One of the most compelling features of Williams syndrome (WS) is the distinctive social profile that holds promise for understanding the underlying neurogenetic systems that provide meaning to human social interaction. Our studies to date suggest that while individuals with WS typically demonstrate an increased appetitive social drive, the social profile is characterized by dissociations (e.g., overly-friendly with a difficulty in making friends; socially fearless but anxious; positive affect with maladaptive behaviors). The aims of Project V focus on the characterization of the social phenotype of WS, enabling links to the genetic and neurobiological pathways of these dissociations. To this end, the Specific Aims are: Aim 1: Insatiable Appetitive Drive for Approaching Strangers will examine the underpinnings and variability of the increased attraction and approachability towards unfamiliar people observed in individuals with WS. Aim 2: The Unique Salience of Faces will elucidate the nature and underlying mechanisms of the atypically high interest in faces in WS, and its relation to the resultant hypersocial phenotype. Aim 3: Unusual Emotional Sensitivity will investigate both (a) the perception and processing of affect of others by those with WS, and (b) the overall affective style of individuals with WS. Using a multi-method design reflecting multiple levels of explanation (electrophysiology, autonomic function, and eye fixation). Project V studies will produce highly nuanced, quantifiable and independent key dimensions of the unique social behavior characteristic of WS. From a theoretical standpoint, a major thrust of the proposed work is to disentangle the processing of key components of social interaction and their respective underpinnings in the context of the enigmatic, yet paradoxical, WS social phenotype, with the ultimate goal of characterizing the complete system of social behavior and understanding the ways it can break down. Such a multileveled approach has not been previously adopted within this domain of inquiry. Results from these studies will add unique knowledge to our understanding of social behavior, by further defining the pathways implicated in gene-brain-behavior linkages, and are designed to contribute to better-informed treatments.


The ubiquitin-dependent N-end rule pathway relates the in vivo half-life of a protein to the identity of its N-terminal residue. We have previously reported UBR1 and UBR2 as its functionally overlapping E3s and elucidated their in vivo roles using mouse knockout approach. Unexpectedly, although both UBR1 and UBR2 strongly bound to N-degrons, UBR1'^'UBR2^'cells still retained the N-end rule E3 activities, indicating the presence of yet unidentified N-recognins (N-degron-Recognizing E3s). The goal of this study is to identify and characterize N-recognins and their substrates and to elucidate the physiological meaning of their E3-substrate interaction. To identify mammalianN-recognins, we developed a novel affinity-based proteomic approach using synthetic peptides bearing N-degron, yielding a novel 570 kDa-protein named UBR4 and a 300 kDa-E3 ligase EDO (termed UBR5). UBR1, -2, -4, and -5 shared a zinc finger-like domain named the UBR box. Mammalian genome appearsto encode seven UBR proteins, named UBR1 through UBR7. Further, by using a functional proteomic approach, we have obtained -35 candidate N-end rule substrates from -14,000 different proteins expressed in the rabbit reticulocyte lysates. Preliminary characterization of candidate substrates unveiled several novel in vivo N-end rule substrates (RGS4, RGS5, RGS16, and CDC6), the first to be identified in mammals. To further extend our current understanding of the N-end rule pathway, we propose the following Aims. Aim 1. To characterize UBR box proteins as candidate N-recoqnins. We will examine: 1) proteolysis of model N-end rule substrates in UBR mutant cells, 2) the interaction and specificity of UBR box proteins with N-degrons, and 3) in vitro ubiquitylation of model substrates by UBR box proteins. Aim 2. To determine whether UBR box motif is the recognition domain for N-degron. We will determine whether the UBR boxes of N-recognins are required and sufficient for direct binding to N-degrons and identify essential residues for recognition of N-degron. Aim 3. To identify physiological N-end rule substrates. We will dissect proteolysis of candidate substrates in reticulocyte lysates and UBR mutant cells, determine the N-recognin-substrate interaction, and test whether N-recognins support substrate ubiquitylation in vitro. Aim 4. To dissect physiological processes underlying identified N-end rule substrates. We will examine the physiological significance underlying the N-end rule dependent proteolysis of RGS4, -5, and -16, emerging in vivo N-end rule substrates.


High Performance Detectors for SPECT P.I.: Dr. Michael R. Squillante Abstract Single photon emission computed tomography (SPECT) is a powerful, noninvasive medical imaging modality that mathematically reconstructs the three dimensional distribution of a radionuclide throughout the body of a human patient or a research animal. Typically, the collected data are displayed and evaluated as a set of two-dimensional images through the organ or diseased area under investigation. SPECT allows quantitative study of the function in the investigated region and therefore is an extremely useful tool for understanding organ and tissue physiology including that in the heart. SPECT is very commonly used in identifying as well as localizing coronary artery disease and as many as 90% of all myocardial perfusion studies are now performed using SPECT. Thus, SPECT is playing a critical role in cardiac imaging, providing both diagnosis as well as prognosis. However, there is urgent need for improvement in the instrumentation that is currently used for this imaging modality and expand its capabilities in order to exploit its full potential. At present, the performance of SPECT systems often is limited by the detectors used in these systems. Modern SPECT systems consist of scintillation crystals coupled to photomultiplier tubes as detectors. Important requirements for scintillators used in SPECT applications include high light output and high energy resolution, reasonably fast response and high gamma ray stopping efficiency. Ideally, the scintillator should also be inexpensive, rugged and easy to manufacture. Currently, NaI(Tl) is the detector of choice in SPECT systems and it is relatively inexpensive and its light output is fairly large. However, the poor energy resolution of NaI(Tl) often limits SPECT performance. The energy resolution of NaI:Tl is limited by its relatively poor proportionality. If scintillators with higher energy resolution at typical SPECT energies (~140 keV) were available, the essential process of scatter rejection would improve. The goal of the proposed effort is to investigate new high resolution detectors for SPECT studies. Enhanced scatter rejection can be expected from the new detectors, which are also expected to be relatively easy to produce. The Phase I project will be aimed at demonstrating the feasibility of the proposed concept, while the Phase II project will be aimed at optimization of the new detectors, implementation of the prototype modules and detailed performance evaluation. PUBLIC HEALTH RELEVANCE: High Performance Detectors for SPECT P.I.: Dr. Michael R. Squillante Project Narrative - The proposed research will investigate a promising detector technology which should have a major impact in health care, in particular, in the development of detectors for in-vivo imaging. Other areas to which this research could be of benefit are: physics research, materials studies, homeland defense, and non-destructive testing.


Research goals for the coming year: We plan to isolate the endotoxin-binding structures from human platelets and lymphocytes in physico-chemically homogeneous form. We will investigate if both major subpopulations of lymphocytes (B and T cells) have endotoxin-binding properties and if in the affirmative, do they have it to a similar extent. We will determine the minimum number of active binding sites per receptor molecule and define the structures on the receptors which carry the active binding sites for endotoxin. We will determine the mode of action the receptors, especially the extent of reversibility of receptor-endotoxin combination. It is one of our major aims to clearly define the chemical structure of the host receptor sites to which endotoxin attaches. We will use the receptor(s) and active receptor fragments in animals to determine if they prevent fever as well as lethal shock. The undersigned agrees to accept responsibility for the scientific and technical conduct of the project and for provision of required progress reports if a grant is awarded as the result of this application.


Studies of the specificity and efficiency of reactions catalyzed by enzymes and the elucidation of a biological control mechanism are the particular subjects of this proposal. The enzymes chosen are of interest to health-related problems. The serine proteins include enzymes involved in blood clotting, nerve transmission, and digestive processes. Some of these enzymes are used clinically in the treatment of inflammations. A possible role in cancer therapy has recently been suggested for lysozyme, an important enzyme in biological defense mechanisms. BIBLIOGRAPHIC REFERENCES: Rubsamen, H., Hess, G.P., Eldefrawi, A.T., and Eldefrawi, M.E. Biochem. Biophys. Res. Comm. 68, 56-63 (1976) Interaction between calcium and ligand-binding sites of the purified acetylcholine receptor studied by use of a fluorescent lanthanide. Rubsamen, H., Montgomery, M., Hess, G.P., Eldefrawi, A.T., and Eldefrawi, M.E. Biochem. Biophys. Res. Comm. 70, 1020-1027 (1976) Identification of a calcium-binding subunit of the acetylcholine receptor.


The yeast mitotic spindle is less complex than its counterparts in larger eukaryotes and has been intensively studied using genetics, biochemistry, cell biology and ultrastructure approaches, providing an opportunity to develop an understanding of its function and regulation at a level that is not currently achievable in any other organism. The proposed studies are critical for attaining this goal. Correct establishment, function and checkpoint monitoring of kinetochore-microtubule attachments are central to mitotic fidelity. Despite much progress identifying key proteins involved in this physical attachment, and assessing their activities in vivo and in vitro, little is understood about mechanisms regulating attachments, about how microtubule-binding outer kinetochore proteins associate with the central kinetochore, or about emergent properties that result when separate subcomplexes like Dam1 and Ndc80 are together in kinetochores. Building upon discovery and analysis of the Dam1 complex, and structure studies of the Ndc80 complex, new studies will identify their binding partners, determine how Dam1 and Ndc80 complex structures relate to function, how post-translational modifications and binding partners affect function, and how ensembles of kinetochore complexes interact dynamically with microtubules. Previous discoveries that Aurora kinase regulates kinetochore attachment via Dam1 complex phosphorylation, identification of an Aurora kinase consensus site, identification of the fourth yeast Aurora kinase complex subunit, and novel implication of casein kinase 2 in inner kinetochore regulation, provide a robust foundation for studies to reveal how protein kinases regulate critical mitotic functions. Proposed studies will determine how the Aurora kinase complex is targeted to specific cellular locations, will identify its targets at each location, and will determine how phosphorylation affects activity of its targets. Molecular genetics and biochemical analysis of the fully reconstituted, four-protein Aurora kinase complex will determine how this complex is regulated. Having recently identified casein kinase 2 as a mitotic regulator of the inner kinetochore protein Ndc10 and the widely conserved Mif2 (CENP-C) linker protein, and obtained in vivo evidence for dual-regulation of these proteins by CK2 and Aurora kinase, powerful in vivo and in vitro tests of how these two kinases regulate kinetochore function will be conducted. Thanks to unique advantages of yeast, strong inroads into a full molecular dissection of mitotic spindle disassembly have already been made, and will be built upon to fully elucidate pathways and mechanisms. As cells exit mitosis, the enormously complex mitotic spindle must be disassembled rapidly, which involves taking apart stabilized microtubule bundles, disassembling protein subcomplexes, reversing post-translational modifications and destroying proteins. Because spindle disassembly mechanisms are largely unexplored, this is an extremely fertile and important area for investigation. Comprehensive genetic screens and interaction analyses will identify genes and pathways, and focused phenotypic and biochemical studies will reveal detailed mechanisms. PUBLIC HEALTH RELEVANCE: These studies will increase understanding of fundamental aspects of mitotic spindle function and regulation, and since many mitotic proteins and mechanisms are evolutionarily conserved, will provide a framework for elucidating mitotic mechanisms in humans. Chromosome instability is a key contributing factor in cancer and birth defects. Therefore, understanding principles of spindle function may suggest novel strategies for prevention, detection and treatment of human diseases.


Alpha1-Antitrypsin (alpha1-AT) is the major proteinase inhibitor in the human plasma, inhibiting pancreatic elastase, trypsin, chymotrypsin and related proteinases. An important relationship between plasma levels of alpha1-AT and two pathological conditions have been observed in humans: unusually low levels of alpha1-AT are found in the plasma of patients with an adult form of pulmonary emphysema and in a childhood form of cirrhosis of the liver. Moreover, an accumulation of alpha1-AT or alpha1-AT-like material have been noted in the livers of these patients, suggesting the glycoprotein is being synthesized but not secreted into plasma. One likely explanation of the problem of transport is the occurrence of abnormality in the attachment and completion of the carbohydrate moiety to the polypeptide chain of alpha1-AT. We have purified, characterized and determined partially the amino acid sequence of alpha1-AT (PiMM) from plasma of normal individuals. We have also studied its mechanism of proteinase inhibitory activity, as well as its survival in animal circulation after removal of sialic acid residues. We propose to perform similar studies on alpha1-At from normal liver (PiMM) and cirrhotic livers (PiZZ). These studies will include determinations of molecular weight, electrophoretic mobility, isoelectric point, amino acid and carbohydrate compositions, peptide mapping, proteinase inhibitory activities, and survival in plasma. The nature of the bond linking carbohydrate unit(s) to the peptide chain will be studied. Ultimately, the amino acid and carbohydrate sequence will be performed. Characterization of the carbohydrate moiety will be of utmost importance since, as in other glycoproteins, it may be vitally important in the transport or survival of the inhibitor in plasma. From these studies, we hope to gain an insight into the relationship between the function and structure of alpha1-At and thus better understand the pathologies of pulmonary emphysema and cirrhosis of the liver on a molecular level.


The extracellular adenosine receptors have a modulatory role in the nervous, circulatory, endocrine and immunological systems. The prospect of harnessing these effects specifically for therapeutic purposes is attractive. We have synthesized the first selective A3 adenosine receptor agonists and antagonists. We recently characterized the first allosteric modulators of this receptor. Imidazoquinoline and pyridinylisoquinoline derivatives were found to enhance the actions of agonists of the A3 receptor, and thus may prove to be suitable leads for the development of therapeutic agents based on this concept. A3 agonists are under development for treating cancer, rheumatoid arthritis, and other diseases. Allosteric modulators of G-protein coupled receptors are a new approach to receptor activation without the limitations of use of directly-acting agonists as therapeutic agents. We have identified two new classes of allosteric modulators of the A3 receptor and are currently exploring the structure activity relationship (SAR).[unreadable] [unreadable] The potential of A3 agonist therapy is of great interest. We are collaborating with Dr. Bruce Liang and Dr. Asher Shainberg on various aspects of the use of adenosine receptor agonists in protection of the heart. We have designed a mixed agonist of A1 and A3 subtypes, both of which are protective in the heart. This mixed agonist MRS3630 is protective in a model of ischemia in the isolated perfused mouse heart. The adenosine A3 agonist IB-MECA is currently in clinical trials for use in cancer treatment by our CRADA partner Can-Fite Biopharma. [unreadable] [unreadable] One of the issues in the development of adenosine receptor ligands is the species dependence. Some compounds that are very potent at a given human adenosine receptor are weak in rat tissue. We have developed adenosine agonists and antagonists that work generally across species. This is important for testing the same compounds in various animal models before suggesting the feasablity of their use in humans. The key to A3 receptor ligands that are potent and selective across species is the use of the nucleoside structure as the starting point in the design process. Nucleosides tend to bind to that receptor subtype with greater consistency across species than nonpurine heterocycles.[unreadable] [unreadable] Adenosine A3 antagonists may be useful for the treatment of glaucoma. Early efforts to identify antagonists of the A3 receptor in our library involved screening of chemically diverse libraries. One of the limitations of this approach is that the antagonists often bind well only at the human, but not murine A3 receptors. We are currently developing other novel A3 antagonists based on nucleotide structures, that have proven to be generally applicable across species. We are currently studying systematically the SAR of adenosine derivatives that affect efficacy as A3 adenosine receptor agonists. Surprisingly, a commonly used A1-selective agonist, cyclopentyladenosine, was found to act as a pure antagonist at the A3 subtype. Other nucleosides may be chemically modified, especially on the ribose moiety, to have reduced efficacy at the A3 receptor. Some of these analogues derived from highly potent A3 agonists, such as 5-dimethylamides, were found to be A3 antagonists. A novel nucleoside-based antagonist of the A3 receptor MRS1292 was found to lower intraocular pressure a mouse model of glaucoma.[unreadable] [unreadable] We are using mutagenesis to study the determinants of recognition of adenosine within the binding site of the A2A and A3 receptors, and proposing conformational factors involved in receptor activation. Since the four subtypes of adenosine receptors have been cloned it has been possible to conduct molecular modeling of the receptor protein, based on sequence analyses and homology modeling using the high resolution rhodopsin structure as template. We intend to use such a modeling approach for the design of more selective adenosine receptor agonists and antagonists. [unreadable] [unreadable] Recently this project has also focused on the effects of adenosine agonists and antagonists in the central nervous system and in the heart and on the possibility of therapeutics for treating neurodegenerative and cardiovascular diseases. An A3 agonist, administered chronically, proved to be highly cerebroprotective in an ischemic model in gerbils. A3 agonists cause morphological and biochemical changes in astroglial cells. Adenosine is released in large amounts during myocardial ischemia and is capable of activating both A1 or A3 receptors that occur on cardiac myocytes to exert a potent cardioprotective effect. We have shown that synthetic adenosine agonists,selective for either the A1 or A3 subtype, protect ischemic cardiac myocytes in culture and in the isolated perfused heart and thus might be beneficial to the survival of the ischemic heart. An acutely administered A3 agonist, Cl-IB-MECA, was cardioprotective in cell culture, through the selective activation of A3 receptors without side effects, such as bradycardia, associated with the A1 subtype. The protection was blocked in the presence of a selective A3 receptor antagonist. Inn summary, highly selective adenosine analogues may have therapeutic potential in treatment of cerebral ischemiastroke and possibly other neurodegenerative disorders as well. It is proposed that modulation of A2B and A3 receptors may be useful in treating asthma and inflammatory diseases. The pharmacolgical properties of novel xanthines developed in our lab that act as selective A2B receptor antagonists are being explored as potential antidiabetic and antiasthamtic agents. The first highly selective A2B receptor antagonist, synthesized in our section, has now been radiolabeled and is used in assaying newly synthesized analogues for affinity at the A2B receptor. We are also screening chemical libraries for novel leads for A2B receptor antagonists.


DESCRIPTION (taken from the application: The concept of orthopaedic research encompasses a very broad area spanning the field of musculoskeletal research from the clinical aspect to both biological and biomechanical basic sciences. The Bioengineering and Orthopaedic Science Gordon Research Conference has been the premier conference for investigators to combine the three disciplines of orthopaedic sciences. The format of these conference, held in a small private school in New Hampshire every other summer, provides a unique environment for the exchange of new and unpublished information. The cross fertilization that can only come from a conference were clinicians, engineers and biologists meet, is more vital to this field than ever before. For the first time, biological principles are being used in the treatment of orthopaedic injuries and diseases and these treatments must confirm to and meet the biomechanical needs of the skeleton. For example, investigators and clinicians are now beginning to utilize molecules such as parathyroid hormone; parathyroid hormone related peptide, insulin-like growth factors, fibroblast growth factor and the hedgehogs. In addition, physical parameters such as ultrasounds and electrical stimulation are currently being used to stimulate bone growth. Bioengineering has moved from the mechanical design of joint implants to the problems associated with long-term use of implant materials and a general understanding of the biological effects of these materials in the living system. For the first time, a session will be held on skeletal tumors. Two noteworthy changes will be made in the format from previous meetings. In the comments on the 1996 Gordon Conference Program, many attendees felt that the junior members of the audience did not feel comfortable speaking out and that the discussion were dominated by only a few senior scientists. In an effort to increase the comfort level of the junior scientists and to increase their participation in general, the following changes were implemented: (1) five to ten scholarships will be designated for junior faculty and postdoctoral fellows and their availability will be announced in conference literature; (2) One evening will be dedicated to talks by junior scientists. Employing these options to encourage the participation of junior scientists has worked very well for other Gordon Conferences such as the Proteoglycan Gordon Conference which uses the scholarship mechanism and Molecular Genetics which uses the junior scientists evening option.


The inflammatory agents, histamine, bradykinin, and serotoin have been shown to increase capillary membrane transport in various tissues. Other studies have suggested a dependence of capillary membrane transport upon vascular calcium concentration. Evidence has been given that both effects are caused by widening of the capillary membrane inter-endothelial clefts. A logical hypothesis is that inflammatory agents alter capillary transport by interfering with calcium in the membrane, thus altering cell-cell contracts. We will test this hypothesis by: 1) quantifying the dependence of capillary membrane transport parameters upon inflammatory agents, 2) quantifying the dependence of transport parameters upon vascular calium concentratin, 3) testing for antagonism of inflammtory agent on transport parameters by calcium, 4) testing for alteration of calcium binding to the capillary endothelium by inflammatory agents, and 5) testing the effect of calcium channel blockers on alteration of transport parameters by inflammtory agents. The experimental preparation is the isolated, perfused (Langendorff) rabbit heart. Measurement of capillary membrane transport parameters (permeability coefficient, filtration coefficient, reflection coefficient) to several nonelectrolytes (urea, sucrose, inulin) will be made by the method of osmotic transients. Calcium binding by capillary endothelium will be observed under electron microscopy using ionic lanthanum as a marker for calcium binding sites. Long-term objectives of the project are to shed light on how normal intercellular junctions are maintained and to point out possible mechanisms for reversing the deleterious effects of inflammatory agents, e.g., edema formation. With particular reference to the heart, edema formation is known to be detrimental to cardiac function and, further, reversal of edema is associated with improvement of that function. If release of inflammatory agents during cardiac damage or shock states contributes to edema and associated reduction in cardiac performance, knowledge of the mechanism of action of inflammatory agents at the capillary level may prove beneficial to its treatment.


The wild-type Rep proteins of adeno-associated virus type 2 (AAV) have been demonstrated in tissue culture to inhibit the replication of HIV-1, the etiological agent of AIDS. Unfortunately, their potential as a therapeutic agent for AIDS is limited because they also appear to block cell division. Several groups have tried and failed to produce cell lines producing high levels of Rep proteins, although we have achieved high levels in transient expression assays in cells transfected with a plasmid expressing the rep gene from the long terminal repeat promoter of HIV-1 (HIV-LTR). Our objective is to create a mutant Rep protein which can block HIV-1 replication without blocking cell division. We have made a series of mutant rep genes expressed from the HIV-LTR. Our collaborators at the New Jersey Center for Advanced Biotechnology and Medicine have already tested several of these mutants for their ability to block HIV-1 replication. Plasmids containing the mutant rep genes are co-transfected with an infectious proviral clone of HIV-1 into human cells and the cells and culture supernatants are assayed for reverse transcriptase activity, HIV-1 RNA and for HIV-1 gag protein. We have identified several portions of the Rep proteins which are important for this inhibition. Several of these mutants inhibit HIV-1 better than the wild-type proteins. If we can identify a mutant Rep protein that blocks HIV-1 production without blocking cell division then we would have the basis for a novel treatment for AIDS. If Rep proteins truly block cell division then they may also be developed into a treatment for cancer. We will continue to produce and test mutant Rep proteins for HIV- inhibition. We will be concurrently attempting to produce cell lines making these mutant proteins. This will allow us to determine which parts of the Rep proteins are involved in the putative cell division block.


The current thrust of our studies of neonatal drug metabolism lie in the area of transplacental carcinogenesis. The model used is the administration late in pregnancy of 3-methylcholanthrene to pregnant mice. It is known that the offspring of such animals have a high incidence of tumors during late adulthood. We plan to evaluate mixed- function oxidase activity in various organs during different developmental stages of such animals. Also studied will be the response of these animals to inducers such as phenobarbital and 3- methylcholanthrene. Another phase of our studies will be to examine the effect of exposure of rats to intense blue light on microsomal drug metabolism in liver and skin. These studies are designed to evaluate possible responses to high intensity light such as those encountered during the clinical practice of phototherapy of jaundiced infants. Bibliographic references: Lampert, R.P., Robinson, D.S., and Soyka, L.F.: A critical look at oral decongestants. Pediatrics 55:550-552, 1975; Soyka, L.F., Robinson, D.S., Lachant, N., and Monaco, J.: The misuse of antibiotics for treatment of upper respiratory tract infections in children. Pediatrics 55:552-556, 1975.


Autonomic dysfunction including orthostatic hypertension (OH) is a major health problem, causing significant morbidity and mortality. Its pathophysiology remains poorly understood and hence its management lacks a solid scientific base. The PPG focuses on the pathophysiology and treatment of autonomic failure. Project 1 (Low) incorporates a novel strategy of cholinesterase inhibition in the treatment of OH, an approach that promises to improve OH without supine hypertension. A second blinded treatment trial will evaluate if sodium chloride will expand plasma volume and if urinary sodium excretion is a suitable surrogate measure of plasma volume status. A series of studies, including the use of microneurography to measure sympathetic impuls3es, will evaluate the pathophysiology of postural tachycardia syndrome (POTS). A novel approach of amplitude modulation of the EEG in POTS shows a selective reduction of a frequency band of 0.02-0.05 Hz; this component is of particular interest since it may have a brainstem origin. The venous capacitance bed will be evaluated (Projects) to determine if there is excessive transcapillary efflux and changes in compliance in POTS and the effects of aging. The relative importance of the mesenteric, systematic and cerebrovascular circulations in OH will be evaluated. Project (Benarroch) will expand its studies on the neurochemical organization of autonomic control regions of the medulla in multiple system atrophy (MSA) and the parkinsonian syndromes. These include quantitative evaluates of new cellular groups (nucleus ambiguus, nuclease retroambiguus) and new receptors (including angiotensin II) that are likely to provide insights into the pathophysiology of autonomic failure in MSA. Project (Joyner) will undertake a detailed evaluation of the effects of denervation (mild in POTS and severe in neurogenic OH) and aging on the venous capacity and compliance. Project (Brimijoin) will focus on the response of the pre-ganglionic neuron to denervation and will study the mechanism of spinal intermediolateral column cell loss, using he model of immune-mediated pre-ganglionic autonomic neuropathy. The roles of apoptosis, excitotoxicity, growth factors, and aging will be evaluated and related to MSA.


DESCRIPTION (from abstract) Many neuroendocrine peptides are synthesized as precursor proteins; post-translational processing of these precursors is a key step in the production of biologically active peptides. The enzymes involved in this processing are important regulators of these intercellular messengers. A variety of endopeptidases and exopeptidases have been proposed to be involved in neuropeptide processing. In this grant application the applicants describe studies to examine the contribution of various processing enzymes in the generation of opioid peptides using transgenic animals and cell lines. The first specific aim is to examine dynorphin processing in animal models that lack specific peptide processing enzymes. They will assess the extent of involvement of carboxypeptidase E (CPE) in dynorphin processing by comparing the profiles of various dynorphin peptides in fat/fat mice (lacking active CPE) with those in control mice. In addition, they will assess the extent of involvement of prohormone convertase 2 (PC2) in dynorphin processing by comparing the profiles in PC2 K/O mice (that lack PC2) with those in control mice. The second specific aim is to examine the contribution of various peptide processing enzymes to dynorphin biosynthesis using a cell culture system that inefficiently processes dynorphin. The investigators will examine whether a dynorphin processing defect in NIT-3 cells (that lack CPE) can be overcome by endocrine carboxypeptidases or by prohormone convertases. These studies will provide information about the interactions of various processing enzymes that could modulate their function. The studies proposed in this grant application will address the physiological role of the processing enzymes in a neuropeptide biosynthesis. The information gained through these studies will provide insights into the multiple regulatory steps (including cross-talk between enzymes) that govern neuropeptide processing which cold modulate the levels of neuropeptides in normal an dysfunctional cellular events such as those seen in several neurological disorders.


The proposed research aims to elucidate the psychosocial and behavioral characteristics of female adolescents who qualify for DSM-III-R diagnosis of Psychoactive Substance Use Disorder (PSUD) as a relatively simple disorder and comorbid Psychoactive Substance Use Disorders and Conduct Disorder (PSUD/CD) as a more complex drug abuse disorder. The latter group of PSUD/CD individuals is at particularly high risk for multiple personal and social problems (e.g., teenage pregnancy, AIDS, prostitution, sexually transmitted diseases, drug teratogenicity). The specific aims of this research are to: 1) identify the biobehavioral characteristics, psychosocial correlates, family liabilities and differential dyadic interaction patterns between parent and adolescent female offspring and; 2) determine the relationship among age of pubertal onset, sexual activity and drug use. In order to accomplish these objectives, five groups of female subjects will be studied: 1) Adolescents with PSUD and no comorbid diagnosis; 2) Adolescents with PSUD/CD; 3) Adolescents with CD only; 4) Control normal adolescents with out PSUD and CD or any other psychiatric disorder, and, 5) Older sisters of the PSUD/CD and PSUD female who do not present PSUD. The results of this study will elucidate the factors associated specifically with a PSUD outcome in female adolescents, identify factors which could potentially protect against a PSUD outcome, and reveal the processes mediating the progression or development from drug use to a diagnosis of PSUD.


The major goal of this Resource is to construct high-resolution expression and functional profiles for orphan receptors in order to elucidate the basis of their tissue and cell-specific functions. Although there are many reports describing the expression patterns of many individual orphans at a given developmental stage or in a specific tissue, systematic analyses of the expression of individual orphans at all stages of development, in all cell types, have yet to be undertaken. It will therefore be important for the proposed Atlas, through this Resource, to rigorously examine the expression pattern of an initial subset of ONRs at the cellular level. The expression profiles of the selected ONRs in all cell types of different organs will complement the expression profiling of ONRs in all organs proposed in Strand A. In addition, we will use transgenic models to determine the spatiotemporal functional profiles for ONRs. These models will help to clarify the role of ligands, regulators and co-regulators in modulating ONR function, and to better understand the developmental, physiological and metabolic roles of these receptors. The transgenic models and the expression of functional profiles will be distributed by this Resource and Bioinformatics Resource to all investigators in the field to facilitate future investigations of various aspects of ONR biology, including the identification of ligands, upstream regulators, co-factors and signaling pathways that modulate the function of the ONRs. This Resource has two objectives 1. Determination of the cell-specific expression profiles of orphan nuclear receptors. To generate transgenic mouse lines which will faithfully recapitulate the in vivo expression of orphan receptors, we plan to use an orphan receptor gene promoter-EGFP system in the context of bacterial artificial chromosomes (BACs) to map the native expression of these orphans. To construct individual functional maps of orphan nuclear receptors in vivo, we will generate BAC-based transgenic models that utilize a functional chimeric receptor and an integrated responsive reporter, EGFP. These BAC transgenic approaches will be used to directly determine the expression and functional profiles of orphan receptors. The goals of the Resource, although long term, are consonant with the original mandate of accelerate the achievement of the long term goals of the Atlas, and acting as a Resource for the entire ONR research community.


Cells of hibernating animals are compared with those of mammals which do not hibernate to examine basic mechanisms of ion transport and interactions of transport mechanisms which ensure cellular ionic balance. The principal adaptation of hibernators studied so far has been the ability of their cells to function at low temperature. Past effort has documented the involvement of the Na/K pump and of general leak in this adaptation in several tissues (kidney, erythrocytes, brain, liver). Future efforts will focus on erythrocytes. Our aim is to define the points of failure in the cold in terms of the pump's kinetics and its overall scheme in cold-sensitive cells of non- hibernators and to exploit unusual features of the pump in hibernator cells for a better understanding of pumps in general. The second objective is to define the differences in the several components of passive permeability to Na between cold-sensitive and cold-tolerant red cells that permit ion balance with reduced active transport at low temperature and to determine how cytoplasmic factors may be involved in bringing pumps and leaks into balance under widely changing conditions. Another adaptation of many hibernators is seasonal inanition and self-starvation. Some species, such as bears and prairie dogs, exhibit this ability even without the concomittant lowering of metabolism by cooling. Studies in human and mammalian red cells have suggested that reduction in energy-dependent membrane transport may be a factor in the energy conservation of the cell during starvation, but this has not been examined in these species which specialize in starvation-adaptation and previous studies have not considered the contribution of reduced permeability as a possibility. We propose to do this. The perceived areas of health-relatedness are, for the low temperature studies, improved viability of cells during storage and, for the seasonal/inantion studies, cellular adaptation to and effects of under-nutrition.


Immune responses are dependent on the coordinated movement of leukocytes from sites of antigen deposition, to secondary lymphoid organs and to sites of infection or inflammation. Chemokines play a critical role in this process by directing leukocyte trafficking throughout the body. Although it is well known that leukocytes express chemokine receptors and can migrate directionally in response to chemokine gradients, the molecular mechanism(s) that control chemokine receptor responses are still largely unknown. CD38, an ADP-ribosyl cyclase, that catalyzes the production of the calcium-mobilizing metabolite cyclic ADP-ribose (cADPR), appears to be a critical regulator of chemokine receptor signaling and leukocyte trafficking. We observed that neutrophil migration is impaired in CD38-deficientmice resulting in attenuated and reduced inflammatory responses. We also found that the cADPPR produced byCD38 modulates calcium mobilization in neutrophils that have been activated with inflammatory chemoattractants such as peptides derived from bacteria and viruses. Furthermore, we showed that cADPR-specific antagonists block the migratory response of neutrophils to these peptides. The data suggest that small molecule inhibitors of CD38could potentially be used to block neutrophil-dependent inflammatory responses. Recently, we observed that the migratory response of dendritic cells is also impaired in CD38 deficient mice. Specifically, we found that CD38-deficient dendritic cells cannot migrate in response to ELC or SLC, chemokines that direct dendritic cells to migrate from sites of damage or injury to lymphoid tissues. This impaired chemotactic response observed inCD38-deficient dendritic cells results in inefficient T cell priming and significantly reduced T cell-dependent immune responses. Based on our previous data, we now hypothesize that CD38, through its production of cADPR, regulates cell-dependent immune responses by modulating the migration of dendritic cells. To test this hypothesis we have proposed the following Specific Aims: (1) we will determine whether CD38 regulates migration of all mature dendritic cell subsets to ELC or SLC, (2) we will determine whether CD38 regulates the migration of dendritic cells to inflammatory chemoattractants and (3) we will determine whether cADPR production by CD38 controls dendritic cell trafficking in vivo. These experiments will validate whether CD38 antagonists have the potential to be used as immunosuppressive agents that attenuate immune responses by modulating leukocyte trafficking.


Most HIV seropositive persons show humoral and cell-mediated immune responses to HIv antigens and in particular to the env gene products of HIV, the external envelope glycoprotein gp120 (EEG). the overall goal of this proposal is to define the role of the carbohydrates of the external envelope glycoprotein in affecting the immune response to the virus. The simian immunodeficiency virus/rhesus macaque model system will be used to focus on a variety of humoral and cell-mediated immune responses to the wild-type EEG and selected mutant and variant EEGs of the cloned SIVmac239 strain of the rhesus macaque simian immunodeficiency virus. Variant and mutant EEGs will be produced using oligonucleotide-mediated site-directed mutagenesis of the EEg DNA sequence and B' mutations in the pathway of protein glycosylation (Objective 1). Purified EEGs will characterized based on their structural and functional properties (Objective 2), and after immunization of small animals with selected mutant and variant glycoproteins the specific immune responses will be assessed (Objective 3). The strength, nature and extent of the responses will be compared with those induced by the wild-type EEg and will be correlated with the carbohydrate structure of the immunogen. The ability of mutant and variant EEG to provide protection from SIV infection will be determined (Objective 4), by challenging immunized monkeys with infectious clones of SIVmac 239 and 251 and with extracts that produce AIDs-like symptoms to determine whether the immune responses are broadly protective. To enhance development of cell mediated immunity, genes specifying the appropriate EEGs will be used to construct replicating simian adenovirus.SIVenv expression vectors, as has been done previously for an HIV env-adenovirus vector. These recombinants will be evaluated as potential vaccine candidates.


This supplementary request for the above named core function under the immediate supervision of Dr. Walter Korytnyk is being submitted for consideration and, hopefully, funding. This collaborative effort involves a number (8) of ongoing projects from groups of investigators as well as many (12) anticipated projects. All of these interests have in common the requirement that the nuclear magnetic resonance spectrometer is essential for the conduct of these individual research proposals. The instrument (NMR) has been purchased with other funds, but in order to function as a core facility we require the personnel and support to effectively operate it. The Core Grant Committee, composed of: Dr. Lucius F. Sinks, Chairman; Dr. Gabor Markus; Dr. Enrico Mihich; Dr. Arnold Mittelman; Dr. E.A. Mirand (Ex Officio), has met with the principal investigator (Dr. Gerald P. Murphy) and Dr. Walter Korytnyk, and have reviewed the proposal herein contained. We feel that the request as written represents a legitimate core function for activities conducted at RPMI and respectfully request that it be considered as a supplement to the present core grant.


This project aims at the visualization via MRI of freezinginterfaces in ice-water mixtures. Specifically, the in-situvisualization of frost and of freezing in porous media will bepursued. MRI is the only solution possible in such systems thatare strongly refracting to light. Attendant MRI thermometrystudies will also be pursued.


This research is directed toward elucidation of the enzymatic mechanisms involved in the assimilatory reduction of nitrate to ammonia. The significance of this pathway is manifest in that the various oxidized inorganic forms of nitrogen provide the ultimate nitrogen source for all life. Nitrate assimilation is a two-step process: the two-electron reduction of nitrate to nitrite, followed by the six-electron reduction of nitrite to ammonia. The enzymes catalyzing these reaction in Neurospora crassa, namely nitrate reductase and nitrite reductase, are adaptively formed in the presence of either NO3 or NO2. Their synthesis is repressed by NH plus 4. Nitrate reductase is a soluble, sulfhydryl-containing molybdoflavohemoprotein, mediating the NADPH-dependent reduction of nitrate via the electron transfer sequence: NADPH yields ((-SH) yields FAD yields cytochrome b-557 yields (Mo)) yields NO3. The nitrite thus formed is stoichiometrically reduced to ammonia by nitrite reductase, also a soluble, sulfhydryl-containing flavo-protein, possessing non-heme iron/sulfur centers and the novel iron tetrahydroporphyrin, siroheme, as prosthetic groups. The suggested electron transfer sequence is: 3NAD (P) H yields ((-SH),FAD, (Fe.S) yields siroheme) yields NO2. The prime objective at this point involves elucidation of the electron transfer catalysis mediated by nitrite reductase through UV/visible spectrophotometric and EPR spectroscopic observations, kinetic studies, and chemical and physical analyses of the nitrite reductase protein and its complement of prosthetic groups. Integration of these results should provide for reliable conclusions regarding the important relationships between structure and function in this multi-electron transferring homodimeric enzyme. The subunit organization of the nitrate reductase and the form and function of its molybdenum cofactor in this organization is also under investigation.


The major objective of the proposed research is the development of an in vitro model for exocytosis. Preliminary investigations indicate that the ways in which Ca2 ion and ATP are involved in this cellular process can be determined from study of the interactions of these molecules with isolated secretion granules. Submillimolar concentrations of Ca2 ion induce total lysis of suspensions of secretion granules isolated from acinar cells of the rabbit parotid. I have identified two processes which precede lysis: 1) the Ca2 ion-specific activation of a granule-associated phospholipase A2 and 2) the large-scale rearrangement of granule membrane proteins (viewed as intramembranous particles in freeze fracture preparations) into interconnecting linear assays. I intend to evaluate the extent to which granule lysis in vitro correlates with exocytosis in vivo and to investigate the relationship of phospholipase activity to granule lysis in a series of comparative kinetic studies. Lysis will be determined by the extent of release of alpha-amylase, a secretory protein found in the granule content. Phospholipase activity will be measured by quantitating the hydrolyzed phospholipids resolved by thin layer chromatography. Membrane protein rearrangements induced by Ca2 ion will be characterized by electron microscopy of freeze fractured preparations. Provided the linear arrays can be stabilized, the molecular species involved will be identified by techniques employing covalent crosslinking and spin labeling.


Abdominal aortic aneurysms (AAA) occur commonly in the elderly population and are a major cause of morbidity and mortality. The mechanisms responsible for aneurysm formation are poorly understood, which has impeded identification of effective medical therapies for this disease. Mounting evidence from studies in human AAA, and in experimental animal models, suggests that oxidative stress plays a key role in the pathogenesis of AAA. While a number of enzymatic pathways are capable of inducing vascular oxidant stress and potentially contributing to AAA, we hypothesis that myeloperoxidase (MPO) is instrumental in this process. MPO is an enzyme expressed primarily in neutrophils (PMNs) and to a lesser extent in monocytes/macrophages, that catalyzes the formation of HOCl, a powerful oxidizing specie, and induces protein nitration. In addition, MPO can be taken up into the blood vessel wall, thus amplifying inflammation, oxidative stress and protease degradation. Using two distinct murine models (elastase-induced and angiotensin II infusion in hyperlipidemic mice), we present data showing that aortic MPO activity and chlorotyrosine expression (a marker of HOCl-mediated oxidative stress) are increased during experimental AAA formation. Our data also suggest a potentially novel role for angiotensin II to exacerbate HOCl-mediated oxidative stress in the blood vessel wall. Moreover, supplementation with taurine, a beta amino acid that reacts with and detoxifies HOCl, prevents AAA formation in these experimental models. To test our hypothesis, we propose three specific aims. In aim 1, we will perform selective immunodepletion and adoptive transfer of PMNs from control or MPO- deficient mice.to test the hypothesis that MPO expression in PMNs contributes to experimental AAA formation in the elastase model. Also, we will immnuodeplete PMNs to determine their role in AAA formation in the angiotensin II infusion model. In aim 2, we will test the hypothesis that genetic deficiency of MPO ameliorates AAA formation in the elastase-induced and angiotensin II infusion models. In aim 3, we will test the hypothesis that transgenic expression of human MPO augments experimental AAA formation. In aims 2 and 3, we will also determine whether angiotensin II upregulates expression and activity of MPO in leukocytes, and whether MPO modulates hypertension and atherosclerosis induced by angiotensin II infusion. Our studies will provide novel insight into mechanisms of AAA formation and linkages between key oxidant stress generating pathways in the pathogenesis of cardiovascular disease.


The overall objective of this project is to obtain a 10 year update and extension of the 1995 National Survey of Adolescents (NSA) by conducting a three wave, longitudinal survey of a new national household probability sample of 12-17 year-old adolescents. The original NSA was conducted via telephone with a national household probability sample of 4,023 male and female adolescents between the ages of 12 and 17. The methods to be used for the proposed project are deliberately modeled after the original NSA. The proposed project will involve a baseline survey of a new cohort of 4,000 12-17 year-olds using identical sample selection and interview procedures as that in the original NSA. The proposed project will also include two additional assessments of adolescent participants to be conducted one and two years after the baseline assessment. As was the case with the odginal NSA, a structured interview will be used to measure a broad range of exposure to witnessed and direct violence victimization (e.g., sexual assault, physical assault, and physically abuse punishment) occurring in family, school, and community settings. Several types of mental health problems will be measured, including PTSD, major depression, suicide ideation and attempts, substance use and abuse, and delinquency. The study will also obtain information from parents as well as adolescents regarding academic performance. The proposed project will provide invaluable comparisons about changes over the past decade in violence exposure, substance use, and related adjustment issues among American youth. Although modeled after the NSA, the proposed project departs from the original in several important respects. First, the assessment of violence exposure in the proposed study is broader and more specific; specifically, assessment of physically abusive punishment will be expanded, and there will be a more comprehensive focus on episodes of school, domestic, and community violence to permit greater description of adolescents' exposure to violence in each of these important settings. Second, the range of outcomes assessed will be diversified beyond mental health, substance abuse, and delinquency to include academic performance, an important developmental marker of child adjustment, as well as risky sexual behaviors. Third, the proposed survey methodology is longitudinal. Thus, specific aims of the proposed project are: 1)To obtain prevalence rates and descriptive data regarding adolescents' exposure to violence (both direct and witnessed) across a range of settings (school, home, community), including sexual assault, physical assault, physically abusive punishment, domestic violence, community violence and school violence; 2) To determine whether there have been changes among a nationally representative sample of 12-17 year olds over the 1995-2005 period with respect to the prevalence of exposure to violence, mental health problems, and risk or protective factors for a range of adverse social and mental health outcomes given exposure to violence;3) To examine the longitudinal trajectory of exposure to violence, development of mental health and substance use, abuse problems, risky sexual behaviors, delinquency and changes in academic performance;4) To test the hypothesis that the relationship between adverse family environment and the outcomes of delinquency and poor academic performance are substantially mediated by exposure to violence and/or violence-related mental health problems; and 5)To test the extent to which the relationships described above are moderated by gender, race, and ethnicity.


Translation of mRNA into protein is thought of as occuring in three stages: initiation, elongaton, and termination. During initiation, the mRNA to be translated and the initiator tRNA are aligned on the 70S ribosome so that peptide synthesis can begin. Three protein factors (IF1, IF2, and IF3), a molecule of GTP, mRNA and fmet-tRNA are required for initiation. In this research, complexes that correspond to intermediates in the initiation pathway will be formed in vitro and probed with hydroxyl radicals generated by both free Fe(II)-EDTA and Fe(II)-EDTA tethered to unique sites on the initiation factors. The sites of protection from hydroxyl radicals and specific cleavages of the rRNA, mRNA, and tRNA will be identified by primer extension. Together, the results of these probing experiments should provide (1) a comprehensive mapping of the RNA environment of each of these factors, (2) sufficient protein-RNA interaction constraints to orient each of the factors with respect to the other components of the initiation complexes to be studied, and (3) insight into the mechanism by which the initiation factors prepare the ribosome to begin protein synthesis.


Brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) play a crucial role in neuronal plasticity and survival. Increasing evidence suggests that deficient BDNF-TrkB signaling may mediate amyloid- (?)-induced neurotoxicity, synaptic dysfunction and memory deficits in Alzheimer's disease (AD). However, it has not been examined whether BDNF-TrkB dysfunction may also contribute to pathogenic mechanisms upstream to A? accumulation. Interestingly, our recent study demonstrated that the selective TrkB agonist 7,8-dihydroxyflavone not only restores impaired memory and TrkB signaling but also lowers A? levels by reducing the ?-secretase enzyme BACE1 in a mouse model of AD. In this R03 application, we will test our focused hypothesis that reduction in TrkB signaling, which is found in early AD or its harbinger mild cognitive impairment (MCI), may be responsible for inducing BACE1 elevation and consequently initiating and/or accelerating disease processes. Specifically, we will use (i) pharmacological (ANA-12, a selective and blood-brain barrier permeable TrkB antagonist) and (ii) genetic (TrkB haploinsufficiency) approaches to experimentally reproduce the impairment of TrkB signaling associated with MCI or incipient AD. In Aim 1-1, these approaches will be applied to young 5XFAD transgenic mice, which develop little or only faint amyloid pathology and are still normal in memory function. We will first compare the levels of BACE1 expression in ANA-12-treated 5XFAD or TrkB+/-.5XFAD bigenic mice (3-month old) with those of age-matched 5XFAD control mice. We will then explore the molecular mechanisms by which pharmacological and genetic reduction of TrkB signaling may cause BACE1 elevation (e.g., changes in transcription, translation, protein stability) and exacerbate AD-like phenotypes including memory deficits in these 5XFAD mice. In Aim 1-2, we will further test whether administration of ANA-12 or TrkB haploinsufficiency may be sufficient to upregulate BACE1 in wild-type mice. Given that A?42 may be important for increasing BACE1 expression in neurons around amyloid plaques during the progression of AD, it will be critical to determine a causal relationship between reduced TrkB signaling and BACE1 elevation in wild- type controls that completely lack toxic human A? species. If the data is positive, we will examine whether the same mechanisms as found in 5XFAD mice may mediate BACE1 elevation following impaired TrkB signaling in wild-type mice, regardless of the presence of human A?. Together, the proposed research is expected to address a key question whether deficient BDNF-TrkB signaling may not only be a mediator of memory impairments as a consequence of A? accumulation (as shown by previous studies) but also have a crucial role in initiating or accelerating the AD process via BACE1 elevation.


This proposal for a Clinical and Translational Science Center at the University of New Mexico Health Sciences Center (HSC) will catalyze the emergence of a transformative, novel, and integrated academic home for clinical and translational science at a flagship institution that serves the entire state of New Mexico and the Southwestern region ofthe U.S. This center will have the infrastructure and consolidated resources to: 1) synergize multi- and interdisciplinary clinical and translational research and researchers to catalyze the application of new knowledge and techniques to clinical practice at the front lines of patient care;2) recruit, train, and advance well-trained interdisciplinary investigators and research teams with strength in cultural sensitivity, health disparity, and biotechnology;3) create an incubator for innovative research and information technologies;and 4) expand existing partnerships between UNM Health Sciences Center (HSC) researchers, practicing clinicians, and communities to speed the development of research. Our vision of the CTSC links and focuses the efforts of our basic, clinical, and translational investigators, community clinicians, clinical practices, health care and research collaborators, and industry partners. Since New Mexico has a high proportion of ethnically diverse (i.e., Hispanic and Native American), rural, medically disenfranchised, and health-disparate populations, a CTSC in New Mexico can uniquely address these problems. The UNM CTSC will draw upon outstanding institutional commitment (at least $57,802,262 ) to achieve its goals. The UNM CTSC possesses authority over the recruiting, hiring, protected time, expectations, promotion and tenure, and evaluation of its faculty members. Evidence of the commitment and effectiveness of the UNM CTSC in these pursuits are apparent from the following achievements made during the planning process: (1) reorganization of the UNM HSC research mission under a new Vice President for Translational Research with authority over research education, compliance and research functions, (2) establishment of an institutionally-funded K12-like program, and recruitment of 5 junior faculty, (3) development of innovative new research education programs, (4) operation of a functionally expanded Participant and Clinical Interactive Resources component that is serving the needs of several community-based studies, including the landmark, new National Children's Study, (5) the redesign of our institutional approach to Biomedical Informatics and development of a regional clinical data warehouse that will be available to investigators using a $5.8 M investment, and (6) construction and renovation of new buildings for the CTSC. Additionally, UNM's commitment includes $14.5 M in new institutional funds committed to CTSC programs, and $8.5 M in matching funds to construct new space to create a physical home for the CTSC. Funding of this proposal will assure sustainability and growth of these transformations that is critically needed to support clinical and translational research at UNM. RELEVANCE (See instructions): The UNM Clinical and Translation Science Center will rapidly escalate transformative scientific discovery into improved human health outcome.


Ubiquitin (Ub) is an essential signal molecule regulating protein degradation, localization and other activities. Generally the C-terminus of the Ub molecule is covalently conjugated to lysine residues of protein substrates by the E1/E2/E3 reaction cascade, and the modification is reversed by the action of deubiquitinating enzymes (DUBs). PolyUb chains can be further assembled on the substrates by the linkage between lysine groups of the first Ub molecule and the C-terminus of the next. Our current proteomic analysis revealed that all seven lysine groups on the Ub molecule can be utilized for the formation of polyUb chains, including Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63. We propose that the formation of functionally distinct polyUb chains is a regulatory step for ubiquitination and deubiquitination. To examine the function of these polyUb linkages, we will first develop a mass spectrometry technology for quantifying the abundance of all seven polyUb linkages, and then study the catalytic specificity between DUBs and polyUb chain topology. Moreover, we will focus on defining the function of Lys11 polyUb chains. Our studies will lead to the development of general proteomic tools for polyUb chain topology and better understanding of the diversity and function of polyUb chains.


Hot flushes during the perimenopausal and post-menopausal periods pose a significant public health concern because they (i) are the primary reason that women seek medical care during the menopausal transition;(ii) often negatively impact the quality of life for women because they are associated with depression, sleep disturbances resulting in fatigue, irritability, and forgetfulness, as well as acute physical discomfort and negative effects on work;and (iii) are thought to be indicative of some impending, serious conditions. Despite the importance of hot flushes in a woman's life, little is known about the underlying mechanism or the risk factors for hot flushes. Although the most efficacious therapy for hot flushes is estrogen replacement therapy, due to the potential risks involved with hormone therapy, many women try to find alternative therapies, such as the selective serotonin and/or serotonin-norepinephrine reuptake inhibitors (SSRIs/SNRIs), clonidine, and gabapentine in spite of their lower efficacy compared to estrogen replacement therapy. Clinical and preclinical data indeed show that these neurotransmitters play critical roles in thermoregulation by changing the sensitivity of the thermostat and subsequently, affecting hot flushes. Although a subpopulation of perimenopausal women do respond to SSRIs/NRIs, the response is heterogenous (some individuals respond well some do not respond at all). The overall goal of this application is to explore why some women with hot flushes do not respond to SSRIs/SNRIs. Our hypothesis is that the heterogenouos response is due to single nucleotide polymorphisms of the serotonin transporter (SERT) and/or the norepinephrine transporter (NET) genes. Therefore, we propose the following specific aims: (1) Genotype a comprehensive set of SNPs within the SERT and NET genes in a sample of 800 perimenopausal women recruited during a previous funding period and 58% of whom experienced hot flushes and genotype these genes in a sample of 200 women being recruited and (2) Determine whether these SNPs are correlated with hot flushes. This proposal matches the R21 funding requirements in that it explores, for the first time, the relationship between SNPs of SERT and NET genes as predictors of hot flush relief. If the heterogeneous response to SSRI/SNRI therapies is due to these SNPs, then a customized, genotype-based therapy may be the ultimate solution to increase efficacy of this approach to treating bothersome hot flushes without hormones. PUBLIC HEALTH RELEVANCE: Although hot flushes during the perimenopausal period pose a significant public health concern. Little is known about the underlying mechanism of hot flushes. Many women try to find alternative therapies, to estrogen such as the selective serotonin and/or serotonin-norepinephrine reuptake inhibitors (SSRIs/SNRIs). However, the response to these is heterogenous and might be due to individual gene variations. This proposal will identify these variations and their relationship to hot flushes.


A limited number of properties separate bacterial pathogens from non-pathogenic species. One such property exhibited by many pathogens is the ability to enter and replicate within eukaryotic cells. Francisella tularensis (FT) is a Gram negative bacterial pathogen that invades and replicates within a myriad of host cell types including, but not limited to, macrophages, dendritic cells and alveolar epithelial cells. The focu of this project is to understand mechanisms by which pathogenic Francisella adapt to, and manipulate the host cell environment and the implications that these properties have on bacterial virulence. In the previous funding period we investigated the roles of specific proteins in FT intracellular growth. During these studies we found that a robust ATG5- independent autophagic response was mounted in FT Schu S4 infected cells and that the bacteria assimilated amino acids derived through autophagic degradation of host proteins. Autophagy is also an anti-microbial response against cytosolic pathogens. However, even chemically induced autophagy did nothing to control FT intracellular growth. Thus, FT Schu S4 simultaneously evades degradation via autophagy while scavenging nutrients that are produced by this process. In this continuing application we propose to determine the function of the pathogenicity island encoded protein PdpC in protecting FT from autophagy and to define the bacterial metabolic processes that make it possible for virulent FT strains to replicate within monocytes.


Phosphatidylinositol-4,5-bisphosphate (PIP2) modulates the actin cytoskeleton, cell migration, invasion and signaling by binding effectors that regulate cytoskeletal dynamics and receptor trafficking. Yet, the mechanism of how PIP2 is generated to regulate these processes is poorly understood. Phosphatidylinositol phosphate kinases (PIPKs) that synthesize PIP2 specifically associate with PIP2 effectors. PIP2 then modulates the proteins and enzymes associated with the PIPK. PIPKI? regulates growth factor stimulated directional cell migration, vesicular trafficking of adhesion receptors and invasion. Here, we show that PIPKI? and PIP2 are required for establishment of cell polarity required for directional cell migration and invasion. In addition, we have discovered a novel signaling nexus that may explain how PIP2 mechanistically regulates many key effectors in adhesion, migration and invasion. Hypothesis: PIPKI? controls key aspects of tumor cell migration and invasion by controlling the assembly of cytoskeletal components and the trafficking of adhesion receptors. Interactions between PIPKI?, the exocyst and IQGAP1 function to integrate and specify the PIP2 generation and signaling required for growth factor stimulated directional cell migration, adhesion and invasion. In vivo the PIPKIg signaling nexus plays a key role in tumor cell intravasation and extravasation, processes required for tumor cell metastasis. Aim 1. The role of PIPKI?i2, the exocyst, and ?1-integrin trafficking in directional cell migration will be investigated. Regulation of integrin trafficking by PIPKI? and the exocyst will be defined with an emphasis on regulation by PIP2, the protein-protein interactions and cell polarity pathways, roles of small G-proteins, and specificity toward integrins. We will determine if a PIPKI?i2 interaction with talin acts as a tether for targeting the PIPKI?i2/?1-integrin/exocyst complex during directional cell migration. Aim 2. Define the mechanism for PIPKI? and IQGAP1 regulation of directional migration. We will study the IQGAP1/PIPKI? interaction and define how signals modulate this nexus. We will reveal the mechanism for PIPKI? control of IQGAP1 association with membrane. We will determine if and how PIPKI? modulates the association or activity of known IQGAP1 interactors, such as small G-proteins and the exocyst. Aim 3. Define the role of the PIPKI?, IQGAP1 and exocyst in cell invasion. The role of PIPKI?, the exocyst and IQGAP1 in formation of invadopodia will be revealed. An emphasis will be on targeting of key proteins required for invasion, such as IQGAP1, the exocyst, ?1-integrin and MMPs to the invadopodia in a 3D matrix environment. Aim 4. Determine if PIPKI?, the exocyst and IQGAP1 are required for extravasation and metastasis. Highly metastatic human breast cell lines will be engineered to over or under express PIPKI?, IQGAP1 or Exo70. We will use a well-established mouse xenograft model to transplant engineered cells into the vasculature. The development and number of metastases will be used to quantify extravasation.


This proposal addresses itself to the study of brain mechanisms involved in visual pattern recognition and perception. We have discovered that it is possible to actually control the development of functional properties of single cells in the visual cortex of kittens by the simple device of controlling their visual experience. We have demonstrated that the "shape" of receptive fields in the visual cortex of controlled experience kittens is directly related to the shape of the visual stimuli they viewed during development. For some units the receptive field shape is a recognizable, though blurred, image of the stimulus! Recently, we have also shown that simple visuo-motor training, during development, powerfully affects somatosensory cortex cells and their polisensory properties in ways which, again, are very directly related to the nature of the experience. These findings form the bases for the proposed experiments. We intend to raise kittens in environments designed to engage specific aspects of visual perception such as texture, line detection and simple recognition tasks. We expect that the kittens will have cells whose functional characteristics will reflect specific aspects or features of the experience thus enabling us to relate function with mechanisms directly. We will continue to try and separate neuronal properties which are inborn from those that are experientially determined. Finally, with the help of computer and neuroanatomical methods we will increase the functional connectivity and trace the structure of networks that have been engendered by controlled experience.


The proposed studies constitute part of a larger effort to understand how the contraction of smooth muscle is regulated at the level of contractile proteins, actin and myosin. The focus of this proposal is to elucidate the mechanism by which the putative regulatory protein calponin interacts with the actin filament to modulate contraction. In particular, thee studies will attempt to determine if calponin regulates a well described state of smooth muscle contraction called a "latch- state". The latch-state allows smooth muscles to remain contracted for long periods with relatively low expenditure of chemical energy. The high-economy of smooth muscle contraction is essential for normal physiologic function. In spite of the central importance of this contractile state for normal function of smooth muscle, the molecular basis for the regulation of the latch-state is unknown. Our central hypothesis is that calponin slows the rate of cross-bridge dissociation from actin, and this leads to activation of unphosphorylated cross bridges via a thin filament-linked mechanism. To test this hypothesis we will measure 1) actin filament sliding velocity, 2) changes in the level of force exerted on regulated actin filaments by a field of immobilized myosin molecules, 3) the force, displacement (step size), and attachment time for single myosin molecules interact with single actin filaments, and 4) the rate of myosin dissociation from actin using stopped-flow techniques. These measurements will provide insights into the physiologic parameters of isometric force and unloaded shortening velocity that characterize the contractile state of intact smooth muscles. These assays, in conjunction with recent x-ray diffraction data and high resolution electron microscopic images of actin myosin, tropomyosin, and calponin allow us to formulate and test specific molecular models for how calponin might interact with actin, tropomyosin, and/or myosin. The proposed studies will begin to address the issue of how calponin might interact with actin, tropomyosin, and/or myosin. The proposed studies will begin to address the issue of how calponin-mediated regulation interacts with the now well established myosin phosphorylation regulatory system. A major goal of the proposed studies will be to elucidate the role of calponin in thin-filament linked regulation of unphosphorylated myosin (i.e. the latch-state).


Treatment of PANC-1 cells with the CB1R agonist Win55,212-2 resulted in a time-dependent decrease in ligand-induced phosphorylation of the EGF receptor and downstream signaling. In contrast, inhibition of CB1R signaling by AM251 led to a significant increase in the expression of EGF ligands (heregulin and betacellulin) and EGF receptor (ErbB1). These results were established by quantitative PCR, Western immunoblotting and EGF/PDGF signaling PCR array. There was no increase in IGF-1 receptor expression by AM251, and inhibition of CB2R with AM630 did not affect expression of the EGF receptor or its ligands, indicating the specific nature of AM251 action in these cells. Flow cytometry analyses and selective labeling of cell surface proteins with biotin probe indicated an increase in EGF receptor at the plasma membrane of AM251-treated PANC-1 cells, which coincided with higher levels of EGF-dependent phosphorylation of EGF receptor and its downstream mediator, Akt, when compared to EGF alone. In addition to the potentiation in EGF signaling, AM251 elicited distinct cellular morphological changes, with the cells changing from a cuboidal shape to a rounded cell with elongated projections. The loss of E-cadherin in response to AM251 suggests that these cells may be undergoing an epithelial to mesenchymal (EMT) transition, a hallmark of invasiveness. Indeed, AM251 enhanced significantly the invasive potential of PANC-1 cells as assessed by Matrigel invasion chambers. Work is underway to systematically examine the signaling pathways and activities of multiple transcription factors that directly control the extent of gene expression and function of the EGF receptor and its ligands in response to AM251. These studies will be expanded to include the use of cell lines from other cancer cell types (e.g., colon, breast, ovaries) and tumor xenograft models in mice.


The bulk of information that has contributed to our understanding of mechanism of progesterone action originates from avian models. During recent years rabbit uteroglobin has been successfully utilized as a model to exploit progestin action in mammalian tissues, too. Previous studies in our laboratory have yielded many interesting features in the regulation of uteroglobin gene activity and its relation to progesterone receptor dynamics, such as inhibition (and potentiation) of progesterone action by simultaneously administered estradiol or tamoxifen; progesterone receptor consumption during progesterone action; multihormonal control of uteroglobin synthesis, and dissociation of receptor changes from the biologic response. The present proposal is a continuation of these studies and utilizes uteroglobin and its mRNA (measured by RIA and cDNA hybridization, respectively) as biological markers for progestin action. To ensure that the regulatory events studied are not peculiar to uteroglobin, other progesterone-induced uterine proteins are concomitantly evaluated. Since uteroglobin is under multihormonal control--progestins, androgens, and estrogens regulate uteroglobin synthesis--this model offers an excellent system for studies of steroidal interactions in the regulation of a specific endometrial gene product. One of the major specific aims is to clarify the mechanism of antiprogesterone action of estradiol in the uterus. Steroid action is mediated via cytosol receptor proteins, but the extent to which biological responsiveness, maintenance and termination of steroid action are related to receptor dynamics are poorly understood. In the case of progesterone, receptor consumption (disappearance of steroid binding site) during hormone action still complicates the above relationships. The second specific aim of this proposal is devoted to clarification of these issues with a special emphasis on progesterone receptor consumption its biological significance. It may well be that an ultimate answer cannot be achieved utilizing current receptor assay techniques based on the use of labeled steroids, and hence progesterone receptors will be purified, characterized, and an immunologic assay for their measurement will eventually be set up.


The balanced growth of a normal cell or the development of a virus in its host cell involves a complex pattern of gene expression in which normal individual genes, or groups of genes, are activated or repressed at appropriate times during the growth cycle. Initial control over this process is effected at the first step of the genetic pathway: the transcription of DNA sequences into RNA. For both bacterial cells and bacteriophages specific transcribing enzymes have been isolated which carry out accurate and regulated reading of genetic sequences in vitro. Since this kind of seective transciption has not yet been observed in vitro for eukaryotic transcribing systems, these prokaryotic transcribing systems serve as essential model systems with which to sudy the factors that govern the regulation of gene expression. We propose to continue our studies on the mechanism of selective transciption--that is, the recognition and utilization of specific promoter and terminator signals on DNA by RNA polymerases. The studies will focus on the transcription of DNA from bacteriophage T7 that is carried out by E.coli polymerase and by T7 phage specific RNA polymerase.


Sun exposure has been consistently implicated in the development of non-melanoma skin cancers, but its role in melanoma etiology remains controversial, and is complicated by poorly understood genetic and behavioral factors. Melanoma incidence continues to increase (it is now the 4th most common cancer among males in California). Efforts to reduce melanoma incidence through sun exposure avoidance have had limited success, underscoring our lack of understanding of the true mechanisms of melanogenesis. Melanoma epidemiology is based on retrospective studies: recall bias probably explains associations between sunburn (almost always self-reported) and melanoma. Moreover, sun burn represents only excessive exposure to solar radiation, while sun-induced DNA damage potentially occurs across all levels of intensity: there is no accepted method for directly measuring sun exposure in epidemiological studies. In order to better understand the etiology of melanoma (particularly the role of sun exposure), valid methods of assessing both current and historical sun exposure are needed. While genetic factors probably play some role in melanogenesis, no study has yet managed to distinguish genetic from sun exposure-related risks. We will address both these issues in a case-control study of identical twins (nested in an established population-based cohort of 52,000 twins in California) assessing the role of sun exposure in melanoma etiology in the absence of genetic effects. Historical sun exposure will be assessed by combining residential history with novel fine-layer interpolated solar radiation level using GIS: we have already shown this method significantly reduces misclassification and recall bias. We will measure the actual impact of solar radiation on the skin, measuring UV- induced epidermal damage in tumor specimens noting differences in risk with varying UV intensity and wavelength. These results have the potential to improve primary prevention of melanoma and other skin cancers, and to direct the research team's future work on gene-environment interactions relevant to melanogenesis. We will measure the actual impact of solar radiation on the skin, measuring ultra-violet (UV)-induced epidermal damage in tumor specimens noting differences in risk with varying UV intensity and wavelength, and estimating the risk of melanoma with lifetime and age- specific exposures to specific wavelengths of UV. These results have the potential to improve primary prevention of melanoma and other skin cancers by indicating the appropriate means of avoiding harmful UV exposure, and to direct the research team's future work on gene-environment interactions relevant to melanogenesis. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]


DESCRIPTION (provided by investigator): We request funds to establish a large-scale (up to 100 liters) bacterial culture facility in Memphis, TN, to be located at St. Jude Children's Research Hospital (St. Jude). This facility will be available to serve a broad community of scientists in Memphis, including those at St. Jude, The University of Tennessee Health Sciences Center (U. Tennessee), St. Jude's partner in graduate education, and The University of Memphis (U. Memphis), a partner in many collaborative research projects. The purpose of this facility is to enable the large-scale production of proteins and other biomolecules for structural, biochemical and drug discovery studies. The proposed Fermentation Facility will impact a wide variety of NIH-funded biomedical research projects at the three institutions noted above, ranging from structural investigations into basic biological function and structure-based drug design, to the production of small molecules as intermediates for the synthesis of new therapeutics and high-throughput small-molecule screening. Overall, the Fermentation Facility will contribute to advancing research funded by more than one dozen R01, P01, U01, or R37 applications distributed between the three institutions. The principal contribution to ongoing research will be to enable large-scale production of biomolecule samples, especially proteins. In some cases, large-scale protein production is already under way but is performed in a highly time inefficient manner. Access to the proposed fermentor will allow scientists to focus much more of their time on biochemical and structural studies rather than on protein production. In other cases, production of certain proteins is currently out of reach due to low expression levels. Studies in this category will be made possible through access to the 100-liter fermentor. An additional benefit associated with creating this facility is the promotion of intra- and inter-institutional collaboration. While many of the participants here already collaborate, use of a shared Fermentation Facility will foster interactions between groups, especially at the level of graduate students and postdoctoral trainees who will actually perform fermentation experiments.


PROJECT SUMMARY The human skin barrier does not always function as intended; in fact, defects in the outer layer of human skin account for a significant number of health problems for people living in the United States. As one example, it is estimated that up to 90 million Americans suffer from some form of atopic dermatitis. Atopic dermatitis and other forms of severely dry skin, such as ichthyosis vulgaris, are associated with defects or mutations in profilaggrin and its processed fragment, filaggrin. Mutations in keratin 1 and keratin 10 proteins also account for a variety of inherited skin disorders manifested by red, dry, scaly skin. Together, profilaggrin and keratins 1 and 10 are critical proteins involved in the stratum corneum of human skin, and elucidating the biochemistry behind their function is important for improving our understanding of how the human skin barrier works. The hope is that advancing our knowledge of how protein structure dictates function in the stratum corneum will generate new methods for treatment of human skin diseases. This proposal aims to address a deficiency in our understanding of the atomic resolution structure of key epidermal proteins, namely profilaggrin and keratins 1 and 10. In particular, it is still unclear (1) what the precise mechanisms are for profilaggrin binding to target proteins; (2) is there correlation between structure and function of the profilaggrin B domain; (3) does profilaggrin interaction with target proteins direct specific events in terminal epidermal differentiation; (4) what are the molecular mechanisms behind keratin intermediate filament aggregation; and (5) can enhanced structural knowledge of key skin barrier proteins pave the way for newly designed topical therapeutics. Given the direct association of profilaggrin and keratins 1 and 10 with multiple human diseases, we believe focusing our studies on these medically important proteins will help us address these outstanding questions. In this project, we examine the biochemical and structural properties of key proteins involved in a functional human skin barrier. Our first aim uses x-ray crystallography and biochemical analysis techniques to determine the structural basis of binding between profilaggrin and one of its targets, annexin II. The second aim examines the x-ray crystal structure of profilaggrin in the absence of bound molecules and performs mutational analysis on key inter-EF-hand linker residues in order to understand the functional role of this protein region. The third aim examines the structural basis for keratin intermediate filament interaction with different parts of the profilaggrin molecule using x-ray crystallography and biochemical techniques. Accomplishing these aims will provide novel insight into the principle biochemistry and atomic resolution structure of key epidermal proteins involved in maintaining the integrity of the human skin barrier.


The correct functioning of the endogenous circadian clock enables organisms to anticipate daily environmental changes and temporally modify behavioral and physiological functions appropriately. All organisms maintain a large number of physiological variables (sleep-wake cycle, locomotor activity, temperature regulation, water/food intake and levels of hormones) under control of the circadian clock. The biological clock readjusts itself by synchronizing to the daily light-dark cycle. Disruption of these rhythms has drastic effects on human health, leading to insomnia, depression, coronary diseases, various neurodegenerative or metabolic disorders and cancer. Recent advances have revealed unexpected links between circadian regulators, chromatin remodeling and cellular metabolism. The HDAC activity of the NAD+dependent SIRT1 enzyme is regulated in a circadian manner and SIRT1 transduces signals originated by cellular metabolites to the circadian clock. We aim to gain new insights into the regulation of chromatin transitions that govern the expression pattern of circadian genes, by understanding the biochemical aspects of the interaction between two chromatin remodelers, SIRT1 and MLL1, a histone methyltransferase. Our preliminary results convincingly indicate that MLL1 is acetylated and it is directly regulated by SIRT1, leading to a global control of circadian gene expression. We will unravel yet unexplored molecular mechanisms in which these two critical epigenetic regulators coordinate their function. This line of research is innovative as it links specific metabolic pathways to the epigenetic control of chromatin remodeling.


Modern techniques in Molecular Biology including cDNA arrays and automated sequencing have made possible the identification of genes expressed by a particular type of cell. The most common strategy to identify novel cell-type specific genes is to sequence cDNA clones obtained from the reverse transcription of mRNA using oligo dT and reverse transcriptase (RT). Due to limitations of the technique, these cDNAs often consist of partial clones of the 3' region of the mRNA. Since this 3' region often consists of non-coding sequences, insights into the function of the protein encoded by the mRNA are not possible. Techniques such as "random priming", optimization of mRNA preparation and improvement of the enzymatic reactions used in cDNA synthesis can increase the yield of full-length product. Even these optimized procedures result in many truncated cDNAs. Furthermore, these procedures require large amounts of mRNA starting material. This laboratory is interested in the developmental regulation of stem cells, a rare population of cells that play a critical role in organogenesis. Genes expressed by these cells are among the most likely to escape identification in the EST database efforts. In this proposal, we propose to develop novel in vitro and in vivo methods to make cDNA from such cell populations. The Specific aims of this project follow: Specific Aim No. 1. To make long cDNA libraries from rare cell populations. Specific Aim No. 2. To develop a method to use random priming to clone the 5' end of the mRNA obtained from rare cells. Specific Aim No. 3. To develop a method to make cDNA in vivo.


In recent decades, the United States has experienced an unabated rise in the number of individuals diagnosed with diabetes;as a result 23 million people are currently living with type 2 diabetes. The pathogenesis of type 2 diabetes involves a complex interplay between insulin resistance and failure of pancreatic [unreadable] cell compensation. [unreadable] cell compensation occurs through improved [unreadable] cell function and [unreadable] cell mass expansion, which are both regulated by the critical pancreatic transcription factor PDXI. Preliminary data show that PDXI deficiency increases [unreadable] cell apoptosis during diet induced insulin resistance by increasing susceptibility to endoplasmic reticulum (ER) stress (M. Sachdeva, unpublished data, 2008). The ER of a [unreadable] cell processes one million insulin molecules per minute, and ER stress has been associated with type 2 diabetes in mice and humans. High throughput gene expression and promoter occupancy analyses suggest that PDXI regulates several genes involved in maintaining ER homeostasis, including EROlip and GRP170. PDXI regulation of EROlip and GRP170 is hypothesized to be important for insulin biosynthesis and [unreadable] cell survival. This hypothesis will be tested in the following specific aims: I. Determine the role of DX1 in insulin biosynthesis, II. Determine the role of PDXI in the regulation of ER homeostasis and p cell survival. Insulin secretion will be studied in mouse insulinoma cells with shRNA silenced EROlip and GRP170. The role of EROlip in insulin disulfide bond formation and the function of GRP170 as a [unreadable] cell chaperone will be analyzed. To determine the effects of EROlip and GRP170 deficiency on ER homeostasis, ER stress markers such as BiP and CHOP will be measured, and Annexin V and TUNEL staining will be used to quantify [unreadable] cell apoptosis. Overexpression of EROlip and GRP170 may partially rescue the PDXI deficiency phenotype suggesting that they are important mediators of the effects of PDXI on insulin secretion and [unreadable] cell survival. Relevance: Failure of [unreadable] cells to sufficiently secrete insulin and death of these insulin-producing cells results in diabetes. Studying the mechanism for PDXI regulation of insulin secretion and [unreadable] cell survival may lead to targets for treatment of type 2 diabetes.


Histone mRNA synthesis is being studied in a cell-free system derived from synchronized mouse myeloma cells. This system carries out many of the reactions of RNA processing as well as transcription. The histone mRNA in mouse cells is analyzed by its ability to hybridize with plasmid DNA coding for the sea urchin histone genes. Our evidence indicates that the primary transcript of the mouse histone genes is a large molecule containing sequences for more than on histone protein. This transcript is synthesized only in early S phase cells. Thus, the primary control is at the level of transcription.


Over the past decade the genetics of development and cancer have converged in the identification of signaling pathways that control embryo patterning and, when aberrantly regulated, lead to cancer. Typically, tumors form in tissues in which the pathways normally operate. Tumors arise due to oncogenic mutations in components of these pathways that lead to ligand-independent constitutive activity. A classic example is the canonical Wnt signaling pathway, an essential developmental pathway that is disrupted in several kinds of tumor. Mouse Wnt1 is a prototypical oncogene first identified when it was shown that activation of Wnt1 by integration of Mouse Mammary Tumor Virus led to cancer of the mammary gland in mice. Mutations in several components of the Wnt/beta-catenin pathway have identified both oncogenes and tumor suppressors in this pathway that lead, in particular, to colorectal cancer. Recently, we have shown that aberrant Wnt signaling may lead to cancers of the immune system, specifically T-cell lymphomas. A large-scale survival study has revealed that relatively late-onset lymphomas and thymomas arise in Tcf mutants older than 6 months of age, however when one copy of Wnt5a is removed, severe, early-onset lymphomas arise as early as 3 months of age. These results suggest that Wnt5a has tumor suppressor activity. Our current experiments are aimed at understanding the underlying mechanisms and suggest that Wnt5a normally antagonises the canonical pathway ie. Wnt/beta-catenin target genes such as the proto-oncogene c-myc. We have also initiated a microarray project to generate a transcriptional profile of tumors so that we can molecularly compare our lymphomas with clinically


Methicillin-resistant S. aureus (MRSA) infections are a major cause of morbidity and mortality in veterans. MRSA is transmitted through direct or indirect contact in the healthcare setting. In acute care settings, we use Contact Precautions (health care workers [HCW] wear gowns and gloves for all patient contact) for patients colonized with MRSA to prevent transmission to other patients. Current Centers for Disease Control and Prevention (CDC) Isolation Guidelines suggest modifying Contact Precautions in long term care facilities (LTCF), but there is little evidence to guide how to modify them. The goal of this revised proposal is to determine the optimal modifications of Contact Precautions for LTCF in order reduce the risk of MRSA transmission and allow care in a home-like, patient-centered environment consistent the Community Living Centers Cultural Transformation. LTCFs provide multiple levels of care including rehabilitation, skilled nursing and maintenance care. In the PI's current VA Merit award, we demonstrated that MRSA transmission is four-fold higher in rehabilitation care than in other long term care. This suggests that the types of care delivered increase the risk of transmission. MRSA transmission to other residents is difficult to study. Recently we developed a novel surrogate measure of MRSA transmission, detection of MRSA on HCW gown and gloves during HCW-patient interaction. Using this new methodology, we will test two major hypotheses based on expert opinion recommendations from the CDC Isolation Guidelines. 1. Risk of MRSA transmission will vary by type of contact with the resident and each activity will have its own risk of transmission. Some activities such as those involving contact with secretions (e.g. draining wounds, ostomy care) will be of higher risk than others (e.g. vital signs, medication administration). 2. For any given type of contact, resident characteristics increases the risk of transmission (e.g. residents totally dependent upon healthcare personnel for healthcare and activities of daily living or residents whose secretions or drainage cannot be contained). Our aim is to estimate the frequency of and risk factors for MRSA transmission to protective gowns and gloves worn by HCW interacting with 400 MRSA colonized VA long term care residents in a multi-site observational study in VA Community Living Centers from four states and the District of Columbia.