensive guide to gene expression in E. coli).","Yeasts, e.g.","Pichia pastoris.","The feasibility of expressing NK-1 receptor in yeast has been demonstrated by Arkinstall et al (1995, FEBS 275 183-187) who successfully expressed therein the closely related receptor, human NK-2.Large-scale production of a human G-protein coupled receptor in yeast was demonstrated by Sizemann et al (1996, Receptors and Channels, Vol 4, 197-203).","In brief, a protocol to express a functional NK-1 receptor in yeast can be represented by: (1) splicing the NK-1 cDNA into a yeast expression vector; (2) Transforming this vector into yeast; and (3) Selecting for yeast containing the NK-1 cDNA and expression of the NK-1 cDNA.","Eukaryotic host cells, e.g.","insect cells, non-mammalian vertebrate cells and mammalian cells.","Non-mammalian vertebrate cell lines include Xenopus (frog) oocytes.","Other cell lines which are contemplated in this invention include HeLa cells (ATCC NoCCL2; NoCCL2.1; NoCCL2.2), Cv 1 cells (ATCC NoCCL70), COS cells (ATCC NoCRL 1650; NoCRL 1651), Sf-9 cells (ATCC NoCRL 1711), C127 cells (ATCC NoCRL-1804), 3T3 cells (ATCC NoCRL-6361), CHO cells (ATCC NoCCL-61), human kidney 293 cells (ATCC No 45504; NoCRL-1573) and BHK (ECACC No84100 501; No84111301); PC12 (ATCC No CRL-1721), NT2, SHSY5Y (ATCC No CRL-2266), NG108 (ECACC No88112302) and F11, SK—N—SH (ATCC No CRL-HTB-11), SK—N—BE(2) (ATCC No CRL-2271), IMR-32 (ATCC No CCL-127).","A preferred system to which the gene of the invention can be expressed are cell lines such as COS cells, 3T3 cells, HeLa cells, 292 cells and CHO cells.","A preferred system for the efficient expression of hNK-1vR involves the use of CHO and COS cell lines.","The gene can be expressed through an endogenous promoter of native CHO or COS, or through an exogenous promoter.","Suitable exogenous promoters include such as SV40 and CMV, or perhaps a eucaryotic promoter such as the tetracycline promoter.","The preferred promoter being CMV.","In some instances, it may be required to tag e.g.","a human NK-1 variant receptor prior to purification.","The tag is then, in most instances, encoded into the nucleotide sequence that is needed to express the polypeptide.","Examples of such tags include, but are not limited to sequences encoding C-myc, FLAG, a sequence of histidine residues, haemaglutin A, V5, Xpress or GST.","Most of these tags can be incorporated directly into the sequence, for instance through PCR amplification by incorporating the appropriate coding sequence in one of the PCR amplification primers.","However, the tag can also be introduced by other means, such as by covalent binding of the appropriate nucleic acid sequence encoding the tag moiety, such as GST, with the 3′ or 5′ end of the nucleic acid sequence encoding the polypeptide sequence.","Purification of the NK variant receptor may, in the case of the use of a histidine tag, then be carried out by passage onto a nickel or copper affinity chromatography column, such as a Ni NTA column.","The polypeptide thus produced may optionally be further characterized, for example by binding onto an immuno-affinity chromatography column on which polyclonal or monoclonal antibodies directed to the NK variant receptor have been previously immobilised.","For some purposes it may be useful to provide mammals e.g.","mice, rats or guinea-pigs in which in which modified human or non-human mammalian tackykinin receptors, e.g.","NK-1v receptors, are present.","Transgenic rats, mice and other mammalian cells may be produced by generating a targeting vector and transfecting the cells to be cultured e.g.","using the gene targeting services of, e.g.","DNX Transgenic Sciences.","In the case of transgenic mammals, stem cells are transfected, cultured to blastocytes and introduced into the uterus of a female mammal.","Because they have in their cell membranes tachykinin receptors that bind to endogenous ligands such as SP but are incapable, or substantially incapable, of initiating their endogenous signal, the animals are useful, in research into the effects of these ligands and the developments of transduction inhibitors, especially because they can indicate the behaviour of the animal the presence of endogenous ligand but in the absence of a transduced signal and in the absence of antagonist, and can therefore provide a true control.","(Silver, Lee M. Title: Mouse genetics—concepts and applications Publisher: New York; Oxford University Press 1995); P De-Felipe-C et al;Nature.","1998 Mar.","26; 392(6674): 394-7).","Assays The present invention further provides a modified tachykinin receptor, such as a human NK-1v receptor, used as a substitute in an assay to identify and evaluate entities that bind to the wild type tachykinin receptor.","The invention also includes human NK-1v receptor used as a substitute in an assay in order to determine the concentration of substance P in body fluids in patients with arthritis, pain, migraine, anxiety, schizophrenia, asthma, rheumatoid arthritis, and in gastrointestinal disorders and diseases of the GI tract, like ulcerative colitis and Crohn's disease.","As previously mentioned three tachykinin receptors have been identified, referred to as NK-1, NK-2 and NK-3 and they have respective endogenous lignads SP, NKA and NKB.","Although each tachykinin has a preferred ligand, each receptor has the ability to interact with the other tachykinin ligands, and the pathology of cross-ligand binding in disease states is poorly understood.","Study of a system where NK-1, NK-2 or NK-3 receptor response either singularly or in tandem has been diminished in vitro or in vivo could significantly aid understanding of the role of tachykinin receptors and their ligands in disease states.","Screening Methods The invention also provides a method for screening for therapeutically active compounds, said method comprising the following steps: (a) providing a cell line expressing the modified tachykinin receptor, e.g.","the human NK-1v receptor; (b) adding test sample to a solution containing labeled SP or other ligand and the cell line from step (a); (c) incubating the cell line, test sample and labeled SP or other ligand mixture from step (b) to allow binding of SP or other ligand and test sample to the modified tachykinin receptor; (d) optionally separate the non-bound labeled SP or other ligand from the labeled SP or other ligand bound to the modified tachykinin receptor; and, if desired, (e) measuring the amount of labeled SP or other ligand that is bound to the modified tachykinin receptor.","Preferably, the assay involves COS-7 cell lines.","Cell membranes containing the modified tachykinin receptor, e.g.","the human NK-1v receptor, can be used instead of whole cells.","The SP or other tachykinin ligand e.g.","NKA or NKB may be labeled by any method known in the screening art, e.g.","by a radioactive label, such as 125I, or by a fluorescent label.","In certain circumstances, (e.g.","fluorescence polarization assays), bound and unbound SP or other tachykinin ligand do not have to be separated to quantify the amount of SP or other ligand bound to the receptor.","Alternatively, the SP or other ligand may be bound to a matrix, and labelled cells may be used to quantify the binding of a modified tachykinin receptor, such as human NK-1 receptor, to the SP.","In this case, the assay procedure may follow the steps set out below: (a) a cell line is provided that expresses a modified tachykinin receptor, such as the human NK-1v receptor; (b) the cell line is labeled; (c) the test sample and labeled cells are added to a matrix binding SP or other ligand; (d) the labeled cells, test sample and matrix-bound SP or other ligand are incubated to allow binding of SP or other ligand and test sample to the expressed modified tachykinin receptor; (e) the labeled non-bound cells are separated from the SP or other ligand bound cells; and, if desired, (f) the amount of labeled cells containing the modified tachykinin receptor, such as the human NK-1v receptor, that has bound to SP or other ligand is measured.","In order to provide transgenic animals or cell lines for use in assays, which animals and/or cell lines comprise a sequence as described herein, general methods are known and may be adapted accordingly.","Different types of vectors including modified retroviruses, adenovirus, adeno-associated virus, herpes virus and plasmid DNA have been proposed as vehicles to introduce foreign genetic material into cells or tissues.","Protein Therapy The invention also encompasses modified tachykinin receptors, especially human NK-1v receptor, for use in protein therapy to reduce the effects of, or an excess of endogenous ligand.","Protein therapy can be used for the suppression of the action of SP in interstitial fluid of the lungs.","For example, a purified preparation of a modified tachykinin receptor, such as human NK-1v receptor, (eg a liquid or powder carrier formulation) can be directly administered to the airways.","Once in the airways, the tachykinin variant receptor can interact and bind to, for example, SP molecules.","This has the effect reducing the amount of SP available for the endogenous NK receptor.","Likewise, a modified tachykinin receptor, such as the human NK-1v receptor, can be directly introduced to body cavities such as joints and interstitial lung space where SP is present.","The variant receptor has the capability to bind SP, therefor reducing the amount of SP available to interact with the wild type receptor and causing down-regulation of the SP cellular response.","The invention provides a modified NK receptor, such as a human NK-1v receptor, for use in removing or suppressing SP in body fluids, e.g.","the interstitial fluid of the lungs and fluid in the cavities of joints.","A purified preparation of a modified NK receptor, such as human NK-1v receptor, (e.g.","a liquid carrier formulation) may be administered directly to a joint.","Once in the fluid- filled joint cavity, the NK variant receptor interacts and binds to SP molecules thereby reducing the amount of SP available to activate the endogenous NK receptor.","The hNK-1v receptor can reduce the effect of excess or inappropriately expressed SP in patients with pain associated with migraine, neuralgia, diabetic, peripheral, AIDS-related and chemotherapy-induced neuropathy, and neuropathies of diverse origin; anxiety and anxiety disorders, such as panic disorder, phobias and obsessive-compulsive behavior; schizophrenia; asthma; rheumatoid arthritis; and in gastrointestinal disorders and diseases of the GI tract, for example ulcerative colitis and Crohn's disease.","Other conditions or disease states that can be treated, ameliorated or prevented include: psychosomatic and psycho-immunological disorders; attention deficit disorder; pre-menstrual (PMT or PMS) or late luteal phase syndrome; mania or hypomania; aggressive behavior disorders; emesis, including motion sickness, migraine-induced sickness and that arising from chemotherapy; postherpetic neuralgia; depression; inflammation; eating disorders, such as obesity, bulimia nervosa and compulsive eating disorders; cognitive disorders, such as dementia and amnestic disorders; movement disorders, such as dyskinesias, akinetic-rigid syndromes, Gilles de la Tourette syndrome, tremor, or dystonia; schizophrenic disorders; substance abuse disorders; bipolar disorder; sexual dysfunction, including impotence; stress; alteration of circadian rhythmicity; Alzheimer's disease; bladder disorders; hypertension; angina; ischaemia; multiple sclerosis; chronic obstructive lung disease; scleroderma; CNS disorders; and other conditions where excess tachykinin peptides such as SP are involved.","Furthermore, the inventors believe that modification to the DRY motif of NK-2 receptor and NK-3 receptor in a similar manner to that described for the NK-1 receptor above, would allow for the production of modified tachykinin receptors which could be used to remove their specific ligands from body fluids.","Several factors need to be taken into account in protein therapies.","These include: solublisation, maintenance of activity and stability of the protein, delivery and dose.","The first three points are closely related.","Proteins are large relative to conventional drugs and their biological activity is dependent on their primary, secondary, tertiary and in some instances quaternary structure being maintained.","They often have labile bonds and numerous chemically reactive groups in their side chains.","Disruptions of their structure by denaturation or aggregation can lead to loss of activity or increase in immunogenicity.","One of the key problems in devising effective formulations for biologically active proteins is to find a formulation that is both stable and biologically active.","The route of administration of the protein also plays a significant role in formulation.","Microsphere formulations can be used for injection, aid in the maintenance of stability and activity of the protein, and offer the possibility of slow release formulations.","Solid large powder formulations are most suitable for use in aerosol and topical treatments.","Putney and Burke (Nature Biotechnology 1998: 153-157) outline various methods for producing microspheres.","One such method is the atomization-freezing process.","In this encapsulation method, the micronised solid protein is suspended in biodegradable polymers of DL-lactic co-glycolic acid (PLGA) solution that is then atomised using sonication or air-atomisation.","This produces droplets that are then frozen in liquid nitrogen.","Addition of ethanol at <−40° C., in which both the protein and the PLGA are insoluble, extracts the organic solvent from the micro-spheres.","This process is further described in U.S. Pat.","No.","5,019,400.An alternative polymer for use in the process is methylene chloride polymer,to encapsulate growth hormone see Johnson et al, Nature Medicine 2: 795-799 (1996).","The following method can be used to incorporate modified NKR, such as human NK-1v receptor, post-purification into microspheres: (a) concentrate the purified, active microsphere modified NKR, such as human NK-1v receptor to >100 mg/ml in the presence of stabilisers; (b) add PLGA or methylene chloride polymer solution and mix; then (c) atomise the frozen suspension in liquid nitrogen to fix the microspheres and extract with ethanol.","Microspheres thus produced should have a diameter in the μM order.","The invention further provides a method for treatment of a patient in need thereof, which comprises administering to said patient a composition in the form of an aerosol that comprises a modified tachykinin receptor as described above, e.g.","an hNK-1Rv receptor.","SP or other ligand such as NKA or NKB can also be removed directly from biological fluids using the modified tachykinin receptors described above.","For example, purified human NK-1v receptor could be bound to a suitable matrix.","Biological fluids containing SP can be passed over the matrix, so that the SP preferentially binds to matrix while other components of the fluid do not and SP becomes preferentially removed from the biological fluid.","Nucleic Acid Therapy The invention further provides a method for gene therapy treatment of a patient in need thereof, which comprises administering to said patient a nucleic acid sequence, virus or plasmid encoding a modified tachykinin receptor as described above, e.g.","an hNK-1Rv receptor.","Different types of vector including modified retroviruses, adenovirus, adeno-associated virus, herpes virus and plasmid DNA have been proposed as vehicles for introducing foreign genetic material into the cells or tissues of patients, and can in principle be used to introduce the modified tachykinin receptor nucleic acid sequences referred to above.","The appropriate strategy for administering the nucleic acid sequence depends on the target tissue, disease state and longevity of the proposed therapy.","Furthermore, down regulation of the effect SP on a cellular system could be achieved by expressing the hNK-1v receptor in cells other than those expressing native NK-1.The expression of SP in these cells (which are in the proximity of cells expressing native NK-1 receptor) could have the effect of “moping” up SP and reducing the available SP for the native receptor to interact with.","Introduction of a modified NK receptor, such as a human NK-1v receptor, (by way of direct introduction of protein or introduction of an expressible gene encoding this protein) into the outer membrane of cells expressing the wild type receptor gives rise to competition between the wild type and variant receptors for available SP.","The variant receptor competes with wild type receptor for binding of available SP and decreases the amount of SP available for the wild type receptor.","As the NK-1v receptor is unable to transduce a signal upon SP binding, the result is a down-regulation of the action of the wild type receptor.","In the treatment of lung tissue, researchers at Stanford University Medical Center, California have initiated a trial of gene therapy for cystic fibrosis in which the active material is delivered to the lungs by aerosol.","The active material consists of a version of the cystic fibrosis trans-membrane conductance regulator gene packed into an adeno-associated virus (AAV) shell.","A similar route can be adopted for delivering the hNK-1v receptor to the lungs of patients where the activation of native hNK-1 receptors by receptor ligands requires to be down-regulated.","A strategy for down-regulating the action between SP and human NK-1v receptor in lung tissue can be adopted which is similar to the strategy proposed for gene therapy in cystic fibrosis.","The same technique of packaging genes into AAV and infection of the lung tissue with aerosol AAV can be used to introduce hNK-1v.","Samulski et al (University of North Carolina, Chapel Hill, N.C. 27599, USA) market a vector which can be used to package human NK-1v receptor gene into AAV.","Alternatively, a plasmid containing the human NK-1v receptor (pCMVNK-1v) may be used directly in gene therapy.","The plasmid may be prepared as a lipid:DNA complex and administered to the lungs as an aerosol, see Pillai et al., Pharm-Res 15(11): 1743-7 (1998), McDonald et al., Pharm-Res 1998 15(5): 671-9(1998).","This strategy can also be used with an NK-1 receptor promoter.","A pre-requisite for the use of gene therapy for humans is the ability to make sufficient pharmaceutical grade plasmid DNA.","This problem has been addressed by Prazeres et al in TIBTECH 17: 169-74 (1999).","Both rAAV and lipid:DNA complex may be administered to the lungs via nebulisers, e.g.","airjet nebulisers.","Methods for administration may generally follow the teachings of McDonald et al, Pharm Res 15(5): 671-9 (1998); Yonemitsu et al, Gene Therapy 4(7): 631-8 (1997); McDonald et al, Human Gene Therapy, 8(4): 411-22 (1997); Bellon et al, Human Gene Therapy 8(1): 15-25 (1997); and Niven, Critical Review of Therapeutic Drug Carrier Systems 12(2-3): 151-231 (1995).","Both viral and plasmid therapeutic compositions can also be delivered to other target sites such as joints and nervous tissue by infusion and injection.","EXAMPLES Embodiments of the invention will now be described in the following Examples Example 1 Preparation of a Modified Human NK-1 Receptor (NK-1v) The starting material used was a plasmid pRc/CMV (Invitrogen Co) containing a cDNA clone encoding the human NK-1 receptor.","The clone encoded 407 amino acids and was flanked by a Nco I site at the 5′ end (around the ATG start codon) and a Xba I site at the 3′ end following the stop codon.","Human NK-1v receptor was made from the above plasmid using a PCR-based strategy that mutated the DRY motif (Asp129, Arg130, Tyr131) at the end of third transmembrane helix to GGA.","Portions of the cDNA clone in the above plasmid were amplified in two separate PCR reactions using Pfu DNA polymerase (Stratagene), 20 cycles of PCR using 1 min at 94° C., 1 min at 55° C., 4 min at 72° C., followed by 10 mins at 72° C., 50 pmol of each primer, 50 ng of plasmid DNA, 100 μl reaction volume.","The first PCR reaction used the following primers to produce a predicted product of 473 bp: (SEQ ID No7) 5′ primer, sense-A: 5′-AAC TAG AGA ACC CAC TGC TTA-3′ (SEQ ID No8) 3′ primer, anti-sense-A: 5′-GCC ATA GCG CCG CCA AAA GCC ACA GCC GT-3′ The second PCR reaction used the following primers to produce a predicted product of 888 bp: (SEQ ID No9) 5′ primer, sense-B: 5′-TTT GGC GGC GCT ATG GCT ATC ATA CAT CC-3′ (SEQ ID No10) 3′ primer, anti-sense-B: 5′-AGC TCT AGC ATT TAG GTG ACA-3′.","Primers anti-sense-A and sense-B contained 17 bases of overlapping complementary sequence at the 5′ ends of each to allow annealing of the two products to form the full length mutated receptor.","All the above primers were custom synthesized by Perkin-Elmer.","The two PCR products were gel-purified (QIAEX, Qiagen) and 50 ng of each purified product was added to a 100 μl Pfu PCR reaction and three rounds of PCR performed without primers to allow the two overlapping regions of the PCR products to anneal and extend.","50 ng of the flanking primers (sense-A and anti-sense-B) were added and 20 cycles of conventional PCR performed.","The resulting full-length product was purified, cloned into pBluescript (Stratagene) and fully sequenced to confirm the mutations had been successfully made.","The clone was modified by site-directed mutagenesis using a Clontech Transformer kit to remove an internal Nco I restriction site using the oligonucleotide sense-C also from Perkin-Elmer.","Sense C: 5′-CGC GGA GGC TTC TAT GGC TGC AT-3′ (SEQ ID No11) The resulting cDNA was excised from the parental vector, spliced into a mammalian expression vector, pCMV3.1 (Invitrogen).","This cDNA was in an orientation which allowed expression of the hNK-1vR polypeptide.","Stock plasmid was prepared by amplification in E. coli and the plasmid DNA subsequently purified using a Qiagen endonuclease free DNA preparation kit, and the fidelity of the construct confirmed by DNA sequencing.","The nucleic acid sequence of the cDNA is SEQ ID No6 (FIG.","2) and the translated protein sequence is SEQ ID No5 (FIG.","1).","Similarly control (negative control) constructs were prepared whereby wild type and variant HNK-1R cDNA was spliced into the vector in an opposite configuration to that described above.","A further control (positive control) was prepared by ligation of the parental wild type hNK-1R cDNA into the vector in a manner which would allow hNK-1R polypeptide to be expressed.","Example 2 COS-7 Cells that Transiently Express Human NK-1v Receptor COS-7 cells were grown in DMEM culture medium supplemented with 10% foetal calf serum and 2mM glutamine and maintained under an atmosphere of 5% CO2.Cells were passaged at approximately 70% confluence by reseeding to a concentration of approximately 10% confluence per 175 cm2 flask.","COS-7 cells were harvested and prepared for electroporation in Equibio electroporation buffer.","Cells (5×106 cells) were electroporated at room temperature in a 4 mm gap cuvette in a final volume of 800 μl containing 30 μg of transforming plasmid DNA, with 250 volts, 1500 μF at infinite resistance using an Equibio EasyJect plus electroporator.","Transformed COS-7 cells were cultured for 2-3 days in a 175cm2 flask prior to assay.","Alternatively cells (approximately 5-50 ul of transfected cells) were cultured in 6 well culture dishes containing 22mm diameter cover slips.","These cover slips coated in cells were used for imaging experiments designed to investigate changes in the concentration of intracellular free calcium ([Ca2+]i).","Cover slips were prepared by first immersing the cover slips in approximately 70% ethanol and then quickly passing through a gas flame to sterilize.","Cover slips were then allowed to air dry in a culture hood prior to placing in the bottom of a culture well of a standard six well culture dish.","Cells were then cultured as described above.","Example 3 COS-7 Cell Membranes Containing Human NK-1v Receptors The transfected COS-7 cells of Example 2 were harvested by treatment with Versene.","(Gibco BRL).","Versene was used, as it allows detachment of the cells from the flask without substantial perturbation to cell membrane proteins, then, the cells were washed once by re-suspending in assay buffer (50 mM Tris HCl pH 7.4, 3 mM MnCl2, 0.02% BSA, 40 μg/ml bacitracin, 2 μg/ml chymostatin, 2 μM phosphoramidon, 4 μg/ml leupeptin) and centrifuged at 1000 g for 5 min.","Cells were re-suspended in 5 ml of the assay buffer and a cell count performed prior to lysing cells, using a Brinkman polytron at setting 6 for 15 seconds.","The homogenate was centrifuged at 20000 g for 10 minutes.","Membrane pellets were re-suspended in assay buffer and stored frozen as 0.5-1 ml aliquots until required for use.","Example 4 Ligand Binding to Receptors in Cell Membranes COS-7 cells were transiently transfected with pCMV3.1 (Invitrogen) containing either parental wild type human NK-1 receptor cDNA or the variant human NK-1 receptor cDNA of Example 1, and cell membranes were prepared 2-3 days post-transfection using the procedure of Example 3.Radioligand binding studies were performed using [125I]BH substance P to label NK-1 receptors.","Non-specific binding was defined by the NK-1 receptor-selective agonist [Sar9, Met(O2)11]substance P (Bachem) at a final concentration of 1 μM.","On the day when they were required, the membrane suspensions were thawed and diluted as appropriate with assay buffer and incubated with varying concentrations of [125I]Bolton-Hunter Substance P (0.05-3 nM, from Amersham Life Sciences) for 50 minutes at 21° C. Saturation analyses were performed to determine the affinity constants and maximum binding capacity for each receptor, by incubating membranes with increasing concentrations of the radioligand, in the presence and absence of [Sar9, Met(O2)11]substance P. Reactions were terminated by rapid filtration under vacuum, onto GFC filters pre-soaked with 0.2% polyethylenimine.","[125I]BH substance P bound with high affinity to both wild type and mutant receptors (Kd values of 2.218±1.107 nM (n=3) and 1.446 nM (n=2) respectively).","Unpredictably, there appeared to be no significant difference between the dissociation constant and the maximum binding capacity of the wild type and variant NK-1 receptors, indicating that the mutation does not affect agonist binding at the NK-1 receptor.","The maximum binding capacity can be directly compared, as shown in Table 1: TABLE 1 Wild Type Variant HNK-1 (n = 3) hNK-1 (n = 2) Kd (nM) 2.218 + − 1.1107 1.446 Bmax 2.201 3.000 Example 5 Analysis of Intra-Cell Receptor Coupling in COS-7 Cells by Changes in Intra-Cellular Free Calcium Concentration Coverslips containing cells were prepared and maintained as described.","Cultured cells were washed twice in a Krebs-Hepes extra-cellular medium buffer (EM, composition in mM: NaCl 118, KCl 4.7, MgSO4 1.2, CaCl2 1.2, KH2PO4 1.2, Hepes 10, glucose 11 and BSA 0.1%, (pH 7.2 at 20° C., (Rossant, et al Endocrinology, 140, 1525-1536), and then loaded with Fura-2 (Rossant, et al Endocrinology, 140, 1525-1536) by incubation for 3 h at 20° C. with EM containing Fura-2-AM (2 μM, Molecular Probes).","This procedure enables the cells to load with Fura-2-AM, which becomes hydrolysed to the free acid form once inside the intact cells.","After loading, coverslips were mounted into imaging chambers and perfused with EM to remove extra-cellular Fura-2-AM and to allow hydrolysis of intracellular Fura-2-AM to occur.","Measurements of changes in the free [Ca2+]i in individual cells were made from the fluorescence ratio (excitations 340 nm/380 nm, emission >510 nm) using a Spectral Wizard monochromator, cooled integrating CCD camera and a dedicated suite of software (Merlin, Life Sciences Resources, Cambridge, UK).","Data are expressed as ratio units 340/380.Genes encoding wild type sense, wild type anti-sense and variant NK-1 receptors described above were transiently transfected into COS-7 (monkey kidney) cell lines.","Two to three days post-transfection, cells were loaded with FURA-2-AM dye (whose fluorescence is [Ca2+]i dependent) and challenged with substance P. The [Ca2+]i was followed by monitoring the change in light emitted (>510 nm) when excitation light of either 340 nm or 380 nm was sequentially used to illuminate the loaded cells and expressed as a ratio (340 nm:380 nm).","Cells challenged with UTP (which increases [Ca2+]i in non-transfected cells) caused a [Ca2+]i response indicating the presence of a functional G-protein Ca2+ linked pathway.","Results plotted in FIG.","3 indicate that cells expressing wild type NK-1 receptor responded to substance P over a 0.01-1000 nM range.","Cells expressing wild type anti-sense and variant NK-1 receptor failed to invoke a Ca2+ response with substance P challenge of 1 nM.","The effect of treatment with varying concentrations of substance-P or with UTP (3μM) for a 1 min time period on the average change in 340:380 ratio levels of COS 7 cultures expressing either wild type or modified NK-1 receptors (FIG.","3, n>50 cells for each treatment group (or on the percentage of COS cells demonstrating a measurable response (Xb) is shown in FIG.","3)."]],"string":"[\n [\n \"Modified tachykinin receptors\",\n \"The invention provides a modified tachykinin receptor in which the three amino acids of the DRY sequence that occurs adjacent to the junction of the TM3 domain with intracellular loop 2 are replaced with amino acids whose side chains are neither lipophilic nor contain charged groups.\",\n \"The receptor exhibits similar ligand binding characteristics to the wild type receptor but is incapable or substantially incapable of initiating an endogenous signal.\",\n \"Thus the ligand exhibits no or substantially no intra-cellular coupling of the receptor to the G protein, whereby there is substantially no transduction of ligand binding signals to the cell, The invention also includes fragments of the receptor containing the modified DRY sequence, and polynucleotides that encode the modified receptor as aforesaid.\",\n \"Therapeutic and diagnostic uses for the receptor are disclosed.\"\n ],\n [\n \"1.A mutant tachykinin receptor in which the three amino acids of the DRY sequence that occurs adjacent to the junction of the TM3 domain with 5 intracellular loop 2 are replaced with amino acids whose side chains are neither lipophilic nor contain charged groups, said receptor exhibiting similar ligand binding characteristics to the wild type receptor but exhibiting substantially no intra-cellular coupling of the receptor to the G-protein, whereby there is substantially no transduction of ligand binding 10 signals to the cell; or a fragment of said receptor containing said modified DRY sequence; or an isolated protein or polypeptide containing an amino acid sequence at least 80% identical to the above sequence; or a variant thereof with sequential amino acid deletions from either the C terminus or the N-terminus; or an allelic variant, heterospecific homologue or biologically active proteolytic or other fragment thereof containing said modified DRY sequence.\",\n \"2.A non-human mammalian receptor according to claim 1.3.A rat or mouse receptor according to claim 1.4.A human receptor according to claim 1.5.A mutant NIL-1 receptor according to claim 1.6.A mutant NK-2 receptor according to claim 1.7.A mutant NK-3 receptor according to claim 1.8.A receptor according to claim 1, wherein the replacement amino acids are selected from G and A.\",\n \"9.The receptor of claim 8, wherein DRY is replaced by GGA.\",\n \"10.A tachykinin receptor of SEQ ID No5, or a receptor having at least 80% amino acid identity with the receptor of SEQ ID No5, and that is capable of binding to substance P but is substantially incapable of initiating its endogenous signal, or a fragment of said receptor.\",\n \"11.An isolated cell membrane incorporating a tachykinin receptor as defined in claim 1.12.Any of the following: (a) an isolated nucleic acid molecule comprising a polynucleotide that encodes a tachykinin receptor as claimed in claim 1; (b) an isolated nucleic acid molecule comprising a sequence that is hybridizable to the above sequence; (c) a gene which is the result of extending the above sequence or any sequence that is hybridizable to the above sequence; (d) a sequence or gene that is functionally equivalent to the above sequence or to a gene that is an extension of the above sequence, i.e.\",\n \"that is not identical to the sequence or gene referred to but functions biologically as equivalent to the sequence or gene referred to, including any allelic variants and heterospecific mammalian homologues, including artificial or recombinant sequences created from cDNA or genomic DNA; (e) a recombinant vector comprising the above gene sequence; and (f) a host cell transformed with the vector.\",\n \"13.Any of the following: (a) an isolated nucleic acid molecule having the nucleotide sequence of SEQ ID No6; (b) an isolated nucleic acid molecule comprising a sequence that is hybridizable to the above sequence; (c) a gene which is the result of extending the above sequence or any sequence that is hybridizable to the above sequence; (d) a sequence or gene that is functionally equivalent to the above sequence or to a gene that is an extension of the above sequence, i.e.\",\n \"that is not identical to the sequence or gene referred to but functions biologically as equivalent to the sequence or gene referred to, including any allelic variants and heterospecific mammalian homologues, including artificial or recombinant sequences created from cDNA or genomic DNA; (e) a recombinant vector comprising the above gene sequence; and (f) a host cell transformed with the vector.\",\n \"14.A method for producing a receptor protein having an amino acid sequenceas defined in claim 1, which method comprises the steps of: (a) inserting said nucleic acid sequence into an appropriate vector; (b) culturing, in an a culture medium, a host cell previously transformed or transfected with the recombinant vector of step (a); (c) harvesting cells containing the receptor protein obtained from step (b); and (d) separating or purifying, from said culture medium or from said host cell, the thus-produced receptor protein.\",\n \"15.A pharmaceutical composition comprising an effective amount of a modified tachykinin ligand as defined in claim 1 or a nucleic acid sequence encoding said ligand and a pharmaceutically and pharmacologically acceptable carrier.\",\n \"16.Use of a modified tachykinin receptor as defined in claim 1 in the preparation of a medicament for the treatment or prophylaxis of a condition associated with substance P or other tachykinin (neurokinin) receptor-binding ligand; 17.A method for the treatment or prevention of a condition associated with over-expression or inappropriate expression of an endogenous tachykinin ligand, which method comprises administration to a patient in need thereof of a non-toxic, effective amount of such a modified tachykinin ligand as defined in claim 1.18.A method for screening for therapeutically active compounds, said method comprising the following steps: (a) providing a cell line expressing a modified tachykinin receptor as defined in claim 1; (b) adding test sample to a solution containing labeled tachykinin ligand and the cell line from step (a); (c) incubating the cell line, test sample and labeled ligand mixture from step (b) to allow binding of said ligand and test sample to the modified tachykinin receptor; (d) optionally, separating the non-bound labeled ligand from the labeled ligand bound to the modified tachykinin receptor; and, if desired, (e) measuring the amount of labeled ligand that is bound to the modified tachykinin receptor.\",\n \"19.Use of a modified tachykinin receptor as defined in claim 1 as a substitute in an assay to identify and/or evaluate entities that bind to the wild type tachykinin receptor.\",\n \"20.Use of a modified tachykinin receptor as defined in claim 1 as a substitute in an assay in order to determine the concentration of ligand in body fluids in patients with arthritis, pain, migraine, anxiety, schizophrenia, asthma, rheumatoid arthritis, and in gastrointestinal disorders and diseases of the GI tract.\",\n \"21.An assay procedure comprising the following steps: (a) providing a cell line is provided that expresses a modified tachykinin receptor as defined in claim 1; (b) labeling the cell line; (c) adding the test sample and labeled cells to a matrix binding SP or other ligand; (d) incubating the labeled cells, test sample and matrix-bound SP or other ligand to allow binding of SP or other ligand and test sample to the expressed modified tachykinin receptor; (e) separating the labelled non-bound cells from the SP or other ligand bound cells; and, if desired, (f) measuring the amount of labelled cells containing the modified tachykinin receptor that has bound to SF or other ligand.\",\n \"22.Use of a modified tachykinin receptor as defined in claim 1 in protein therapy to reduce the effects of an excess of or inappropriately produced endogenous ligand.\",\n \"23.A method for treatment of a patient in need thereof, which comprises administering to said patient a composition in the form of an aerosol that comprises a modified tachykinin receptor as defined in claim 1.24.A method for gene therapy treatment of a patient in need thereof, which comprises administering to said patient a nucleic acid sequence, virus or plasmid encoding a modified tachykinin receptor as defined in claim 1.\"\n ],\n [\n \"<SOH> BACKGROUND TO THE INVENTION <EOH>Tachykinins are important in the mediation of many physiological and pathological processes including inflammation, pain, migraine, headache and allergy induced asthma.\",\n \"They belong to an evolutionary conserved family of peptide neurotransmitters that have an established role in neurotransmission.\",\n \"They share the C-terminal sequence Phe-Xaa-Gly-Leu-Met-NH 2 (SEQ ID NO 12) in which Xaa represents a hydrophobic residue.\",\n \"That sequence is characteristic of tachykinins and believed to be mainly responsible for their biological activity at neurokinin receptors.\",\n \"Mammalian tachykinins include substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) which exert their effects by binding to specific receptors.\",\n \"SP is the most prominent member of the tachykinergic system and is released from sensory nerve endings throughout the body.\",\n \"Its amino acid sequence is: in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"lead\\\"?\",\n \"H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH 2 (SEQ ID N o 1) in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"tail\\\"?\",\n \"The amino acid sequence for NKA is: in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"lead\\\"?\",\n \"H-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH 2 (SEQ ID N o 2) in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"tail\\\"?\",\n \"The amino acid sequence for NKB is: in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"lead\\\"?\",\n \"H-Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH 2 (SEQ ID N o 3) in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"tail\\\"?\",\n \"SP has been implicated in the pathology of numerous diseases.\",\n \"For example, it has been shown to be involved in the transmission of pain, in conditions associated with vasodilation, smooth muscle contraction, bronchoconstriction, activation of the immune system and neurogenic inflammation.\",\n \"It has been implicated in migraine, as well as in disorders of the central nervous system, such as anxiety and schizophrenia; in respiratory and inflammatory diseases, such as asthma and rheumatoid arthritis; and in gastrointestinal (GI) disorders and diseases of the GI tract, such as ulcerative colitis and Crohn's disease.\",\n \"Tachykinin receptors include NK-1, NK-2 and NK-3 and are membrane proteins of the super-family of guanine nucleotide-binding protein (G-protein)-coupled receptors (GPCR).\",\n \"They have extra-cellular binding sites that have preferential affinities for the ligands SP, NKA and NKB respectively.\",\n \"Like other G-protein coupled receptors, they possess an extra-cellular N-terminus, an intra-cellular C-terminus and seven trans-membrane (TM) α-helices of 20-30 amino acids connected by first, second and third extra-cellular loops and by first, second and third intra-cellular or cytoplasmic loops.\",\n \"The greatest homology is found in the membrane-spanning α-helices of trans-membrane domains TM1-TM7 while the N- and C-termini show greater diversity between the three types of neurokinin receptor.\",\n \"Cloned NK-1 receptors have been reported, including those for the Rana catesbeina , (Simmons et al., Neuroscience, 79: 1219-1229 (1997)), Mus musculus , (Sundelin et al, Eur.\",\n \"J. Biochem.\",\n \"203: 625-631 (1992)), Rattus norvegicus , (Hershey et al, J. Biol Chem., 266: 4366-4374 (1991) and Yokota et al, J. Biol.\",\n \"Chem., 264: 17649-17652 (1989)), Cavia porcellus , (Gorbulev et al., Biochem.\",\n \"Biophys.\",\n \"Acta 1131: 99-102 (1992)), and Homo sapiens (human), (Takeda et al, Biochem.\",\n \"Biophys.\",\n \"Res.\",\n \"Comm.\",\n \"179: 1232-1240 (1991) and Fong et al U.S. Pat.\",\n \"No.\",\n \"5,525,712 and U.S. Pat.\",\n \"No.\",\n \"5,584,886).\",\n \"Cloned rat and bovine neurokinin-2 receptors have been reported (Y. Sasi et al., Biochem.\",\n \"Biophys.\",\n \"Res.\",\n \"Comm., 165: 695 (1989), and Y. Masu, et al., Nature 329: 836 (1987)).\",\n \"Cloned rat neurokinin-3 receptor has also been reported (R. Shigemoto, et al., J. Biol.\",\n \"Chem., 265:623 (1990)).\",\n \"All three receptors share the signal transduction mechanisms of a G-protein coupled receptor.\",\n \"The receptor is in an OFF state when no ligand is present, but, is triggered into an ON state when an agonist ligand binds to the receptor.\",\n \"The G-protein when in its ON state triggers a downstream signaling pathway or signal cascade.\",\n \"The G-protein comprises α-, β- and γ-units that are bound together in the OFF state, the α-subunit then having GDP bound to it.\",\n \"In the ON state GTP replaces the GDP bound to the α-subunit.\",\n \"The α-subunit becomes dissociated from the β- and γ-subunits and becomes available for activating the signal cascade.\",\n \"After a short period, the GTP becomes hydrolyzed to GDP and the G-protein returns to its non activated OFF state.\",\n \"Hydrolysis provides a negative feedback mechanism that ensures that the G protein is only in its activated ON state for a short period.\",\n \"Various studies have been undertaken, involving different G-protein receptors, to determine how the various regions of the protein structure affect intra-cellular coupling of the receptor to the G-protein and consequential transduction of ligand binding signals to the cell.\",\n \"G-protein receptors have a well-conserved sequence in the second intracellular loop where the loop joins the third trans-membrane domain that is known as the DRY sequence.\",\n \"It has the residues in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"lead\\\"?\",\n \"5′-DRYXXV(P)XXPL-3′ (SEQ ID N o 4) in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"tail\\\"?\",\n \"in which L represents Leu, Ile, Val, Met or Phe and X represents any amino acid.\",\n \"It has been suggested that the DRY sequence contributes to the efficient binding and activation of G-proteins.\",\n \"Fraser et al., Proc.\",\n \"Natl Acad Sci.\",\n \"USA, 85: 5478-5482 (1988) report a change of Asp to Asn at position 130 of the human β-adrenergic receptor (i.e.\",\n \"the D of the DRY sequence) resulting in human β-adrenergic receptor that exhibits high affinity binding of agonist whilst being unable to interact effectively with G-protein.\",\n \"However, a second paper from the same laboratory reports that the previous very high agonist binding efficiency in human β-adrenergic receptor mutated at position 130, upon which the above mentioned conclusion had been based, had not been reproduced (Wang et al., Mol Pharmacol 40(2): 168-79 (1991)).\",\n \"In a review article Savarese and Fraser said that this locus may be important for coupling to some, but not all, G-proteins ( Biochem J., 283: 1-19 (1992)).\",\n \"Moro et al made mutants of the Hm1 muscarinic cholinergic receptor with changes towards the 5′-end of the DRY sequence and found that replacing L at position 131 with A gave the strongest reduction in coupling efficiency ( J. Biol.\",\n \"Chem.\",\n \"268: 22273-22278 (1993)).\",\n \"Subsequently, Shibata et al made a mutant of the angiotensin II receptor type I in which DRY at positions 125-127 is replaced by GGA and M at position 134 is replaced by A, resulting in uncoupling of the mutant A receptor from G-proteins ( Biochem.\",\n \"Biophys.\",\n \"Res.\",\n \"Com.\",\n \"218: 383-389 (1996)).\",\n \"The authors concluded that DRY sequence as a whole including the final lipophilic amino acid L serves as a general site for G-protein coupling but they did not go on to consider what effects might be obtained by change confined to the DRY portion of the sequence.\",\n \"Comparing the binding affinities of the two known isoforms of the human NK-1 receptor shows the importance of the cytoplasmic tail.\",\n \"The long form (407 amino acids) and short form (311 amino acids) differ in the length of the C-terminus.\",\n \"The long form has similar substance P binding characteristics to the rat NK-1 receptor, while the short form of the receptor has an apparent substance P binding affinity 10-fold less than the rat NK-1 receptor.\",\n \"Furthermore, studies on these and other receptors have shown that the effect of mutations is unpredictable and specific to certain families of receptors.\",\n \"Hence, a change in one G-protein coupling family will not necessarily have the same effect on another.\",\n \"Other attempts to distinguish protein binding and signaling effects bear this out.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides a mutant tachykinin receptor in which the three amino acids of the DRY sequence that occurs adjacent to the junction of the TM3 domain with intracellular loop 2 are replaced with amino acids whose side chains are neither lipophilic nor contain charged groups, said receptor exhibiting similar ligand binding characteristics to the wild type receptor but being incapable or substantially incapable of initiating an endogenous signal.\",\n \"Thus the ligand exhibits no or substantially no intra-cellular coupling of the receptor to the G-protein, whereby there is substantially no transduction of ligand binding signals to the cell.\",\n \"The way in which tachykinin receptors attach to cell membranes, the trans-membrane domains extra-cellular and cytoplasmic loops and the place where the DRY sequence referred to above occurs are apparent by inspection of FIG.\",\n \"5 of the accompanying drawings.\",\n \"The invention further provides any of the following: a fragment of said mutant tachykinin receptor containing said modified DRY sequence; an isolated protein or polypeptide containing an amino acid sequence at least 95% identical to the above sequence; a variant thereof with sequential amino acid deletions from either the C terminus or the N-terminus; and an allelic variant, heterospecific homologue or biologically active proteolytic or other fragment thereof containing said modified DRY sequence.\",\n \"The invention also comprises an isolated cell membrane in which a modified tachykinin receptor as aforesaid is incorporated as membrane protein.\",\n \"Such cell membrane material finds utility for research and in particular for screening for therapeutically useful compounds as described below.\",\n \"The invention yet further provides any of the following: (a) an isolated nucleic acid molecule comprising a polynucleotide that encodes a modified tachykinin receptor as aforesaid; (b) an isolated nucleic acid molecule comprising a sequence that is hybridizable to the above sequence; (c) a gene which is the result of extending the above sequence or any sequence that is hybridizable to the above sequence; (d) a sequence or gene that is functionally equivalent to the above sequence or to a gene that is an extension of the above sequence, i.e.\",\n \"that is not identical to the sequence or gene referred to but functions biologically as equivalent to the sequence or gene referred to, including any allelic variants and heterospecific mammalian homologues, including artificial or recombinant sequences created from cDNA or genomic DNA; (e) a recombinant vector comprising the above gene sequence; and (f) a host cell transformed with the vector.\",\n \"The invention also provides a method for producing one of the amino acid sequences described herein and especially the receptor protein defined by SEQ ID N o 5, which method comprises the steps of: (a) inserting said nucleic acid sequence into an appropriate vector; (b) culturing in a culture medium a host cell previously transformed or transfected with the recombinant vector of step (a); (c) harvesting cells containing the receptor protein obtained from step (b); and (d) separating or purifying from said culture medium or from said host cells the thus-produced receptor protein.\",\n \"In step (d) of the above method, the receptor protein may be obtained either from the culture medium and/or by lysing the host cell, for example by sonication or osmotic shock.\",\n \"The invention yet further provides a method for screening for therapeutically active compounds, said method comprising the following steps: (a) providing a cell line expressing a modified tachykinin receptor as aforesaid; (b) adding test sample to a solution containing labeled SP or other tachykinin ligand and the cell line from step (a); (c) incubating the cell line, test sample and labeled SP or other ligand mixture from step (b) to allow binding of SP or other ligand and test sample to the modified tachykinin receptor; (d) optionally separating the non-bound labeled SP or other ligand from the labeled SP or other ligand bound to the modified tachykinin receptor and, if desired, (e) measuring the amount of labeled SP or other ligand that is bound to the modified tachykinin receptor.\",\n \"The invention further provides: (a) the use of a modified tachykinin receptor according to the invention in the preparation of a medicament for the treatment or prophylaxis of a condition associated with substance P or other tachykinin (neurokinin) receptor-binding ligand; (b) the use of such a modified tachykinin receptor in therapy; (c) a method for the treatment or prevention of a condition associated with over-expression of an endogenous tachykinin ligand, which method comprises administration to a patient in need thereof of a non-toxic, effective amount of such a modified tackykinin ligand as described above; (d) a composition comprising a modified tachykinin ligand as described above in association with a pharmaceutically and pharmacologically acceptable carrier therefor; and (e) a use, method or composition according to any one of (a) to (d) above, in which the modified tachykinin ligand becomes generated in vivo from a nucleic acid sequence encoding such a ligand.\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention relates to a nucleic acid sequence encoding a tachykinin receptor, the tachykinin receptor encoded by said sequence, methods for its preparation and its use in therapy and screening.\",\n \"In particular, the invention relates to a modification to tachykinin receptor proteins that gives rise to unexpected and useful properties.\",\n \"BACKGROUND TO THE INVENTION Tachykinins are important in the mediation of many physiological and pathological processes including inflammation, pain, migraine, headache and allergy induced asthma.\",\n \"They belong to an evolutionary conserved family of peptide neurotransmitters that have an established role in neurotransmission.\",\n \"They share the C-terminal sequence Phe-Xaa-Gly-Leu-Met-NH2 (SEQ ID NO 12) in which Xaa represents a hydrophobic residue.\",\n \"That sequence is characteristic of tachykinins and believed to be mainly responsible for their biological activity at neurokinin receptors.\",\n \"Mammalian tachykinins include substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) which exert their effects by binding to specific receptors.\",\n \"SP is the most prominent member of the tachykinergic system and is released from sensory nerve endings throughout the body.\",\n \"Its amino acid sequence is: H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (SEQ ID No1) The amino acid sequence for NKA is: H-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 (SEQ ID No2) The amino acid sequence for NKB is: H-Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2 (SEQ ID No3) SP has been implicated in the pathology of numerous diseases.\",\n \"For example, it has been shown to be involved in the transmission of pain, in conditions associated with vasodilation, smooth muscle contraction, bronchoconstriction, activation of the immune system and neurogenic inflammation.\",\n \"It has been implicated in migraine, as well as in disorders of the central nervous system, such as anxiety and schizophrenia; in respiratory and inflammatory diseases, such as asthma and rheumatoid arthritis; and in gastrointestinal (GI) disorders and diseases of the GI tract, such as ulcerative colitis and Crohn's disease.\",\n \"Tachykinin receptors include NK-1, NK-2 and NK-3 and are membrane proteins of the super-family of guanine nucleotide-binding protein (G-protein)-coupled receptors (GPCR).\",\n \"They have extra-cellular binding sites that have preferential affinities for the ligands SP, NKA and NKB respectively.\",\n \"Like other G-protein coupled receptors, they possess an extra-cellular N-terminus, an intra-cellular C-terminus and seven trans-membrane (TM) α-helices of 20-30 amino acids connected by first, second and third extra-cellular loops and by first, second and third intra-cellular or cytoplasmic loops.\",\n \"The greatest homology is found in the membrane-spanning α-helices of trans-membrane domains TM1-TM7 while the N- and C-termini show greater diversity between the three types of neurokinin receptor.\",\n \"Cloned NK-1 receptors have been reported, including those for the Rana catesbeina, (Simmons et al., Neuroscience, 79: 1219-1229 (1997)), Mus musculus, (Sundelin et al, Eur.\",\n \"J. Biochem.\",\n \"203: 625-631 (1992)), Rattus norvegicus, (Hershey et al, J. Biol Chem., 266: 4366-4374 (1991) and Yokota et al, J. Biol.\",\n \"Chem., 264: 17649-17652 (1989)), Cavia porcellus, (Gorbulev et al., Biochem.\",\n \"Biophys.\",\n \"Acta 1131: 99-102 (1992)), and Homo sapiens (human), (Takeda et al, Biochem.\",\n \"Biophys.\",\n \"Res.\",\n \"Comm.\",\n \"179: 1232-1240 (1991) and Fong et al U.S. Pat.\",\n \"No.\",\n \"5,525,712 and U.S. Pat.\",\n \"No.\",\n \"5,584,886).\",\n \"Cloned rat and bovine neurokinin-2 receptors have been reported (Y. Sasi et al., Biochem.\",\n \"Biophys.\",\n \"Res.\",\n \"Comm., 165: 695 (1989), and Y. Masu, et al., Nature 329: 836 (1987)).\",\n \"Cloned rat neurokinin-3 receptor has also been reported (R. Shigemoto, et al., J. Biol.\",\n \"Chem., 265:623 (1990)).\",\n \"All three receptors share the signal transduction mechanisms of a G-protein coupled receptor.\",\n \"The receptor is in an OFF state when no ligand is present, but, is triggered into an ON state when an agonist ligand binds to the receptor.\",\n \"The G-protein when in its ON state triggers a downstream signaling pathway or signal cascade.\",\n \"The G-protein comprises α-, β- and γ-units that are bound together in the OFF state, the α-subunit then having GDP bound to it.\",\n \"In the ON state GTP replaces the GDP bound to the α-subunit.\",\n \"The α-subunit becomes dissociated from the β- and γ-subunits and becomes available for activating the signal cascade.\",\n \"After a short period, the GTP becomes hydrolyzed to GDP and the G-protein returns to its non activated OFF state.\",\n \"Hydrolysis provides a negative feedback mechanism that ensures that the G protein is only in its activated ON state for a short period.\",\n \"Various studies have been undertaken, involving different G-protein receptors, to determine how the various regions of the protein structure affect intra-cellular coupling of the receptor to the G-protein and consequential transduction of ligand binding signals to the cell.\",\n \"G-protein receptors have a well-conserved sequence in the second intracellular loop where the loop joins the third trans-membrane domain that is known as the DRY sequence.\",\n \"It has the residues 5′-DRYXXV(P)XXPL-3′ (SEQ ID No4) in which L represents Leu, Ile, Val, Met or Phe and X represents any amino acid.\",\n \"It has been suggested that the DRY sequence contributes to the efficient binding and activation of G-proteins.\",\n \"Fraser et al., Proc.\",\n \"Natl Acad Sci.\",\n \"USA, 85: 5478-5482 (1988) report a change of Asp to Asn at position 130 of the human β-adrenergic receptor (i.e.\",\n \"the D of the DRY sequence) resulting in human β-adrenergic receptor that exhibits high affinity binding of agonist whilst being unable to interact effectively with G-protein.\",\n \"However, a second paper from the same laboratory reports that the previous very high agonist binding efficiency in human β-adrenergic receptor mutated at position 130, upon which the above mentioned conclusion had been based, had not been reproduced (Wang et al., Mol Pharmacol 40(2): 168-79 (1991)).\",\n \"In a review article Savarese and Fraser said that this locus may be important for coupling to some, but not all, G-proteins (Biochem J., 283: 1-19 (1992)).\",\n \"Moro et al made mutants of the Hm1 muscarinic cholinergic receptor with changes towards the 5′-end of the DRY sequence and found that replacing L at position 131 with A gave the strongest reduction in coupling efficiency (J. Biol.\",\n \"Chem.\",\n \"268: 22273-22278 (1993)).\",\n \"Subsequently, Shibata et al made a mutant of the angiotensin II receptor type I in which DRY at positions 125-127 is replaced by GGA and M at position 134 is replaced by A, resulting in uncoupling of the mutant A receptor from G-proteins (Biochem.\",\n \"Biophys.\",\n \"Res.\",\n \"Com.\",\n \"218: 383-389 (1996)).\",\n \"The authors concluded that DRY sequence as a whole including the final lipophilic amino acid L serves as a general site for G-protein coupling but they did not go on to consider what effects might be obtained by change confined to the DRY portion of the sequence.\",\n \"Comparing the binding affinities of the two known isoforms of the human NK-1 receptor shows the importance of the cytoplasmic tail.\",\n \"The long form (407 amino acids) and short form (311 amino acids) differ in the length of the C-terminus.\",\n \"The long form has similar substance P binding characteristics to the rat NK-1 receptor, while the short form of the receptor has an apparent substance P binding affinity 10-fold less than the rat NK-1 receptor.\",\n \"Furthermore, studies on these and other receptors have shown that the effect of mutations is unpredictable and specific to certain families of receptors.\",\n \"Hence, a change in one G-protein coupling family will not necessarily have the same effect on another.\",\n \"Other attempts to distinguish protein binding and signaling effects bear this out.\",\n \"SUMMARY OF THE INVENTION The invention provides a mutant tachykinin receptor in which the three amino acids of the DRY sequence that occurs adjacent to the junction of the TM3 domain with intracellular loop 2 are replaced with amino acids whose side chains are neither lipophilic nor contain charged groups, said receptor exhibiting similar ligand binding characteristics to the wild type receptor but being incapable or substantially incapable of initiating an endogenous signal.\",\n \"Thus the ligand exhibits no or substantially no intra-cellular coupling of the receptor to the G-protein, whereby there is substantially no transduction of ligand binding signals to the cell.\",\n \"The way in which tachykinin receptors attach to cell membranes, the trans-membrane domains extra-cellular and cytoplasmic loops and the place where the DRY sequence referred to above occurs are apparent by inspection of FIG.\",\n \"5 of the accompanying drawings.\",\n \"The invention further provides any of the following: a fragment of said mutant tachykinin receptor containing said modified DRY sequence; an isolated protein or polypeptide containing an amino acid sequence at least 95% identical to the above sequence; a variant thereof with sequential amino acid deletions from either the C terminus or the N-terminus; and an allelic variant, heterospecific homologue or biologically active proteolytic or other fragment thereof containing said modified DRY sequence.\",\n \"The invention also comprises an isolated cell membrane in which a modified tachykinin receptor as aforesaid is incorporated as membrane protein.\",\n \"Such cell membrane material finds utility for research and in particular for screening for therapeutically useful compounds as described below.\",\n \"The invention yet further provides any of the following: (a) an isolated nucleic acid molecule comprising a polynucleotide that encodes a modified tachykinin receptor as aforesaid; (b) an isolated nucleic acid molecule comprising a sequence that is hybridizable to the above sequence; (c) a gene which is the result of extending the above sequence or any sequence that is hybridizable to the above sequence; (d) a sequence or gene that is functionally equivalent to the above sequence or to a gene that is an extension of the above sequence, i.e.\",\n \"that is not identical to the sequence or gene referred to but functions biologically as equivalent to the sequence or gene referred to, including any allelic variants and heterospecific mammalian homologues, including artificial or recombinant sequences created from cDNA or genomic DNA; (e) a recombinant vector comprising the above gene sequence; and (f) a host cell transformed with the vector.\",\n \"The invention also provides a method for producing one of the amino acid sequences described herein and especially the receptor protein defined by SEQ ID No5, which method comprises the steps of: (a) inserting said nucleic acid sequence into an appropriate vector; (b) culturing in a culture medium a host cell previously transformed or transfected with the recombinant vector of step (a); (c) harvesting cells containing the receptor protein obtained from step (b); and (d) separating or purifying from said culture medium or from said host cells the thus-produced receptor protein.\",\n \"In step (d) of the above method, the receptor protein may be obtained either from the culture medium and/or by lysing the host cell, for example by sonication or osmotic shock.\",\n \"The invention yet further provides a method for screening for therapeutically active compounds, said method comprising the following steps: (a) providing a cell line expressing a modified tachykinin receptor as aforesaid; (b) adding test sample to a solution containing labeled SP or other tachykinin ligand and the cell line from step (a); (c) incubating the cell line, test sample and labeled SP or other ligand mixture from step (b) to allow binding of SP or other ligand and test sample to the modified tachykinin receptor; (d) optionally separating the non-bound labeled SP or other ligand from the labeled SP or other ligand bound to the modified tachykinin receptor and, if desired, (e) measuring the amount of labeled SP or other ligand that is bound to the modified tachykinin receptor.\",\n \"The invention further provides: (a) the use of a modified tachykinin receptor according to the invention in the preparation of a medicament for the treatment or prophylaxis of a condition associated with substance P or other tachykinin (neurokinin) receptor-binding ligand; (b) the use of such a modified tachykinin receptor in therapy; (c) a method for the treatment or prevention of a condition associated with over-expression of an endogenous tachykinin ligand, which method comprises administration to a patient in need thereof of a non-toxic, effective amount of such a modified tackykinin ligand as described above; (d) a composition comprising a modified tachykinin ligand as described above in association with a pharmaceutically and pharmacologically acceptable carrier therefor; and (e) a use, method or composition according to any one of (a) to (d) above, in which the modified tachykinin ligand becomes generated in vivo from a nucleic acid sequence encoding such a ligand.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be further described with reference to the accompanying drawings, in which: FIG.\",\n \"1: describes a protein sequence, being a translation of the sequence shown in FIG.\",\n \"1 and being the sequence of a modified human NK-1 receptor according to the invention (hNK-1Rv1).\",\n \"FIG.\",\n \"2: is a consensus cDNA sequence encoding a modified human NK-1 receptor according to the invention.\",\n \"FIG.\",\n \"3: is a bar chart showing the results of Calcium imaging experiments on NK-1 wild type receptor and the modified receptor of FIG.\",\n \"1 (hNK-1Rv1), and wild type receptor antisense transfected COS cells.\",\n \"FIG.\",\n \"4: shows the sequence alignments between NK-1 and other NK receptors in various species in the region of the DRY motif in TM3, demonstrating high conservancy (In the Figure, NK-1 sequences 1-4 provide SEQ ID NO 13, NK-1 sequence 5 provides SEQ ID NO 14, NK-2 sequences 1-2 and 4-7 provide SEQ ID NO 15, NK-2 sequence 3 provides SEQ ID NO 16 and NK-3 sequences 1-3 provide SEQ ID NO 17).\",\n \"FIG.\",\n \"5: is a diagram showing the nNK-1Rv1 receptor and portions of the cell membrane into which it is incorporated (SEQ ID NO 18; in addition to the DRY motif, L223 is changed to I; this change is believed to be inconsequential).\",\n \"DESCRIPTION OF PREFERRED EMBODIMENTS Definitions Definitions for a number of terms used in this specification are given below: Alleles or allelic sequences mean alternative forms of the receptor genes referred to above resulting from one or more variations in the nucleic acid sequence, resulting in altered mRNAs and proteins or polypeptides Functional equivalence when used in relation to gene sequences or amino acid sequences covers sequences that are not identical to the sequence referred to but function biologically or chemically as equivalents of the disclosed sequence.\",\n \"Incapable of initiating an endogenous signal in the present context means that, within experimental error (±5% of control), the modified NKR does not result in conversion of GTP to GDP in the cell or a recombinant construct; and/or does not evoke a calcium flux therein; and/or does not evoke other components of the signal transduction pathway.\",\n \"Isolated, when used in relation to a polynucleotide sequence, means such a sequence that has been removed from its natural environment, i.e.\",\n \"from the organism in which it occurs in nature and/or from genes that are immediately contiguous (one at the 5′ end and the other at the 3′ end) in the naturally occurring genome of the organism from which it is derived.\",\n \"Isolated when used in relation to a cell membrane refers to the membrane as a discrete entity substantially separate from cytoplasmic material.\",\n \"Operably linked refers to a linkage of polynucleotide elements in a functional relationship.\",\n \"For instance, a promoter or an enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.\",\n \"More specifically, two DNA molecules (such as a polynucleotide containing a promoter region and a polynucleotide encoding a desired polypeptide or polynucleotide) are said to be “operably linked” if the nature of the linkage between the two polynucleotides neither results in the introduction of a frame-shift mutation nor interferes with the ability of the polynucleotide containing the promoter to direct the transcription of the coding polynucleotide.\",\n \"Stringent hybridisation conditions is a recognized term in the art and for a given nucleic acid sequence refers to those conditions which permit hybridisation of that sequence to its complementary sequence and not to a substantially different sequence.\",\n \"It generally implies at least about 97% identity between the sequences.\",\n \"Protein and Nucleic Acid Sequences In the above mentioned mutant receptors, amino acids for modifying the DRY sequence that are non-polar and have the correct hydrophilic/lipophilic balance include glycine and alanine which are preferred.\",\n \"However, the inventors also envisage the use of other non polar amino acids.\",\n \"In a preferred aspect, the invention provides a nucleic acid sequence as shown in FIG.\",\n \"2 [SEQ ID No6], which encodes the consensus amino acid sequence for the modified hNK-1v or a sequence that hybridizes thereto under stringent hybridization conditions.\",\n \"The invention also provides a preferred hNK-1v receptor sequence shown in FIG.\",\n \"1 [SEQ ID No5].\",\n \"The invention further provides a nucleic acid sequence encoding a receptor protein having at least 80%, preferably 90%, more preferably 95%, and most preferably 98% amino acid identity with the hNK-1Rv1 encoded by the nucleic acid sequence of SEQ ID No6, and which encodes protein that can bind to SP, but cannot initiate its endogenous signal, or encodes a peptide fragment thereof according to sequence (d), or encodes a sequence complementary thereto according to sequence (e), as defined hereinabove.\",\n \"The invention also provides a protein that: (a) has at least 80%, preferably 90%, more preferably 95%, and most preferably 98% amino acid identity with the hNK-1Rv1 protein having the amino acid sequence of SEQ ID No5; and (b) can bind to SP or to a peptide fragment thereof, or to a sequence complementary thereto, but cannot initiate its endogenous signal to G protein.\",\n \"The hNK-1Rv1 variant of the NK-1 receptor shown in SEQ ID No5 has similar ligand binding characteristics to the wild type receptor, but is deficient in cell signaling capabilities, as demonstrated by the results of tests described in Examples 4 and 5.Some mammalian receptors may exhibit overall homology with the hNK-1 receptor of less than 80% (for example, rat NK-2 receptor has about 48% homology to rat NK-1 receptor), but are nevertheless included within the scope of this invention in view of their conservancy in the intracellular DRY region of TM3.Accordingly, the present invention provides a polypeptide having as low as 40% overall amino acid identity with the hNK-1Rv1 protein, but having at least 75%, such as at least 80%, preferably 90%, more preferably 95%, and most preferably 98% amino acid identity with the TM3 intracellular loop of the bNK-1Rv1 protein in the vicinity of the DRY motif, and having the properties previously specified.\",\n \"In view of the high level of conservancy demonstrated by other NK receptors in the DRY region of TM3 intracellular loop, and also the high level of conservancy observed in that region between human and other species, the present invention also provides a nucleotide sequence that encodes a modified neurokinin receptor, wherein the modification is or includes substitution of the DRY motif within the intracellular loop of TM3, namely, Asp129, Arg130 and Tyr131, by Gly, Gly and Ala, respectively, for example wherein the receptor is a modified NK-2 or modified NK-3 mammalian receptor.\",\n \"Vectors The invention further provides a vector (e.g.\",\n \"a plasmid or virus) comprising a nucleic acid encoding the modified tachykinin receptor, especially the hNK-1Rv1 receptor or any other modified hNK-1R of this invention.\",\n \"A recombinant vector of the invention comprises an expression vector comprising a nucleic acid sequence encoding the modified NK-R, especially the hNK-1Rv1, polypeptide.\",\n \"One suitable vector for the expression of a human variant NK-1 receptor of the invention is a baculovirus vector that can be propagated in insect cells and in insect cell-lines.\",\n \"Expression requires that appropriate signals are provided in the vector, said signals including various regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells.\",\n \"The regulatory sequences of the expression vectors are operably linked to the nucleic acid encoding the modified tachykinin receptor, especially the human NK-1 variant receptor.\",\n \"Generally, recombinant expression vectors include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence.\",\n \"The heterologous structural sequence is assembled in an appropriate frame with the translation, initiation and termination sequences, and preferably a leader sequence capable of directing sequences of the translated protein into the periplasmic space or the extra-cellular medium.\",\n \"Where the vector is adapted for transfecting and expressing desired sequences in eukaryotic host cells, preferred vectors comprise an origin of replication from the desired host, a suitable promoter and an enhancer, and also any necessary ribosome binding sites, polyadenylation site, transcriptional termination sequences, and optionally 5′-flanking non-transcribed sequences.\",\n \"Suitable promoter regions used in the expression vectors according to the invention are chosen taking into account the host cell in which the heterologous nucleic acids have to be expressed.\",\n \"A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression, or may be endogenous to the native polynucleotide containing the coding sequence to be expressed.\",\n \"Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.\",\n \"A recombinant vector of the invention may be used to amplify a polynucleotide derived from the nucleic acid sequence encoding the modified NKR, especially the hNK-1Rv1 polypeptide that has been inserted in a suitable host cell, this polynucleotide being amplified every time the recombinant vector replicates.\",\n \"DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, and polyadenylation sites may be used to provide the require non-transcribed genetic elements.\",\n \"The suitable promoter regions used in the expression vectors according to the invention are chosen taking into account the host cell in which the heterologous nucleic acids are to be expressed.\",\n \"A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively it can be endogenous to the native polynucleotide containing the coding sequence to be expressed.\",\n \"Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.\",\n \"Preferred bacterial promoters are the LacI, LacZ, T3 or T7 bacteriophage RNA polymerase promoters, the lambda PR, PL and trp promoters (EP 0 036 776), the polyhedrin promotor, or the p10 protein promoter from baculovirus (Kit Novagen; Smith et al., 1983); O'Reilly et al., 1992, Baculovirus expression vectors: A Laboratory Manual.\",\n \"W.H.\",\n \"Freeman and Co., New York).\",\n \"Preferred selectable marker genes contained in the expression recombinant vectors of the invention for selection of transformed host cells are preferably dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, TRP1 for S. cerevisiae or tetracyclin, rifampicin or ampicillin resistance in E. coli, or Levan saccharase for mycobacteria, this latter marker being a negative selection marker.\",\n \"Preferred bacterial vectors of the invention are listed hereafter as illustrative but not limitative examples: pQE70, pQE60, pQE-9 (Qiagen), pD10, fephagescript, psiX174, p.Bluescript SK, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (Qiagen).\",\n \"Preferred bacteriophage recombinant vectors of the invention are P1 bacteriophage vectors such as described by Sternberg N. L. (1992;1994).\",\n \"A suitable vector for the expression of hNK-1vRa polypeptide of the invention or a fragment thereof, is a baculovirus vector that can be propagated in insect cells and in insect cell-lines.\",\n \"A specific suitable host vector system is the pVL 1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC NoCRL 1711) which is derived from spodoptera frugiperda.\",\n \"The recombinant expression vectors of the invention may also be derived from an adenovirus such as those described by Feldman and Steg.\",\n \"(1996) or Ohno et al.\",\n \"(1994).\",\n \"Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type two or five (Ad 2 or Ad 5) or an adenovirus of animal origin (French Patent Application noFR 93 05 954).\",\n \"Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vivo gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, murine sarcoma virus, and Ross Sarcoma Virus.\",\n \"Other preferred retroviral 5 vectors are those described in Roth et al.\",\n \"(1996), in PCT Application WO 93/25 234, in PCT Application WO 94/06920, and also in Roux et al.\",\n \"(1989), Julan et al.\",\n \"(1992) and Nada et al.\",\n \"(1991).\",\n \"Yet, another viral vector system that is contemplated by the invention consists in the adeno associated virus (AAV) such as those described by Flotte et al.\",\n \"(1992), Samulski et al.\",\n \"(1989) and McLaughlin et al.\",\n \"(1996).\",\n \"Expression Systems For expression of the modified neurokinin receptors, the invention provides host cells transformed (prokaryotic cells) or transfected (eukaryotic cells) with such a vector; and any cell, or live organism, including a non-human mammal, that has been genetically engineered to produce such a polypeptide, said cell or live organism incorporating expressibly therein a nucleic acid sequence according to this invention.\",\n \"Heterologous expression systems may be used to express cloned NK-1 receptor, NK-2 receptor and NK-3 receptor, including human and non-human mammalian variants thereof.\",\n \"The choice of expression system depends on a number of factors including stability of protein expression, post translational modification and required yield.\",\n \"However, as a general rule, the more complex the organism the lower the yield of expressed receptor, but the greater the likelihood that the receptor will be in its native conformation.\",\n \"Several expression hosts are commonly available Prokaryotic host cells, e.g.\",\n \"Escherichia coli DH5-α (see Sambrook et al., for a comprehensive guide to gene expression in E. coli).\",\n \"Yeasts, e.g.\",\n \"Pichia pastoris.\",\n \"The feasibility of expressing NK-1 receptor in yeast has been demonstrated by Arkinstall et al (1995, FEBS 275 183-187) who successfully expressed therein the closely related receptor, human NK-2.Large-scale production of a human G-protein coupled receptor in yeast was demonstrated by Sizemann et al (1996, Receptors and Channels, Vol 4, 197-203).\",\n \"In brief, a protocol to express a functional NK-1 receptor in yeast can be represented by: (1) splicing the NK-1 cDNA into a yeast expression vector; (2) Transforming this vector into yeast; and (3) Selecting for yeast containing the NK-1 cDNA and expression of the NK-1 cDNA.\",\n \"Eukaryotic host cells, e.g.\",\n \"insect cells, non-mammalian vertebrate cells and mammalian cells.\",\n \"Non-mammalian vertebrate cell lines include Xenopus (frog) oocytes.\",\n \"Other cell lines which are contemplated in this invention include HeLa cells (ATCC NoCCL2; NoCCL2.1; NoCCL2.2), Cv 1 cells (ATCC NoCCL70), COS cells (ATCC NoCRL 1650; NoCRL 1651), Sf-9 cells (ATCC NoCRL 1711), C127 cells (ATCC NoCRL-1804), 3T3 cells (ATCC NoCRL-6361), CHO cells (ATCC NoCCL-61), human kidney 293 cells (ATCC No 45504; NoCRL-1573) and BHK (ECACC No84100 501; No84111301); PC12 (ATCC No CRL-1721), NT2, SHSY5Y (ATCC No CRL-2266), NG108 (ECACC No88112302) and F11, SK—N—SH (ATCC No CRL-HTB-11), SK—N—BE(2) (ATCC No CRL-2271), IMR-32 (ATCC No CCL-127).\",\n \"A preferred system to which the gene of the invention can be expressed are cell lines such as COS cells, 3T3 cells, HeLa cells, 292 cells and CHO cells.\",\n \"A preferred system for the efficient expression of hNK-1vR involves the use of CHO and COS cell lines.\",\n \"The gene can be expressed through an endogenous promoter of native CHO or COS, or through an exogenous promoter.\",\n \"Suitable exogenous promoters include such as SV40 and CMV, or perhaps a eucaryotic promoter such as the tetracycline promoter.\",\n \"The preferred promoter being CMV.\",\n \"In some instances, it may be required to tag e.g.\",\n \"a human NK-1 variant receptor prior to purification.\",\n \"The tag is then, in most instances, encoded into the nucleotide sequence that is needed to express the polypeptide.\",\n \"Examples of such tags include, but are not limited to sequences encoding C-myc, FLAG, a sequence of histidine residues, haemaglutin A, V5, Xpress or GST.\",\n \"Most of these tags can be incorporated directly into the sequence, for instance through PCR amplification by incorporating the appropriate coding sequence in one of the PCR amplification primers.\",\n \"However, the tag can also be introduced by other means, such as by covalent binding of the appropriate nucleic acid sequence encoding the tag moiety, such as GST, with the 3′ or 5′ end of the nucleic acid sequence encoding the polypeptide sequence.\",\n \"Purification of the NK variant receptor may, in the case of the use of a histidine tag, then be carried out by passage onto a nickel or copper affinity chromatography column, such as a Ni NTA column.\",\n \"The polypeptide thus produced may optionally be further characterized, for example by binding onto an immuno-affinity chromatography column on which polyclonal or monoclonal antibodies directed to the NK variant receptor have been previously immobilised.\",\n \"For some purposes it may be useful to provide mammals e.g.\",\n \"mice, rats or guinea-pigs in which in which modified human or non-human mammalian tackykinin receptors, e.g.\",\n \"NK-1v receptors, are present.\",\n \"Transgenic rats, mice and other mammalian cells may be produced by generating a targeting vector and transfecting the cells to be cultured e.g.\",\n \"using the gene targeting services of, e.g.\",\n \"DNX Transgenic Sciences.\",\n \"In the case of transgenic mammals, stem cells are transfected, cultured to blastocytes and introduced into the uterus of a female mammal.\",\n \"Because they have in their cell membranes tachykinin receptors that bind to endogenous ligands such as SP but are incapable, or substantially incapable, of initiating their endogenous signal, the animals are useful, in research into the effects of these ligands and the developments of transduction inhibitors, especially because they can indicate the behaviour of the animal the presence of endogenous ligand but in the absence of a transduced signal and in the absence of antagonist, and can therefore provide a true control.\",\n \"(Silver, Lee M. Title: Mouse genetics—concepts and applications Publisher: New York; Oxford University Press 1995); P De-Felipe-C et al;Nature.\",\n \"1998 Mar.\",\n \"26; 392(6674): 394-7).\",\n \"Assays The present invention further provides a modified tachykinin receptor, such as a human NK-1v receptor, used as a substitute in an assay to identify and evaluate entities that bind to the wild type tachykinin receptor.\",\n \"The invention also includes human NK-1v receptor used as a substitute in an assay in order to determine the concentration of substance P in body fluids in patients with arthritis, pain, migraine, anxiety, schizophrenia, asthma, rheumatoid arthritis, and in gastrointestinal disorders and diseases of the GI tract, like ulcerative colitis and Crohn's disease.\",\n \"As previously mentioned three tachykinin receptors have been identified, referred to as NK-1, NK-2 and NK-3 and they have respective endogenous lignads SP, NKA and NKB.\",\n \"Although each tachykinin has a preferred ligand, each receptor has the ability to interact with the other tachykinin ligands, and the pathology of cross-ligand binding in disease states is poorly understood.\",\n \"Study of a system where NK-1, NK-2 or NK-3 receptor response either singularly or in tandem has been diminished in vitro or in vivo could significantly aid understanding of the role of tachykinin receptors and their ligands in disease states.\",\n \"Screening Methods The invention also provides a method for screening for therapeutically active compounds, said method comprising the following steps: (a) providing a cell line expressing the modified tachykinin receptor, e.g.\",\n \"the human NK-1v receptor; (b) adding test sample to a solution containing labeled SP or other ligand and the cell line from step (a); (c) incubating the cell line, test sample and labeled SP or other ligand mixture from step (b) to allow binding of SP or other ligand and test sample to the modified tachykinin receptor; (d) optionally separate the non-bound labeled SP or other ligand from the labeled SP or other ligand bound to the modified tachykinin receptor; and, if desired, (e) measuring the amount of labeled SP or other ligand that is bound to the modified tachykinin receptor.\",\n \"Preferably, the assay involves COS-7 cell lines.\",\n \"Cell membranes containing the modified tachykinin receptor, e.g.\",\n \"the human NK-1v receptor, can be used instead of whole cells.\",\n \"The SP or other tachykinin ligand e.g.\",\n \"NKA or NKB may be labeled by any method known in the screening art, e.g.\",\n \"by a radioactive label, such as 125I, or by a fluorescent label.\",\n \"In certain circumstances, (e.g.\",\n \"fluorescence polarization assays), bound and unbound SP or other tachykinin ligand do not have to be separated to quantify the amount of SP or other ligand bound to the receptor.\",\n \"Alternatively, the SP or other ligand may be bound to a matrix, and labelled cells may be used to quantify the binding of a modified tachykinin receptor, such as human NK-1 receptor, to the SP.\",\n \"In this case, the assay procedure may follow the steps set out below: (a) a cell line is provided that expresses a modified tachykinin receptor, such as the human NK-1v receptor; (b) the cell line is labeled; (c) the test sample and labeled cells are added to a matrix binding SP or other ligand; (d) the labeled cells, test sample and matrix-bound SP or other ligand are incubated to allow binding of SP or other ligand and test sample to the expressed modified tachykinin receptor; (e) the labeled non-bound cells are separated from the SP or other ligand bound cells; and, if desired, (f) the amount of labeled cells containing the modified tachykinin receptor, such as the human NK-1v receptor, that has bound to SP or other ligand is measured.\",\n \"In order to provide transgenic animals or cell lines for use in assays, which animals and/or cell lines comprise a sequence as described herein, general methods are known and may be adapted accordingly.\",\n \"Different types of vectors including modified retroviruses, adenovirus, adeno-associated virus, herpes virus and plasmid DNA have been proposed as vehicles to introduce foreign genetic material into cells or tissues.\",\n \"Protein Therapy The invention also encompasses modified tachykinin receptors, especially human NK-1v receptor, for use in protein therapy to reduce the effects of, or an excess of endogenous ligand.\",\n \"Protein therapy can be used for the suppression of the action of SP in interstitial fluid of the lungs.\",\n \"For example, a purified preparation of a modified tachykinin receptor, such as human NK-1v receptor, (eg a liquid or powder carrier formulation) can be directly administered to the airways.\",\n \"Once in the airways, the tachykinin variant receptor can interact and bind to, for example, SP molecules.\",\n \"This has the effect reducing the amount of SP available for the endogenous NK receptor.\",\n \"Likewise, a modified tachykinin receptor, such as the human NK-1v receptor, can be directly introduced to body cavities such as joints and interstitial lung space where SP is present.\",\n \"The variant receptor has the capability to bind SP, therefor reducing the amount of SP available to interact with the wild type receptor and causing down-regulation of the SP cellular response.\",\n \"The invention provides a modified NK receptor, such as a human NK-1v receptor, for use in removing or suppressing SP in body fluids, e.g.\",\n \"the interstitial fluid of the lungs and fluid in the cavities of joints.\",\n \"A purified preparation of a modified NK receptor, such as human NK-1v receptor, (e.g.\",\n \"a liquid carrier formulation) may be administered directly to a joint.\",\n \"Once in the fluid- filled joint cavity, the NK variant receptor interacts and binds to SP molecules thereby reducing the amount of SP available to activate the endogenous NK receptor.\",\n \"The hNK-1v receptor can reduce the effect of excess or inappropriately expressed SP in patients with pain associated with migraine, neuralgia, diabetic, peripheral, AIDS-related and chemotherapy-induced neuropathy, and neuropathies of diverse origin; anxiety and anxiety disorders, such as panic disorder, phobias and obsessive-compulsive behavior; schizophrenia; asthma; rheumatoid arthritis; and in gastrointestinal disorders and diseases of the GI tract, for example ulcerative colitis and Crohn's disease.\",\n \"Other conditions or disease states that can be treated, ameliorated or prevented include: psychosomatic and psycho-immunological disorders; attention deficit disorder; pre-menstrual (PMT or PMS) or late luteal phase syndrome; mania or hypomania; aggressive behavior disorders; emesis, including motion sickness, migraine-induced sickness and that arising from chemotherapy; postherpetic neuralgia; depression; inflammation; eating disorders, such as obesity, bulimia nervosa and compulsive eating disorders; cognitive disorders, such as dementia and amnestic disorders; movement disorders, such as dyskinesias, akinetic-rigid syndromes, Gilles de la Tourette syndrome, tremor, or dystonia; schizophrenic disorders; substance abuse disorders; bipolar disorder; sexual dysfunction, including impotence; stress; alteration of circadian rhythmicity; Alzheimer's disease; bladder disorders; hypertension; angina; ischaemia; multiple sclerosis; chronic obstructive lung disease; scleroderma; CNS disorders; and other conditions where excess tachykinin peptides such as SP are involved.\",\n \"Furthermore, the inventors believe that modification to the DRY motif of NK-2 receptor and NK-3 receptor in a similar manner to that described for the NK-1 receptor above, would allow for the production of modified tachykinin receptors which could be used to remove their specific ligands from body fluids.\",\n \"Several factors need to be taken into account in protein therapies.\",\n \"These include: solublisation, maintenance of activity and stability of the protein, delivery and dose.\",\n \"The first three points are closely related.\",\n \"Proteins are large relative to conventional drugs and their biological activity is dependent on their primary, secondary, tertiary and in some instances quaternary structure being maintained.\",\n \"They often have labile bonds and numerous chemically reactive groups in their side chains.\",\n \"Disruptions of their structure by denaturation or aggregation can lead to loss of activity or increase in immunogenicity.\",\n \"One of the key problems in devising effective formulations for biologically active proteins is to find a formulation that is both stable and biologically active.\",\n \"The route of administration of the protein also plays a significant role in formulation.\",\n \"Microsphere formulations can be used for injection, aid in the maintenance of stability and activity of the protein, and offer the possibility of slow release formulations.\",\n \"Solid large powder formulations are most suitable for use in aerosol and topical treatments.\",\n \"Putney and Burke (Nature Biotechnology 1998: 153-157) outline various methods for producing microspheres.\",\n \"One such method is the atomization-freezing process.\",\n \"In this encapsulation method, the micronised solid protein is suspended in biodegradable polymers of DL-lactic co-glycolic acid (PLGA) solution that is then atomised using sonication or air-atomisation.\",\n \"This produces droplets that are then frozen in liquid nitrogen.\",\n \"Addition of ethanol at <−40° C., in which both the protein and the PLGA are insoluble, extracts the organic solvent from the micro-spheres.\",\n \"This process is further described in U.S. Pat.\",\n \"No.\",\n \"5,019,400.An alternative polymer for use in the process is methylene chloride polymer,to encapsulate growth hormone see Johnson et al, Nature Medicine 2: 795-799 (1996).\",\n \"The following method can be used to incorporate modified NKR, such as human NK-1v receptor, post-purification into microspheres: (a) concentrate the purified, active microsphere modified NKR, such as human NK-1v receptor to >100 mg/ml in the presence of stabilisers; (b) add PLGA or methylene chloride polymer solution and mix; then (c) atomise the frozen suspension in liquid nitrogen to fix the microspheres and extract with ethanol.\",\n \"Microspheres thus produced should have a diameter in the μM order.\",\n \"The invention further provides a method for treatment of a patient in need thereof, which comprises administering to said patient a composition in the form of an aerosol that comprises a modified tachykinin receptor as described above, e.g.\",\n \"an hNK-1Rv receptor.\",\n \"SP or other ligand such as NKA or NKB can also be removed directly from biological fluids using the modified tachykinin receptors described above.\",\n \"For example, purified human NK-1v receptor could be bound to a suitable matrix.\",\n \"Biological fluids containing SP can be passed over the matrix, so that the SP preferentially binds to matrix while other components of the fluid do not and SP becomes preferentially removed from the biological fluid.\",\n \"Nucleic Acid Therapy The invention further provides a method for gene therapy treatment of a patient in need thereof, which comprises administering to said patient a nucleic acid sequence, virus or plasmid encoding a modified tachykinin receptor as described above, e.g.\",\n \"an hNK-1Rv receptor.\",\n \"Different types of vector including modified retroviruses, adenovirus, adeno-associated virus, herpes virus and plasmid DNA have been proposed as vehicles for introducing foreign genetic material into the cells or tissues of patients, and can in principle be used to introduce the modified tachykinin receptor nucleic acid sequences referred to above.\",\n \"The appropriate strategy for administering the nucleic acid sequence depends on the target tissue, disease state and longevity of the proposed therapy.\",\n \"Furthermore, down regulation of the effect SP on a cellular system could be achieved by expressing the hNK-1v receptor in cells other than those expressing native NK-1.The expression of SP in these cells (which are in the proximity of cells expressing native NK-1 receptor) could have the effect of “moping” up SP and reducing the available SP for the native receptor to interact with.\",\n \"Introduction of a modified NK receptor, such as a human NK-1v receptor, (by way of direct introduction of protein or introduction of an expressible gene encoding this protein) into the outer membrane of cells expressing the wild type receptor gives rise to competition between the wild type and variant receptors for available SP.\",\n \"The variant receptor competes with wild type receptor for binding of available SP and decreases the amount of SP available for the wild type receptor.\",\n \"As the NK-1v receptor is unable to transduce a signal upon SP binding, the result is a down-regulation of the action of the wild type receptor.\",\n \"In the treatment of lung tissue, researchers at Stanford University Medical Center, California have initiated a trial of gene therapy for cystic fibrosis in which the active material is delivered to the lungs by aerosol.\",\n \"The active material consists of a version of the cystic fibrosis trans-membrane conductance regulator gene packed into an adeno-associated virus (AAV) shell.\",\n \"A similar route can be adopted for delivering the hNK-1v receptor to the lungs of patients where the activation of native hNK-1 receptors by receptor ligands requires to be down-regulated.\",\n \"A strategy for down-regulating the action between SP and human NK-1v receptor in lung tissue can be adopted which is similar to the strategy proposed for gene therapy in cystic fibrosis.\",\n \"The same technique of packaging genes into AAV and infection of the lung tissue with aerosol AAV can be used to introduce hNK-1v.\",\n \"Samulski et al (University of North Carolina, Chapel Hill, N.C. 27599, USA) market a vector which can be used to package human NK-1v receptor gene into AAV.\",\n \"Alternatively, a plasmid containing the human NK-1v receptor (pCMVNK-1v) may be used directly in gene therapy.\",\n \"The plasmid may be prepared as a lipid:DNA complex and administered to the lungs as an aerosol, see Pillai et al., Pharm-Res 15(11): 1743-7 (1998), McDonald et al., Pharm-Res 1998 15(5): 671-9(1998).\",\n \"This strategy can also be used with an NK-1 receptor promoter.\",\n \"A pre-requisite for the use of gene therapy for humans is the ability to make sufficient pharmaceutical grade plasmid DNA.\",\n \"This problem has been addressed by Prazeres et al in TIBTECH 17: 169-74 (1999).\",\n \"Both rAAV and lipid:DNA complex may be administered to the lungs via nebulisers, e.g.\",\n \"airjet nebulisers.\",\n \"Methods for administration may generally follow the teachings of McDonald et al, Pharm Res 15(5): 671-9 (1998); Yonemitsu et al, Gene Therapy 4(7): 631-8 (1997); McDonald et al, Human Gene Therapy, 8(4): 411-22 (1997); Bellon et al, Human Gene Therapy 8(1): 15-25 (1997); and Niven, Critical Review of Therapeutic Drug Carrier Systems 12(2-3): 151-231 (1995).\",\n \"Both viral and plasmid therapeutic compositions can also be delivered to other target sites such as joints and nervous tissue by infusion and injection.\",\n \"EXAMPLES Embodiments of the invention will now be described in the following Examples Example 1 Preparation of a Modified Human NK-1 Receptor (NK-1v) The starting material used was a plasmid pRc/CMV (Invitrogen Co) containing a cDNA clone encoding the human NK-1 receptor.\",\n \"The clone encoded 407 amino acids and was flanked by a Nco I site at the 5′ end (around the ATG start codon) and a Xba I site at the 3′ end following the stop codon.\",\n \"Human NK-1v receptor was made from the above plasmid using a PCR-based strategy that mutated the DRY motif (Asp129, Arg130, Tyr131) at the end of third transmembrane helix to GGA.\",\n \"Portions of the cDNA clone in the above plasmid were amplified in two separate PCR reactions using Pfu DNA polymerase (Stratagene), 20 cycles of PCR using 1 min at 94° C., 1 min at 55° C., 4 min at 72° C., followed by 10 mins at 72° C., 50 pmol of each primer, 50 ng of plasmid DNA, 100 μl reaction volume.\",\n \"The first PCR reaction used the following primers to produce a predicted product of 473 bp: (SEQ ID No7) 5′ primer, sense-A: 5′-AAC TAG AGA ACC CAC TGC TTA-3′ (SEQ ID No8) 3′ primer, anti-sense-A: 5′-GCC ATA GCG CCG CCA AAA GCC ACA GCC GT-3′ The second PCR reaction used the following primers to produce a predicted product of 888 bp: (SEQ ID No9) 5′ primer, sense-B: 5′-TTT GGC GGC GCT ATG GCT ATC ATA CAT CC-3′ (SEQ ID No10) 3′ primer, anti-sense-B: 5′-AGC TCT AGC ATT TAG GTG ACA-3′.\",\n \"Primers anti-sense-A and sense-B contained 17 bases of overlapping complementary sequence at the 5′ ends of each to allow annealing of the two products to form the full length mutated receptor.\",\n \"All the above primers were custom synthesized by Perkin-Elmer.\",\n \"The two PCR products were gel-purified (QIAEX, Qiagen) and 50 ng of each purified product was added to a 100 μl Pfu PCR reaction and three rounds of PCR performed without primers to allow the two overlapping regions of the PCR products to anneal and extend.\",\n \"50 ng of the flanking primers (sense-A and anti-sense-B) were added and 20 cycles of conventional PCR performed.\",\n \"The resulting full-length product was purified, cloned into pBluescript (Stratagene) and fully sequenced to confirm the mutations had been successfully made.\",\n \"The clone was modified by site-directed mutagenesis using a Clontech Transformer kit to remove an internal Nco I restriction site using the oligonucleotide sense-C also from Perkin-Elmer.\",\n \"Sense C: 5′-CGC GGA GGC TTC TAT GGC TGC AT-3′ (SEQ ID No11) The resulting cDNA was excised from the parental vector, spliced into a mammalian expression vector, pCMV3.1 (Invitrogen).\",\n \"This cDNA was in an orientation which allowed expression of the hNK-1vR polypeptide.\",\n \"Stock plasmid was prepared by amplification in E. coli and the plasmid DNA subsequently purified using a Qiagen endonuclease free DNA preparation kit, and the fidelity of the construct confirmed by DNA sequencing.\",\n \"The nucleic acid sequence of the cDNA is SEQ ID No6 (FIG.\",\n \"2) and the translated protein sequence is SEQ ID No5 (FIG.\",\n \"1).\",\n \"Similarly control (negative control) constructs were prepared whereby wild type and variant HNK-1R cDNA was spliced into the vector in an opposite configuration to that described above.\",\n \"A further control (positive control) was prepared by ligation of the parental wild type hNK-1R cDNA into the vector in a manner which would allow hNK-1R polypeptide to be expressed.\",\n \"Example 2 COS-7 Cells that Transiently Express Human NK-1v Receptor COS-7 cells were grown in DMEM culture medium supplemented with 10% foetal calf serum and 2mM glutamine and maintained under an atmosphere of 5% CO2.Cells were passaged at approximately 70% confluence by reseeding to a concentration of approximately 10% confluence per 175 cm2 flask.\",\n \"COS-7 cells were harvested and prepared for electroporation in Equibio electroporation buffer.\",\n \"Cells (5×106 cells) were electroporated at room temperature in a 4 mm gap cuvette in a final volume of 800 μl containing 30 μg of transforming plasmid DNA, with 250 volts, 1500 μF at infinite resistance using an Equibio EasyJect plus electroporator.\",\n \"Transformed COS-7 cells were cultured for 2-3 days in a 175cm2 flask prior to assay.\",\n \"Alternatively cells (approximately 5-50 ul of transfected cells) were cultured in 6 well culture dishes containing 22mm diameter cover slips.\",\n \"These cover slips coated in cells were used for imaging experiments designed to investigate changes in the concentration of intracellular free calcium ([Ca2+]i).\",\n \"Cover slips were prepared by first immersing the cover slips in approximately 70% ethanol and then quickly passing through a gas flame to sterilize.\",\n \"Cover slips were then allowed to air dry in a culture hood prior to placing in the bottom of a culture well of a standard six well culture dish.\",\n \"Cells were then cultured as described above.\",\n \"Example 3 COS-7 Cell Membranes Containing Human NK-1v Receptors The transfected COS-7 cells of Example 2 were harvested by treatment with Versene.\",\n \"(Gibco BRL).\",\n \"Versene was used, as it allows detachment of the cells from the flask without substantial perturbation to cell membrane proteins, then, the cells were washed once by re-suspending in assay buffer (50 mM Tris HCl pH 7.4, 3 mM MnCl2, 0.02% BSA, 40 μg/ml bacitracin, 2 μg/ml chymostatin, 2 μM phosphoramidon, 4 μg/ml leupeptin) and centrifuged at 1000 g for 5 min.\",\n \"Cells were re-suspended in 5 ml of the assay buffer and a cell count performed prior to lysing cells, using a Brinkman polytron at setting 6 for 15 seconds.\",\n \"The homogenate was centrifuged at 20000 g for 10 minutes.\",\n \"Membrane pellets were re-suspended in assay buffer and stored frozen as 0.5-1 ml aliquots until required for use.\",\n \"Example 4 Ligand Binding to Receptors in Cell Membranes COS-7 cells were transiently transfected with pCMV3.1 (Invitrogen) containing either parental wild type human NK-1 receptor cDNA or the variant human NK-1 receptor cDNA of Example 1, and cell membranes were prepared 2-3 days post-transfection using the procedure of Example 3.Radioligand binding studies were performed using [125I]BH substance P to label NK-1 receptors.\",\n \"Non-specific binding was defined by the NK-1 receptor-selective agonist [Sar9, Met(O2)11]substance P (Bachem) at a final concentration of 1 μM.\",\n \"On the day when they were required, the membrane suspensions were thawed and diluted as appropriate with assay buffer and incubated with varying concentrations of [125I]Bolton-Hunter Substance P (0.05-3 nM, from Amersham Life Sciences) for 50 minutes at 21° C. Saturation analyses were performed to determine the affinity constants and maximum binding capacity for each receptor, by incubating membranes with increasing concentrations of the radioligand, in the presence and absence of [Sar9, Met(O2)11]substance P. Reactions were terminated by rapid filtration under vacuum, onto GFC filters pre-soaked with 0.2% polyethylenimine.\",\n \"[125I]BH substance P bound with high affinity to both wild type and mutant receptors (Kd values of 2.218±1.107 nM (n=3) and 1.446 nM (n=2) respectively).\",\n \"Unpredictably, there appeared to be no significant difference between the dissociation constant and the maximum binding capacity of the wild type and variant NK-1 receptors, indicating that the mutation does not affect agonist binding at the NK-1 receptor.\",\n \"The maximum binding capacity can be directly compared, as shown in Table 1: TABLE 1 Wild Type Variant HNK-1 (n = 3) hNK-1 (n = 2) Kd (nM) 2.218 + − 1.1107 1.446 Bmax 2.201 3.000 Example 5 Analysis of Intra-Cell Receptor Coupling in COS-7 Cells by Changes in Intra-Cellular Free Calcium Concentration Coverslips containing cells were prepared and maintained as described.\",\n \"Cultured cells were washed twice in a Krebs-Hepes extra-cellular medium buffer (EM, composition in mM: NaCl 118, KCl 4.7, MgSO4 1.2, CaCl2 1.2, KH2PO4 1.2, Hepes 10, glucose 11 and BSA 0.1%, (pH 7.2 at 20° C., (Rossant, et al Endocrinology, 140, 1525-1536), and then loaded with Fura-2 (Rossant, et al Endocrinology, 140, 1525-1536) by incubation for 3 h at 20° C. with EM containing Fura-2-AM (2 μM, Molecular Probes).\",\n \"This procedure enables the cells to load with Fura-2-AM, which becomes hydrolysed to the free acid form once inside the intact cells.\",\n \"After loading, coverslips were mounted into imaging chambers and perfused with EM to remove extra-cellular Fura-2-AM and to allow hydrolysis of intracellular Fura-2-AM to occur.\",\n \"Measurements of changes in the free [Ca2+]i in individual cells were made from the fluorescence ratio (excitations 340 nm/380 nm, emission >510 nm) using a Spectral Wizard monochromator, cooled integrating CCD camera and a dedicated suite of software (Merlin, Life Sciences Resources, Cambridge, UK).\",\n \"Data are expressed as ratio units 340/380.Genes encoding wild type sense, wild type anti-sense and variant NK-1 receptors described above were transiently transfected into COS-7 (monkey kidney) cell lines.\",\n \"Two to three days post-transfection, cells were loaded with FURA-2-AM dye (whose fluorescence is [Ca2+]i dependent) and challenged with substance P. The [Ca2+]i was followed by monitoring the change in light emitted (>510 nm) when excitation light of either 340 nm or 380 nm was sequentially used to illuminate the loaded cells and expressed as a ratio (340 nm:380 nm).\",\n \"Cells challenged with UTP (which increases [Ca2+]i in non-transfected cells) caused a [Ca2+]i response indicating the presence of a functional G-protein Ca2+ linked pathway.\",\n \"Results plotted in FIG.\",\n \"3 indicate that cells expressing wild type NK-1 receptor responded to substance P over a 0.01-1000 nM range.\",\n \"Cells expressing wild type anti-sense and variant NK-1 receptor failed to invoke a Ca2+ response with substance P challenge of 1 nM.\",\n \"The effect of treatment with varying concentrations of substance-P or with UTP (3μM) for a 1 min time period on the average change in 340:380 ratio levels of COS 7 cultures expressing either wild type or modified NK-1 receptors (FIG.\",\n \"3, n>50 cells for each treatment group (or on the percentage of COS cells demonstrating a measurable response (Xb) is shown in FIG.\",\n \"3).\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10451304"}}}],"truncated":false,"partial":false},"paginationData":{"pageIndex":2,"numItemsPerPage":100,"numTotalItems":4495096,"offset":200,"length":100}},"jwt":"eyJhbGciOiJFZERTQSJ9.eyJyZWFkIjp0cnVlLCJwZXJtaXNzaW9ucyI6eyJyZXBvLmNvbnRlbnQucmVhZCI6dHJ1ZX0sImlhdCI6MTc3NjY3MjQ3NCwic3ViIjoiL2RhdGFzZXRzL2hvd2V5L2h1cGQiLCJleHAiOjE3NzY2NzYwNzQsImlzcyI6Imh0dHBzOi8vaHVnZ2luZ2ZhY2UuY28ifQ.Wh06FjAqlh_FZ9yrmyREV2aiIlxbt2QVwX5awJ27wisFpTihYq8JZTvaVhidOGep5PxvcAveXDIknU7546YlCg","displayUrls":true,"splitSizeSummaries":[{"config":"default","split":"train","numRows":4495096,"numBytesParquet":108339066781}]},"discussionsStats":{"closed":0,"open":0,"total":0},"fullWidth":true,"hasGatedAccess":true,"hasFullAccess":true,"isEmbedded":false,"savedQueries":{"community":[],"user":[]}}">
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"Storage medium convey apparatus and convey method and recording and/or reproduction apparatus",
"Apparatus that carries or transfers a memory medium having a solid-state memory over the inside and outside portions of a recording and/or reproducing apparatus is adapted so that, by selective engagement wit... | Patent_10399003 |
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"Apparatus and method for hydroforming a tubular part",
"A method and apparatus for shaping a raw tube into a formed part.",
"The part can be configured within a die assembly including a hydroforming die structure and a pair of tube-engaging punches.",
"The punches are inserted into the ends of th... | Patent_10399192 |
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"Method and system for online sales and purchase",
"Internet-based commercial network connects multiple qualified participant buyers and sellers.",
"Items are made available for defined periods of time, referred to as an event (139).",
"Event terms and conditions include item types, price, quantit... | Patent_10399196 |
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"Urine test for the diagnosis of prion diseases",
"The present invention relates to a method for detecting the presence of the abnormal isoform of prion protein (PrPSC) in a urine sample of a subject.",
"The method of the invention comprising the steps of: (a) providing a urine sample of said subject;... | Patent_10399321 |
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"Method of and system for controlling the ratio of a variable lead parameter and an adjustable lag parameter for a lag-lead process",
"In a method of and system for controlling the air/gas ratio in a lag-lead combustion plant the lead and lag parameters are monitored to provide lead and lag signals repres... | Patent_10399374 |
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"Gel-type polymer electrolyte and use thereof",
"A gel-type polymer electrolyte, wherein said polymer comprises (A) an ethylene-unsaturated carboxylic acid copolymer or a derivative thereof and (B) a polyalkylene oxide having a hydroxyl group at one terminal thereof or a derivative thereof, which are bond... | Patent_10399377 |
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"Urotensin-II agonists and antagonists",
"The present invention features a novel class of cyclic polypeptides that have U-II antagonist and agonist activity.",
"The invention also features methods for treating physiological or psychological conditions characterized by an excess or under expression of ... | Patent_10399542 |
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"Method for verifying a perforation pattern serving as a security characteristic",
"A method of checking the authenticity of a perforation pattern which serves as a security feature and which when viewed in daylight back-lighting shows image information in the substrate of a document in particular in card... | Patent_10399741 |
[
[
"Lightweight aggregate binder formulation",
"The invention relates to a thermally stable composition, and methods for the preparation thereof, for use in the manufacture of a lightweight aggregate including a continuous bituminous phase, a discontinuous aqueous phase, an anionic oxide suspended in the dis... | Patent_10399873 |
[
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"Reengineering event-driven field processes with a self-managed team approach",
"The invention provides a globally scalable, business-to-business (B2B) Mobile Process Service (MPS) that creates value for users and furthermore, through a franchise arrangement, allows value to be captured especially by tele... | Patent_10405937 |
[
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"Security feature",
"The invention concerns a process for producing a security feature and a print medium which is equipped in that respect, which affords the possibility of not having to implement any change and in particular mechanical change in the surface of the card body.",
"In that respect the s... | Patent_10415199 |
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"System and method for estimating cash flow at risk for a non-financial institution",
"A method for estimating cash flow at risk for a non-financial entity over a particular future time period, including receiving quarterly data associated with at least two of a plurality of non-financial entities, genera... | Patent_10415206 |
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"Non-photochromic, colored, borosilicate inorganic glasses which absorb ultraviolet, and preparations",
"The object of the present invention is non-photochromic, colored, borosilicate inorganic glasses, which absorb ultraviolet and which contain effective amounts of silver, of copper, of halogen(s), of re... | Patent_10415237 |
[
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"Preparation of 4-methyl-2, 3, 5, 6-tetrafluorobenzyl alcohol",
"A process for the preparation of 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene comprising fluorinating 2,3,5,6-tetrachloroterephthalonitrile to obtain 2,3,5,6-tetrafluoroterephthalonitrile, hydrogenating 2,3,5,6-tetrafluoroterephthalonit... | Patent_10415258 |
[
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"Medicament dispenser",
"A medicament dispenser comprising: (i) a housing having an outlet; (ii) a medicament container locatable within said housing; (iii) an electronic dose counter associated with said outlet, wherein said dose counter comprises a first sensor for directly detecting a medicament releas... | Patent_10415279 |
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"Carpet stretcher",
"A sliding hammer apparatus which is attachable to a gripper head part of a carpet stretching device (10), the apparatus having a body portion (14) having an elongate substantially horizontal member (15) which has a front end and a rear end, a hand operable sliding hammer (16) slidably... | Patent_10415337 |
[
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"Latch device for securing cargo containers together and/or to vehicle decks",
"A cargo container hold down device that includes a housing defined by a base which forms a planar surfacing thereabout on which cargo container corner fittings results in the applied relation on the container relative to the s... | Patent_10415394 |
[
[
"Telephone directory assistane method and telephone directory assistance system",
"This telephone directory assistance system accepts a request for a telephone number of a registrant, and judges whether the registrant is registered in a telephone number data base.",
"When the registrant is registered ... | Patent_10415439 |
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"Software development",
"A method of developing software capable of performing a task is disclosed.",
"The task comprises at least one task step required to perform the task.",
"The task step has a resultant effect.",
"The method comprises: recording a use case comprising one or more scenarios... | Patent_10415466 |
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"Bridge for attaching auxiliary lenses",
"An auxiliary spectacle apparatus is provided wherein auxiliary lenses have a mechanical bridge fastener for releasably engaging the bridge portion of the primary frames in a manner which provides security of attachment in all directions."
],
[
"1-21.",
... | Patent_10415467 |
[
[
"Means for detecting pathological transformation of the app protein and their uses",
"A method of intra-articular drug delivery may include selecting an attachment zone in a synovial joint; affixing a drug release device in the attachment zone, the drug release device comprising a base affixable in the at... | Patent_10415479 |
[
[
"Food grade transglutaminase inhibitor and uses thereof",
"Transglutaminase inhibitor containing composition obtainable from milk, in particular from a whey fraction obtainable from skimmed milk by forming a curd from the milk, separating the curd from the whey in a centrifugation step followed by subject... | Patent_10415584 |
[
[
"Aminoglycosides as antibiotics",
"The present invention provides aminoglycosides and pharmaceutical compositions that include the aminoglycosides.",
"The aminogylcosides are useful to treat or prevent infectious diseases (e.g., bacterial infections) in a mammal (e.g., human)."
],
[
"1.A compo... | Patent_10415732 |
[
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"Method for the detection of viable microorganisms",
"Method for the detection and enumeration of viable microorganisms.",
"A liquid that comprises one or more markers incorporated in a liquid sol-gel precursor, is provided.",
"A transparent slide is coated with a thin uniform layer of the liquid ... | Patent_10415844 |
[
[
"Extension arm for a sheet-processing printing machine",
"A sheet delivery mechanism for a sheet-fed printing machine which is adapted to reduce changes in the position of sheet material in a stack during a stack changeover operation and which facilitates the formation of a stack with properly aligned edg... | Patent_10415871 |
[
[
"Arrangement in ventilatory treatment of the lungs",
"The invention relates to a valve arrangement in the ventilatory treatment of the lungs of a living creature (2) under a state of anaesthesia, comprising valve equipment (4) devised to carry an insufflation gas and/or an insufflation gas mixture from a ... | Patent_10415942 |
[
[
"Diamido alkoxides as polymerisation initiators",
"The present invention relates to a complex having the formula (I) wherein M is selected from Sn(II), Sn(IV), Al(III) and Mg(II); each of R1 and R2 is independently hydrocarbyl; R3 is H or hydrocarbyl; and X is a linker group.",
"A further aspect of in... | Patent_10416284 |
[
[
"System, process and software arrangement for recognizing handwritten characters",
"A process and system are provided for determining a most likely combination of characters.",
"In particular, character data which includes information indicative of at least one handwritten character is obtained.",
... | Patent_10416319 |
[
[
"Sucking atomizer for gasoline-oxygen cutting-welding",
"A jet-inducing atomizer for gasoline-oxygen cutting-welding comprises a main body, a diffusion tube, a preheating oxygen fine-tube and a gasoline fine-tube.",
"The diffusion tube and the preheating oxygen fine-tube are provided at the center of ... | Patent_10416374 |
[
[
"Deflection fitting for the safety belt of a motor vehicle",
"A deflection fitting for the safety belt of a motor vehicle consists of a belt guide (17, 18) which surrounds the safety belt (7) and a pivot bearing (13) about which said belt guide is pivoted, about a pivot pin (14) and parallel to the direct... | Patent_10416605 |
[
[
"Method and system for tubing a borehole in single diameter",
"The invention concerns a method for casing a borehole which consists of successively lowering an unlimited plurality of strings of casing having a common diameter, the next one through the preceding one, to be installed one beneath the other a... | Patent_10416773 |
[
[
"Exoenzyme toxin of aeromonas salmonicida, and uses thereof",
"A protein toxin named Aeromonas salmonicida exoenzyme T (AcxT), which belongs to the family of ADP-ribosylating toxins, is disclosed as is a Calcium (or other cation concentration) dependent promoter of A. salmonicida.",
"Also disclosed ar... | Patent_10416981 |
[
[
"Rapid curing anaerobic compositions",
"A curable composition having enhanced cure speed which includes a curable component, a cure system and an additive for enhancing cure speed.",
"The additive includes an alkali metal halide salt and the reaction product of: (a) a polyfunctional isocyanate and a m... | Patent_10432199 |
[
[
"Mewthod and device for analysing the surface of a substrate",
"The object of the invention is a process for scanning a surface of a substrate (2), which process consists in taking at least one reflected image of at least one test pattern (1) on the said surface and extracting by digital processing local ... | Patent_10432269 |
[
[
"Radio receiver",
"A radio receiver (30) comprises an ADC (13) including a clip counter.",
"The power of digitised signals provided by the ADC (13) is estimated by a power estimator (31), and an ideal gain value is computed from the power so estimated by a gain computation device (32).",
"Gain com... | Patent_10432312 |
[
[
"Information storage apparatus and electronic device in which information storage apparatus is installed",
"There are disclosed an information storage apparatus and an electronic device mounting the information storage apparatus, wherein when the electronic device mounting an information storage element u... | Patent_10432330 |
[
[
"Fabrication of integrated circuit",
"A method of fabricating an integrated device on a chip comprising first and second features (A, B), the second feature, B, having greater dimension and/or being of coarser design than the first feature A.",
"The method involves the steps of: depositing a resist on... | Patent_10432526 |
[
[
"Differential gear for vehicle",
"There is disclosed an automotive differential device for transferring a rotational drive force from an automotive engine to a drive axle.",
"This differential device includes: a differential gear mechanism equipped with a pair of side gears, three pinions meshing with... | Patent_10432539 |
[
[
"Hinge arrangment for a cabinet door",
"A hinge arrangement for a cabinet door, comprising: a pair of hinge seats (10), which are affixed to one of the parts defined by the door (P) and the cabinet (G); hinge pins (20), which are affixed to the other of said parts and which are each designed to be fitted ... | Patent_10432540 |
[
[
"Method for identifying compounds or lead structures against rna target motifs and rna/protein interactions",
"Disclosed is a method for identifying compounds (lead structures) which specifically bind to (a) desired RNA target motif and can inhibit or eliminate the function thereof or (b) suppress a compo... | Patent_10432560 |
[
[
"Electrochemical double-layer energy storage cells with high energy density and high power density",
"The invention concerns a method for preparing activated carbons based on wood, preferably softwood and in particular pine wood, for making electrodes for energy storage cells, particularly for super-capac... | Patent_10432590 |
[
[
"Derivatives of 4-hydroxybutanoic acid and of its higher homologue as ligands of $g(g)-hydroxybutyrate (ghb) receptors, pharmaceutical compositions containing same and pharmaceutical uses",
"The invention concerns the field of synthesis organic chemistry applied to the pharmaceutical field and concerns no... | Patent_10432692 |
[
[
"Process for preparing siloxane-filler compositions using an extruder mixer",
"Methods describing a process for the production of polysiloxane containing masses, containing surface treated filler material, by continuously feeding materials to a screw extruder (12) in which they are mixed and conveyed to a... | Patent_10432749 |
[
[
"Self-closing packaging for moisture-sensitive material",
"Packaging for moisture-sensitive material, in particular detergent material, comprising an outer container comprising a container body and a lid cover hingedly fixed to the container body, and an inner bag made of water-impermeable flexible sheet ... | Patent_10432765 |
[
[
"System and process for validating, aligning and reordering one or more genetic sequence maps using at least one ordered restriction map",
"A method and system are provided for comparing ordered segments of a first DNA restriction map with ordered segments of a second DNA restriction map to determine a le... | Patent_10432766 |
[
[
"Thinners for invert emulsions",
"A method of reducing the viscosity of oil-based drilling fluids and well service fluids at low temperatures and a thinner compound for use in such drilling fluids and well service fluids is disclosed.",
"The method comprises adding to said drilling fluids or well serv... | Patent_10432787 |
[
[
"Latex products",
"The present invention finds that a detackified natural rubber latex product can be provided by a combination of treatment with a hydrophilic group sealant, coating with a detackifying polymer, halogenation and the like.",
"The present invention also finds that a detackified natural ... | Patent_10432879 |
[
[
"Acoustic communication system",
"There is described a number of encoders for encoding a data signal within an audio signal.",
"In some of the encoders, the audio signal is separated into a tonal part and a residual part, and the data signal is shaped based on the residual part.",
"In other encode... | Patent_10432889 |
[
[
"Electronic Data Storage Medium",
"The invention concerns an electronic data storage medium including a body (30) made from insulated plastic material which has two main parallel sides, a semiconductor component (34) and a number of external electrical contact pads flush with the first main side (30a) of ... | Patent_10432926 |
[
[
"Display memory, driver circuit, display, and cellular information apparatus",
"A display memory able to reduce power consumption, able to generate graphics at a high speed, and not needing memory mapping, a driver circuit, a display using the driver circuit, and a portable information apparatus, wherein ... | Patent_10432937 |
[
[
"Phase transfer of nanoparticles",
"The invention relates to phase transfers of nanoparticles and to a catalysis using said nanoparticles.",
"The aim of the invention is to facilitate a transfer of nanoparticles from an organic solution to an inorganic, especially, aqueous solution.",
"To this end... | Patent_10432942 |
[
[
"Ultrasonic reflux system for one-step purification of carbon nanostructures",
"Reflux systems and methods for purifying carbon nanostructures using same are provided.",
"The reflux system includes a solvent flask, an extraction tube connected to the solvent flask by a siphon tube and a vapor tube eac... | Patent_10433125 |
[
[
"Method and system to automatically qualifying a party to participate in a network-based commerce transaction",
"A system to facilitate network-based commerce includes receiving information relating to an item to be transacted via a network-based commerce system from a first party.",
"Criterion inform... | Patent_10433173 |
[
[
"Devices and methods for controlled delivery from a drug delivery device",
"The invention features a plug for use with a drug delivery device, wherein the plug defines an expansion control channel, which accommodates thermal expansion of a formulation in a reservoir of a drug delivery device, and an exit ... | Patent_10433196 |
[
[
"Membrane molecule indicator compositions and methods",
"The invention provides membrane molecule indicators, including polypeptides, encoding nucleic acid molecules and cells containing such polypeptides and nucleic acid molecules.",
"The invention membrane molecule indicators are characterized in th... | Patent_10433245 |
[
[
"Growth of human dendritic cells for cancer immunotherapy in closed system using microcarrier beads",
"A method and apparatus for reproducibly generating dendritic cells are provided.",
"Blood mononuclear cells are loaded into a cell culture container containing microcarrier beads therein.",
"Tiss... | Patent_10433387 |
[
[
"Spinal intervertebral implant adjustable in situ",
"The invention concerns a spinal intervertebral implant (100) comprising at least a first element (101′) having a first end (112′), and a second element (101) having a second end (112), each end having successive ramps (108, 116), the ramps of the two en... | Patent_10433531 |
[
[
"Seal for a loading dock bumper",
"At a loading dock for a truck, a compressible bumper seal extends across the front face of a dock bumper to help seal an air gap that may otherwise exist between the bumper face and the rear of the truck.",
"Without the seal, the gap may be created by the truck stopp... | Patent_10433636 |
[
[
"Method, computer program product and use of a computer program for stabilizing a multiphase flow",
"The present invention relates to a method for stabilizating multiphase flow through a pipeline, riser or well flow line (8a), characterized by implementation of a dynamic feedback controller (9) by control... | Patent_10433653 |
[
[
"System and method for the management of genomic data",
"A system and method is disclosed for managing users' genomic data, including providing and offering access to genomic-based services, routing genomic data to providers of genomic-based services, brokering financial transactions related to the manage... | Patent_10433700 |
[
[
"Collapsing chair with solid armrest and tensioned seat",
"A chair (100) with solid armrests (150) and a tensioned seat collapses in a single movement, in which the front legs (110) approximate each other when the front and rear legs (120) pivot towards each other and when the seat and the back pivot towa... | Patent_10433703 |
[
[
"Method for quantifying cytosine methylations in genomic dna that is amplified in a complex manner",
"A method is described for preparing demethylated DNA as reference material for the analysis of cytosine methylations in genomic DNA samples with the use of complex amplification."
],
[
"1.A method... | Patent_10433742 |
[
[
"Spinal intervertebral implant adjustable in situ comprising hard pass point",
"The invention concerns a spinal intervertebral implant (200) comprising at least an upper element (203), a lower element (205), and an intermediate member (201) adapted to co-operate with the upper and lower elements by helica... | Patent_10433769 |
[
[
"Brush part for electric toothbrush",
"A brush part for an electric toothbrush in which tufts of bristles are arranged in polygons, and in which the tufts of bristles lean, either around the polygons or inwardly and outwardly, so that the tuft alignments converge and preferably cross."
],
[
"1-12.... | Patent_10433850 |
[
[
"Package, particularly a cigarette package, and method and device for the production thereof",
"A cuboidal cigarette pack with a large-surface-area pack side, namely rear side (13), is provided with a blank (23), which serves as a printing carrier, in the region of this rear side (13).",
"Said blank i... | Patent_10433892 |
[
[
"Emergency release assembly and sliding door incorporating the same",
"An emergency release assembly for a door includes a latch mechanism to retain the door in a normally closed position yet releases to let the door move to an emergency open position.",
"A release mechanism triggers the latch mechani... | Patent_10433974 |
[
[
"Method for control of a diagnosis of a catalyst in the exhaust of an internal combustion engine",
"A control for diagnosing a catalytic converter in the exhaust gas of an internal combustion engine having means for determining the catalytic converter temperature is presented.",
"The diagnostic method... | Patent_10433982 |
[
[
"Sliding litter scoop",
"The present invention provides an animal refuse scoop comprising a scoop, and a handle attached to the scoop.",
"The scoop defines a top and bottom surface, and a plurality of openings therein.",
"The openings are defined to allow the passage of litter granules therethroug... | Patent_10438966 |
[
[
"Universal clutch puller and installer",
"The materials needed for making the universal clutch puller and installer (UCPI) consist of flat steel stock and metal pins.",
"After outlining, cutting, drilling, and bending you will end up with a device that makes the installation and removal of clutches ea... | Patent_10442901 |
[
[
"Information processing apparatus and program",
"The present invention is associated with an information processing apparatus and a program adapted to provide content to a terminal via a server without otherwise notifying the terminal thereof.",
"A personal computer 1 transmits an image taken by a CCD... | Patent_10450041 |
[
[
"Relating to fabric care",
"The invention is concerned with improvements relating to fabric care and in particular to means by which the apparent ageing of clothes can be reduced or retarded.",
"Colour loss during laundering, as opposed to during wear, is a significant source of colour loss.",
"We... | Patent_10450077 |
[
[
"Method for producing acrylate adhesive materials",
"A process for preparing pressure sensitive adhesives based on acrylate hotmelt, in which a monomer mixture including at least the following monomers (a) 70 to 100% by weight of compounds from the group of (meth)acrylic acid and the derivatives thereof c... | Patent_10450365 |
[
[
"Dual-pivot steering system and method",
"A system for guided travel over a surface between parallel guide rails includes a first frame including at least one rotatable element for travel over the surface; a first pin connected to the first frame, for rotation of the first frame thereon; a second frame in... | Patent_10450376 |
[
[
"Dual polarisation antenna",
"A dual polarisation antenna comprising: one or more radiating elements (20); a pair of side walls (11,12) arranged on opposite sides of the radiating element(s); and one or more conductive isolating elements (44, 51, 70, 40, 41), each isolating element being at least partiall... | Patent_10450480 |
[
[
"Sound Reproducing Device",
"The sound-reproducing device according to the first embodiment of the invention comprises a front and back covers fastened together with sheets located therebetween, a sound message reproducing unit, a impact sensors unit and sheet position detecting unit the output of which i... | Patent_10450552 |
[
[
"Considerations, evaluations, investigations and searching",
"Methods of considering DNA based links between two or more situations are provided.",
"Amongst the methods is a method including obtaining a plurality of test results, each test result relating to a situation, each test result including inf... | Patent_10450597 |
[
[
"Microspray column, mass spectrometer, and mass spectrometry",
"A problem is to provide a microspray column capable of increasing the ionization efficiency, and a high sensitive mass spectrometer and a mass spectrometric method using such a microspray column.",
"For solving this problem, we provide a ... | Patent_10450608 |
[
[
"Imaging head with laser diode array and a beam-shaping micro light-pipe array",
"An optical imaging heads that produce a plurality of light spots on light sensitive media such as photographic film or printing plate.",
"The optical head incorporates an array of multi-mode laser diodes as a light sourc... | Patent_10450660 |
[
[
"Focussing of compound libraries using atomic electrotopological values",
"The present invention relates to a method for generating a focussed compound library containing an enriched amount of ligand compounds being capable of binding to a predetermined receptor."
],
[
"1.A method for generating a... | Patent_10450665 |
[
[
"Transmitter and transmission control method ,and receiver and reception control method",
"Radio resources are saved, and power consumption is reduced.",
"A base station 101 carries out training for obtaining correlation information that correlates the reception quality of a packet channel transmitted... | Patent_10450689 |
[
[
"Oxidation dyeing composition for keratinous fibres on amphiphilic polymers of at least an ethylenically unsaturated monomer with sulphonic group and comprising a hydrophobic part",
"The invention concerns an oxidation dyeing composition for keratinous fibres, in particular for human keratinous fibres and... | Patent_10450703 |
[
[
"Traversing firewalls and nats",
"An incoming UDP packet is allowed to traverse a network address translation (NAT) device or a firewall, wherein first, a TCP connection is opened and a Raw-IP interface is utilized to build the UDP-like packet using the parameters of the TCP connection (e.g., session numb... | Patent_10450751 |
[
[
"Novel nucleic acids and polypeptides",
"The present invention provides novel nucleic acids, novel polypeptide sequences encoded by these nucleic acids and uses thereof."
],
[
"1.An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-30368, a... | Patent_10450763 |
[
[
"Novel means for the diagnosis and therapy of ctcl",
"A means for the diagnosis and therapy of T lymphomas, in particular Cutaneous T Cell Lymphomas (“CTCL”) are provided.",
"Tumor markers which are universal for CTCL are described.",
"More particularly, a molecule, termed SC5 by the inventors, an... | Patent_10450818 |
[
[
"Anti-Cd28 Antibody",
"The invention concerns an antibody directed against the CD28 receptor and capable of blocking CD28/B7 interaction, and proteins derived from said antibody, for use in particular to block CD28-dependent activation of lymphocytes."
],
[
"1.A protein capable of binding specific... | Patent_10450832 |
[
[
"Deflector Devices",
"A deflector device (22) for use with a tow line between a seismic survey vessel and a tow, in particular a seismic streamer or streamer array, in the water behind the vessel comprises a vertically oriented wing-shaped body (28) shaped to produce in use a sideways force which urges th... | Patent_10450862 |
[
[
"Methods for the production of multimeric proteins and related compositions",
"Improved methods for the production of multimeric-protein-complexes, such as redox proteins and immunoglobins, in association with oil bodies are described.",
"The redox protein is enzymatically active when prepared in asso... | Patent_10450903 |
[
[
"Adaptive comparator circuit and acoustic distance sensor comprising said circuit",
"The invention relates to an adaptive comparator circuit comprising a comparator with a threshold value input which receives a threshold voltage and a signal input which receives a voltage signal.",
"The threshold valu... | Patent_10450932 |
[
[
"Fuel injector and method for installing a fuel injector in a valve seat",
"A fuel injector, which, at its inflow-side end, has an intake connector at whose outer circumference at least two sealing rings are provided, which seal the fuel injector from a valve seat.",
"Due to an intermediate ring, the ... | Patent_10450961 |
[
[
"PROCESS FOR THE PRODUCTION OF GRAIN ORIENTED ELECTRICAL STEEL STRIPS",
"A process for the production of grain oriented electrical Fe—Si strips in which a Si-containing alloy is directly cast as a strip between 2.5-5.0 mm thick and cold rolled in one stage, or in more stages with intermediate annealing, t... | Patent_10450969 |
[
[
"Generic architecture for adaptable software",
"This invention concerns adaptable software.",
"In particular it concerns a method for constructing an adaptable soft-ware application.",
"A first step involves instantiating a first set of knowledge elements (kitems) related to the type of business o... | Patent_10450974 |
[
[
"Generic knowledge agents",
"This invention concerns a generic knowledge software agent, or kuark, existing within a knowledge environment.",
"The kuark comprises: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark, and a desti... | Patent_10450975 |
[
[
"Support and guiding structure for friction element in disc brakes",
"Method and apparatus for friction element control in spot-type automotive disc brakes provides for use in a disc brake (10) of the kind comprising a fixed caliper (32) and at least one axially slidable disc (12, 14), a friction element ... | Patent_10451031 |
[
[
"Lugged cap forming system",
"Transfer apparatus and method are provided from first (15A, 16A) to second (15B, 16B) stations of tooling in a cap (11) making press.",
"This cap is biased against the first upper tools by a first airstream (50) introduced under the cap and moves upward with the first sta... | Patent_10451057 |
[
[
"Diving aid",
"Diving aid for divers, at least comprising a power supply, alarm sound-generating means (3) and activating means (2) for activating the sound-generating means (3), which activating means (2) are designed in such a way that when the alarm device is in use said activating means can be activat... | Patent_10451113 |
[
[
"Composition comprising an oxidizing and complexing compound",
"The present invention is related to a composition comprising an oxidizing compound and a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.",
"Th... | Patent_10451230 |
[
[
"Level sensor",
"A level sensor (2) comprising a support (6) with a bearing (11) for a lever arm (3) with a float (4) and a guide rail (16) pointing in the direction of the bearing (11).",
"The lever arm (3) has a stirrup (5) made of plastic and a lever wire (7) clipped thereto.",
"The stirrup has... | Patent_10451269 |
[
[
"Inks for in-mould decoration",
"An energy-core ink or varnish composition is provided for use in an in-mould decoration (IMD) process, comprising an energy-curable resin, additional reactive monomers and/or oligomers and, optionally and in an amount not exceeding 10% by weight, a solvent, wherein the res... | Patent_10451279 |
[
[
"Map kinase integrin-binding domain",
"Dislosed is the integrin-binding domain of MAP kinase.",
"Interaction between MAP kinase and integrin via this domain activates MAP kinase and initiates cellular activity.",
"Methods of modulating cellular activity by inhibiting direct MAP kinase-integrin bin... | Patent_10451291 |
[
[
"Modified tachykinin receptors",
"The invention provides a modified tachykinin receptor in which the three amino acids of the DRY sequence that occurs adjacent to the junction of the TM3 domain with intracellular loop 2 are replaced with amino acids whose side chains are neither lipophilic nor contain cha... | Patent_10451304 |