Mycobacterium avium-intracellulare complex organisms are the most common cause of mycobacterial lung disease other than tuberculosis and are the leading cause of morbidity and mortality in the HIV-infected AIDS patients. Infections caused by M. avium patients with AIDS often go untreated as existing antibiotics are ineffective in adequately controlling these infections. This necessitates the need for novel molecular targets for chemotherapy. Replication of the bacterium leading to its multiplication is one of the necessary steps to establish an infection. Initiation is the first committed step in the replication, and the replication process is believed to be regulated at the level of initiation. Thus, understanding the basic steps involved in initiation of DNA replication in M. avium will help define important molecular targets against which new generation drugs can be developed. Initiation of replication occurs once per cell cycle at a specific site on the chromosome called oriC. Initiation is believed to be triggered by the interactions of dnaA with its recognition sequences present in oriC called the dnaA-boxes. These interactions are thought to facilitate recruiting of other proteins resulting in the completion of initiation. This research proposal focuses on identification and characterization of the oriC and Dna protein of M. avium. To obtain oriC, chromosomal DNA fragments of M. avium that support autonomous replication when present in nonreplicative plasmids will be identified, cloned and their nucleotide sequence will be determined. Sequential deletions from both the 5' and 3' regions will be carried out to identify the minimal DNA region that is essential for oriC activity. Site directed mutagenesis will be carried out to identify the putative DnaA boxes that are essential for oriC activity. The ability of M. avium oriC to function as autonomously replicating sequences in other bacteria will be determined. The dnaA gene will be over-expressed, the gene product will be purified and its interaction with the wild type and mutant sequences will be investigated. Antisense dnaA oligonucleotides that target to the dnaA mRNA to affect the expression of M. avium dnaA gene will be explored in an effort to evaluate the role of the M. avium gene. Using oriC plasmids and cell free extracts, an in vitro replication system will be established. The ultimate long-term goal of these experiments is to utilize the knowledge thus gained to develop rational drugs that affect the initiation of replication thereby preventing growth and resulting M. avium infections.


The goal of this project is to develop much-needed new tools for study of the Kallikrein-Kinin system. This system appears to play a critical role in regulation of blood pressure, counteracting the pressor effects of the renin-angiotensin system, and as a principal mediator of the inflammatory response. We have developed a new assay system for kallikreins, and will use it to try to find inhibitors of kallikrein, particularly of urinary kallikrein, which appears to be the critical enzyme in blood pressure regulation. The potential inhibitors will be analogs of the natural substrate of the enzyme. Potential inhibitors of bradykinin at its receptors will be sought among analogs of bradykinin modified so as to increase binding affinity and decrease intrinsic activity. Peptides will be synthesized by the solid phase method and purified by standard techniques. Biological activity of the potential kallikrein inhibitors will be determined in our kallikrein assay using fluorescence for quantitation and HPLC for characterization of products. Biological activity of kinin analogs as agonists and antagonists will be determined in rat uterus, guinea pig ileum, and rat blood pressure assays. Binding affinity of the kinin analogs will be determined by measuring their ability to compete with 125I-Tyrosine1-kallidin for receptors on renal membranes. Studies to define completely the solution conformation of bradykinin will be completed, and conformational changes in the bradykinin molecule in combination with antibodies and membrane receptors will be studied with fully enriched 13C-bradykinins.


Considerable evidence demonstrates that pain disproportionately affects racial and ethnic minorities in the United States, especially African Americans (AAs), who experience frequent, severe and disabling chronic pain compared to their non-Hispanic white (NHW) counterparts. These racial and ethnic disparities extend to many chronic pain conditions, but are perhaps best documented in people with osteoarthritis (OA). Previous research revealed that AAs with OA experience more frequent and severe clinical pain and disability and show a quantitative sensory testing profile suggesting impaired pain inhibition and enhanced generalized pain facilitation compared to NHWs. In addition, previous findings demonstrated higher pain catastrophizing (a tendency to negatively evaluate one's ability to cope with pain and to respond to anticipated or actual pain in a heightened negative cognitive and emotional manner) among AAs compared to NHWs. While multiple factors inevitably contribute, cognitive and affective processes and ethnic group differences in central pain processing represent potentially important determinants of greater clinical pain among African Americans. Despite its pervasive negative effects, no neuroimaging study to date has experimentally manipulated pain catastrophizing and measured cerebral activity during experimentally-induced pain to determine the neural mechanisms whereby catastrophizing impacts pain responses. Moreover, the extent to which the influence of pain catastrophizing on these central pathways contributes to ethnic group differences in the experience of pain remains unexplored. Therefore, the overall goal for this mentored career development proposal (K22) is to elucidate the neural mechanism involved in pain catastrophizing and its influence on pain processing in different ethnic groups. Primary training goals for the current proposal are to: 1) develop a comprehensive knowledge base in neuroimaging techniques, methodological designs, data acquisition, data analyses, and interpretation of findings; 2) expand knowledge of advanced pain assessment skills including quantitative sensory testing and clinical models of pain; 3) Obtain expertise in principles of experimental design and statistical methodology utilized for biomedical research, including neuroimaging studies; and 4) enhance translational research skills to function as an independent investigator. Study 1 (Phase I) will determine whether pain catastrophizing contributes to ethnic group differences in pain-related brain function, clinical pain, and pain sensitivity among AAs and NHWs with knee OA. Study 2 (Phase II) will characterize the impact of an anti-catastrophizing manipulation on central pain processes and pain among AAs and NHWs with knee OA. The proposed career development plan extends from the PI's prior work on pain catastrophizing and mechanisms of pain processing, and will also provide the neuroimaging training and expertise to propel a promising young investigator at the intersection of pain and neuroscience research.


PROJECT SUMMARY/ABSTRACT The majority (>60%) of clinically important drug targets in humans are either integral or peripheral membrane- bound proteins, including G protein-coupled receptors, ion channels, cytochrome P450 enzymes, and P- glycoprotein. Partitioning of drugs within the lipid cell membrane or membranes of subcellular organelles are known to affect their interactions with such membrane-bound proteins, influencing their efficacy and fate in the body. While membrane interactions can result in better efficacy, selectivity and longer duration of action, the excessive membrane accumulation of drugs causes undesirable toxicities through off-target interactions. Therefore, merely increasing the lipophilicity of drugs to increase potency for membrane-associated targets is often detrimental in terms of the overall drug profile. Thus, the knowledge of quantitative estimate of membrane distribution, preferred location (depth), orientation, and conformation of drug molecule within the bilayer is crucial in understanding its target binding kinetics, onset and duration of drug action, and disposition. Our central hypothesis, formulated based on extensive literature and our preliminary data, is that application of this knowledge to lead optimization in rational drug discovery will result in improved efficacy, selectivity and safety. Hence, the long-term goal is to understand the ?specifics? of the structure-membrane interaction relationship and apply this knowledge to rational design of new therapeutics aimed for membrane-associated targets. To this end, the overall objective of this application is to develop, validate, and apply an integrated in silico approach for fast and accurate prediction of membrane partitioning characteristics, combining all-atom MD simulations and a fragment-based continuum solvent model. The objective of this project will be accomplished by two specific aims: (1) Develop an ?integrated in silico approach? for fast and accurate prediction of percentage distribution, most preferred location, orientation, and conformation of drugs in phospholipid bilayer. The current continuum solvent model will be recalibrated using MD simulation results of 27 structurally diverse chemicals with experimentally known bilayer locations to obtain optimized solvatochromic fragment constants for bilayer distribution prediction. (2) Determine whether the membrane-partitioning characteristics of 17 clinically relevant ?2-adrenergic receptor (?2-AR) agonists and antagonists, quantified by the integrated approach, correlate to their experimental association rates (kon) to the ?2-AR and thus affect their onset and duration of action. This study is innovative because it uses atomistic details of membrane-drug interactions from combined molecular dynamics (MD) simulations and our1 continuum solvent model to understand the structural determinants of receptor-binding kinetics of the studied short- and long-acting anti-asthmatic drugs with diverse structural and physicochemical properties. This proposed study is significant because the expected results will fundamentally advance our knowledge on how specific membrane-drug interactions can be exploited in drug discovery of new therapeutics binding to any membrane-associated receptors, enzymes, and transporters with optimal efficacy and safety.


This project consists of two parts: the synthesis of peptides of biological interest, especially those with enzymatic activity; and the modification and improvement of solid phase peptide synthesis. We are developing resin supports of improved stability and protecting groups that can be selectively removed. Several side reactions are under study and ways to eliminate them are being devised. Methods to monitor the coupling and deprotection step, especially the picric acid method, are being developed. We are using the synthetic approach to study structure-function relations in various hormones and enzymes. Bibliographic references: Detection and Prevention of Urethane Acylation During Solid Phase Peptide Synthesis by Anhydride Methods. Merrifield, R.B., Mitchell, A.R. and Clarke, J.E., J. Org. Chem. 39, 660-668 (1974); Synthetic Approaches to the Study of Proteins. Merrifield, R.B. and Hodges, R.S., in "Proceedings of the International Symposium on Macromolecules", Rio Janeiro, July 26-31, 1974, E.B. Mano, ed., Elsevier, New York, 1975, pp. 417-431.


The threat of terrorist attacks involving radioactive material and the potential for radiation accidents require the development of improved treatment strategies for victims of radiation exposure. Hematopoietic cells are highly sensitive to radiation damage, and their loss after radiation exposure results in lethal infections. Development of treatment that prevents immune damage from radiation and reconstitutes immune function after radiation exposure would be a significant advance. The aim of this project is to study four promising, clinical-grade cytokines that are likely to significantly improve immune reconstitution after lethal dose irradiation in the well-established dog model. The four cytokines are fms-like tyrosine kinase-3 ligand (Flt3 ligand [FL]), keratinocyte growth factor (KGF), interleukin (IL)-7, and transferrin (Tf) given alone or in combination either before or after exposure to lethal doses of total body irradiation (TBI). All four cytokines have anti-apoptotic activity after gamma irradiation and have direct or indirect beneficial effects on lymphocytes and other immune cells. We will study these cytokines in the dog since (1) the dog model of radiation exposure and hematopoiesis has been highly predictive of human clinical outcomes, (2) there is extensive preliminary data with cytokines for radioprotection of the dog after acute radiation, (3) all four of the human cytokines we have proposed are cross-reactive in the dog, and (4) these cytokines have been studied in humans and are in various stages of clinical development. The goal is to achieve survival of dogs after an otherwise lethal dose of irradiation with sustained immune reconstitution without hematopoietic stem cell (HSC) transplantation. In Specific Aim 1, cytokines will be given after TBI and in Specific Aim 2, cytokines will be given before and after TBI. In Aim 1, we will give 500 cGy TBI and treat dogs with G-CSF plus each study cytokine. In this model, a radioprotective cytokine is defined as achieving significantly improved survival compared to G-CSF alone. In the subsequent experiments, the TBI dose will be successively increased by 100 cGy increments and dogs will be treated with a combination of radioprotective cytokines. The primary endpoint is recovery of hematopoiesis and survival beyond day 30. The secondary endpoint is immune reconstitution. Upon study completion, we will have identified the optimal cytokine treatment and the highest dose of TBI that can be reliably survived without HSC support.


PROJECT SUMMARY (ABSTRACT) Helicobacter pylori is a Gram-negative bacterium that colonizes the gastric mucosa of humans. Although most H. pylori-infected persons remain asymptomatic, potentially serious sequelae of infection include gastric adenocarcinoma, duodenal ulceration, gastric ulceration, and gastric lymphoma. Gastric cancer is the second leading cause of cancer-related death worldwide, and H. pylori has been classified as a type I carcinogen by the World Health Organization. One of the major secreted proteins of H. pylori is a toxin known as VacA. VacA causes multiple alterations in gastric epithelial cells, and inhibits activation and proliferation of T lymphocytes. Most cellular effects of VacA are dependent on its ability to form anion-selective membrane channels. There is a high level of genetic variation among vacA alleles from unrelated H. pylori strains, and the encoded VacA proteins exhibit marked differences in their ability to cause alterations in human cells. The molecular basis for the observed differences in activities is not yet completely understood. A large body of literature indicates that H. pylori strains containing certain forms of vacA (termed s1, i1, or m1) are associated with a higher risk of gastric cancer or peptic ulcer disease than are strains containing other forms of vacA (termed s2, i2, or m2). Thus, VacA is considered to be an important H. pylori virulence factor. The long-term goals of this work are to understand the mechanisms by which H. pylori infection can lead to disease, to understand the basis for variation in clinical outcomes among H. pylori-infected persons, and to develop effective means for prevention and treatment of illnesses associated with H. pylori infection. The specific aims are (i) to investigate VacA structural features that are required for intracellular toxin activity and membrane channel formation, (ii) to analyze differences in functional properties of VacA proteins encoded by different H. pylori strains, and (iii) to identify and analyze host cell components that are required for VacA cytotoxicity. Methods will include cryo-electron microscopy, crystallography, molecular genetics, and analysis of gene trap and shRNA libraries. This work is relevant not only for the study of H. pylori-associated diseases, but will also increase our understanding of bacterial pore-forming toxins, chloride-conducting membrane channels, beta- helical passenger domains secreted by an autotransporter pathway, and protein targeting of mitochondria.


Patients identified as having non-A, non-B hepatitis will be followed as long as possible. Liver biopsy will be obtained when the SGPT is elevated for more than 6 months and then as indicated thereafter. Biopsy material will be saved for fluorescent studies when reagents for non-A, non-B hepatitis become available. All patients developing non-A, non-B hepatitis following open heart surgery will be followed with serial studies of SGPT to determine the incidence of chronic liver disease and to compare this incidence with those having type B hepatitis. BIBLIOGRAPHIC REFERENCE: Alter, H.J., Seeff, L.B., Kaplan, P.M., McAuliffe, V.J., Wright, E.C., Gerin, J.L., Purcell, R.H., Holland, P.V. and Zimmerman, H.J.: Type B Hepatitis: The infectivity of blood positive for e antigen and DNA polymerase after accidential needlestick exposure. New Engl. J. Med. 295: 909-913, 1976.


Central to synaptic transmission are the family of ionotropic neurotransmitter receptors, which are responsible for the rapid responses to neurotransmitters in nerve and muscle. In this proposal, a class of receptors that are members of this family, the neuronal nicotinic acetylcholine receptors (AChRS), will be studied. As the site where nicotine binds in the brain, these receptors are responsible for nicotine addiction and may also play a role in neurodegenerative diseases such as Alzheimers disease. Neuronal AChRs are composed of a number of subtypes as classified by their diverse pharmacology and distribution. Further evidence of the diversity has been the cloning of a large number neuronal AChR subunit isoforms. While neuronal AChRs are found throughout the central and peripheral nervous system, neither the subunit composition nor the function of single nAChR subtype is known. The neuronal alpha-bungarotoxin binding receptor (BuTxR), a neuronal AChR subtype, has been selected for study because recent studies suggest that these receptors are a Ca2+ influx pathway, which at presynaptic sites stimulates neurotransmitter release and underlie nicotine addiction. The first objective is to examine whether BuTxRs are homomers composed only of alpha7 subunits or heteromers composed of subunits in addition to alpha. In addition, the number of BuTx sites per BuTxR will be determined. The next objective is to begin to define the function of BuTxRs. Towards this objective, we will test how permeable BuTxRs are to Ca+2 and if Ca2+ entering through BuTxRs contributes to secretion in PC12 cells. We will also address why so few BuTxRs functional in our undifferentiated PC12 cells and test the hypothesis that the number of functional BuTxRs are regulated. The last part of the grant is concerned with questions about BuTxR expression. Heterologous expression of alpha7 homomers in mammalian cells is poor to nonexistent. In contrast, alpha7/5HT3 chimeric subunits readily express as homomers in mammalian cells. Using alpha7/5HT3 chimeric subunits, we will determine alpha7 subunit regions that prevent homomer formation. In PC12 cells,different subunit processing at folding events will be characterized and their effect on BuTxR expression tested. Finally, we will further characterize intracellular BuTxR subunit pools, which appear to be precursors of the cell-surface BuTxRs.


DESCRIPTION: This proposal is for continued support for a program of research that has explored the relationship between visual structure and attentional functioning in mentally retarded and nonretarded individuals. Because basic properties of multi-element stimulus arrays are intrinsically relational and are embedded in ubiquitous environmental contexts, the ability to detect interstimulus relations like similarity/dissimilarity is essential for adaptive attentional functioning. Although children who have low mental ages (both mentally retarded and young nonretarded children) typically exhibit markedly deficient performances on tasks that demand relational responding, the present investigators have demonstrated that appropriate modifications of the visual array, designed to increase the perceptual salience of relevant stimulus relationships, can rapidly facilitate the performances of mentally retarded individuals in such experimental contexts. Informed by the findings obtained concerning the effects of perceptual structure on stimulus detection, the investigators are now proposing to extend their approach to several new paradigms, including (a) an integration of oddity and match-to-sample methodologies, and (b) assessing the visual processing abilities of mentally retarded individuals at the critical preattentional stage. This "front-end" perceptually-based approach of "guiding" attention is based on manipulations that have been used successfully in much prior research, particularly that of the current investigators. The proposed studies involve an examination of a range of experimental contexts that share common perceptual variables (e.g., symmetry and contiguity), and assess the relative contribution of these variables to attentional and preattentional processing in individuals with and without mental retardation. The findings of the proposed studies are expected to inform both applied and theoretical issues with respect to the attentional functioning of individuals with mental retardation. The proposed experiments include an integration of oddity and match-to-sample methods (i. e., oddity-based comparison arrays) with implications for generalized performances and extension of that paradigm to the investigation of set size and spatial contiguity in the matching/oddity framework. In a match-to-sample paradigm, stimulus variables including stimulus structure, symmetry of stimulus pattern, and adjacency of elements will be investigated and an analysis of the relationship between stimulus inspection time and stimulus variables will be conducted. Experiments on visual search are proposed that address stimulus variables of element contiguity and symmetry, and are followed by an investigation of guided search. The remaining experiments proposed utilize random dot kinematograms to evaluate stimulus organization variables in motion-defined forms, as well as the influences of training on detection and motion direction discrimination.


Project Summary The understanding of basic DNA repair mechanisms is crucial, as altered regulation of these systems are observed in cancerous cells and also allow for antibiotic resistance in prokaryotes. In E. coli, two major mechanisms of transcription-coupled nucleotide excision repair (TCR) are responsible for removing bulky lesions in DNA that transcribing RNA polymerase encounters- the Mfd-mediated ?forward translocation? model and the UvrD-mediated ?backtracking? model. In this project I will use a genome-wide assay called XR-Seq to further understand E. coli TCR across the genome by determining locations of the genome repaired by each pathway. Further, I will perform NET-Seq in -/+ UV conditions to correlate transcription of both the sense and antisense nascent transcripts to TCR. Together, these studies will provide important information concerning the two known TCR pathways in E. coli by helping us understand how they may work together or separately across the genome and how their absence impacts genome integrity. Ultimately, this work will contribute to further defining the mechanisms of important repair processes that occur in prokaryotes and provide a guideline for future genome-wide studies of other important DNA repair processes.


Epithelial ovarian cancer is the leading cause of death in women with gynecologic cancer. Although it represents only one-fourth of all new cases of gynecologic cancer, it causes over one-half of all deaths due to these diseases. Memorial Sloan-Kettering Cancer Center (MSKCC) is an institution with extensive clinical and laboratory research facilities. The Gynecology Service of the Department of Surgery in collaboration with the Breast/Gynecology Service of the Division of Solid Tumor Oncology of the Department of Neurology, the Department of Pathology, the Department of Epidemiology and Biostatistics and the Immunology Program of the Sloan- Kettering Institute with their principal interest being directed towards ovarian cancer. Since approximately 140 new cases (both untreated and previously treated) of ovarian cancer are seen annually, an adequate patient population exists. For these reasons, a program project to develop new therapeutic strategies for the treatment of epithelial ovarian cancer has been developed. The aim of this program project is 1) to conduct phase I and II clinical trials of innovative chemotherapy regimens 2) to collect a bank of ovarian cancer tissues and body fluids, 3) to evaluate the symptomatology of cancer pain and to assess quality of life as related to therapy, 4) to perform pharmacokinetic studies of new monoclonal antibodies against ovarian cancer antigens in animal models and humans with the aim of being able to target ovarian cancers for diagnostic imaging and radioisotope therapy, and 5) to study in a systematic fashion the immunology and immunochemistry of ovarian cancer in order to characterize malignant and normal ovarian and mesothelial tissues and develop a better group of monoclonal antibodies for clinical use. Based on initial collaboration, the working structure of this program project has been developed. Progress to date warrants funding of a program project to expand this research and carry it forward.


The goals of this program are to improve California State University's (CSUN) research capacity and infrastructure and to increase minority participation in basic biomedical research. The majority of the students who attend CSUN are ethnic minorities (39.8% are underrepresented minorities; University enrollment 31,312, fall 2004). There will be a direct effect on enhancing minority participation in science. Improving research capacity of faculty and students: This proposed MBRS SCORE program includes 18 full SCORE proposals and 3 pilot SCORE proposals. The proposed projects are from diverse fields including theoretical and materials physics, cellular, physiological, genetic, and developmental biology, chemistry and biochemistry, disease processes and social and neuropsychology. Improving the research infrastructure: Cutting-edge research requires instrumentation that is expensive and complex. The successful completion of projects included in this proposal requires specialized instruments. We are requesting support to purchase equipment that will bring the DNA sequencing facility up-to-date and to begin its transformation into a Center for Cellular, Genetic, and Molecular Analysis. Meeting the specific outcomes presented in the following project proposal will fulfill the mission of the MBRS SCORE program which is to "....significantly improve the research capabilities of minority and minority-serving institutions....to increase the number of underrepresented minorities participating in biomedical and behavioral research." The funding provided by the NIH for the CSUN SCORE program will improve the quality and quantity of biomedical research conducted in the laboratories of the PIs and in the affected departments. We expect that 12 of the proposed SCORE PIs will arrive at a point in their research careers after this round of SCORE that they will be able to successfully compete for R01-type of research support. The students, many of whom are underrepresented in basic biomedical sciences, in these departments will have the opportunity to become engaged in top-notch research that will be conducted in SCORE-supported laboratories. [unreadable] [unreadable] [unreadable]


This is an application for partial funding of the Summer Conference on Physiology and Pathophysiology of the Splanchnic Circulation, to be held under the auspices of the Federation of American Societies for Experimental Biology (FASEB), from July 24 to July 29, 1993, at Copper Mountain, Colorado. The participants will be limited to 150 basic and clinical scientists, who will be selected on the basis of their expertise and interests. There will be 9 major scientific sessions, each with 4 to 5 oral presentations by the experts in the field and open discussion. In addition, there will be 4 poster sessions, which will allow junior participants to present their data and discuss them with the experts in the field. The topics to be discussed will be recent advances in the following subjects: 1) non-adrenergic, non-cholinergic regulation of gastrointestinal circulation; 2) imaging technology for study of the vascular system; 3) endothelial cell biology; 4) role of leukocytes in modulation of gastrointestinal inflammation; 5) ischemia/reperfusion and tissue injury; 6) systemic toxins of gut origin; 7) nitric oxide and the splanchnic circulation; 8) vasomodulators of endothelial cell injury; 9) hepatic circulation and heterogenous liver injury. The areas of controversy, uncertainty and agreement in these fields will be defined. The conference will provide a unique opportunity for interaction among basic scientists (physiologists, pharmacologists, etc.) and clinical scientists (gastroenterologists, surgeons, etc.) with an interest in the splanchnic circulation and gastrointestinal and hepatic diseases. The interactive environment should stimulate collaborative research efforts and help to identify new and more productive directions for future research on pathophysiology, pathogenesis, and treatment for gastrointestinal and hepatic diseases.


To determine the safety and optimal dose of DNA/liposome compound when injected directly into colorectal cancers which have spread to the liver and to transfer this gene to the cancer cells in the liver thereby making them susceptible to destruction by the normal immune cells of the patient.


The National Medical Association (NMA) is the nation's oldest and largest organization representing African American physicians and health professionals in the United States. It was founded in 1985 and is the collective voice of more than 25,000 African American physicians and the patients they serve. The NMA extends equal right and privileges to all physicians. This meeting is targeted to clinical practitioners, academic researchers and professionals interested in reaching a large population of minority physicians. The NMA Ophthalmology Section meets at the annual meeting. The meeting is targeted to clinical practitioners and research clinicians and fellowship trained ophthalmologists. Subspecialty scientific sessions are given over the annual meeting with a Symposium targeted on the first day which offers issues that are cross-sectional in their appeal to ophthalmology. The Sunday Symposium allows the top 7 awardees residents/medical students to present their scientific papers. The presenters are chosen from the scientific committee in the early part of the year for the Rabb-Venable Outstanding Research Award. The presentation of honorable mentions and winner of the Rabb-Venable awards is conducted on Sun. & Mon. evening at a dinner symposium. The winner of the Rabb Venable Award will go to ARVO the following year. The awards will increase by 3 awards a year over the next 5 years. The scholarship funds are supported by the pharmaceutical industry. The deadline for applications is Jan. 31st of the convention year. This grant allows the student/resident awardees to present their research, attend the grant writing session given by the NIH at the NMA meeting, interact with the NEI representative and travel expenses to the annual meeting. The submitted abstracts are encouraged to be in any of the subspecialties of ophthalmology emphasizing the latest developments in understanding, diagnosing, preventing and treating the many sight-threatening diseases and conditions which affect minority patients as well as the general population. This is the premier eye meeting for minority physicians in the U.S. Today's NMA fights for better medical care and opportunities for all Americans. The NMA is on the frontlines of evolving issues that affect the profession, including: burgeoning health care costs; the eroding autonomy of physicians; and the caring for uninsured patients who need immediate medical attention. These are many of the issues that affect our patients today. [unreadable] [unreadable]


The goal of this proposed collaborative R01 randomized controlled trial is to test the effectiveness of a novel intervention, the Treatment Initiation and Participation (TIP) program, on depression treatment adherence and depression outcomes among geriatric primary care patients. TIP is a brief, psychosocial intervention aimed at reducing the multifaceted personal barriers to adhering to depression treatment. Adherence is a challenge across the lifespan, but among older adults with depression, this challenge is compounded by medical and psychiatric co-morbidity, medical regimen complexity, and skeptical attitudes towards mental illness and its care. For older adults, the effect of stigma on seeking care for mental health issues such as depression is particularly strong. The decision to begin treatment for depression entails both countering the ageist notion that depression is a normal outgrowth of aging and confronting the stigma of mental health treatment particularly prevalent in this cohort. The treatment gap created by non-adherence in later life is becoming an even more prominent issue as the nation's demographic profile shifts. The proposed RCT will be conducted with diverse community samples from two geographically complementary primary care centers (Ann Arbor, Michigan and New York City). The study will recruit 260 older adults who have been newly prescribed antidepressant medication by their primary care physician, and randomize participants to receive either the TIP intervention or usual care. To test the study hypotheses, research assessments will be conducted at study entry, and at 6, 12 and 24 weeks after enrollment. If the proposed intervention is useful in improving antidepressant adherence, it has the potential to decrease the deleterious effects of untreated depression in a growing number of older adults. As a brief manualized intervention, TIP-PC is designed to fit easily within primary care practices and to be delivered by non-MD staff. PUBLIC HEALTH RELEVANCE: Depression and its treatment in later-life presents many challenges for the growing number of older adults in our society; beyond personal suffering, untreated depression worsens the outcomes of many medical illnesses and increases the risk for falls, cognitive decline, and death. Older adults experience many barriers that interfere with their choice or ability to follow the treatment their doctor recommends. The purpose of this research is to test a personalized and flexible primary care-based program designed to help patients experience successful depression treatment.


RNA-directed DNA polymerase (reverse transcriptase) is found in animal oncornaviruses and some human cancer cells. The avian myeloblastosis virus (AMV) DNA polymerase is the most readily available and best studied such enzyme and has been shown to have an unusually high degree of infidelity of transcription. Examples of this infidelity include random initiation, premature termination, a double strand reaction, incorporation of noncomplementary bases, a slippage reaction and inability to transcribe certain homopolymers. These phenomenon have not yet been studied with the human enzyme, in part because of the limited amounts of enzyme purified. By doing a large scale purification of RNA-directed DNA polymerases from human malignant tissues, it will be possible to do a number of studies on these enzymes which have already been done by us with the AMV DNA polymerase, compare the human enzyme to the AMV DNA polymerase, and compare enzymes from patients with different cancers. If the human enzyme behaves qualitatively the same as AMV DNA polymerase, studies with AMV DNA polymerase can be extrapolated to the human enzyme. If there are differences, these studies will be the start of detailed investigations of the human enzyme which may lead to more specific therapy of cancer.


Bacterial cells have evolved multiple regulatory mechanisms to ensure that their subcellular structures and organelles, such as pili and flagella, are assembled and positioned correctly. Our goal is to determine how the core components of a conserved regulatory system, and their interactions, have been adapted to serve the particular needs of different organisms. In Caulobacter crescentus, the protein components of this regulatory system (PodJ, DivJ, and PleC) follow defined patterns of subcellular localization that contribute to asymmetric cell morphology: organelles specifically develop at one pole but not the other. The regulatory components are conserved in Sinorhizobium meliloti, a related bacterium that induces nodule formation in plant roots during symbiosis. However, S. meliloti cells are morphologically symmetric compared to C. crescentus. We will examine how these conserved components function in S. meliloti to regulate the assembly of surface organelles and how this regulation affects host-microbe interaction. (1) We will first determine the localization patterns of the regulatory proteins and the phenotypes of null mutants that lack the proteins. (2) We will then detect and analyze changes in the mutants' transcriptional profiles to see if the proteins control expression of similar genes in different species. (3) We will also assess whether the mutations disrupt interaction between the bacterium and its hosts, preventing effective nodulation. In addition to these research objectives, the principal investigator aims to achieve the following developmental objectives under the guidance of a senior scientist: (4) establish an independent research group, (5) enhance mentoring skills, and (6) improve the quality of research. [unreadable] [unreadable] Relevance to Public Health: The proposed research is expected to reveal the driving principles of a regulatory pathway that exists in multiple bacterial species, including pathogens. Components of the pathway may serve as targets for new antimicrobial compounds. [unreadable] [unreadable] [unreadable]


Ischemic brain injuries are leading causes of morbidity and mortality to the aging population, but current therapy is poor in part because of our limited understanding of pathogenic mechanisms leading to neuronal loss. A critical trigger of the injury process seems to be acute energy loss, leading to membrane depolarization, excessive release of the excitatory neurotransmitter glutamate and neuronal Ca2+ accumulation. A large and persistent Ca2+ rise (Ca2+ deregulation) seems to be indicative of neuronal death. Recent evidence implicates critical contributions of another divalent cation, Zn2+, which is abundant in the brain and is normally very tightly regulated. However after ischemia or prolonged seizures, free Zn2+ accumulates in neurons, and observations that Zn2+ chelation is protective implicates a role in neuronal death. Culture studies have revealed that exogenously applied Zn2+ can enter neurons and accumulate in mitochondria and powerfully disrupt their function. However, little is known about mechanisms of injury caused by the accumulation of endogenous Zn2+ in native brain tissues. The proposed project thus seeks to address the following hypothesis: Accumulation of Zn2+ in hippocampal pyramidal neurons contributes critically to the initiation of ischemic neuronal injury, in part via deleterious interactions with mitochondria. Preliminary studies indicate that endogenous Zn2+ accumulates in pyramidal neurons in hippocampal slices subjected to oxygen glucose deprivation (OGD), prior to detectable Ca2+ accumulation, and that the Zn2+ appears to enter mitochondria and contribute to the induction of Ca2+ deregulation and cell death. Aim I will apply fluorescent imaging techniques (using both single cell and bulk loaded indicators) to acute hippocampal slices to examine Zn2+ accumulation in CA1 neurons during OGD, examine its interactions with mitochondria and determine its contributions to Ca2+ deregulation and cell death during acute OGD, and the subsequent reperfusion period. This key aim will seek to provide the first rigorous examination of the above hypothesis, and examine a range of interventions that may offer protection while helping to elucidate the sequence of events involved in the triggering and expression stages of injury. Aim II will use a range of approaches to determine the sources and routes of the injurious Zn2+ accumulation. These issues of where the Zn2+ comes from are complex, yet crucial to development of optimal interventions. Aim III will use organotypic slice culture models to examine roles of Zn2+ in the triggering of delayed neurodegeneration (up to 3 days after the OGD), in order to examine downstream injury processes and test therapeutic interventions that may offer protection when delivered well after the ischemia. It is hoped that these studies will provide new insights as to the sequence of events involved in the triggering of ischemic neuronal injury which will lead to new and effective neuroprotective strategies.


In vivo imaging of cellular and molecular structures in the intact brain provides a powerful tool for wideranging investigations in normal physiology, or in experimental models of disease processes. We have recently developed methods using a light microscope-based technique, multiphoton microscopy, to image microscopic structures in the brains of living transgenic mice over periods of months. Multiphoton microscopy utilizes a near-infrared laser for excitation of fluorophores deep within scattering tissue, with high spatial and temporal resolution. The spatial resolution of this imaging technique is about 1micrometer, several orders of magnitude better than other in vivo techniques, like PET, or MRI. In this application, we propose to develop new techniques that will provide important in rive readouts for biological imaging. This research will also lay the groundwork for development of contrast reagents suitable for use in human brain imaging with PET or MRI. We will develop, in Aim 1, techniques for high-resolution, in vivo imaging of structural reporters in the brain. We will investigate procedures to image individual neurons and microglia with high spatial resolution in the intact brain. In Aim 2, we propose to develop imaging techniques that exploit functional reporters in these living cells in the brain. Development of these molecular imaging techniques will build upon techniques accomplished in Aim 1. We have been using an experimental, transgenic mouse model of Alzheimer's disease that develops senile similar to those found in patients with Alzheimer's disease (AD). Our imaging techniques have allowed us to image the senile plaques in vivo in these mice with high spatial resolution. We will apply our new imaging techniques to this mouse model and address important questions that will provide insight into the pathophysiology of this disease. Our current techniques, however, rely on invasive procedures to gain access to the brain for imaging. In Aim 3, we will develop new techniques for non-invasive, in rive detection of senile plaques. New techniques using nearinfrared contrast reagents, and IR-sensitive detectors will allow non-invasive detection of plaques in the intact animal, and may also lead to clinically relevant diagnostic procedures for AD patients. In summary, the proposals outlined in this application will lead to generally applicable new techniques for cellular and molecular imaging in the intact brain.


This application seeks further support for the development of a wayfinding system specifically for building interiors of the facilities of public accommodations (e.g. office buildings, transportation facilities, hospitals, etc.) that will increase accessibility of those facilities to visually impaired users and serve as an environmental rehabilitative intervention for vision loss. This computerized system, called Pathfinder, interacts with users using artificial speech, voice recognition, electronic tactile touch tablet, and a high contrast visual CRT display. Embodying principles of universal design and intended to be useful for all users, including fully-sighted, the system will also increase accessibility for wheelchair users and hearing-impaired persons. Phase I results demonstrated feasibility with blind users unfamiliar with one floor of a Manhattan office building, and showed that the system is capable of training users with no experience with visual or tactile maps on its use without human intervention. Phase II research will address further development of the physical design of the product, the user training module, the tactile and voice recognition interfaces, and the visual display for low vision. Additionally, computer programming tools for building map and signage design professionals will be developed and tested. The product, along with an instructional package will be commercialized in Phase III.


Reports on HIV treatment trials in primates rarely publish any statistical analysis. VacMan is user-friendly computer program intended for a virologist's use on Macintosh, IBM Windows, and SUN X-Windows. VacMan's windowing interface was made possible by the NCBI Coretools and Vibrant software packages. VacMan calculates statistics for in vivo and in vitro infectivity data. The VacMan's statistical techniques are based on Bayesian analysis, but have a novel means of statistical inference: Bayesian predictive distributions are used like classical direct probabilities to make decisions about null hypotheses. The resulting decisions agree remarkably well with virologists' opinions about primate trials, but are objective and quantitative. Quantitative assessment of primate trials leads to several unappreciated principles of effective animal use: dilutions other than 10-fold can improve titration accuracy; infecting titration animals at the lowest doses possible can lower challenge doses; and finally, challenging test animals in small trials with more virus than controls safeguards against false successes, "reuses" animals, and strengthens experimental conclusions. These principles take on greater significance when one considers that chimpanzees in HIV trials cost about $50,000 each to use, and are an endangered species.


Project summary/Abstract: Alzheimer?s disease (AD) is a devastating disease that affects more than five million people in the U.S. alone. Early neurodegeneration biomarkers can play an important role in clinical trials of AD, especially biomarkers that are non-invasive, inexpensive, and radiation-free. Our laboratory has recently developed and validated a technique, T2-Relaxation-Under-Spin-Tagging (TRUST), to measure the brain?s oxygen extraction fraction (OEF) and metabolism with MRI. The technique does not require any exogenous tracer, can be completed within 5 minutes on a standard 3T, has high test-retest reproducibility, and has been successfully evaluated in a multi-site setting. Mounting evidence suggests that OEF is a potential marker of neurodegeneration in AD. First, we have shown that patients with amnestic Mild Cognitive Impairment (MCI), a mild form of AD, have significantly diminished OEF. Second, cognitively normal elderly individuals with genetic risk to developed AD, e.g. APOE4 carriers, have reduced OEF. Finally, brain oxygen extraction and metabolism have also been found to be associated with cognitive dysfunction and tauopathy measured from the CSF. To validate the oxygen metabolism biomarker using gold-standard measures of neurodegeneration, human studies often require a long follow-up period and a relatively high cost. Therefore, it is logical to first conduct an exploratory/developmental (R21) study to demonstrate the plausibility of such hypothesis in animal models. Toward this goal, we propose a longitudinal study in AD mouse model using a novel oxygenation MRI technique, and validate its association with neurodegeneration as measured by histology. This study has two specific aims. Aim 1 is to determine cross-sectional and longitudinal characteristics of brain oxygenation and metabolism in AD mouse model relative to control mice. We will measure OEF and cerebral metabolic rate of oxygen (CMRO2) in a novel AD model that our co-investigator, Dr. Wong, recently developed. We hypothesize that cross-sectionally OEF will be lower in the AD mice compared to control mice, and the difference will be more pronounced at an advanced age. Longitudinally, OEF in the AD mice will show progressive decrease starting 6 months of age. Aim 2 is to validate the imaging biomarkers with histological measurement of neurodegeneration. The animals undergoing imaging will be sacrificed at three time points (a sub-sample at each time point) and histology will be performed to measure neuron count and tau burden. Imaging markers will be compared to these histological measures to determine the degree to which they can predict current and future neurodegeneration. Impact: Upon the completion of this study, we will have established a concrete relationship between imaging measures of brain oxygen metabolism and hallmarks of AD pathology such as tauopathy and amyloidosis, which will provide a strong foundation for human validation studies in a larger-scale project.


Newly formed blood vessels fail to mature into fully functional vessels in tumors due to the chronically angiogenic microenvironment. The functional impairment of these vessels hampers drug delivery, thereby diminishing the efficacy of antitumor therapies. Excessive vessel permeability associated with tumors also allows tumor cell invasion into the circulation facilitating metastatic spreading. Therefore, the ability to conrol vessel maturity in tumors provides a potential therapeutic opportunity. The long-term goal of this study is to understand the molecular basis for vascular maturation. Our recent studies uncovered an essential role of the small GTPase R-Ras in establishment of mature, functional blood vessels in tumors. Thus, R-Ras promotes normalization of the tumor vasculature. Now, the important new objective of this investigation is to determine the molecular pathway for R-Ras-mediated vessel regulation so that new molecular targets may be identified for controlling the tumor vasculature for therapeutic advantage. R-Ras enhances vascular integrity through regulation of VE-cadherin. Our recent studies also show that R-Ras attenuates VEGF signaling in endothelial cells by inhibiting VEGFR2 internalization upon VEGF stimulation. Furthermore, R-Ras not only promotes endothelial cell-pericyte association but also facilitates intercellular signaling between the two cell types via TGF-beta and Jagged1-mediated Notch signaling. These pathways promote endothelial cell quiescence and mural cell differentiation; therefore, they are important for vessel maturation. Based on these observations, we propose a hypothesis that R-Ras orchestrates these pathways to redirect nascent tumor vessel formation from an angiogenic sprouting/branching process to a maturation process. In this proposal, we will investigate the significance and precise roles of the R-Ras pathways during tumor vascularization. In Aim 1, we will identify and characterize the key signaling pathways mediating the R-Ras effects on endothelial cells and pericytes in a series of in vitro experiments using various culture/coculture systems. In Aim 2, we will validate the findings from Aim 1 in various animal models to determine the role of these mechanisms during the establishment of functional tumor vessels. The proposed studies will provide an important insight into the molecular basis for the vascular normalization phenomenon and its implications in tumor malignancy and therapies.


DESCRIPTION: The overarching goal of this application is to investigate the face recognition deficits of individuals with autism. Initially, this will entail documenting the behavioral as well as functional and structural cortical signatures associated with this impairment and, in doing so, contrasting face processing with that of other stimulus classes, such as objects or words. To understand the nature of the face impairment further, two additional lines of investigation will be pursued. In the first, the investigators will characterize the topography and function of striate and extrastriate visual areas in these individuals using functional magnetic resonance imaging of visual eccentricity and meridian maps. In addition, they will undertake analyses of the structural morphometry of regions such as fusiform and lingual gyrus to explore the neural substrate further. In the second, the investigators will explore the intermediate level vision of these individuals, with specific emphasis on their ability to derive configurations from local elements using sensitive behavioral assays. The investigators will also explore the developmental aspects of these perceptual processes by comparing the performance of young and older high-functioning autistic individuals, along with matched controls.


This project will study the mechanisms by which individual cells maintain control over their own internal calcium milieu while transporting substantial quantities of this element. Two calcium transporting epithelia, embryonic chick chorioallantoic membrane and the small intestine of rat and chicken, will be studied. Parallel studies will be pursued involving measuring calcium uptake and transport by intact tissue preparations either in vivo or in vitro, using conventional radioisotope counting procedures; and studies at the cellular level employing electron probe microanalysis and transmission electron microscopy. We intend to elucidate the mechanism by which actively transported calcium enters a transporting cell, is sequestered within the cell, transferred to another surface, and how the sequested calcium leaves the transporting cell. We hope to discover whether calcium in transit is complexed with phosphate or other counterion; and whether calcium can diffuse between adjacent epithelial cells, in a passive mannner, through junctional complexes.


The overall objective of this project is to explore the potential of nuclear magnetic resonance for providing detailed information about the structure and dynamic properties of enzymes, particularly those of high molecular weight. Enzymes specifically labeled with C13 or F19 nuclei will be obtained by in vivo incorporation of labeled amino acids or by in vitro chemical modification of intact enzymes with labeled reagents. Carbon-13 and F19 nuclei were chosen because their wide chemical shift range and the relative narrowness of their nmr signals should result in high resolution spectra. The labeled enzymes and their noncovalent complexes with substrates, inhibitors, and coenzymes will be studied by pulse nmr techniques. The enzymes to be studied include E. coli alkaline phosphate containing 4-fluorotryptophan or m-fluorotyrosine, horse liver alcohol dehydrogenase amidinated or guanidinated with a C13 enriched reagent, E. coli tryptophan synthetase alpha subunit iwht C13 enriched histidine residues, and E. coli aspartate transcarbamylase labeled with C13 in phenylalanine, tyrosine, cysteine or tryptophan side chains


We have recently established several functional clones of normal rat anterior pituitary cells. These clones have exhibited good growth characteristics and have retained a normal diploid karyotype after 20-24 passages. One of these clones (2B8) appears to produce only prolactin since no other anterior pituitary hormones have been detected in the conditioned media. We propose to carry out baseline studies on the 2B8 clone using light microscopy, immunocytochemistry, transmission and scanning electron microscopy and time-lapse cinematography. Polyacrylamide gel electrophoresis will be carried out on conditioned media after incorporation of (3H) leucine into newly synthesized hormone. 2B8 cells will be implanted under the kidney capsule of immature, hypophysectomized rats in order to determine if the prolactin secreted is biologically active. The direct effect of selected steroids, hypothalamic hormones, neurotransmitters, and putative pineal hormones on prolactin synthesis and release from 2B8 cells will be determined. Finally, we will attempt to induce the transformation of 2B8 clonal cells by prolonged exposure to hydrocortisone. Hydrocortisone has been shown to markedly stimulate growth hormone secretion and inhibit the production of prolactin from GH3 pituitary tumor cells.


Recent studies suggest that surgical exposure of the sciatic nerve prior to assessment of nerve blood flow (NBF) may account for discordant reports of increased versus decreased NBF in diabetic and galactosemic rats. Regardless of whether NBF is observed to be increased or decreased, it is normalized by inhibitors of aldose reductase (ARI). To clarify these paradoxical findings, NBF was examined in rats (7-8 rats per group) fed normal chow (C), chow containing 50% galactose (G), or 50% galactose + the ARI zopolrestat (Z) at 100 mg/kg/day. After 3 weeks the rats were anesthetized and one sciatic nerve was surgically exposed (SE) without touching the nerve. The incision was immediately closed and 2 h later NBF (ml/g wet wt/min) was assessed in the SE nerve and in the contralateral unexposed nerve (UE) by injection o 10 um 46Sc-microspheres. In UE nerves NBF was 37% higher in G than in C. This increase was prevented by Z. In SE nerves NBF was 49% lower in G than in C and was virtually normalized by Z. These data indicate that 1) galactosemia increases NBF by 37% in UE nerves, 2) SE markedly increases NBF by 4 fold in C rats but only 1.5 fold in G rats, and 3) the reduced NBF in SE nerves in G vs C are largely prevented by ARI. These observations are consistent with numerous reports that vasodilatory responses to various stimuli are blunted by aldose reductase-linked mechanisms in diabetic rats. These observations provide an explantation for most previous discordant reports of increased versus decreased sciatic NBF induced by galactosemia and diabetes.


Defining the complex mechanisms of viral oncogenesis requires a broad understanding in at least three areas of research: molecular and cellular biology, immunology, and virology. It is the goal of the proposed training program to provide graduate students and postdoctoral fellows a challenging and enriched scientific environment in which both formal and informal training within these three broad, but interrelated areas can occur. The breadth of the scientific interests of the training faculty create a blend of research areas, many of which incorporate virus systems to dissect the molecular events that occur within the normal versus the cancer cell. The specific research programs include (a) molecular and cellular biology-regulation of cell growth and gene expression, oncogenes, signal transduction, and mechanisms of neoplastic transformation; (b) immunology-expression, oncogenes, signal transduction, and mechanisms of neoplastic transformation; (b) immunology; and (c) virology-molecular studies of virus replication, assembly, oncogenesis, pathogenesis, latency, and gene regulation using adeno, cytomegalo, hepatitis B, herpes simplex, papilloma, papova, and retroviruses. The trainers associated with the proposed training grant have their primary academic appointments within the Department of Microbiology and Immunology. However, each of the trainers is associated with multiple interdisciplinary graduate programs which cross departmental and college boundaries. Graduate students eligible for appointment on the training grant may be associated with one of several graduate programs including Chemical Biology, Cell and Molecular Biology, Genetics, Microbiology and Immunology, Molecular Medicine, and Neurosciences. The potential of the trainers to recruit students from such a variety of programs helps to insure that a significant pool of highly qualified students will be available for appointment to the training grant. The training program for graduate students and postdoctoral fellows includes a dynamic research environment that has excellent research and core support facilities and faculty research laboratories with productive and well funded research programs. The research and training environment is further enhanced by numerous interactions among the various laboratories as well as journal clubs, student seminars, and visiting scientist seminar programs. Finally, the research areas of emphasis within the training program provide a broad foundation on which a creative student and postdoctoral fellow can base a productive career in research in the area of citruses and cancer.


Dopamine (DA), norepinephrine (NE), and epinephrine (EPI) and their metabolites and dopamine-beta-hydroxylase (DBH) are present in human plasma and cerebrospinal fluid (CSF). Levels of these substances reflect the balance between their release and removal. The increases in levels of NE and EPI with standing, exertion, stress, drugs, administration, etc., are used to examine sympathetic neuronal responsivity. The responses and level of activity of the sympathetic nervous system in cardiovascular disorders (hypertension, hypotension) in alcoholism, a variety of psychiatric disorders, and after treatment with drugs are being examined. Dopamine-beta-hydroxylase (DBH) is the enzyme which converts dopamine (DA) to NE. NE and DBH can be measured in cerebrospinal fluid (CSF) and may be useful to estimate in disease in which a defect is suspected, i.e., essential hypertension, Shy-Drager syndrome, amyotrophic lateral sclerosis (ALS), enuresis and hyperactivity in children, and schizophrenia.


The process of cytoskeleton assembly and disassembly is essential for many critical phagocyte functions, including locomotion, phagocytosis, adhesion, egress from the vasculature, and cytokine production. Understanding the mechanisms by which cytoskeleton assembly is regulated and acts in turn to direct the functions of the phagocytic cell is at the core of understanding the mechanisms of augmentation of host defense at sites of inflammation. The molecular details of this regulation and it's influence on leukocyte activation has been difficult to study. This laboratory has developed a system which has significantly advanced the understanding of the role of the cytoskeleton in the mechanism of leukocyte activation. This lab has found that the pathway initiated by ligation of FcR by Ig6 in phagocytes increases [Ca2+]i by stimulating release of Ca2+ from intracellular stores through a novel IP3-independent pathway that requires the actin cytoskeleton. A specific actin-binding protein called l-plastin is required for this release of Ca2+. Our hypothesis is that l-plastin is an important link between cell surface receptors, such as FcR, and the cytoskeleton in phagocytes. In order to test this hypothesis, we will study the cell biology and biochemistry of l-plastin in phagocytes that naturally express the protein, as well as develop an in vitro transfection system to allow us to study the effects of altering or mutating l-plastin on l-plastin cell biology and biochemistry as well as IgG and integrin-mediated signal transduction. Actin binding, phosphorylation, and immunolocalization of l- plastin and IgG-mediated calcium responses and phagocytosis in phagocytic cells will be studied as models of normal l-plastin function. L-plastin function will be defined further by applying these assays to cell transfection systems using mutated forms of l-plastin. Cells that do not normally express l-plastin as well as mouse cell lines that express endogenous l-plastin will be transfected with the human form of l-plastin. The function of wild type human l-plastin in these cell lines will be validated by comparing l-plastin function and IgG-mediated responses in cells transfected with normal l-plastin and normal phagocytes.


This project involves studying the biochemistry and genetics of Drosophila melanogaster RNA polymerases to determine how the different forms of this enzyme are involved in the regulation of transcription and thus the development of eucaryotic organisms. The method of analysis will involve (1) locating in the D. melanogaster genome the structural genes coding for polymerases I and II and (2) biochemically isolating and characterizing these forms of the enzyme and any factors which may affect their activity. This information will be used as an aid in isolating temperature conditional (both heat and cold) mutants of polymerases I and II for studies concerning (1) the correlation of genetic data with biochemical data on the subunit structure of the polymerases, (2) determining how the forms may be related with respect to the subunit structure, (3) the regulation of the levels of polymerases I and II during development and in response to hormones, (4) the regulation of transcription and metabolism of hnRNA and rRNA, (5) the effect of inhibiting transcription on developmental processes.


[unreadable] Inadequate dietary intake assessment tools hamper studying relationships between diet and disease. Methods suitable for use in large epidemiologic studies (e.g., dietary recall, food diaries, and food frequency questionnaires) are subject to considerable inaccuracy, and more accurate methods (e.g., metabolic ward studies, doubly-labeled water) are prohibitively costly and/or labor-intensive for use in population-based studies. A simple, inexpensive and convenient, yet valid, dietary measurement tool is needed to provide more accurate determination of dietary intake in populations. We therefore propose a three-phase project to develop and test a new assessment tool called FIVR (Food Intake Visualization and Voice Recognizer) that uses a novel combination of innovative technologies: advanced voice recognition, visualization techniques, and adaptive user modeling in an electronic system to automatically record and evaluate food intake. FIVR uses cell phones to capture both voice recordings and photographs of dietary intake in real-time. These dual sources of data are sent to a database server for recognition processing for real-time food recognition and portion size measurement through speech recognition and image analysis. The user model will allow for enhanced identification of food and method of preparation in situations that images alone might not produce accurate results as the system learns through experience and adapts to the individual's food patterns. Objectives are to fuse existing voice and image recognition techniques into a system that will recognize foods by food type and unique characteristics and determine volume by film clip showing an image from at least two angles. The item identified will be matched to an appropriate food item and amount within a food composition database and nutrient intake computed. Researchers will be able to view the resulting analysis through an adapted version of the dietary analysis program, ProNutra. The proposed protocol will incorporate three discrete phases: 1) technology development, integration, and testing; 2) validity testing with a controlled diet (metabolic ward study); and 3) real-world utility testing. Validity of the data collected will be judged by how closely the nutrient calculations match the known composition of the metabolic ward diets consumed. FIVR has the potential to establish a method of highly accurate dietary intake assessment suitable for cost-effective use at the population level, and thereby advance crucial public health objectives. [unreadable] [unreadable] [unreadable] [unreadable]


Current investigations are being conducted on the events leading to the formation of several types of cataracts. Diabetic or sugar cataract formation initiated by the enzyme aldose reductase is being studied. Methods for controlling the onset of these cataracts through the regulation of this enzyme are being developed. Hereditary cataract formation is also being studied in a strain of mice developed in our laboratory. Known as the Philly mouse, these mice develop osmotic cataracts by an as yet unknown mechanism.


Biliary atresia results from an inflammatory and fibrosing obstruction of extrahepatic bile ducts in young infants. Despite prompt diagnosis and surgical treatment, the disease progresses and leads to end-stage cirrhosis in most infants. The etiology and pathogenic mechanisms of disease are largely undefined. In the first period of this award, we searched for prominent molecular processes in livers of infants with biliary atresia and found a dominant interferon-gamma (IFN?)-rich proinflammatory circuit at the time of diagnosis. We then used a novel mouse model of experimental atresia to directly examine the role of IFN? in regulating the biliary atresia phenotype. This model displayed a similar proinflammatory process in the liver. Most notably, the in vivo loss of IFN?, completely prevented duct obstruction and improved long-term outcome. In this competing renewal application, we propose an overriding hypothesis that the pathogenic mechanisms of biliary atresia begin with an epithelial injury by the innate immune system and progress to duct obstruction by the activation of an exuberant adaptive immune response. This hypothesis will be tested in three closely related but independent aims. In Aim 1, we will determine the role of hepatic CD8+ T cells in neonatal injury of bile ducts. This will be done by investigating how CD8+ T cells engage cholangiocytes through recognition and binding to the MHC-I complex, and by dissecting the intracellular signals trigged by IFN? to render cholangiocytes susceptible to apoptosis induced by tumor necrosis factor-alpha. In Aim 2, we will establish the mechanisms by which hepatic NK cells injure the neonatal duct epithelium. Using a similar experimental approach, we will examine the mechanisms of NK cell-mediated cytotoxicity and how NK cells work in synergy with CD8+ T cells to induce epithelial injury and duct obstruction. And in Aim 3, we will define how hepatic macrophages trigger the innate immune response following a neonatal viral challenge that targets the bile ducts. In this aim, we will determine whether hepatic macrophages are targeted by rotavirus. In related experiments, we will also investigate the molecular mechanisms by which infected macrophages induce chemotaxis to neutrophils and, possibly, cytotoxicity to cholangiocytes. Upon completion, the proposed experiments will advance our understanding of the biological basis for experimental atresia and potentially identify new therapeutic targets to stop progression of disease and improve long-term outcome in children with biliary atresia. Project Narrative: This project studies biliary atresia, the most common cause of chronic liver disease in children and the leading indicator for pediatric liver transplantation in the United States. With the aim to determine key cellular and molecular mechanisms of disease pathogenesis, the proposed experiments will use a unique mouse model of neonatal biliary injury to dissect the biological processes regulating the injury and obstruction of extrahepatic bile ducts. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]


The kallikrein-like enzyme activity of the toad bladder was measured after 24 hour incubations with either aldosterone 10 to the minus seven, vasopressin 10 to the minus seven, or Ringer's solution. There was no significant change in kallikrein-like acitvity after aldosterone or vasopressin incubation when compared with the control.


The above research project is concerned with certain aspects of the enzymology of nitrate metabolism, namely, a continuing investigation at the molecular levels of (a) in vitro assembly of assimilatory nitrate reductase by incubation of a cell-free extract of the Neurospora nitrate reductase mutant (nit-1), induced by nitrate, and that of other mutants, uninduced wild type and other molybdenum-containing proteins. It includes the purification and characterization of both the nitrate- inducible protein of the nit-1 mutant and the presumed molybdenum- containing component or cofactor (including its ultimate identification), (b) Neurospora assimilatory NADPH-nitrate reductase, elucidating its subunit and cofactor interrelationships both structurally and functionally, (c) the purification and characterization of nitrate reductase-related proteins of several non-allelic nitrate reductase Neurospora mutants, and (d) the purification and characterization of the enzymes involved in the stepwise reduction of nitrate to ammonia (i.e., nitrate assimilation) and in nitrate respiration (where nitrate replaces molecular oxygen as the terminal electron acceptor) including denitrification. In addition to its academic value, the data could be helpful in evaluating the relationship between nutritive value and yield with respect to the use of different nitrogen compounds in agricultural practice. It may also prove to have a bearing on the normal and abnormal metabolic states of mammaliam tissue in view of the known occurrence of the molybdenum cofactor in mammalian xanthine oxidase, liver aldehyde oxidase, and liver sulfite oxidase.


This research aims to study mechanisms of synaptic function with emphasis on the origins and locations of individual synaptic membrane polypeptides. By reacting epsilon-amino groups of lysines which are exposed on membrane surfaces with sodium boro (H3) hydride in the presence of pyridoxal phosphate it is possible to selectively label exposed peptide areas of intact synaptosomal membranes. Combining this technique with purification and subsequent gel electrophoresis of synaptosomal sub-fractions from chick brain allows classification of membrane polypeptides into four groups: those occurring entirely within the membrane, those exposed on both the internal and external faces, and those exposed only at one surface, either external or internal. The nature of the peptides around the exposed lysine residues will be characterized by labeling polypeptides eluted from the gels with I125, cleaving with trypsin or cyanogen bromide, and performing autoradiographic peptide mapping on the resulting peptide mixtures. Whether or not synaptic vesicles fuse with the external membrane will be investigated by comparing labeled gels and peptides of these two fractions. Distances between polypeptides in the membrane will be determined with bifunctional reagents with various bridge lengths between the functional groups. When basic parameters are defined synaptosomes prepared from synapses in differing states of activity will be examined. Alterations of membrane conformation and subsequent protein exposure may reflect neural activity. Synaptosomal membranes will be prepared from electrically stimulated brain slices and chick optic lobes that are either denervated (by enucleation) or receive minimal input (by eyelid suture). Comparing such preparations with normal synaptosomal membranes may reveal a relation between synaptic membrane structure and its functional use. Such data will have important implications for theories both of normal brain function and of the etiology of neurological and psychiatric illness.


Project 1 Determinants of disease progression and role of latent infection in transmission will study a series of factors that will help understanding why only a limited number of individuals infected with Leishmania donovani develop Visceral Leishmaniasis (VL) on the Indian subcontinent. Project 1 will also contribute to assess the role of asymptomatically infected individuals (those who do not progress to disease) in L. donovani transmission. This project will combine field and laboratory work. Two serological surveys of leishmaniasis infection will be done in high transmission areas to identify recent asymptomatic seroconvertors and controls in the study area (see Core C for details). Similariy, VL cases and controls (i.e healthy household contacts) will also be identified and included in the different studies. Project 1 is divided in 4 specific aims. Aim 1 will examine the association of HLA-type with seroconversion and disease progression controlling for several confounders identified in the previous risk factor studies. Aim 2 will use a recently developed quantitative PCR to assess parasite load in different subpopulations (i.e.seroconvertors and seronegative individuals) ahd assess its influence in progression to VL taking into account genetic and environmental factors. Aim 3 will examine the association between co-infections with other Neglected Tropical Diseases (NTD) and VL in two case-control studies. (1) An unmatched case-control study comparing Leishmania seropositives with the general population and (2) a matched case-control study comparing VL cases to community controls will be used to assess the effect of filaria- or geo-helminths-L. donovani co-infection on progression to VL. Finally, Aim 4 will contribute to study the role of asymptomatically infected persons by (1) validating a modified SLA-based Quantiferon to detect cellular immune response in asymptomatic individuals and (2) selecting individuals (peripheral blood mononuclear cells (PBMC) culture positive) to be included in the xenodiagnosis experiments (see Project 2 for details) . RELEVANCE (See Instructions): Results generated in this project will be of importance for individual as well as for public health. Identifying risk factors for progression from VL infection to disease may allow for secondary prevention. Evidence on the potential role of asymptomatic infections in L. donovani transmission may have major implications for the current VL control strategy.


Somatic stem cells residing in many organs are responsible for tissue maintenance and repair and also contribute to aging and cancer. While the role of stem cells and cell turnover is obvious in rapidly renewing tissues, their significance in the gastrointestinal neuromuscular compartment has only recently been recognized. The classic view of gastrointestinal motility disorders including diabetic gastroparesis is that they are due to impaired tissue function and degeneration of terminally differentiated cell types such as enteric neurons, smooth muscle cells and interstitial cells of Cajal (ICC). This is also reflected in current treatment modalities, which focus on symptom control and stimulation of residual function. However, these treatments are not curative and to a large extent ineffective. Recent studies demonstrating considerable plasticity of postnatal enteric neurons, smooth muscle cells and ICC indicate that this old view is untenable. We recently identified a stem cell residing in the gastric musculature of adult mice that can give rise to ICC (ICC-SC). This paradigm shift has opened entirely new opportunities to unravel the mechanisms of ICC maintenance and differentiation and to develop new, rational therapies to regenerate ICC networks from endogenous or transplanted tissue stem cells in these disorders. However, there are several barriers that must be overcome before this goal can be realized including a lack of understanding of the mechanisms that control ICC-SC self- renewal and differentiation, as well as the role of the tissue microenvironment in these processes. Therefore, the overall goals of the current project are to determine the key cell-intrinsic and cell-extrinsic factors that control ICC-SC proliferation and differentiation and to test pharmacological agents that target ICC-SC via these factors in preclinical disease models. Our first specific aim is to determine the role and mechanisms-of-action of polycomb group proteins as cell-intrinsic factors in the epigenetic control of ICC-SC maintenance and differentiation and to test the in vivo utility of indirect histone methyltransferase inhibitors to stimulate, through the inhibition of polycomb activity, the differentiation of transplanted and endogenous ICC-SC. Our second specific aim is to determine the role and molecular mechanisms-of-action of glycogen synthase kinase 3 (Gsk3) as an extrinsic factor in regulating ICC-SC through stem cell factor expression in smooth muscle cells and to determine the in vivo utility of Gsk3 inhibitors to reverse gastroparesis by supporting ICC-SC self- renewal and differentiation. We will use novel models and new technology developed in our laboratory including freshly isolated and cultured ICC-SC, transplantation of genetically labeled ICC-SC, and quantitative analysis of key stages of ICC development by flow cytometry. We will also employ state-of-the-art molecular biological and recombinant DNA techniques to study epigenetic control of gene transcription and mouse models for preclinical testing. The proposed studies will determine the mechanistic basis for, and set the stage for clinical trials of, novel pharmacological interventions for loss of ICC in motility diseases.


This project involves the application of microprocessor technology and improved man-machine interface methods to permit physicians and their associates to more directly communicate with computer record systems. A pilot study involving medical transactions input directly by practicing physicians is underway. The goal is to develop better ways to automate the essential physician contribution to the health care record that is used in both research and patient care.


Epidemiological studies provide compelling evidence for a link between low birth weight, arising either from fetal growth restriction, preterm birth, or both, and adult diseases such as coronary heart disease, Type 2 diabetes, obesity and stroke. In growth restricted pregancies the fetus often experiences periods of hypoxemia. We have begun examining the effects of hypoxia during specific develomental stages of pregnancy, and have preliminary data suggesting that 30 day old postnatal rats whose dams were hypoxic from day 12 to 15 of pregnancy, exhibit elevated plasma insulin, normal plasma glucose, reduced liver glycogen and glycogen synthase concentrations. These characteristics were not altered in postnatal rats exposed to prenatal hypoxia from either day 15 to 18 or day 18 to 21 of gestation. Based on these studies, our overall hypothesis is: Development stage-specific prenatal hypoxia is a key regulator of "fetal programming" setting the stage for the onset of insulin resistance, obesity and ultimately Type 2 diabetes. The specific hypothesis to be tested is: Prenatal hypoxia from fetal day 12 through 15 will alter postnatal metabolism resulting in insulin resistance in the young adolescent rat. To test this hypothesis we will determine if hypoxia during discrete windows of development induces insulin resistance at postnatal day 30. Time-mated rats will be exposed to hypobaric-hypoxia from day 12-15, day 15-18 or day 18-21. Offspring from these pregnancies will be compared to offspring from ad-lib fed controls, controls pair-fed during the window of hypoxia, and controls pair-fed during the window of hypoxia through parturition. Pups derived from hypoxic pregnancies, as well as those derived from the pair-fed, and continuously pair-fed controls will be fostered onto additional ad-lib fed dams at birth. At 30 days of age, rats will receive intravenous glucose or insulin tolerance tests, or will undergo euglycemic, hyperinsulinemic clamps to assess insulin resistance. Rats undergoing glucose tolerance or insulin resistance tests, following a 48 hr recovery, will be sacraficed for collection of liver, skeletal muscle and visceral adipose tissue. If peripheral insulin resistance is conferred, alterations in insulin receptor and GLUT 4 mRNA and protein concentrations will be examined. If insulin resistance is liver specific, we will focus on the insulin signalling cascade (e.g. insulin receptor, IRS's, PIS kinase, protein kinase B, glycogen synthase kinase and glycogen synthase), in liver. These studies will provide insight in to the role fetal hypoxia alone may play in the development of Syndrome X. [unreadable] [unreadable]


PROJECT ABSTRACT With sepsis screening and increased compliance with evidence based, guidelines-driven standard operating procedures for early diagnosis and treatment of sepsis in surgical intensive care unit (ICU) patients, early mortality has been dramatically reduced and late-onset fulminant multiple organ failure has virtually disappeared. A new phenotype of chronic critical illness (CCI), however, has emerged in surgical ICU patients characterized by prolonged ICU stays, recurrent nosocomial infections, poor wound healing, progressive cachexia and manageable organ dysfunctions. We have coined the term persistent inflammation, immunosuppression and catabolism syndrome (PICS) to reflect the pathophysiologic hallmarks of this growing epidemic. Many of these patients (especially the elderly) are discharged to long-term acute care facilities where they often suffer an indolent death. We hypothesize that CCI characterized by morbid long-term outcomes is now a predominant clinical trajectory of surgical ICU patients who survive sepsis. The overall objective of Project #1 is to characterize the epidemiology of CCI in surgical ICU patients who develop sepsis and to determine its long-term consequences. The challenge is to return those surgical ICU patients who survive sepsis to a functional, productive life, and to reduce their burden to the healthcare system and to society through early interventions. We need to identify early, however, which patients are at highest risk for morbid long-term outcomes and might benefit from novel interventions. Project #1's main functions will be the following: ? Define the epidemiology and long-term consequences of sepsis in surgical ICU patients. Clearly, there is a compelling need to better understand the long-term consequences of sepsis in surgical ICU patients, especially those who progress into CCI who are at high risk for PICS. ? Identify clinical indices and biomarkers that can predict CCI in surgical ICU patients early (within 48 hours) after sepsis. These prediction models could help provide insight into underlying pathophysiology and design entry criteria in future trials. ? Identify clinical indices and biomarkers on day 14 in patients with CCI after sepsis that predict morbid outcome (defined as death or full functional dependency at 1 year). These findings could be used to gain insight into underlying pathophysiology by comparing novel biomarkers at earlier time points in patients at highest risk for morbid long-term outcomes versus those patients at lowest risk. A CCI score could then be developed as a composite endpoint in future interventional trials. To accomplish the above goals, Project #1 will perform, over 5 years, a single-site, prospective, longitudinal study of 400 adult surgery and trauma ICU patients who develop sepsis.


The ovum is surrounded by the cumulus, the corona, the zona and vitelline membrane. Capacitated rabbit sperm undergo the acrosome reaction during the passage through the cumulus. Enzymes released following the acrosome reaction or enzymes still bound to the vesicles composed of the plasma (PM) and the outer acrosomal membranes (OAM) hydrolyse the cumulus matrix and allow sperm to reach the corona cells and sperm which are left with a limiting inner acrosomal membrane (IAM) encounter the zona pellucida. The sperm binding to the zona and its subsequent penetration may involve a sequential action of binding to the zona and its subsequent penetration may involve a sequential action of acrosomal hydrolases bound to IAM. The fusion of spermatozoa with vitelline membrane probably involves phospholipases of the persistent PM and OAM on the equatorial segment (ES) and the post acrosomal region (PAR). The subsequent exocytosis of corticle granules may also be triggered by sperm enzymes. This sequence of events provides the rationale for this proposal. The overall objective of this proposal is to elucidate further the role of acrosomal enzymes in fertilization. Specifically, we will isolate and compare enzymes associated with acrosomes; determine the localization of key enzymes involved in sperm-egg binding; isolate acrosomal enzymes from rabbit spermatozoa and determine their effects on rabbit ova. After the extraction of acrosomal enzymes, sperm still contain nearly one-half of neuraminidase, one third of arylsulfatase, and one third of hyaluronidase. these enzymes can be extracted by further extraction of the denuded sperm with 1% Triton X-100 and 0.1 M CaCl2. This treatment also solubilizes the equatorial segment. We will follow this extraction procedure by studying simultaneous changes in sperm morphology at the ultrastructural level. In view of increasing importance of glycosyltransferase and sialic acid in gamete interactions, we will attempt localization of these and other acrosomal enzymes by immunofluorescence, radiolabeling, and cytochemical techniques. The enzyme purification will employ affinity chromatography on hydrophobic columns, high pressure liquid chromatography coupled with conventional methods. The role of acrosomal enzymes in sperm-egg binding, and the ovum penetration will be studied. Enzymes will also be tested on isolated zona.


A small amount of mercury is released from dental amalgam restorations and may be absorbed in the human body. Although restorations are believed not to be a health hazard to a large majority of patients, some concern remains because of the toxicity of mercury. The goal of this research is to gain a scientific understanding of the processes involved and to use the knowledge to minimize the release. The study will be performed in the laboratory using a simulated oral environment. The objective is to measure the rate of the processes, which are believed to play major roles in the mercury release: dissolution of mercury from a surface, which has been stripped of the protective film, repassivation of the amalgam surface after film stripping, and changes in the chemical form of mercury after dissolution in the liquids. The rates of these processes will be determined for representative dental amalgams and as a function of major variables, such as pH, temperature, and chemistry of the environment. The experimental techniques will include solution analysis by Atomic Absorption Spectrophotometry, and electrochemical measurements. The results of the study will identify the factors that have major roles in the mercury release, and provide quantitative information on the rates of the reactions involved. They will serve as a basis for a selection of measures to lower the release.


Cardiovascular calcification is a common consequence of aging, diabetes, hypercholesterolemia, hypertension, abnormal valve mechanics &inflammation, &chronic renal insufficiency. Once thought benign, the deleterious clinical consequences of calcific vasculopathy are now becoming clear;it is a key component of pathophysiology leading to CVA, Ml, and PVD -- with amputation and cardiovascular mortality portended by the anatomy and extent of calcific vasculopathy. The ability to cure or substantially reverse macrovascular calcification (MVC) represents an unmet clinical need. A better understanding of MVC initiation and progression is required. Osteogenic &inflammatory gene regulatory programs are activated in MVC-- variably involving adventitial, medial, intimal, and valvular tissues -- via mechanisms overlapping those that control bone physiology. We've shown that a pro-osteogenic program -- a "feed-forward" BMP2-Msx2-Wnt signaling cascade -- is activated in aortic myofibroblasts by diabetes and dyslipidemia. By contrast, PTH/PTHrP receptor agonists (e.g., teriparatide) suppress vascular myofibroblast calcification and aortic Msx2-Wnt gene expression. We now study the role and regulation of myofibroblast Msx2-Wnt signaling in MVC. In Aim 1, we use LDLR-/-.TOPGAL+ reporter mice (LacZ transgene under control of Wnt-responsive TCF/LEF element) to relate spatial activation of aortic Wnt signaling to diet-induced Msx2-Wnt expression and recruitment of osteoprogenitors from adventitial, medial, &valvular myofibroblasts. We also test if the enhanced aortic calcification and Wnt expression we observe in CMV-Msx2 transgenic mice activates aortic LacZ expression. Co-culture studies of primary aortic myofibroblasts will test if Msx2--activated paracrine Writ signaling is inhibited by PTH (1-34). In Aim 2, using TOPGAL mice, we test if PTH(1-34) inhibits canonical Wnt signaling in vivo. In Aim 3, we map Msx2 promoter protein-DNA interactions that convey transcriptional suppression to PTH(1-34) in myofibroblasts, emphasizing elements that mediate BMP2 and Wnt activation.


Obese patients are at increased risk for kidney disease due to circulation of pro-inflammatory mediators and underlying renal metabolic and hemodynamic disturbances, including glomerular hyperfiltration. These patients also have unfavorable lithogenic urinary metabolic profiles, such as low urinary pH, hypocitrituria, hyperuricosuria, that predispose them to kidney stone disease. Gastric operations, in particular Roux-en-Y-gastric bypass (RYGB), are increasing as an interventional strategy to facilitate weight loss in this population. Despite the theoretical renal advantages gained during weight loss for glycemic and hypertension control, up to 75% of RYGB patients will develop sustained hyperoxaluria post-operatively, and a subset will develop kidney stones, nephrocalcinosis, impaired renal function, or even renal failure due to oxalate nephropathy. Currently, the mechanisms behind these adverse renal events are unknown. Preliminary data from an obese rat model of RYGB surgery indicates that gastric bypass causes more glomerular injury, interstitial macrophage migration, increased osteopontin production than sham surgery or pair-fed obese controls. Therefore, we hypothesize that obesity is a low-grade inflammatory process that amplifies renal host responses to RYGB-associated hyperoxaluria. In this setting, glomerular, interstitial, and papillary cells are chronically exposed to high amounts of oxalate and/or calcium oxalate crystals, leading to the production of reactive oxygen species, oxidative stress, renal injury, and inflammation. The objective of this application is to provide a training environment for the principal investigator to examine the role of RYGB-associated hyperoxaluria in the development of renal injury and nephrolithiasis, with special focus on renal histology, transport physiology, tissue proteomics, and genomics. Our specific aims are: 1) To further characterize renal effects of RYGB surgery in a diet-induced obese rodent model versus controls by comparing metabolic profiles, pro-inflammatory mediators, histology, protein expression, and pathway-focused gene expression profiles;2) To further our understanding of RYGB induced hyperoxaluria by investigating segmental differences in intestinal oxalate handling and the effect of Oxalobacter colonization on urinary oxalate levels;3) To compare protein and gene expression profiles from renal papillary tips of human RYGB stone formers with and without Randall's plaque deposition. Understanding the mechanisms of hyperoxaluria following RYGB will provide insights into the pathogenesis of oxalate nephrolithiasis associated with obesity. My K08 program is structured to allow me to further explore relevant rodent models of obesity, to validate preliminary hypothesis generated by a recent minority supplement award, to enhance my understanding of oxalate transport within the gut and kidney, and then, most importantly, translate these efforts into mechanisms of disease within human renal tissue. PUBLIC HEALTH RELEVANCE: Morbidly obese patients who undergo Roux en Y gastric bypass (RYGB) may develop sustained post-operative hyperoxaluria that leads to kidney stones, impaired renal function with calcium oxalate deposition within kidney collecting ducts associated with inflammation and fibrosis, or even renal failure due to oxalate nephropathy. The objective of this application is the establishment of a training environment for the candidate to develop an animal model that explores gut and renal mechanisms of RYGB-associated hyperoxaluria in the development of renal injury and nephrolithiasis, with special focus on renal histology, transport physiology, tissue proteomics, and genomics. As a boarded urologic surgeon with a strong track record of publications in urologic stone disease, the candidate is professionally trained to develop a surgical model of stone disease and renal metabolism, and his K08 program is structured to allow him to further explore relevant rat models of obesity, to validate preliminary hypothesis generated by a recent minority supplement award, to enhance his understanding of oxalate transport within the gut and kidney, and to translate these efforts into mechanisms of disease within human renal tissue.