Potassium channels in the renal cortical collecting tubule (CCT) play an important role in potassium (K) secretion by the kidney and help to maintain the body's overall K balance. In this nephron segment, potassium secretion rates depend on both the apical membrane K permeability and the electrochemical driving forces for K across the apical membrane. The apical membrane K permeability relevant for K secretion appears to arise from a small conductance, mildly inward rectifying channel which has a high open probability (P-O) under normal conditions. However, the P-O of this channel is strongly dependent on intracellular pH. This largely explains the clinical observation that alkalosis is often associated with substantial K loss and hypokalemia. Recently two closely related renal K channels have been cloned from rat, designated ROMK1 and ROMK2. The physiological relevance of the ROMK family is its functional similarity to the small conductance apical K channels that are responsible for K secretion from mammalian CCT and K recycling in TALH. The predicted primary sequences and membrane topologies of ROMK are quite different from the voltage-gated K channels of excitable cells. However, both of these ROMK clones share significant homologies with the inward rectifier (IRK) family of channels that maintain resting potential near EK and permit long depolarizing responses in excitable cells. The present proposal exploits the structural and functional similarity between ROMK and IRK families to address some important issues regarding structure-function relations in ROMK2. Site directed mutagenesis will be used to determine the regions and specific residues of ROMK2 that are important for: (1) mild inward rectification, (2) strong pH dependence, (3) permeation path and selectivity characteristics, (4) site and affinity of barium block. The proposal also addresses structural issues involving the proximity of specific residues in the amino and carboxy termini to the putative pore region of the channel and to known "inward rectifier" sites in the second transmembrane spanning region. Interactions between K and barium within the conduction pathway will also be investigated as a method for characterizing the structure of the permeation path. The project will enhance our knowledge of basic processes underlying K homeostasis in health and disease.


The objectives of this proposal are to develop an understanding of the mechanisms by which protease activity in coagulation, fibrinolysis, and inflammation is regulated. To accomplish these objectives the interaction of proteases with the plasma protease inhibitor, a2-Macroglobulin (a2M), and the interaction of the resultant complexes with cell surface receptors will be characterized. Major areas of focus will be to elucidate the mechanism by which proteinases react with this inhibitor, to access the importance and role of a2M as a regulator of protease activity in vivo, and to isolate and characterize the a2M receptor from fibroblasts. Conformational changes occurring in a2M are central to the function of the inhibitor, and not only result in inhibition of protease activity, but also generate receptor determinants on the molecule. Studies are proposed to determine the relationship between conformational changes, protease inhibition, and the generation of the receptor binding sites on the inhibitor-protease complex. Specific probes for monitoring each process have been developed, and will be extensively utilized in these studies. The specific in vivo processes in which a2M plays a key role will be addressed by isolating complexes formed under physiological conditions and identifying which specific enzymes associate with the inhibitor. Complexes will be isolated by immunoaffinity chromatography using a monoclonal antibody that is specific for a2M-protease complexes. Studies are also proposed to characterize the interaction of the inhibitor-protease complex with specific receptors. To accomplish this goal the receptor determinants on the complex will be identified by preparing anti- bodies against synthetic peptides representing regions of the molecule. The receptor will also be isolated from fibroblasts, and the properties characterized. Studies will be initiated on the amino acid sequence of regions of the receptor. Monoclonal antibodies prepared against the purified receptor will assist in characterizing subunits with the goal of identifying regions that participate in receptor function, and will be useful for comparing the structure of the receptor from various cell types and species.


The purpose of this study is to determine the safety, tolerability and efficacy of pain relief of recombinant human nerve growth factor (rhNGF) for HIV-associated sensory neuropathy. Five patients were enrolled and active in this clinical trial during the above time period (recruitment started during 8/96). Patients can be randomized to either a low dose rhNGF, a high dose rhNGF or placebo. All five patients have tolerated the study medication well. Two of the patients have reported a benefit in their neuropathy while the other three patients have reported no change. Since 11/30/96, the recruitment for this study continues to grow. We now have 7 patients active in this study and more in the screening process. Nationwide, there are now 147 patients enrolled among 15 sites. At the current rate, the national recruitment goal of 180 participants will be reached by approximately 2/28/97. Due to the rapid accrual rate and to statistical reasons, the study is currently being amended to: 1) increase the number of participants in this study to 260 or 270 and 2) to provide a 48 week open-label phase of this study that would be open to all participants who successfully completed the initial double-blind phase of the study. Participants will either receive low dose or high dose rhNGF during this phase of the trial. No interim analysis has been conducted on the data collected thus far at this point in time.


This is a single-blind placebo controlled trial identifying patients with life-threatening ventricular arrhythmias.


Project Summary/Abstract Stress echocardiography is a clinically established, cost-effective technique for detecting and characterizing coronary artery disease by imaging the left ventricle (LV) of the heart at rest and then after either exercise or pharmacologically-induced stress to reveal ischemia. However, acquisitions are heavily operator dependent, two-dimensional (2D), and interpretation is generally based on qualitative assessment. While a variety of quan- titative 2D approaches have been proposed in the research literature, none have been shown to be superior to the still highly variable qualitative visual comparison of rest/stress echocardiographic image sequences for detecting ischemic disease. Here, we propose that the way forward must focus on a new computational im- age analysis paradigm for quantitative 4D (three spatial dimensions plus time) stress echocardiography. Our strategy integrates information derived from both radiofrequency (RF) and B-mode echocardiographic images acquired using a matrix array probe. The integrated analysis system will yield accurate and robust measures of strain and strain rate - at rest, stress and differentiallly between rest and stress - that will identify my- ocardial tissue at-risk after dobutamine-induced stress. This work will involve the development of novel (1) phase-sensitive, correlation-based RF ultrasound speckle tracking to estimate mid-wall displacements, (2) ma- chine learning techniques to localize the LV bounding surfaces and their displacements from B-mode data, (3) a meshless integration approach based on radial basis functions (RBFs) and Bayesian reasoning/sparse coding to estimate dense spatiotemporal parameters of strain and strain rate and (4) non-rigid registration of rest and stress image sequences to develop unique, 3D differential deformation parameters. The quantitative approach will be validated with implanted sonomicrometers and microsphere-derived flows using an acute canine model of stenosis. The ability of deformation and differential deformation derived from 4D stress echocardiography to detect new myocardial tissue at-risk in the presence of existing infarction will then be determined in a hybrid acute/chronic canine model of infarction with superimposed ischemia. The technique will be translated to hu- mans and evaluated by measuring the reproducibility of our deformation and differential deformation parameters in a small cohort of subjects. Three main collaborators will team on this work. A group led by Matthew O'Donnell from the University of Washington will develop the RF-based speckle tracking methods. An image analysis group led by the PI James Duncan at Yale University will develop methods for segmentation, shape tracking, dense displacement integration and strain computation. A cardiology/physiology group under Dr. Albert Sinusas at Yale will perform the acute and chronic canine studies and the human stress echo studies. A consultant from Philips Medical Systems will work with the entire team to bridge the ultrasound image acquisition technology.


Diffuse intrinsic pontine gliomas (DIPG) comprise 70-80% of all brainstem gliomas in children. Approximately 300 children develop brainstem gliomas per year. Prognosis for patients with DIPGs remains dismal with a median survival of less than 1 year and no improvement in survival has been realized in more than three decades. No effective chemotherapeutic regimens are currently available.Little is known about the biology of these lesions because they are typically not biopsied and surgical resection is impossible given their location.We are establishing an International Diffuse Intrinsic Pontine Glioma Registry to provide a comprehensive database of clinical, radiologic and pathologic data linked to a bioinformatics repository of molecular data of patients. This is a new project, currently under development. Two initial studies are planned.


Guidelines for providing quality care to patients with acute myocardial infarction (AMI) based on randomized controlled trial (RCT) evidence may not be appropriate in higher risk groups because of limited external validity imposed by stringent RCT exclusion criteria. Like the Agency for Healthcare Research and Quality (AHRQ) priority populations, RCT ineligible populations are often characterized by risk factors that typically indicate the need for additional or specialized care. Chronic conditions and comorbidities, disability, special health care needs and advanced age are all considered priority populations by AHRQ and are standard exclusion criteria for RCTs. The VA patient population shares a number of these risk factors, i.e. increased comorbidites and disability, in addition to high proportions of low income and rural patients also considered priority populations by AHRQ. This dilemma of less quality evidence for those in more need of care is amplified by the methodological challenges and ethical constraints of randomization in high risk groups. The overall objective of this project will use veterans as an example of a population typically excluded from RCTs to examine how they differ from the general population and to specifically examine outcomes for this population and as they relate to characteristics that make veterans ineligible for most RCTs. This objective is divided into three specific aims: 1) To describe the population of VA patients admitted with AMI and compare it to those patients represented in community based AMI registries using standard descriptive statistics; 2) to compare outcomes for VA patients with an ECG diagnosed ST-elevated myocardial infarction (STEMI) receiving reperfusion within guideline recommended time to those receiving later reperfusion using multivariable regression; 3) to describe the population of VA patients based on time from symptom onset to hospital arrival and evaluate outcomes for those presenting more than 12 hours from symptom onset using instrumental variable analysis. Because veterans often have one or more characteristics typically making them ineligible for RCTs, guidelines for treatment based on this evidence may not represent the best quality care for them. Prior to investing additional resources implementing quality improvement interventions to meet guideline recommendations, an indepth examination of characteristics and outcomes specific to the veteran population with AMI is important to better inform resource allocation and provide optimal individualized care. [unreadable] [unreadable] [unreadable]


Colorectal Cancer (CRC) incidence and mortality rates are highest in African Americans (AA's) compared with all other ethnic groups. One factor that may contribute to this trend is the lower rate of participation in CRC screening among AAs, which is critical to the prevention and early detection of CRC. Recent data indicate that the removal of precancerous polyps (via colonoscopy) decreases CRC incidence by 75-90 percent. Despite the implementation of national policy changes to increase CRC screening (through Medicaid/Medicare reimbursement for CRC screening and easier "open" access to colonoscopy) adherence remains alarmingly low. Our preliminary data show that, even after implementation of standard patient navigation (SPN) (i.e., assisting patients with making/keeping their appointments), only 40 percent of low-income minorities followed-through on their physician recommendation. Guided by Cognitive-Behavioral Social Learning Theory as a conceptual framework and cultural targeting as an intervention strategy, the proposed randomized clinical trial will investigate integrating within SPN a targeted discussion of intrapersonal and cultural barriers to colonoscopy (i.e., fear, lack of knowledge, medical mistrust, fatalism and fear) prevalent with low-income AAs. Based on research on source credibility and reference group-based social identity theory, we will also explore navigator status as a peer on the impact of culturally targeted PN. Thus, we will compare three PN strategies: SPN carried out by a professional navigator, Culturally Targeted PN carried out by a professional (CTPN-Pro) and Culturally Targeted PN carried out by a peer who has undergone colonoscopy (CTPN-Peer). Specific Aims: Aim 1: Compare the efficacy of SPN, CTPN-Pro, and CTPN-Peer on adherence to colonoscopy CRC screening in average risk, low-income AAs who have a primary care physician referral for colonoscopy. Aim 2: Explore potential mechanisms (i.e., mediators) underlying the beneficial effects of CTPN-Pro and CTPN-Peer and to examine for whom the CTPN-Pro and CTPN-Peer are most effective (i.e., moderators). Aim 3: Compare the cost effectiveness of CTPN-Pro and CTPN-Peer. Cost effectiveness will be examined in terms of direct clinical costs of screening (i.e., savings associated with more efficient use of personnel, space, and equipment) and patient costs (i.e., costs of CRC treatment and the opportunity costs to the patient per life-year saved). Results from the proposed work will facilitate the broad dissemination of PN to reduce ethnic and racial health disparities in CRC incidence mortality and will advance our understanding of PN. [unreadable] [unreadable] [unreadable]


The purpose of this project is to conduct two intervention trials using multiple vitamin-mineral supplements to evaluate the relationship between such supplements and esophageal cancer incidence and mortality. One trial is being conducted in patients diagnosed with esophageal dysplasia (n = 3400) and the other in the general population in a high-risk region (n = 30,000). The effect of these supplements on regression/progression of esophageal dysplasia and total cancer incidence, total cancer mortality, and total mortality will be evaluated. These two studies are being conducted in Linxian (Henan Province) in the People's Republic of China (PRC). Linxian, a rural country with population 800,000, was selected because it has the highest rate of esophageal cancer in the world (>100/100,000) and because there is suspicion that the population's chronic deficiencies of multiple nutrients may be etiologically involved. This study is being conducted jointly by the Biostatistics Branch of the Division of Cancer Etiology and the Cancer Prevention Studies Branch of the Division of Cancer Prevention and Control at the NCI in collaboration with the Cancer Institute of the Chinese Academy of Medical Sciences.


Research suggests that a substantial proportion of women who describe themselves as contraceptive users are using contraception ineffectively, thereby putting themselves at risk of unintended pregnancy. Numerous studies attest to the importance of women's beliefs about contraception and pregnancy in explaining ineffective contraceptive use. The relationship between beliefs and contraceptive use can be conceptualized in the Health Belief model, which posits that contraceptive behavior is a function of perceived susceptibility to pregnancy, perceived severity of pregnancy, and perceived benefits and barriers to contraceptive use. Due to time and personnel constraints, family planning providers have little opportunity to elicit and discuss these beliefs with their clients. This project designs and evaluates a method by which counselors can efficiently address those beliefs that may put clients at risk of unintended pregnancy. The method uses a questionnaire, filled out by clients waiting for services, as a risk assessment tool; counselors will be given access to this risk assessment tool and will be trained to use it to guide their counseling. Subsequent contraceptive behavior will be compared between three groups: 1) women who receive standard counseling according to the current protocol, 2) women whose counselors were trained to address "risky" beliefs but were not given access to the tool, and 3) women whose counselors had access to the tool. 525 women visiting Planned Parenthood of Metropolitan Washington who are seeking oral contraception but are not currently using it will be asked to participate in the study. The first 175~participants will receive standard counseling. The next 175 will receive standard counseling by specially trained counselors, and the final 175 will receive individualized counseling from the same trained counselors who will now have access to their risk assessment tools. All participants will complete the risk assessment tool, a client satisfaction questionnaire, and be called after three months to assess contraceptive behavior and changes in beliefs. Behavioral outcomes to be assessed include 1) continuation of oral contraceptive use, 2) consistency of oral contraceptive use, and 3) consistency of overall contraceptive use, including women who discontinue oral contraceptives or use them inconsistently. Staff perceptions of the interventions will be assessed, as will the efficiency and feasibility of the interventions. If the tool is successful, it will help family planning providers improve both the efficiency and the quality of services at a negligible cost.


The overall goal of this proposed work is to develop a system for delivering therapeutic ionizing xradiation along a flexible, curved path from an extracorporeal x-ray source to an internal body cavity or lumen and depositing this radiation into a desired volume of tissue. Such a device would operate in a manner comparable to the solid optical fiber methods used to transmit laser radiation into interior body cavities. It is indeed based upon similar physical principles, but utilizes reflection along the hollow fiber interior and hence is not affected by any material in contact with the capillary outside surface. A flexible conduit/catheter for transmitting x-rays is possible because of the phenomenon termed total surface specular x-ray reflection, which allows x-ray radiation incident at grazing angles along the interior surface of a hollow capillary to be transmitted without loss of energy. The possibility of using this phenomenon in a medical device does not appear to have been previously recognized. As the interior diameter of the hollow capillaries through which the x-rays are transmitted is reduced, increasingly large bending angles are permitted without loss of the transmitted intensity. Curvatures as sharp as 1 cm are possible using existing lead glasses. Such a conduit/catheter assembly can be used in conjunction with an endoscope or bronchoscope or other flexible assemblies. Radiation could also be delivered via an angiographic catheter or directly via a trochar needle into a lesion. Three specific aims are proposed: 1) to construct such an x-ray carrying conduit/catheter assembly; 2) to measure the deliverable dose rate as a function of x-ray photon energy, capillary diameter, bending radius, and the intensity of the extracorporeal x-ray source; 3) to assess quantitatively the feasibility of this device in clinical practice, particularly with existing endoscopes and bronchoscopes, and as a treatment modality generally.


ABSTRACT The current equipment supplement request is for an upgrade of the current mass spectrometry platform to enable nano-scale liquid chromatography. Active grants R01 GM099989 (PI: Lee), and R01s GM130810 and GM121603 (PI: Atkins) jointly request the funds for a Waters M-class HPLC with the corresponding NanoLockSpray ionization source. The upgrade will provide the enhancement in sensitivity to overcome the limits in sensitivity and material requirements that have hindered analytical characterization of numerous the proteins of interest. This will dramatically increase the range of systems and samples we can analyze. The equipment will be shared equally among three NIGMS-funded PIs while being managed and maintained by the School of Pharmacy Mass Spectrometry Facility.


The overall goal of this proposal is to understand how clostridial neurotoxins interact with their target cells and to use this knowledge to develop effective inhibitors of the toxin-cell interaction. Three botulinum toxins (A,B and E) will be studied to determine whether observations made with one toxin (botulinum toxin A) might have general applicability. If the botulinum toxins behave similarly, comparisons will be made to tetanus toxin to determine what similarities exist between the two classes of neurotoxins. Specifically, studies of the binding of botulinum toxins B and E to G1b gangliosides and derivatives thereof will be carried out. Results will be compared to those obtained for botulinum toxin A and tetanus toxin. Accumulating evidence suggests that gangliosides may not be the only cell surface receptor for these neurotoxins. In recent studies, we have found that botulinum toxin A, its heavy chain and the carboxy terminal half of the heavy chain bind to synaptosomal proteins. Tetanus toxin also adhered to synaptosomal proteins but it had to be preincubated with GT1b in order to bind to protein. The protein adhered to by both neurotoxins has an apparent molecular weight of approximately 77 kDa and is glycosylated. Experiments designed to purify and characterize the approximately kDa protein will be continued, and the site(s) adhered to by the toxin will be identified as will the portion of the neurotoxin responsible for the binding, and in in vitro experiments designed to identify the functional aspects of specific interactions.


The primary mammalian pacemaker, located in the suprachiasmatic nuclei (SCN) of the hypothalamus, drives circadian rhythms in physiology and behavior that are synchronized with the environment. In temperate climates, animals use annual changes in day length, or photoperiod, to adjust their physiological state with changes in season. The way in which the clock encodes photoperiod is unknown, but two coupled oscillators, one synchronized to morning (M) and the other synchronized to evening (E), whose phase relationship varies with day length would allow the SCN to compute seasonal changes in photoperiod. These dual oscillators may be embedded in the molecular transcriptional and translational feedback loops that participate in endogenous circadian rhythm generation in the SCN (4). The clock gene, Period 1 (Perl), is an important component of rhythm generation and also participates in the synchronization of the clock to light. The current proposal will investigate role of the isolated SCN in encoding day length in photoperiodic Perl reporter rats generated by crossing the photoperiodic Fischer 344 (F344) strain with transgenic Per1::luciferase (Per1::luc) rats that express luciferase under the control of the Perl promoter. The anatomical locations of putative M and E oscillators will be investigated by imaging real-time Perl promoter activity in subdivisions of the SCN in photoperiodic rats exposed to either short or long day lengths. Singlecell luminescence imaging will be used to examine photoperiod-dependent differences in Perl promoter activity within an individual SCN neuron. This proposal will also investigate the role of the molecular clock in encoding the critical photoperiod permissive for reproduction by comparing the Perl promoter activity profile of rats exposed to either inhibitory or permissive day lengths for reproduction. Furthermore, it will be determined whether photoperiod encoding in the SCN differs between photoperiodic F344 and nonphotoperiodic Wistar rats. Seasonal variations in photoperiod may also affect neurological processes in humans. PUBLIC HEALTH RELEVANCE Many patients diagnosed with seasonal affective disorder (SAD) experience depressive episodes only during the fall and winter, and treatment with early morning bright light corrects the delay in their circadian rhythms. Therefore, investigating the role of the SCN in encoding photoperiod may lead to the development of novel therapeutic treatments for SAD patients. [unreadable] [unreadable] [unreadable]


SUPPRESSION OF ENDOCRINE FUNCTION IN HUMAN BREAST CANCER


The long-term goals of this project are to elucidate the molecular mechanisms used by bacteriophage T4 to initiate DNA replication, and to understand the relationships between phage replication, recombination and transcription. As judged by striking similarities in the DNA metabolic machineries of T4 and eukaryotic cells, the phage T4 model system may be particularly relevant to studies of human chromosomal replication, recombination and repair, and thus to human health. Two well-characterized T4 replication origins contain a middle-mode promoter and a downstream segment of DNA that readily unwinds. The roles of the promoter and downstream region in initiation will be explored, using origin DNA mutations and T4 mutants that lack particular replication proteins. One interesting model is that the combination of a promoter and an unwinding region leads to a stable RNA-DNA hybrid in the unwinding region. This model will be directly tested in vitro, and the results could have important implications for both transcription and DNA replication. A new system of transposon mutagenesis will also be used to find additional genes involved in origin-dependent replication and in T4 recombinational processes. The mechanism of recombination-dependent T4 replication will be explored using a novel system in which DNA replication is triggered by site-specific double-strand breaks in the viral genome. Specific intermediates in recombination-dependent replication will be analyzed in wild type and various mutant infections, and the requirement for homologous DNA segments will be further explored. Direct studies of T4 recombination will also utilize site-specific double-strand breaks in the phage genome. A single-strand annealing model for genetic recombination has long been postulated for phage T4 and has recently emerged for recombination between repeated eukaryotic genes. This model will be tested by analyzing the recombination of DNA molecules that could provide complementary single-stranded regions adjacent to double-strand breaks. The mechanism of recombination at native T4 recombination hotspots will also be examined, particularly because these hotspots are triggered by the above-mentioned replication origins of the virus.


Hematopoietic stem cell (HCT) transplantation offers curative therapy for a variety of malignant and nonmalignant disorders. It is limited by donor availability, transplant related toxicity, graft vs. host disease (GVHD), malignant relapse, infections, and for some patients, reduction in their post-transplant quality of life. Continuing progress from the previous funding period, the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) proposes to develop and execute scientifically meritorious, prospective clinical trials addressing key issues in HCT. We will conduct multicenter Phase II and more importantly, prospective Phase III trials in six key areas including: alternative donors and graft sources; regimen related toxicity; GVHD; disease recurrence; infection and immune reconstitution; and late effects and quality of life. Due to the complexity of pediatric transplantation, especially in those with rare inherited disorders, which are complex and difficult to study, we will devote special attention and focused scientific expertise to using the coordinated strength of the Network to improve BMT outcomes for this unique population. The BMT CTN will also amplify and leverage Network resources through collaborative research and ancillary studies of biologic endpoints that will be integrated with and complement clinical endpoints. The Network will also seek active collaboration with other scientific bodies including NCI- funded Cancer Cooperative Groups to improve the efficiency of clinical transplant studies for the large number of patients who could benefit from BMT and to maximize our successful completion of high quality and high priority clinical trials. (End of abstract.)


The proposal aims to define the molecular boundaries of size, shape, structure, and relevant physicochemical properties for a volatile organic compound (VOC) to act as an effective trigeminal (i.e., chemesthetic) stimulus in humans. Chemesthetic responses in the mucosae of the face to airborne chemicals include nasal pungency (i.e., irritation) and eye irritation, two frequently mentioned adverse symptoms arising from indoor (e.g., sick building syndrome) and occupational environments. This proposal is a follow-up of our previous work exploring the physicochemical basis for the production of these chemesthetic responses by VOCs. Systematic chemosensory testing of members of homologous chemical series has revealed that a homolog can be reached where the ability to evoke these trigeminal responses begins to fade, and finally disappears for all ensuing homologs, constituting a "cut off" effect. The proposal focuses on the two or three homologs from each series at the boundary of the cut off effect, and will measure the sensory responses of nasal pungency, nasal localization, and eye irritation. From each substance selected to be tested, we will obtain stimulus-response (psychometric) functions spanning the range from chance detection to virtually perfect detection in order to identify the precise homolog reaching the cut off point in each homologous series. By means of chemical modeling and by additional sensory testing of rigid chemical structures molded on the molecular parameters of the cut off homologs, we will define the molecular and physicochemical boundaries for any volatile compound to be able to act as an effective trigeminal chemosensory stimulus. Knowledge of such boundaries has important implications for both basic and applied aspects of trigeminal chemoreception. From a basic perspective it will contribute to the chemical characterization of the trigeminal reception process(es), from an applied perspective it will allow to understand and prevent adverse sensory reactions from air pollutants.


PROJECT SUMMARY/ABSTRACT Drug addiction is a chronic disease that produces significant costs to individuals and society. While almost one million Americans suffer from cocaine use disorders, there are currently no approved pharmacotherapies for this disease. Our laboratory has implemented animal models of cocaine abuse that analyze motivation to take cocaine (behavioral economics demand curve analysis) and the propensity for cocaine relapse (cued reinstatement of extinguished cocaine seeking). I will use those behavioral methods in conjunction with DREADD chemogenetics and morpholino antisense approaches to determine the roles of orexins (hypocretins) in nucleus accumbens shell (AcbSh) in cocaine abuse. Orexins are importantly involved in the motivation for cocaine, and orexin neurons provide direct projections to AcbSh. In addition, all drugs of abuse increase dopamine signaling in this brain region, and in vitro studies found that orexin in AcbSh increases dopamine concentration. These and other findings point to a role for orexin signaling in AcbSh in cocaine abuse. However, this possibility has received little examination. The hypothesis of this proposal is that orexin in AcbSh drives cocaine demand and is necessary for cued-reinstatement of cocaine seeking behavior. Aim 1 will use pharmacology to inactivate AcbSh orexin receptors OX1R and OX2R in behavioral economics (BE) and reinstatement paradigms. Previous work with BE found that OX1R signaling is associated with cocaine demand (motivation). OX2R is more abundant in AcbSh and has been recently implicated in drug seeking. I expect that inactivation of both receptors will decrease demand and cocaine cued-reinstatement. Aim 2 will use a disconnection approach with DREADDs and morpholino antisense to confirm results in Aim 1. A hM4Di DREADD will inactivate lateral or dorsomedial hypothalamic neurons projecting to AcbSh unilaterally while morpholino antisense microinjected into the lateral hypothalamus or dorsomedial hypothalamus will downregulate orexin on the contralateral side to the DREADDs during BE and cued-reinstatement. I predict that inactivation of AcbSh- projecting lateral hypothalamic neurons in rats that receive orexin morpholino antisense in LH will result in decreased cocaine demand during BE and increased lever pressing in cued-reinstatement. This would support previous suggestions that lateral hypothalamic orexin neurons mediate reward, whereas dorsomedial orexin neurons mediate arousal. The expected results would indicate that this lateral hypothalamic orexin-AcbSh circuit drives cocaine demand and reinstatement. Together, the proposed studies will increase our understanding of the orexin system, as well as the neural circuitry underlying cocaine addiction. This fellowship will train the applicant in orexin neurobiology and in several innovative techniques in systems and behavioral neuroscience, including chemogenetic and pharmacological approaches; self-administration and behavioral economics paradigms; cannula and catheter surgeries; morpholino knockdown; and immunohistochemical techniques. These skills will be critical for the applicant to become an independent investigator in the drug addiction field.


The NIAMS IRP Animal Program has oversight for 25 Animal Study Proposals (ASPs) using mouse, rat, fish, and rabbit models. At NIAMS, there is a great deal of research around arthritis, recombination/chromatin remodeling using AID transgenic mice, familial Mediterranean fever (FMF) and Tnf receptor associated periodic syndrome. There is also a great deal of research involving muscle and related diseases (experimentation with: N-RAP protein, glycogen storage disease Type II, SIRT1 gene, myofibril assembly, myositis including drug induced, and the voice generating muscle in Midshipman fish) and the study of sub-cellular organelles and cytoskeleton in skeletal muscle. Studies at NIAMS also include in vitro experiments studying retroviral transduction, bone marrow transplantation, and epidermal differentiation studies using fetal and adult skin tissues. Research involving allergic rhinitis and other transmit receptor-induced cellular responses is conducted at the institute. Many of the institutes studies involve apoptosis and autoimmunity. Also of interest within NIAMS are cartilage studies focused on tissue-engineered cartilage, chondrogenesis, GDF-F, COMP and osteoarthritis.


Musculoskeletal soft tissue injuries represent a large portion of sports- related injuries. The frequency of occurrence of these injuries has increased rapidly over the last decade as participation in athletic activities and physical conditioning have increased in the United States. The junction between skeletal muscle cells and tendon collagen fibers has been implicated in a common type of soft tissue injury which occurs when active muscle is passively extended. These injuries apparently occur most frequently in inadequately trained participants which suggests that training and, conversely, disuse can influence the structure or physical properties of these myotendinous junctions (MTJs). This proposed investigation is directed toward elucidating the effects of exercise and disuse on the structure, molecular composition and mechanical strength of MTJs using frog skeletal muscle as a model. These experimental animals were selected because: 1) they are unusual in that single cell mechanical testing can be easily performed, and 2) the molecular composition and quantitative ultrastructure of their MTJs has been studied previously. In the proposed investigation, these animals will be subjected to strength-training, increased-frequency exercise, reduced-frequency exercise or denervation atrophy. At selected times in their training, representative animals from each group will be sacrificed and hind limb muscles analyzed for changes in: 1) mass, 2) aerobic capacity, 3) anaerobic capacity, 4) quantitative MTJ structure, 5) concentration of structural proteins at the MTJ, or 6) breaking strength at the MTJ. These data will provide the first information on the response of MTJs to exercise and disuse. These data will also provide the basis of a comprehensive description of the relationships between modifications in muscle cell structure and molecular composition to load-bearing at MTJs in ways that can be related to clinically-observed defects in humans.


Voltage-activated Ca channels serve two critical functions: the regulation of cellular excitability and the regulation of Ca entry. Alterations in the density, or function, of L-type Ca channels are implicated in a variety of cardiovascular diseases. Thus, elaborating the basic mechanisms that regulate Ca channels is important for understanding both fundamental channel physiology and for therapeutic intervention. During the past funding period, we have identified the Rem GTPase as a novel modulator of I(Ca). It was originally thought that Rem association with CaVbeta- subunits chronically regulated I(Ca) by inhibiting channel trafficking. Our studies have disproved this hypothesis, demonstrating Rem-mediated Ca channel regulation without changes in surface density. Instead, Rem seems to modulate Ca channel activity through interactions with both CaVbeta and the proximal CaV1.2 C-terminus near the CB/IQ domain. Two of the most physiologically relevant controls of I(Ca) are PKA-modulation and calmodulin (CaM)-modulation. Our most recent studies suggest that Rem modulates I(Ca) responses to each of these signaling pathways. Thus, Rem appears to contribute to both beta-adrenergic and Ca-CaM control of I(Ca). The specific hypothesis to be tested is that Rem GTPase regulates Ca channel activity in cardiac muscle through interactions with both CaVb-subunits and the CaV1.2 C- terminus. Three hypothesis driven aims focus our studies and advance knowledge of this novel regulatory mechanism. Aim 1 will explore the nature of Rem-mediated channel regulation by examining the effect of Rem loss on Ca channel regulation. Initial characterization of Rem knockout mice indicates that i) Rem functions in vivo to regulate I(Ca);ii) contributes to the cardiac response to pressure-overload;and iii) contributes to cardiac myocyte growth/maturation homeostasis. Aim 2 will determine whether interaction of Rem with CaVbeta or CB/IQ is critical for modulation of I(Ca). Aim 3 will investigate how Ca- calmodulin modulates Rem-mediated channel blockade, and determine whether PKA phosphorylation alters Rem membrane trafficking or the interaction between Rem and its binding partners. RGK G-proteins function as modulators of Ca channel activity, contributing to regulation of I(Ca), excitation- contraction coupling, and the cardiac response to pressure-overload. The goal of this research is to generate a deeper understanding of the physiological ramifications and molecular mechanism of RGK/Ca channel modulation, to speed progress toward the therapeutic exploitation of RGKs in cardiovascular disease. PUBLIC HEALTH RELEVANCE: Voltage-activated calcium channels serve two critical functions: the regulation of calcium entry and the control of heart contraction. Changes in the number, or function, of L-type calcium channels are implicated in a variety of cardiovascular disease, including atrial fibrillation, heart failure, and ischemic heart disease. Thus, understanding the basic mechanisms that regulate calcium cannels is important in understanding both fundamental heart muscle performance and for therapeutic intervention. We have identified a family of proteins that regulate calcium channels and the goal of this research is to speed progress toward the day when these proteins can be used to treat heart disease.


Individuals who develop rapid progression of HIV-1 infection have been shown to have a high burden of relatively homogeneous viruses (quasi species) and a low frequency of cytotoxic T lymphocytes that respond to HIV-1, whereas individuals who progress more slowly or not at all have a lower burden of a much more diverse viral quasi species and evidence of vigorous CTL response to HIV-1. We hypothesize that these individuals' antiviral cellular immune responses (CTL and T helper) are effectively exerting selective pressure upon the viral quasi species, leading to amino acid changes, quasi species diversification, and modified interaction with the host immune system due to altered binding of MHC-binding regions. This proposal describes the development of EpiMatrix, a computer-driven T cell epitope prediction algorithm, and the application of the algorithm to the evaluation of quasi species evolution. It is proposed that the replacement of anchor- based motifs with extended matrix-based motifs (as in EpiMatrix) will improve the predictive capacity of T cell epitope prediction algorithms for selected MHC alleles and will permit evaluation of quasi species evolution. The specific aims of the proposed work are: To develop EpiMatrix, a second generation T cell epitope prediction program. To apply EpiMatrix to HIV-1 quasi species; predict, synthesize, & test MHC-binding regions/T epitopes. To evaluate the impact of quasi species evolution on T cell response to epitopes defined in the previous aim. The use of EpiMatrix of predict MHC-binding regions and T cell epitopes within variable regions will allow us, for the first time, to accurately assess the influence of sequence diversity within both conserved and variable regions upon the ability of the host cellular immune response to recognize and respond to HIV-1 quasi species. The successful completion of this project will lead to an enhanced understanding of the interplay between the antiviral pressure of the cellular immune responses, viral burden, and diversity of quasi species within infected individuals. These fundamental interactions that define HIV-1 infection are critical to our understanding of HIV-1 pathogenesis. In addition, the epitope prediction method and epitopes defined during these investigations will aid in the development of a vaccine against AIDS.


Of great contemporary concern in the U.S., as well as in other countries, is the question of what constitutes a healthful dietary intake of the B-vitamin, folate. Although adequate amounts of folate intake protect against colorectal cancer, accumulating evidence indicates that overly abundant quantities among those who harbor pre-cancerous or cancerous lesions may produce a paradoxical acceleration of tumorigenesis. There are many sources of folic acid in the american diet that supplement the quantities naturally present in foods: voluntarily-fortified foods, vitamin supplements and mandatory fortification. The seminal question is whether these sources are collectively sufficient to produce the cancer-promoting effect of folate;a grave concern for which there is already supportive evidence from several pre-clinical and clinical studies. Due to ethical and chronological constraints, this dilemma cannot be resolved by human studies alone. Consistent with this reality are the conclusions of a recent international conference on the topic, which included among the high priority recommendations the need to pursue the issue in appropriate animal models. The overall goal of this proposal, therefore, is to answer these questions within the context of a highly innovative and powerful animal model. This novel animal model of colorectal carcinogenesis, GEM-1, allows one to monitor development of colonic neoplasms in vivo over a period of several months. This will enable us to determine critical parameters such as the threshold of dose at which dietary folate has a promotional effect and to develop a mathematical model that defines the relationship. Our proposed studies will: 1) characterize the GEM-1 model's response to supraphysiological quantities of dietary folic acid in regard to cancer promotion, 2) define the quantitative nature of the dose-response relationship between supraphysiological folic acid intake and acceleration of tumorigenesis, 3) determine whether delivery of supraphysiological quantities of a natural form of the vitamin, 5-methyltetrahydrofolate, lacks the cancer-promoting effects observed with the synthetic form, folic acid, and 4) provide evidence that the molecular pathway by which excess folic acid is driving carcinogenesis is by providing abundant quantities of the rate-limiting substrate for DNA synthesis, thymidine. PUBLIC HEALTH RELEVANCE: There is great debate in the U.S. and in other countries as to what constitutes a healthful dietary intake of the B-vitamin, folate, because many sources of the vitamin have been introduced into the food stream and there is accumulating evidence to suggest that overly abundant quantities of the vitamin may promote the risk of colorectal cancer. The existing data concerning the possible promotion of cancer is rather equivocal, and yet the potential ramifications are very great, making this a particularly pressing public health issue that needs to be addressed with some urgency. The studies proposed in this application will be conducted in a novel and particularly well-suited animal model and will provide important answers as to whether the intake of folate that is helping optimize health for one segment of the population might be presenting potential risks to other segments of the society.


HIV-1 RT continues to be a key target for anti-HIV therapy. The long term goal of our project is to comprehensively define HIV RT function and the mechanism of drug resistance. There is an increasing realization that proper positioning of template-primer is central to several key aspects of RT function. We intend to delineate the functional role of three specific interactions between HIV-1 RT and the template-primer. These contact points lie along the template-primer cleft, in front of the active site (contacting the template 5'-overhang), behind the active site (contacting the template at penultimate basepair), or further away in the minor groove (contacting the primer at 3rd to 6th basepairs) respectively. Three specific aims are listed. First, we will study the fingers subdomain-template contacts near the active site via nested deletions of 33-[M loop of HIV-1 RT created to disrupt the proposed interactions with the template overhang and/or the dNTP. In addition to testing these mutants for dNTP-binding, accuracy of dNTP insertion, processivity and strand transfer, we will also test the role of J33-j34 loop as a determinant of the natural high susceptibility of HIV-1 RT to 2',3'-dideoxyNTPs. The mutation rate of multi-dideoxynucleoside analog resistant RT variants and whether a proofreading-like activity contributes to the overall fidelity will be tested. Second, we will investigate the role of the template grip 135a, which forms a close contact with the penultimate basepair of the template-primer, in RT function. We will both attempt to delineate the mechanism of frameshift mutagenesis by HIV-1 RT, the precise role of template grip 135a in frameshift fidelity and the mechanistic basis for its global influence on fidelity. Altered template grip mutants of HIV-1 RT will be tested for effects on affinity to the template-primer duplex, processivity, RNase H activity and strand transfer. Lastly, we will test whether the thumb helix clamp involving aH and cxl helices indeed serves a critical function in enzyme translocation subsequent to each dNTP addition cycle. We will also test its role in processive polymerization and in frameshift fidelity.


The CITAR of the University of Texas School of Public Health (UTSPH) is a program for advanced training on HIV prevention research. Fellowships are for foreign health professionals leading to a 4 year doctoral degree at the UTSPH in Houston followed by a 5th post-doctoral year doing supervised research in their home country. Support for CITAR Fellows may come from a variety of sources but to date has been almost exclusively from the Vietnam Education Foundation (VEF) which provides 2 year of stipends and institutional support. An AITRP grant would support the remaining 3 years and travel expenses between Houston and our research and training sites in Vietnam. The goals of CITAR are: 1. Education &training of outstanding [foreign health professionals for research careers in public health in their home country on HIV and HIV related issues and to help them obtain funding to carry out those careers;2. Foster research and research capacity in developing countries;3. Promote understanding and cooperation between the United States and other countries. We chose Vietnam because the combination of the early but rapidly expanding AIDS epidemic, commitment of the Vietnamese government but severe limitations of its current public health manpower make it uniquely suited to benefit from our faculty who are highly experienced in AIDS research and international health. .Presently we have 7 Vietnamese Fellows (6 from VEF and 1 on a VN government scholarship) and 3 more VEF Fellows starting Fall 2005. We expect to have 2 to 4 new additional students in each subsequent year. There are 6 elements to the CITAR training experience: 1. Faculty supervised HIV field research leading to a thesis;2. Didactic academic courses;3. Weekly seminar in which faculty and fellows share presentations and discussions about HIV and related issues, 4. Practica;5. Course on practical issues of international research, and;6. Close mentoring of all aspects of the educational experience. A critical cross-cutting element will be ethics, focusing on the ethical conduct of science which will occur in all of the teaching elements. An AITRP would not only fit the philosophy and goals of CITAR for these Vietnam trainees, it would leverage Fogarty resources by supporting fellows already two years into their doctoral programs, with proven academic performance &commitment to research in Vietnam. We will hold student-faculty activities in VN aimed at successful reentry including short courses &VN based.


[unreadable] We have developed a water-based gel that changes color in response to doses of ionizing radiation, producing a stable, non-diffusing image of the radiation dose distribution. We have also developed an optical scanner system to record and manipulate the three-dimensional images. The system has significant advantages over competing methods. In this Phase I project we will demonstrate whether this system is suitable for use as a quality assurance method for radiation therapy treatment planning. The research program will determine the practical limits of radiation dose sensitivity under clinical conditions, and investigate how changes in gel composition and scanning techniques will affect the sensitivity and reproducibility, as well as cost. We will obtain much of the necessary data using clinical irradiation facilities and procedures. In Phase II we plan to establish the accuracy of the system for dose measurement under clinical conditions, develop software to enhance the user-friendliness of the system and perform clinical trials to support a possible application for FDA approval as a 510(k) device. Our long-term commercial objectives are to bring this technology to market by licensing the gel technology to a specialty chemical company, and licensing the scanner technology to a company that currently sells radiation therapy equipment. [unreadable] [unreadable]


Our goal is to understand the molecular mechanisms through which obesity and excessive fat consumption increase the risk of cancer development. Epidemiological studies have shown that of all cancers, obesity has the most dramatic effect on the common form of liver cancer - hepatocellular carcinoma (HCC). In addition to its important metabolic functions, the liver is an immune organ that is severely impacted by obesity, resulting in non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH), inflammatory conditions that greatly increase HCC risk. To genetically and molecularly investigate how obesity increases HCC risk, we established mouse models in which liver fat accumulation (hepatosteatosis) strongly enhances development of chemically-induced HCC;models that a recent review found to be highly relevant to the etiology and pathogenesis of human HCC. Using these models, we had demonstrated that obesity-induced liver inflammation plays a key role in HCC pathogenesis by activating the oncogenic transcription factor STAT3. However, hepatosteatosis also results in chronic activation of Target of Rapamycin (TOR) complex 1 (TORC1), thereby suppressing autophagy. We postulate that TORC1 activation and suppressed autophagy are additional mechanisms that contribute to the molecular pathogenesis of obesity-promoted HCC. To investigate this central hypothesis of the current proposal, four specific aims will be pursued: 1) Determine whether inhibition of TORC1 prevents obesity-promoted hepatocarcinogenesis;2) Investigate the contribution of chronic TORC1 activation to DEN-induced hepatocarcinogenesis;3) Identify effector pathways that mediate TORC1 effects on obesity-promoted hepatocarcinogenesis;and 4) Examine whether TORC1 feedback regulation by Sestrin has a role in obesity-promoted hepatocarcinogenesis. Whereas the first two aims will ask whether TORC1 activation is required and sufficient for obesity-promoted hepatocarcinogenesis, the third aim will explore the hypothesis that the most critical pathogenic function of chronic TORC1 activation is suppression of basal autophagy. The last aim will investigate the connection between tumor suppressor p53 and the regulation of TORC1 activity and autophagy, which depends on expression of Sestrins, a small family of p53-regulated proteins that activate AMPK. Studies will be performed mainly in vivo using both constitutive and inducible gene targeting as well as a novel approach for isolation and specific manipulation of pre-neoplastic HCC initiating cells. We will also develop new tools for uncoupling TORC1 from inhibition of autophagy. Collectively, the proposed experiments will shed new light on the pathogenic mechanisms that link hypernutrition and obesity to tumor development and progression via TORC1 and its ability to suppress basal autophagy. The proposed studies will also lead to new preventive and therapeutic strategies that will reduce the toll of obesity-promoted liver cancer as well as other cancers, such as pancreatic cancer, that are also strongly impacted by the obesity epidemic. PUBLIC HEALTH RELEVANCE: The obesity epidemic has precipitated a marked increase in the incidence of hepatocellular carcinoma (HCC) or liver cancer. The mechanisms by which obesity increases HCC risk are not fully known but their understanding is required for the design of effective therapeutic and preventive strategies. We will use recently developed mouse models and innovative technology for isolation of pre-neoplastic cells to fully understand how obesity promotes HCC development.


Objectives: Evaluate the risk of arsenic exposure and mitigation effectiveness in an Arizona mining/smelting community through speciated analysis of environmental media and urine samples. Background: The predominantly Hispanic populations of the Hayden- Winkelman community live adjacent to an open pit copper mine/smelter complex. Previous studies in these towns measured arsenic exposures that exceeded the 95th percentile for Arizona, although the sample size was limited. Community concerns have been expressed over arsenic exposure and the potential for adverse health effects. Specific Aims: To 1) Measure a representative sample of households for multi-media, multi-pathway, exposure to arsenic species; 2) Measure individual adsorbed dose using speciated urine arsenic; 3) Model bioavailability of arsenic species by media and pathway; 4) Evaluate the contribution of individual factors (age, socioeconomic status, ethnicity, lifestyle, diet, and disease) to arsenic absorbed dose; 5) Measure the effectiveness of interventions to decrease arsenic exposure; and 6) Educate the community through the establishment of a computer learning center linked with the University. Study design: 130 randomly selected and 20 community designated households will be evaluated for speciated arsenic exposure (HG-AAS) in indoor and outdoor air (PM10, PM25, vapor phase), vacuumed house dust, surface soil, water (drinking and tap), food and beverage collected using trained community members with quality assurance audits. In association with questionnaire data, regression analysis will be used to determine the effect of media, route of exposure, and individual factors on urine arsenic species concentration, and compared with existing statewide data (NHEXAS). Sixty-five intervention and 20 control households will be reevaluated for the effect of substitute or treated water and HEPA vacuuming. A public computer learning center will be developed and staffed by the community. Expected Outcome: Community members will earn the extent of their exposure to arsenic, the effectiveness of low-tech interventions, and how to obtain information on environmental exposures. There is also potential for future research, including evaluation of biological markers of effect of low-level arsenic exposure.


Stroke is the 3rd leading cause of death and the primary cause of severe, long term disability. Each year 795,000 Americans have a stroke and the annual costs to the economy are $57.9 billion. The vast majority of acute ischemic strokes are caused by a thrombus (blood clot) which occludes the blood vessel and stops blood flow to the brain. Tissue plasminogen activator (TPA), an agent that catalyzes the dissolution of blood clots, is the only effective, FDA-approved treatment for ischemic stroke. Unfortunately, TPA is associated with significant risks, delays in treatment, and is unsuccessful in up to 70% of patients at dissolving blood clots in sufficient time to protect the brain. There is a need for a safer, more effective therapy that facilitates early treatment, saves lives, reduces disability and lowers health care costs. In pre-clinical studies, we have shown that these goals might be achieved by a molecule that inactivates the major inhibitor of plasmin and, dissolves clots through a unique mechanism that avoids the risk of hemorrhage and neurotoxicity associated with TPA therapy. Following FDA guidance, we converted this molecule into a biologic drug candidate for stroke (stromab) that potently accelerates the dissolution of human clots. The goal of this Fast Track application is to move stromab further towards human trials by following FDA guidance to: 1) determine the optimal formulation and therapeutic time window for treatment, 2) produce and purify stromab under GLP conditions, 3) investigate the safety, pharmacokinetics and pharmacodynamics of stromab and, 4) submit an IND to the FDA. PUBLIC HEALTH RELEVANCE: Each year 795,000 Americans have a stroke and the annual costs to the economy are $57.9 billion. Tissue plasminogen activator, the only approved treatment for ischemic stroke, is associated with significant risks, delays in treatment, and is unsuccessful in up to 70% of patients at dissolving blood clots in sufficient time to protect the brain. This project seeks to develop a novel therapy for stroke that could markedly reduce death, disability and costs.


ABSTRACT Complex lymphatic anomalies, which include a variety of diagnoses: lymphangiectasia, Central Conducting Lymphatic Anomaly (CCLA), Generalized Lymphatic Anomaly (GLA), Kaposiform Lymphangiomatosis (KLA), and Gorham Stout Disease (GSD), are chronically debilitating and often life-threatening diseases. Unfortunately, for most patients, physicians can offer only palliative care that requires multiple outpatient visits and hospital admissions. The absence of data on the molecular etiology, and lack of understanding of the underlying molecular mechanisms in lymphatic anomalies have greatly hampered further research and precision medicine focused clinical trials. Our long-term goal is to identify efficacious therapies for complex lymphatic anomalies. The objective of this application is to uncover novel disease-causing genes/mutations and use in vitro and in vivo models established in our previous studies to determine optimal treatment strategies. Our preliminary studies have revealed multiple genes converging on the Mitogen-Activated Protein Kinase (MAPK) signaling pathway and modeling mutations in cellular and zebrafish systems have recapitulated the essential morphological features seen in the lymphatic anomaly patients. We have found that a handful of MEK/ERK inhibitors showed the biochemistry and morphological reversal of the effects of mutations in RASopathy genes. This proposal will test the hypothesis that sequencing of highly informative patients referred by an integrated multidisciplinary lymphatic anomalies clinic will unveil novel RASopathy genes and mutations, and these can be rapidly interrogated through our established cellular and zebrafish models to further investigate the mutation phenotype spectrum effect and correlating molecular biomarkers. The specific aims of this proposal are to: 1) Discover additional RASopathy genes and mutations through exome sequencing of patients with complex lymphatic anomalies; 2) Delineate the molecular mechanisms of newly identified genes and mutations in cellular and zebrafish models; and 3) Leverage novel disease models for therapeutic rescue to explore potential future therapeutic targets for human disease. The results from these experiments will have a significant impact on the field because they will answer fundamental questions regarding the genetic etiology, molecular mechanisms, and treatment options, and most importantly, provide validated pre-clinical data for molecularly implemented precision-based therapies for clinical trials. This knowledge will significantly advance our understanding of different types of lymphatic anomalies.


The simian immunodeficiency virus, SIVmne, has a high degree of genetic homology to the human lentivirus HIV-2 and a lower but still quite significant homology to HIV-l, both of which are etiologic agents of human AIDS. SIVmne, like the other SIVs isolated recently causes a severe immunodeficiency in the nonhuman primate, Macaca nemestrina, which selectively involves CD4+ cells and is virtually identical to AIDS in man. We have found a linkage between the expression on a subset of CD4+ lymphocytes of adhesive receptors for high endothelial venules (HEV) and selective depletion and perhaps susceptibility to infection with SIVmne. This arose during our work to develop the macaque as a model system for analysis of the role such receptors have in directing organ- specific migration of lymphocytes through lymph nodes, Peyer's patches, and inflammatory sites. With the monoclonal antibodies (Mabs) Hermes-l and recently with Hutch-l we have defined two distinct types of CD4+ lymphocytes, HEV receptor/hi and lo, which differ markedly in response to mitogens, and capacity to recirculate through lymph nodes. HEV-receptor high cells are lost selectively in simian AIDS before the onset of clinical illness. Our future goals are a) to determine the mechanisms which underly the selective affects of SIVmne on these two cells, b) determine if Mabs to HEV receptors can be used as sensitive prognostic indicators in AIDS, and e) further define the role of HEV-receptors in directing lymphocyte migration and their structural features which confer organ-specific recognition of endothelial cells. We will also explore the capacity of interleukins to regulate expression of a) HEV-receptors on lymphocytes and b) their ligands on endothelium. We propose to examine linkage of these homing receptors to specialized lymphocyte functions and determine the in vivo migratory properties of normal lymphocyte subsets and T cell clones. Various aspects of the project use cellular immunology techniques as well as hybridoma technology and molecular biological methods such as subtractive hybridization cDNA cloning and in situ hybridization with SIV and interleukin specific probes.


SUMMARY Computational Chemical Biology (CCB) Core will provide comprehensive computational support, including chemical virtual screening, physical property prediction, target validation, hits identification, lead optimization, pharmacophore identification, and pharmacokinetic and dynamic modeling. In addition, the CCB core will provide information management infrastructure supporting the creation and management of scientific data analysis workflows, project reporting, and database management for chemicals including their structures and biological properties. These activities will be fully integrated with projects and with Cores B and D. For example, the analysis of high-throughput screening data and chemical structure-activity relationship data generated by Core B will be stored, indexed, computationally analyzed, and visualized at the CCB Core.


The objectives of this project are to make homogeneous preparations of the enzymes undecaprenyl (C55) pyrophosphate synthetase and undecaprenol phosphokinase for Lactobacillus plantarum and Micrococcus lysodeikticus. They will be characterized with respect to their substrate specificity, phospholipid or membrane reqirements and the stereochemical structure of their substrates and products. Application of various alkyl-agarose columns will be made for possible purification of the partially purified C55-PP synthetase. Synthetase specificity will be determined with stereochemically pure C15 and C20 pyrophosphates and kinetic constants will be compared fo the two bacterial systhetases. The stereochemical purity of the enzyme products will be evaluated using 2R-23H and 2S-2 3H-IPP. Analogues of isopentenyl pyrophosphate will be synthesized and tested as substrates for the synthetase. Phospholipid and detergent requirements will also be compared for these two bacterial synthetases. Undecaprenol kinase will be further studied to improve the stability of the solubilized enzyme, greatly increase the degree of purification, and investigate more thoroughly the detergent and phospholipid activation of the enzyme.


This grant has supported studies on various aspects of class switching and lymphokine induction in the spontaneous murine B leukemia, BCL1. Our findings during the last period have prompted a broader investigation of B cell differentiation using this model system and normal B cells. Based on significant supporting data, we have condensed our continuing efforts into three projects. First, we want to determine the precise mechanism and its control for allelically excluded, dual synthesis of mu and gamma1 chains in BCL1 subclones. We know that this occurs by some form of discontinuous transcription, and not by production of a 'long transcript' covering the entire VDJ to C-gamma1 region. Second, we want to determine precisely how IL-5 and IL-6 in conjunction with antigen induce increased rates of u transcription and DNA replication in BCL1 cells rendered antigen-specific by transfection. We have identified the target sequences and the protein-DNA interactions for IL-5+Ag. We want to do the same with IL-6 plus go after the factors induced by both lymphokines. Third, we want to study proteins expressed only at the immature/mature B cell differentiation stage based on the premise that functions relevant to Ig gene expression will be discovered. We have cloned from BCL1 two mRNAs that satisfy the criteria of cell type- and stage-specificity.


Kinetic and thermodynamic studies of various antibody aggregates interacting with mobile Fc receptors on cell surfaces.


For fifteen years the Biomedical Computing Shared Resource (BCSR) has promoted high quality innovate cancer research by providing members of the UWCCC with an advanced biomedical computing environment. As a matter of policy, computing support within the University and Medical School is decentralized and largely the responsibility of individual departments and centers such as the UWCCC. Within this context, the BCSR plays an essential role by providing both technical and intellectual resources that address the unique and specific needs of the UWCCC in an efficient and cost effective manner. The BCSR supports the UWCCC research enterprise in five years. First, it is responsible for much of the network and I/T infrastructure used on a daily basis by every connected UWCCC member. Second, it provides an array of computing services to UWCCC investigators, other shared resources, and to UWCCC administration. Third, it develops and maintains specialized research resources targeted to the particular needs of UWCCC investigators. Fourth, BCSR staff provide critical software engineering, project management and computer science expertise for specific projects. Fifth, the staff of the BCSR fulfill an important leadership role by tracking or anticipating the needs of the UWCCC research community and then developing the resources to meet these needs. The facilities of the BCSR have been used by Drs. Gould, Moser and Newton to build genetic maps of mice and rats. Drs. Newton and Reznikoff have used them to test new methods for assessing gene loss. Drs. Lindstrom and Kinsella have used these facilities to assess models for therapeutic radiation damage to tumors. Drs. Love and Sondel have used these facilities to build and maintain research databases. In addition, the facilities and staff of the BCSR are used by scientific-oriented shared resources such as the Clinical Trials Research Office for patient and protocol tracking, the Analytical Instrumentation Laboratory and Flow Cytometry for computing support and consultation, and the Biostatistics Shared Resource for computational and database resources. The BCSR also provides essential support and services to UWCCC Administration.


This proposal is concerned with mutagenesis and carcinogenesis induced by aromatic polycyclic hydrocarbons which are environmental pollutants. The experiments described will show whether, and how, DNA adducts formed from the proximate carcinogens produce mutations. Our study offers a new approach: we will use the mammalian virus SV40, with its DNA modified in vitro by treatment with the proximate hydrocarbon carcinogen. The modified and characterized SV40 DNA vector will then be studied in infected cells from which we will learn the molecular nature (base change, insertion, deletion) and location of mutations caused by the hydrocarbon adducts. Our studies will also help to elucidate the role of DNA repair in mutagenesis. We will study both pre- and post-replicative repair and will determine whether the repair process itself is error-prone. A close correlation between mutagenesis and carcinogenesis will be made possible by examining the extent of mutagenesis of SV40 in the same cells in which transformation by the hydrocarbons will be studied. This last approach will also provide a basis for the screening of potential carcinogens.


Ascaris lumbricoides var. suum is a parasitic intestinal nematode which possesses an anaerobic energy metabolism. Carbohydrates are dissimilated to a mixture of volatile acids and succinate. Studies are continuing on the pathway of formation of the volatile acids, particularly alpha-methylbutyrate and alpha-methylvalerate. Ascaris muscle differs biochemically from mammalian muscle in many respects. It functions anaerobically. Pyruvate kinase activity is barely detectable. Lactate is not formed by intact worms. In spite of the anaerobic nature of Ascaris muscle, mitochondria are present. Studies are in progress to better define the relationships between cytoplasmic and mitochondrial metabolisms. Malate, formed in the cytoplasm via CO2 fixation into phosphoenolpyruvate, serves as the mitochondrial substrate. In the mitochondrion, malate dismutates to form succinate and pyruvate with the concomitant anaerobic formation of ATP. A number of anti-cestodal agents have been found to inhibit the P32-ATP exchange reaction in both cestode and Ascaris mitochondria. It is proposed to continue these studies on phosphorylating Ascaris mitochondrial systems in an attempt to better localize the possible mode of action of these drugs. Effects of these agents on net ATP forming systems in both Ascaris and Hymenolepis diminuta are being investigated. Studies of the intermediary metabolism of the two filarial worms, Litomosoides carinii and Dipetalonema witei (vitae) and Brugia pahangi are being initiated.


DESCRIPTION: The interactions of molecular tracks and motors result in movements in cells. Fundamental to our understanding of the way in which motility is accomplished in cells is a detailed description not only of the atomic structures of the complexes involved, but also the way in which the activity of these complexes is regulated in response to the needs of the cell. The long term goals of the work described in this application are to understand in structural terms, the way in which myosin motors are regulated. Three types of myosin-based regulation will be investigated: (I) Regulation mediated by light chain phosphorylation will be investigated by examining a number of smooth muscle myosin constructs. (ii) Regulation by light chain dissociation will be examined in brush border myosin l and myosin V. (iii) Regulation by heavy chain phosphorylation will be investigated in Acanthamoeba and Dictyostelium myosin ls. The methods to be used include cryoelectron microscopy, helical image analysis and modeling using the x-ray structures of the myosin head and regulatory domain. The experiments have been designed to visualize actomyosin conformations which are the basis of the regulatory mechanism. Atomic models of the regulated and activated states will be built from the EM and X-ray data. The results should provide detailed insights into the structural basis for regulation in myosin involved in uterine contractions, blood pressure maintenance and intestinal peristalsis (smooth muscle myosin), and in intracellular trafficking and endocytosis (myosin ls and Vs). Some exploratory experiments on dynein are planned.


ZetrOZ has developed a prototype novel, miniature, portable, high-power, ultrasound system for would healing. Wound healing is a major clinical issue, and can present significant clinical challenges both when wounds are acute--occurring due to trauma, burns, surgery, or due to more chronic health problems. Wound healing is an intricate fibroproliferative response to repairs damaged tissue following injury. This process is fragile and susceptible to interruption or failure, leading to non-healing wounds. Historically, wounds have been historically treated in a sub-optimal fashion with basic wound care products designed to cover wounds and absorb exudates. Ultrasound (a safe, commonly used, FDA-approved treatment modality) has been shown to have beneficial effects on factors associated with tissue healing in both in vitro and in vivo animal models, including promotion of histamine release, angiogenesis, and collagen deposition (thereby increasing wound breaking strength), and ultimately results in a reduction in wound size. Although ultrasound therapy has been previously demonstrated to accelerate wound healing, conventional techniques utilize shorter bursts of high-intensity treatments. Such treatment is typically limited to administration by a medical provider. In contrast to current technologies, our device significantly reduces the size, cost, and power requirements of ultrasound therapy when compared to traditional ultrasound devices. The system can deliver therapeutic acoustical energy waves at lower voltages than those in conventional ultrasound systems. During this project we aim to demonstrate that a human wearable low intensity therapeutic ultrasound system enhances wound healing. This will be accomplished through testing the system on a single battery charge in an animal cadaver. We will also demonstrate that low intensity therapeutic ultrasound improves wound healing, as measured by therapy on the rate and extent of epithelialization and granulation tissue formation (the major components in human wound healing) in wild-type and diabetic murine excisional wound models. The proposed experiments will investigate two different power levels to further understand impact of intensity on would healing.


Melanins (eumelanins, pheomelanins) are photoprotective pigments. They remove light energy, dissipating it either as heat or in a chemical reaction in which oxygen is consumed. Recent work has shown that they also scavenge toxic species, e.g. superoxide and singlet oxygen. Protection may arise either from light filtering, removal of oxygen, or such scavenging. The cost of the protection is measured in terms of the chemical or thermal damage that may result. If the damage outweighs the protection afforded by the pigment, phototoxicity results: it has been hypothesized that damage may be more pronounced with pheomelanins than with eumelanins and may promote skin cancer in red-haired individuals. New experiments are proposed to: (i) measure the effectiveness of isolated pigments in scavenging superoxide and singlet oxygen and the resulting damage to the melanin (e.g. bleaching, irreversible free-radical production); (ii) determine whether scavenging reactions are effective in melanosomal and cellular systems; (iii) determine the effect of melanin on cell respiration and survival following exposure either to visible or UV light in the absence or presence of sensitizers. These experiments will include a comparison of eumelanins and pheomelanins, and an assessment of the utility of sensitizers in promoting chemical or thermal damage in pigmented cells will be made. In these studies use will be made of recently-developed electron spin resonance (ESR) procedures for measuring oxygen consumption and superoxide production in photolytic and cellular systems and of a new model pigmented cell system: Chinese hamster ovary (CHO) cells containing phagocytized melanin.


Project Summary and Relevance The aim of this Phase II SBIR proposal from Applikate Technologies, LLC, is to build on the successful completion of our Phase I milestones with the design, construction, and testing of prototype devices and software for implementing the ClearView tissue histology system in clinical settings by laboratory personnel. ClearView is a novel approach to performing complete and non-destructive imaging of tissue specimens for lung and other cancers. Lung cancer biopsy diagnosis error rates may run as high as 15%. Although there are many potential sources of error, important aspects relate to limitations of current methods for tissue processing and analysis. Standard methods limit the amount of tissue that can be visualized by the diagnostic pathologist and that is available for personalized-medicine testing. They also are not amenable to consultation with off-site experts and are dependent on manual processing by adequately trained histotechnologists. Applikate Technologies has developed ClearView, a complete, non-destructive, fast, tissue processing and imaging approach to histology that improves on limits of slide-based histology by providing complete tissue visualization, reducing artifacts of preparation, permitting improved quantitative interpretation, showing additional growth pattern information, and eliminating extensive manual preparation. The ClearView system yields images that are, in many cases, superior even to traditionally processed histology slides with pseudo-coloring that is virtually indistinguishable from conventional staining, all in an entirely digital format ready for remote access and storage. And ClearView is fast enough to provide diagnostic quality images in under two hours for same day diagnosis. We have also demonstrated, during our Phase I funding, that the ClearView process yields orders of magnitude more DNA for molecular analyses and is compatible with immunohistochemical staining. Furthermore, the methodology is applicable not only to lung cancer diagnosis, but to evaluation of practically any solid tissue cancer. The specific aims of the proposal are to: 1) Develop an application-specific, high-speed multiphoton microscope and associated software; 2) Develop the ClearView prototype tissue chamber, and 3) Develop the ClearView prototype tissue processor. These three elements, along with proprietary reagents, form the core of the ClearView system. Following in-house testing, two complete prototype systems will be placed in two hospitals, one a large tertiary care center and the second a mid-sized hospital, for on-site testing and feedback by non-expert users in a clinical setting. Successful completion of these aims will set the stage for a Phase III transition to regulatory approval and provide a proven platform for early implementation through service work for academic research and clinical research groups.


Background: Essential to high-quality palliative care assessment and management is the availability of psychometrically-sound and clinically-relevant screening, diagnosis, and outcome evaluation tools that will enable healthcare providers to attain optimal patient evaluation, prognosis, treatment selection, patient satisfaction, and quality of life improvement. We propose to develop a patient-centered, model-driven, evidence-based system called the System for Interactive Assessment and Management in Palliative Care (SIAM-PC) using innovative methodologies and technologies. Aims: Our four distinct but related specific aims are to: 1) identify and refine the domains of palliative care, using existing theoretical frameworks as a guide; 2) compile the items for multidimensional palliative care item bank, drawing from existing measures and supplementing with newly written items; 3) empirically construct a palliative care item bank using item response theory (IRT); and 4) pilot test a computerized adaptive testing (CAT) platform to dynamically administer palliative care assessment in clinical settings. Methods: First, we will identify and refine the key domains of palliative care, guided by existing frameworks, from in-depth individual interviews (n=50; 20 patients, 20 family members and 10 clinicians) and literature review (Aim 1). Second, we will compile items pertinent to these domains from existing measures, writing new items for un-covered domains (Aim 2). Third, we will test these items by collecting and analyzing data from patients (n=200) receiving palliative (Aim 3). Finally, we will pilot test the CAT system (n=40) in clinical practice (Aim 4). Outcomes: At the end of this R21 project, we expect that (1) the conceptual model of palliative care measurement can be improved; (2) the initial sets of palliative care domains/items can be constructed; (3) items displaying DIF can be identified and revised; and (4) the CAT prototype will demonstrate how the system should be implemented in clinical settings. Palliative care is a major public health concern. There is significant consensus and ample evidence in the literature to call for a comprehensive, interdisciplinary approach to address this concern. This comprehensive assessment and care is demanding in that it depends upon multiple sources of information, and yet the practicalities of real-life care depend upon concise information. Our proposed study is to create valid methods to connect comprehensive assessment with concise information in patient assessment and care. [unreadable] [unreadable] [unreadable]


Cerebrovascular amyloid beta-protein (ABeta) deposition is a hallmark of Alzheimer's disease (AD) and related disorders including hereditary cerebral hemorrhage with amyloidosis Dutch-type (HCHWA-D). Cerebrovascular ABeta deposition in these disorders is prominently associated with smooth muscle cellular pathology in the cerebral vessel wall and can lead to a loss of vessel integrity and hemorrhagic stroke. The reasons for this remain unclear. The overall hypothesis that forms the basis of this competing continuation grant proposal is that ABeta deposition in the cerebral vessel wall can modulate the function of hemostatic enzymes thus creating a microenvironment that is conducive to hemorrhagic stroke. The objectives of this proposal are to characterize the effects of ABeta on the regulation of certain hemostatic enzymes at the biochemical and cellular levels. The specific aims of this proposal are as follows: First, the effect of soluble ABeta, solution-assembled fibrillar ABeta, and cell-surface-assembled, fibrillar ABeta on the inhibition of factor XIa (FXIa) and certain other coagulation enzymes by PN-2/AbetaPP will be characterized. The inactivation rates of FXIa and other coagulation enzymes by PN-2/AbetaPP in the presence of soluble and the different fibrillar ABeta peptides will be determined. The proteolysis of ABeta peptides by coagulation enzymes will be investigated. Second, studies will be conducted to determine the effect of soluble ABeta, solution-assembled fibrillar Abeta, and cell-surface-assembled fibrillar ABeta on the stimulation of tissue plasminogen activator (tPA) activity. The effect of ABeta peptides on cellular tPA levels and plasminogen activation activity will be explored. In addition, proteolysis of ABeta peptides by tPA and plasmin will be investigated. Third, investigations will compare the pathologic effects of intact ABeta and hemostatic enzyme proteolyzed ABeta on cultured human cerebrovascular smooth muscle (HCSM) cells, a cell type intimately associated with the cerebrovascular pathology of AD and HCHWA-D. The proposed studies will provide a better biochemical understanding of the influence that ABeta has on the inhibition of certain coagulation proteases and the stimulation of plasminogen activation, two processes that together can create a milieu which could result in loss of vessel integrity and hemorrhagic stroke.


This project involves the development and testing of a novel MHC based vaccine designed to provide both humoral and cellular immunity against viral attack. Such vaccines have particular relevance for protection against AIDS. These vaccines are based on linking viral epitopes through a tether to B2-microglobulin. This tripartite construct can replace normal peptides present in MHC-l binding sites, leading to the generation of virus-specific T-cell responses. Initial studies with a model mouse Sendai virus construct have provided proof of principle for this approach. This includes the cloning, refolding and purification of the vaccine and demonstration that it binds/exchanges with B2-microglobulin on cell surfaces, upregulates MHC expression on cells expressing empty class I MHC and stimulates T-cell hybridomas that are reactive to the Sendai virus epitopes present on the vaccine. The proposed studies involve: A. Further studies with the Sendai virus model including 1) additional investigation with the Beta2-linker-Sendai epitope construct to determine the effect of different linker sizes on binding to the MHC and on subsequent CFL responses 2) development and testing of a DNA version of the vaccine construct 3) determining whether vaccination of mice with these constructs will provide protection against challenge with the Sendai virus. As part of these studies the optimization of the route, dosage and duration of effectiveness will be determined. B. Development and testing of AIDS MHC-vaccine constructs, including I) using a p17 (Gag) sequence 2) using a Pol sequence 3) using a Nef sequence. These epitopes were chosen because of their highly conserved nature, their cross-clade reactivity, and their role in the spread of the disease.


The 1980s might be called the decade of awakening private sector interest in containing medical care costs. Employers have begun to take initiatives to control their outlays for health insurance premiums paid as a fringe benefit. Employers are redesigning benefit plans to increase consumer cost sharing, offering alternative health care plans, and changing the methods used to finance premiums. Since insurance benefits provided by employers constitute about one-third of all health care expenditures, these private sector initiatives are viewed as a new potential mechanism to moderate the spiralling rate of inflation in the health care sector. The proposed study has four objectives: (1) to describe what employers are doing to control their health benefit costs, (2) to estimate the savings associated with alternative cost cutting measures, (3) to estimate the demand for particular innovations by medium and large sized firms, and (4) to assess the implications of our findings for policy reforms relating to employer-based health insurance. The project will utilize Bureau of Labor Statistics data, 1981-1984, on the benefit offerings of some 1200-1500 firms nationwide. Three analyses are planned. The first is a cross tabular examination of the extent and kinds of initiatives that employers have taken over the period 1981 through 1984. The second is a regression analysis of premiums that includes benefit provisions and other plan characteristics among the explanatory variables. The third is a regression analysis of the demand for selected benefit characteristics. The implicaitons of our results for policy reforms affecting employers will be examined in detail.


The objective of this study is to develop an in vitro potency assay to replace the guinea pig death test portion of the official release test for the diptheria component. VERO cells will be used as a target for the diptheria toxin. After screening several lot of fetal bovine serum for diptheria antitoxin, a lot has been approved and purchased. After examining several methods for determining viable cells, the Neutral Red Assay has been selected. The U.S. Standard Diptheria Toxin Lot 35119 will be used for the cell death assay. It is currently used in the animal death test and will be used in the rabbit skin test. A reproducible minimum cytopathic dose is being established for the 4 day assay. Inhibition of cell death will be measured using the U.S. Standard Diptheria Antitoxin, Immune Serum Globulin, IGIV and guinea pig immune serum.


Aggressive/disruptive behavior in the elementary school years is a strong indicator of antisocial behavior, drug abuse and low educational and occupational attainment in adolescence and young adulthood. The Good Behavior Game (GBG) and Promoting Alternative Thinking Strategies (PATHS) represent two of a handful of universal, elementary school, preventive interventions which have been shown in large scale, randomized controlled trials to have an immediate and beneficial impact on aggressive/disruptive behavior. PATHS seeks to accomplish reductions in such behavior via teacher led instruction aimed at facilitating emotion regulation and social problem-solving, whereas the GBG provides teachers with an efficient means of reducing aggressive/disruptive behavior using social learning principles within a game-like context. Importantly, however, the effects of the GBG on aggressive/disruptive behavior proved modest in our 1st and 2nd generation Johns Hopkins University Preventive Intervention Research Center (JHU PIRC) field trials. This has been the case for PATHS as well. We recently completed a 27-school, randomized controlled trial examining whether the combination of these interventions, which we refer to as PATHS to PAX, would yield significantly greater impact on aggressive/disruptive behavior than the GBG alone. Our rationale for expecting greater impact was that the use of the GBG should result in reductions in aggressive/disruptive behavior, which should then facilitate the acquisition of the emotion regulation and social problem-solving skills taught in PATHS. PATHS to PAX did result in a modestly greater reduction in aggressive/disruptive behavior than the GBG alone at 1-year post-test. Yet, the most aggressive/disruptive students still failed to sufficiently benefit from te PATHS to PAX intervention. Accordingly, in this application, we propose to examine whether the addition of the Incredible Years (IY), an evidence-based preventive and treatment intervention aimed at reducing aggressive/disruptive behavior, would yield greater impact on these behaviors than the PATHS to PAX intervention alone. We also propose to examine whether the combination of the PATHS to PAX + IY results in increased frequency of implementation of the PATHS to PAX intervention. We hypothesize that relative to teachers in the PATHS to PAX alone condition, teachers in the PATHS to PAX+IY condition will perceive PATHS to PAX as more efficacious and will therefore be more likely to implement it. Four cohorts of 12 schools each will be recruited with schools randomly assigned to 1 of 3 intervention conditions: 1) Control; 2) PATHS to PAX Alone; or 3) PATHS to PAX+IY. Assessments of student outcomes will be carried out at pre-test and post-test in the fall and spring of the initial school year foreach cohort and at a 6-month and one year follow-up.


[unreadable] [unreadable] This application is for competitive renewal of a PHS Institutional National Service Award at the David Geffen School of Medicine at UCLA to prepare fellows for an academic career in Hematology. This award was initiated in July, 2002, and supports 4 fellowship slots in years 2 and 3 of Hematology/Medical Oncology training. Of 11 fellows who have entered the program, 4 are currently in training and 7 have completed their fellowship, of which 4 are on the full-time faculty at UCLA, two are in clinical trial development with industry, and one has entered clinical practice. The faculty consists of 31 academic scientists and physicians, grouped into four areas of mutual research interest, Hematopoiesis and Stem Cell Biology (Kenneth Dorshkind, PhD, Director), Leukemia (Gary Schiller, MD, Director), Lymphocyte Biology and Lymphoma (Alan Lichtenstein, MD, Director) and Hemostasis, Thrombosis and Vascular Biology (Victor Marder, MD, Director). The entire faculty have extramural research grant support, including 24 (77%) with NIH grants. Interaction with other training programs in complementary disciplines emphasizes our goal of interactive basic and translational research careers. A research retreat is; held each spring, during which time each mentor presents an overview of the laboratories' research interest, followed by a report of the respective fellows' progress. Formal review of the mentor's effectiveness and the fellow's progress and goals are prepared by the Steering Committee, made up of the four Focus Directors. A core curriculum is offered to all fellows, including a "Clinical Research Course", which covers subjects on research design and analysis, ethics, regulatory, issues, grant preparation and responsible conduct of research. Fellows are encouraged to obtain an advanced degree as part of the STAR program, with formal course work in appropriate departments for one or two additional years. Of the 7 fellows who have completed their program during the interval 2002 - present, 2 have obtained the Master's degree and 1 has received a PhD degree. The training of hematologists with expertise in basic, clinical and translational research is an important priority for the David Geffen School of Medicine at UCLA and this application seeks support for the pivotal period between clinical training and an eventual career in academic hematology. [unreadable] [unreadable] [unreadable]


A non-hemin regulated translational repressor protein has been purified partially from the postribosomal supernatant of Friend leukemia cells grown in the absence of dimethylsulfoxide. This repressor inhibits globin synthesis in lysates from rabbit reticulocytes and in a highly active fractionated system using brine shrimp ribosomes, rabbit globin mRNA, peptide elongation enzymes from reticulocytes and initiation factors from either reticulocytes or Friend cells. It has no effect on poly(U) directed peptide synthesis, nor on the extension or release of nascent globin peptides that were initiated in intact reticulocytes. In contrast to the hemin controlled repressor from reticulocytes, it does not block mRNA independent synthesis of methionylpuromycin but it does inhibit mRNA dependent synthesis of methionylvaline. A block in binding of mRNA to ribosomes appears to be involved. Current work is directed towards evaluating the physiological role that this repressor protein plays in the virus induced block in differentiation of murine cells of the erythroid line. Such a block in differentiation may account for the accumulation of proerythroblasts and the pathological state defined as Friend virus induced leukemia in infected mice. BIBLIOGRAPHIC REFERENCES: Globin mRNA Translation on Artemia salina Ribosomes with Components from Friend Leukemia Cells. G. Kramer, P. Pinphanichakara, D. Konecki and B. Hardesty. Eur. J. Biochem. 53: 471-480 (1975). Aminoacyl-tRNA Specificity of a 40S Ribosomal Subunit Binding Factor from Rabbit Reticulocytes, J. Cimadevilla and B. Hardesty, Biochem. Biophys. Res. Comm. 63: 16-23 (1975).