Human retinal pigment epithelial (RPE) cells accumulate fluorescent lipofuscin granules with age and this phenomenon has been implicated in several age-related degenerative retinopathies. The standing hypothesis of lipofuscin accumulation implicates lipid peroxidation processes in the formation of indigestable fluorescent (460 nm peak emission) residues which are sequestered and concentrated in tertiary lysosomes of the cellular phagolysosomal system. In preliminary studies utilizing human RPE, blue-emitting flurophores were not found within the lipofuscin granules as this hypothesis predicts. Rather, at least two yellow-emitting compounds of unknown identity have been extracted and isolated from purified lipofuscin granules. Yellow-emitting fluorophores are not normally considered indicative of lipid peroxidation byproducts, but their presence is more in keeping with subjective descriptions of yellow fluorescent emissions observed by fluorescence microscopy. I have further isolated at least two age-related blue-emitting fluorophores from cellular sources outside the lipofuscin compartment, and another compount which is present only in aged human RPE and which produces an intensely fluorescent compount upon reaction with ninhydrin. The aims of the present proposal are to utilize thin layer chromatography (TLC) to isolate and purify these compounds from human RPE extracts and to characterize and classify or identify them by means of corrected spectrofluorometry and uv-vis, infrared, nuclear magnetic resonance and mass spectrometry. Standardized high performance TLC and quantitative densitometry will be used to track the age-related appearnce and accumulation of these compounds in relation to one another in RPE from a wide age-range of human donors, and to survey a range of species in search of appropriate animal models. Fluorescence assays will be developed and deployed in further subcellular fractionation studies on the acute effects of the lipofuscinolytic drug, centrophenoxin, on these compounds. These studies will serve to focus the direction of future research and will yield new information essential for the reformulation of the hypothesis of lipofuscin formation and accumulation, and of our understanding of aging phenomena in postmitotic cells in general.


The purpose of this study is to (1) quantify the recovery of labeled CO2 in breath during a primed constant infusion of sodium 13C bicarbonate in human pregnancy, during fasting and in response to feeding and (2) to examine the effect of advancing gestation on these parameters.


Acanthamoeba keratitis is a painful and debilitating infection of the eye caused by an opportunistic amoeba that occurs ubiquitously in nature. From the discovery of this disease in 1973 until 1984, there were only 11 published cases. At present, an estimated 20 new cases are confirmed per month and the majority are associated with contact lens wear. Both awareness and incidence of the disease are thought to be increasing. The success of medical treatment is inconsistent and repeated surgical intervention often is required. There is considerable confusion over identification of clinical isolates. Cell morphology and affinity for specific antisera are the criteria used for identification, but these approaches often give ambiguous results. There is an urgent need for methods to detect small numbers of amebas and to identify the infectious organisms reliably. This project proposes the use of small subunit ribosomal RNA genes (18S rDNA) as a basis for development of genus-, species-, and strain- specific oligonucleotide probes suitable for laboratory and clinical identification of isolates. This one-year pilot project will focus on sequencing of 18S rDNA isolated from amoeba strains associated with human eye infections. Nuclear rDNA will be amplified by the polymerase chain reaction and then sequenced. Sequences will be determined using oligonucleotide primers, primer extension, and dideoxynucleotide chain termination. rDNA sequences then will be analyzed to determine the extent of sequence variability among strains and searched for subsets of sequences that would be suitable targets for probes with different levels of specificity (i.e. genus, species, or strain). Development of clinical probes would occur in subsequent projects if suitable sequence variability is discovered in this study.


The mechanisms mediating kidney damage in diabetes likely stem from chronic hyperglycemia; however, the steps involved are not completely understood. Our main hypothesis is that a high ambient glucose level, independent of genetic and hemodynamic factors, exerts a powerful influence on renal cell growth and extracellular matrix biosynthesis in tissue culture. An exciting finding has been the elucidation that Transforming Growth Factor-Beta (TGF-Beta) is an autocrine mediator of many of the renal effects of high glucose. The specific objective of this proposal are to focus on two major aspects of the effects of high glucose on renal glomerular mesangial cells: the modulation of key proteins which regulate transcription, synthesis and bioactivity of TGF- Beta; and the high glucose-induced perturbations in growth factor- mediated cellular signaling and the role of different protein kinase C isozymes. In vivo correlates will be sought by examining kidney expression and cell localization of TGF-Beta in experimental DN in rodents, and attempt to intercept renal TGF-Beta in vivo using neutralizing anti-TGF-Beta antibodies. The significance of these studies is that they will provide a detailed mechanistic understanding of the harmful effects of high glucose concentration on the kidney, and also pave the way for designing innovative means of prevention of DN, even when glucose control is suboptimal.


In this revised application for a Mentored Patient-Oriented Research Career Development Award (K23), Dr. David Kimhy will pursue training and research in the cognitive and neurobiological mechanisms of stress regulation and psychosis in schizophrenia. The goals of the training and research plan are integrated around the hypothesis that cognitive coping strategies mediate the link between stress and psychotic symptoms during recovery from psychosis in schizophrenia. Under the sponsorship of Dr. Dolores Malaspina, the training plan capitalizes on the resources of NYSPI/Columbia University, combining formal coursework, direct supervision, and clinical research experience and training. The plan involves mentorship and didactic instruction in key areas including: 1) psychosis research in schizophrenia using of Experience Sampling Method (ESM), 2) clinical methodologies for assessment of stress-regulating systems, 3) data analysis using multilevel linear modeling, and 4) cognitive-behavior therapy for psychosis (CBTp). Current methodologies have limited ecological validity to establish causal links between momentary changes in stress and psychosis. Dr. Kimhy will study simultaneously the real-time physiological and psychological correlates of psychosis using ambulatory assessment of cardiac autonomic regulation (Heart Rate Variability; HRV) synchronized with data collected using Experience Sampling Method (ESM) with Palm handheld computers. The research plan involves simultaneous probing of momentary psychotic symptoms, subjective stress, HRV, and cognitive coping strategies. Assessments will be conducted before and after pharmacological and CBTp treatments of psychosis. This methodology translates basic science into use in real-time, real-world environments (in-vivo and in-situ), allowing for the assessment of psychosis as part of the ebb-and-flow of daily functioning. Understanding these interactions will advance our understanding of the mechanisms of recovery from psychosis in schizophrenia. The proposed training and research plan will provide Dr. Kimhy with the knowledge, training, and experience necessary to develop as an independent researcher focusing on the neurobiological and psychological mechanisms of stress regulation and psychosis in schizophrenia. [unreadable] [unreadable] [unreadable]


We propose to build a multidisciplinary Zebrafish Cardiac Development Research Center with a collaborative group of zebrafish developmental biologists, zebrafish cardiac physiologists, and experts in chromatin structure, genome-wide gene network profiling, bioengineering and bioinformatics. We will investigate gene regulatory networks at distinct steps in development that control normal cardiac development at multiple molecular levels. Several diverse perturbations of cardiac development will serve as "inputs" to assess changes in cardiac development gene regulatory networks, including genetic mutants and morphants, embryological cell lineage manipulations and pharmacological treatments. The "outputs" will be analyses of chromatin structure that regulates transcriptional programs, epigenetic modifications including DNA methylation and chromatin marks that contribute to transcriptional activation (H3K4me3) or transcriptional repression (H3K9me3, and H3K27me3), genome-wide gene expression profiles (including mRNAs, microRNAs, SINE RNAs, other small RNAs) and transcriptional regulators involved in the patterning, morphogenesis and physiology of cardiomyocytes during development. Bioinformatics comparisons of normal and a large number of aberrant profiles within zebrafish will uncover molecular signatures of cardiac developmental defects. This multi-layered molecular profiling of cardiac development has not been performed in any organism. As new candidate genes or other perturbations (including pharmacological or environmental) arise within the Cardiac Development Consortium and Pediatric Cardiac Genomics Consortium, they will be incorporated into this multi-layered molecular profiling program to rapidly obtain genome-wide molecular signatures. Our infrastructure will make these datasets readily accessible to the consortia for trans-species comparisons to uncover conserved molecular signatures relevant to human developmental heart defects. RELEVANCE (See instructions): Obtaining multi-layered molecular profiles of cardiac developmental defects will reveal the underlying causes of cardiac developmental defects, with the long term goal of applying these insights to treatment of children with heart defects.


Patients with systemic lupus erythematosus have been found to have disturbances of cell-mediated immune responses. Alterations of B and T lymphocytes as well as monocyte function, natural killer cell and interferon responses, have been observed both in patients with SLE and certain of their relatives. The major goal of these studies is to further characterize the types of immunological distrubances in SLE and to elucidate the mechanisms which lead to alterations of immune regulation and are responsible for the pathogenesis of this disease. The ability of immunosuppressive drugs to alter cell mediated immune function and possibly to re-establish more nearly normal immunoregulation is being actively studied.


TRANSGENIC AND CHIMERIC MOUSE CORE: Director-N. Cooke The Transgenic and Chimeric Mouse Core Facility (TCMF) of the University of Pennsylvania has been in operation since February 1992 under the continuous co-direction of Dr. Nancy Cooke and Dr. Stephen Liebhaber and under the day-to-day technical direction of Dr. Jean Richa. The initial components of the TCMF were assembled from financial contributions of the School of Medicine and resources from the laboratories of Drs. Cooke and Liebhaber in 1989 with subsequent phased expansions in facilities, capabilities, personnel, and services. Over the past five years the Core has generated a wide range of transgenic and gene-targeted mouse lines that have proven to be powerful tools for the study of the molecular biology and physiology of Type I and Type II diabetes, obesity, and developmental and mechanistic bases for a wide variety of other disorders of endocrinology and metabolism. The Core laboratory is located in a pathogen-free microbiologic barrier facility in the basement of the Clinical Research Building. A primary function of the TCMF is to provide a centralized laboratory that will generate infectionfree strains of mice carrying transgenes or targeted genetic alterations of specific interest to individual projects in the DERC. The centralization of these technically demanding procedures in the Core laboratory results in enhanced efficiency and significant cost reduction for each project. During the current project period the TCMF has grown significantly in service offerings and its facilities have been improved and expanded. A major increase in the throughput potential of the Core has been achieved by the doubling in the size of the Core microinjection laboratory, funded by the School of Medicine. This expansion has doubled the capacity in line with the continuing increase in demand for services. In addition, the Core is now finalizing a new Cryopreservation Facility for the long-term storage of embryos and sperm. This addition to the Core will be opening in the summer of 2006. This newly configured, secure facility is off-site from the main Core laboratory, and has been established by funds from the School of Medicine. Additional services that have been established during the current funding period include sperm cyropreservation, intracytoplasmic sperm injection (ICSI), DNA diagnostics, and integration with the School of Medicine gene knock-out service. These services have substantially increased the utility of the Core to Center members. The Core will continue to maintain the highest level of quality service and will continue to acquire new techniques and expertise to maintain services at a 'state-of-the-art' level.


The overall goal of our research is to understand the molecular and cellular events that lead to cancer. Our lab in particular studies the actions of a family of proteins called STATs, which are transcription factors. Transcription factors are proteins that bind to DNA and regulate the expression of genes, which then go on to determine the phenotype and behavior of the cell. Originally, all genes were thought to code for proteins, but it is now clear that certain genes are not made into proteins. This type of gene, called a microRNA because of its relatively short length, was only recognized fairly recently. We now know that microRNAs act by inhibiting the conversion of protein-coding genes into proteins. Since their discovery, microRNAs have been found to play a part in all aspects of cancer development and progression. At present, very little is known about microRNA genes regulated by STATs and their relevance to cancer. Recently, we have identified a novel microRNA whose expression is controlled by STATs. Our proposed research will investigate the functional significance of STAT regulation of this microRNA as it relates to cancer. In our first set of experiments, we will initially characterize where STATs bind to the DNA relative to the microRNA. This will reveal how the microRNA is molecularly regulated. Next, we will assess if regulation of this microRNA is intact in cancer cells. Since the microRNA may have anti-cancer effects, we believe that STAT regulation of the microRNA goes awry in cancer. Lastly, we will analyze the causes for dysregulation of the microRNA, which could be genetic (a change in the DNA sequence) or epigenetic (other properties of the DNA change, making it harder to read). In our second set of experiments, we hypothesize that the microRNA helps to sensitize cancer cells to a stimulus, called TRAIL, that is capable of selectively killing cancer cells. First, we wll enhance or inhibit the activity of the microRNA and see if that increases or decreases sensitivity to TRAIL, respectively. Next, we will test if the microRNA acts by inhibiting a specific survival pathway. We believe that enforcing the activity of this pathway will rescue cells with enhanced activity of the microRNA. Lastly, we will test the involvement of specific genes in the actions of the microRNA.


Objectives: Providers and patients face barriers to accomplishing the complete communication needed to make complex decisions together about diabetes treatments and self-management tasks. Providers often have limited time and patients often have limited resources. Peer mentoring models have been found in two recent VA research trials to be more effective than usual care, financial incentives, and nurse care management in improving glycemic control in high-risk Veteran patients with diabetes. While peers can be trained in effective approaches to support other Veterans' self-management behaviors, such coaches necessarily lack diabetes and diabetes medications content expertise to help Veterans better share in treatment decisions and goal-setting with their health care providers. Accordingly, in a recent research study we developed and tested a tailored, interactive diabetes and diabetes medication information tool that outreach workers can use to facilitate discussions with patients. Such tools can enhance the sustainability and effectiveness of coaching programs to better prepare patients to set self- management goals, action plans, and to discuss treatment options with their providers. Because this intervention addresses barriers to disease management for chronically-ill patients, physicians, and case managers, the study may have broader impact on management practices for other chronic illnesses. Project Methods: 348 diabetes patients with poor glycemic control (A1c>8.0% or A1c>8.5% if older than 70) will be recruited from the Detroit VA and randomized to either Peer Support with TEC or Peer Support alone. All participants will be assigned to one of 87 peer coaches, who also are Detroit VA diabetes patients who previously had poor glycemic control but are currently in good control. Each peer coach will be assigned about 4 participants on the basis of race and approximate age. Peer coaches will undergo training in motivational interviewing-based counseling approaches and instruction in use of the iPad-platform decision aid tool. After their baseline assessment, participants in both arms will receive information on their lab and blood pressure values and will be randomized to one of the two study arms. Participants randomized to the control group will be scheduled for an initial visit wit their coach. The coach will then help them list questions and concerns they wish to discuss with their health care provider, practice raising their questions and concerns and develop an action plan to address barriers to self-management they have identified. Participants in the TEC arm will be scheduled for an initial visit with their coach to review the decision aid, which has incorporated their personal baseline data. The coach will then provide the same intervention that the Control group receives. During the next six months the coach will call their assigned peers once a week to provide support for their action steps. These calls will be placed using a confidential IVR system that connects the callers without sharing phone numbers. The research will measure changes in HbA1c, BP, patient-centered outcomes, mediators and moderators of intervention effects and cost-effectiveness.


Both DNA replication and centrosome duplication are semi-conservative replication processes that must be coordinated to occur once-and-only-once per cell cycle. Recent findings have suggested that both cyclin dependent kinases and the SCF and APC ubiquitin ligases conspire to regulate both DNA replication and centrosome duplication. Biochemical studies show that the conserved Cdc14 family of phosphatases are selective for dephosphorylating substrates of cyclin-dependent kinases. We have recently discovered that a form of the human Cdc14 phosphatase also has a critical role at the centrosome. Our preliminary studies show that the two known forms of human Cdcl4, called Cdcl4A and Cdcl4B, localize to the centrosome and nucleolus, respectively, We have mapped targeting sequences directing each form to these sites and find that there are sequences that target Cdcl4A and B to their respective sites, but that both proteins partition between centrosomes and nucleoli and that this partition is affected by nuclear import and export signals. Importantly, perturbing the normal function of Cdcl4A by overexpression strongly disrupts centrosome duplication and causes mitotic catastrophe (including chromosome missegregation, a failure to reform nuclei following mitosis, and a block to cytokinesis). Thus, Cdcl4A may play a critical role in mitotic progression and genome stability. Supporting this idea, we find that in Xenopus eggs, the Xenopus Cdcl4 protein is important for mitotic exit. These studies will further test the role of Cdc 14 in mitosis and genome stability. Our specific alms are: (1) to test the role of Cdc 14 and cycin-dependent kinases in specific biochemical steps in the centrosome duplication cycle; (2) to map the centrosomal targeting sequences of Cdcl4A and to use these mutants to determine whether Cdcl4 has additional roles in centrosome maturation or maintenance and later steps in mitosis; (3) to study the specific biochemical requirements for Cdc 14 in mitotic exit using an in vitro system for mitotic exit in Xenopus egg extracts; and (4) to purify and identify Cdcl4A and B interacting proteins using an affinity purification procedure and mass spectrometry to sequence the associated proteins. By purifying proteins associated with both wild type and catalytically inactive ("substrate-trapping") mutants of Cdcl4, we hope to identify both Cdcl4 regulators and substrates. These aims will both map Cdc 14-regulated pathways and begin to define the critical Cdk and Cdcl4 targets for regulating steps in mitosis and genome stability.


Abstract: Emerging and established viral diseases take an enormous toll on human health. Current treatment approaches are unlikely to halt epidemic spread of many viruses, notably HIV-1, due to prohibitive costs of treatment (i.e. access), compliance issues, rapid viral mutation, and the influence of hard-to-reach high-risk viral 'superspreaders'. We propose to shift the treatment paradigm toward developing Therapeutic Infectious Pseudoviruses (TIPs) that require the pathogen to replicate. TIPs would transmit along a pathogen's normal transmission route, reaching precisely those high-risk populations that most require therapy. TIPs co-opt wild-type virus packaging elements, decreasing disease-progression in vivo and reducing disease transmission on a population scale. We have demonstrated that an anti-HIV TIP could mutate with equal speed and under evolutionary selection to maintain its parasitic relationship with wild-type virus, thereby overcoming viral mutational escape. Since TIPs replicate conditionally (i.e. piggyback) treatment compliance and cost issues are eliminated. A precedent for the safety of TIPs exists in the oral polio vaccine (a live-attenuated vaccine) which exhibits limited spread and is being used in the polio eradication campaign. To develop candidate TIPs we will capitalize upon our expertise in HIV-1 transcriptional circuitry. We discovered that HIV-1 exploits stochastic gene-expression to control entry into a dormant state (proviral latency). By targeting a cellular gene (SirT1) essential for viral feedback, we have biased HIV-1 toward dormancy and diminished reactivation. We will exploit this innovative strategy of forcing viruses into dormancy by utilizing our single-cell imaging methods to conduct high-throughput imaging screens for therapeutic candidates that promote viral latency. Next, these candidate TIPs will be analyzed in novel microfluidic chemostats that maintain homeostatic infection and allow viral evolution in an in vivo-like setting. By integrating these approaches with predictive models, we will develop a revolutionary therapy to halt the spread of HIV/AIDS and other infectious diseases. Public Health Relevance: Emerging and established viral diseases are major health concerns. Many viral diseases lack effective treatments or preventative vaccines and even when available these treatments are unable to halt epidemic spread due to viral mutational escape and the presence of infectious superspreader individuals. Clearly, new and more effective antiviral strategies are needed and this proposal presents a multi-pronged approach to identify and develop an innovative new antiviral approach.


Project Summary: Core A The Administrative Core provides for essential administration, communication, and compliance responsibilities of the Exploratory Grant. It acts to promote the integration, support the Program Director and Program Administrator, organizes meetings of the Program faculty and staff, and facilitate participation in national external activities. The Administrative Core is directed by the Program Director, Dr. Standaert. He is assisted by the Center Administrator, Ms. Katherine Belue. The Core will be responsible for the coordination of the Program. Although the investigators do have established collaborative interactions, achieving the integration needed for a successful P50 Udall application will require a higher level of integration and interaction among the PI?s, their laboratory trainees and staff, and the broader community of neuroscientists and immunologists who may participate in a P50 application. The approach to coordination will include electronic communication (video conferencing and a web-based content management system for document sharing, messaging, and calendaring) as well as in person meetings. These will include biweekly meetings of the project investigators and their team. The Core will support an annual ?mini-retreat? to assist in expanding collaborations beyond the initial Exploratory Grant investigators and achieving the scope required for a successful P50. The Core will oversee sharing, regulatory compliance, and financial management. The Core will establish a web site, to communicate the activities of the Exploratory Program and maintain ties with our community partners. Training is a key component of our plans for a future Udall Center plan. All three project PI?s have current pre- and post-doctoral fellows, and close relationships with important training. We will use the support from the Exploratory Grant to strengthen these connections and promote the participation of trainees and faculty at all levels in the activities of the Exploratory Grant.


Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe persistent skin infection which has been recently designated an emerging infection in West Africa. The disease has a unique pathology. Despite extensive tissue damage and the presence of a heavy bacterial load, there is little acute inflammatory response to the organism. A single Buruli ulcer may cover 15 percent of a person's body surface; the only cure is surgery and skin grafting. A polyketide-derived macrolide toxin designated mycolactone has been identified in M ulcerans. Macrolides are produced as secondary metabolites by soil bacteria and fungi. They have enormous pharmaceutical value as cytostatins, immunosuppressants, antifungal agents, antihelminthic agents and antibiotics. Mycolactone is the first macrolide identified from a pathogen. Evidence suggests that mycolactone is responsible for most of the pathology in Buruli ulcer. Mycolactone-mediated phenotypes include cell cycle arrest, immunosuppression and death via apoptosis. Neither the genetics of mycolactone synthesis nor its mechanism of action are known. The goals of this proposal are: 1) to clone and sequence genes for mycolactone biosynthesis, 2) to construct mutants defective in mycolactone production, and 3) to begin characterizing events in the cellular pathways involved in mycolactone mediated cell death and immunosuppression using micro-array gene-expression technology. Results from these studies should have a significant impact on the treatment and prevention of Buruli ulcer as well as provide insight into potential role of polyketides in other mycobacterial diseases such as tuberculosis. In addition, this work may provide useful insight into macrolide cell biology.


The goals of the University of Iowa as members of the Cancer and Leukemia Group B over the next five years are: 1) to sustain our pattern of increasing accrual to CALGB trials; 2) to continue to develop programs in both clinical and basic science areas at the University of Iowa which will lead to innovative clinical trials for the group; and 3) to add further affiliates in rural areas in Iowa so that state-of-the-art cancer care can be brought to these communities too small to support cancer programs. Over the last four years of the grant, our accrual went from 98/year to 148/year. Our stated goal in the last grant was to accrue approximately 100+ patients per year. In the first three months of 1992 we have accrued 52 patients (a rate of 208/yr). We hope to average 175 pts/year in this grant period. Current or soon to be open CALGB trials initiated from the University of Iowa include: 1) subcutaneous IL-2 in advanced small cell lung cancer with assay at the University of Iowa of sIL-2 receptor and LAK cell generation; and 2) the use of stem cells to allow multiple cycles of intensive chemotherapy without autologous bone marrow transplant. Basic science programs with potential for future CALGB trials are: 1) the development of bispecific monoclonal antibodies to lymphoma cell antigens; 2) studies in vitro of HMG-CoA reductase inhibitors to block the farnesylation and geranylation necessary for RAS expression; and 3) the use of PCNA as an intermediate marker for the development of neoplasia in the lung. Medical oncologists from the University of Iowa are now serving Mount Pleasant, Iowa and Muscatine, Iowa. Affiliates are going in Mason City, Davenport, and Waterloo. We are exploring further outreach programs in towns too small to ever support a fulltime oncologist and affiliates in larger towns to bring CALGB programs to underserved rural areas of the state. This will allow accrual of minority populations in towns such as Muscatine and the poor in rural areas served by these programs. As the physicians carrying out the care will be University of Iowa faculty, we will be able to provide quality care and data in these areas.


Two cortical pathways originate from primary core areas of auditory cortex: a ventral pathway subserving identification of sounds, and a dorsal pathway, which was originally defined - similar to the visual system - as a processing stream for space and motion. We have recently proposed that this dorsal pathway should be redefined in a wider sense as a processing stream for sensorimotor integration and control (Rauschecker, 2011). This broader function explicitly includes spatial processing but also extends to the processing of temporal sequences, including spoken speech and musical melodies in humans. In this project, we will test the expanded model of the auditory dorsal stream by training rhesus monkeys to produce fixed sound sequences on a newly designed behavioral apparatus (monkey piano). By pressing a lever the monkey will produce a musical tone of a specific pitch; by pressing several levers in succession, the monkey will produce a melody. After a monkey has learned to reliably play the same melody, we will perform functional magnetic resonance imaging (fMRI) of auditory-responsive brain regions in the awake monkey while it listens to the learned self-generated sequence. Control stimuli include melodies the monkey has been passively exposed to by listening to another monkey play for the same amount of time, and novel melodies that the monkey never heard before. Preliminary data suggest that areas activated by the self-generated melody include a region in inferior parietal cortex as well as one focus each in dorsal and ventral premotor cortex. The locations of activated regions will guide subsequent electrophysiological recording with linear microelectrode arrays (LMAs). Each recording site will be tested with the same sequences. Next we will record neuronal responses in premotor cortex to passive listening of the sound sequences and compare them to neuronal activity obtained when the monkey actively produces the sequence with and without sound. Finally, we will add video of a monkey playing the sound sequence on the monkey piano and study multisensory interactions along the dorsal stream using fMRI and LMAs. In particular, responses in caudal auditory belt and parabelt will be compared with those in inferior parietal and premotor cortex in simultaneous recordings. Our studies, using alert monkeys trained in a behavioral task, will contribute to the understanding of unified principles of perception and cognition across sensory systems and their interactions with the motor system. Investigating the auditory dorsal stream in a nonhuman primate will provide valuable information about the evolution of speech and music in humans. Our studies are highly relevant for higher-order processing disorders of audition and speech, such as dysarthria, apraxia of speech, aphasia and specific language disorders which involve inadequate coordination between sensory and motor systems. The results will also improve our understanding of disorders of sensory-motor integration, such as ataxia, which may be caused by stroke or neurodegenerative disease, thus, leading to better therapies and rehabilitation strategies.


Project Summary/abstract Herpes simplex virus (HSV) persists for life in peripheral neurons in the form of a latent infection. In response to neuronal stress, the virus reactivates from latency to permit reinfection. Reactivation is associated with significant disease. For example, replication in the cornea following reactivation results in keratitis. Transmission to the central nervous system following reactivation can result in herpes simplex encephalitis (HSE). Without treatment, HSE has a fatality rate of 70%, and even with treatment, many survivors exhibit long-term sequelae. Although anti-viral drugs are available that limit HSV productive replication, no therapies target the latent stage of infection to prevent reactivation and there is no vaccine against HSV. Therefore, our long-term goals are to understand how HSV responds to neuronal stress and develop strategies to prevent reactivation occurring. Our lab and others have shown that the mechanism by which viral gene expression initiates during reactivation is distinct from de novo infection with the virus. We have found that a neuronal stress pathway resulting in activation of c-Jun N-terminal kinase (JNK) triggers changes to the viral chromatin and permits reactivation. Specifically, the histones associated with viral promoters maintained a modification associated with heterochromatin (H3K9me3) but also became phosphorylated on H3S10 in a JNK-dependent manner. Using both a primary neuronal model of HSV-1 latency that we have developed and mouse models of infection we will determine how activation of JNK permits viral gene expression to be induced during reactivation. By performing ChIP-seq and shRNA knock-down of candidate proteins, we will examine how JNK gets recruited to viral promoters and identify additional cellular proteins involved in HSV-1 reactivation. We will also determine how JNK signaling overcomes the H3K27me3 repressive histone medication to permit reactivation from genomes associated with this modification. Finally, we will examine the mechanism that ATRX restricts HSV-1 reactivation and test the hypothesis that genomes associated with ATRX are non- permissive for reactivation. These studies into the intimate interaction between the latent viral genome and initiation of a neuronal stress response are especially significant as they provide mechanistic insight into how the virus undergoes reactivation. Because the virus has likely co-opted cellular pathway to achieve reactivation, we will also uncover process that are important for the host response to neuronal stress. Importantly, by understanding the very earliest events in HSV reactivation, our long-term goals are to develop therapies that target the latent genome and make it unresponsive for reactivation.


Engagement of the multicomponent antigen receptor in T cells (TCR) results in rapid activation of a protein tyrosine kinase pathway. A major TCR- associated protein tyrosine kinase is ZAP-70, a protein that binds to the activated TCR. Under conditions of TCR activation, the ZAP-70 bound to the TCR is itself tyrosine phosphorylated and activated. We have expressed this enzyme using a baculovirus system and have developed a purification scheme enabling us to obtain sufficient ZAP-70 for enzymatic analysis. Our studies of the mechanism of ZAP-70 activation includes further analysis of mutants of the enzyme that lack critical tyrosine residues. Expression of these mutants in T cells that lack endogenous ZAP-70 leads to the conclusion that phosphorylation of this enzyme is critical to its regulation. Additional studies focus on a substrate of tyrosine kinases in T cells, p120cbl, a proto-oncogene, which may be involved in the Ras pathway in T cells. Earlier studies demonstrate that this protein can be found in a complex with the linker protein Grb2. We have mapped the interaction and have demonstrated that the Grb2-p120cbl complex is regulated by TCR activation. Engagement of the Fc receptor for IgE on mast cells also results in p120cbl tyrosine phosphorylation. In these cells we have demonstrated that the Syk protein tyrosine kinase is primarily responsible for this phosphorylation. Moreover, Syk and p120cbl can be found in a complex whose function is under investigation.


Most oral leukoplakic lesions represent hyperkeratotic epithelium (benign), dysplasia, or squamous cell carcinoma. The lesions must be biopsied and examined microscopically for diagnosis. But, morphology alone does not answer the question about which non-invasive lesions will progress to cancer. The uncertainty of whether a given lesion will progress to cancer or not often compromises the use of the best therapeutic option which is complete excision because of the complications of surgery. So, the question to be answered by the proposed research is, "Will a given leukoplakic oral lesion likely progress to cancer?" The research will begin to answer the question by examining the genomic sequences of pp32r1 (GenBank AF008216) present within existing biopsy specimens. In a pilot sequencing survey of the pp32r1 gene in tobacco-associated, oral, leukoplakic lesions, the principal investigator found a mutant, growth-accelerating pp32r1 gene in an oral squamous cell carcinoma. The first specific aim of the project is to amplify and sequence the pp32r1 gene within formalin-fixed, paraffin embedded specimens of oral leukoplakia. The basic hypothesis is that certain neoplastic cells within those lesions contain mutations encoding non-conservative amino acid substitutions within residues 136-172. These mutations define malignant cells long before they clonally enlarge and form a clinical cancer. Another question concerns the discrete molecular activities of mutant, carcinoma-associated PP32R1 proteins as they accelerate cellular growth. The pp32r1 gene is a member of a family of genes that encode proteins with myriad activities. All the family members contain N-terminal domains with leucine-rich repeats that form adapter sites for protein-protein interactions. And, all the family members contain extremely acidic C-terminals that form alternative adapter sites for protein-protein interactions by ionic forces. Another common physical characteristic is that other family members, particularly PP32, often appear in nature bound to other intracellular proteins. It is thought that mutant, carcinoma-associated, growth-accelerating PP32R1s differentially bind intracellular proteins in comparison to wild-type PP32R1s and other members of the PP32 family. The resultant abnormal macromolecular complexes change cellular homeostasis in ways that accelerate growth. The second specific aim of the project is co-immunoprecipitation of PP32R1 binding proteins, separation of the binding partners by 2-dimensional (2D) gel electrophoresis, and identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Identification of the intracellular protein ligands will provide a specific functional context for mutant PP32R1S.


: Structural biology and, in recent years, structural proteomics have yielded tremendous insight into protein mechanism and function. However, a high percentage of proteins remain off-limits to high-throughput structure determination methods. For example, solving structures of membrane-bound proteins is a difficult and idiosyncratic art. Furthermore, proteins susceptible to aggregation are simply not amenable to current methods for structure determination. The first long-term goal of the research proposed here is the development of a high-throughput method for converting insoluble proteins, both membrane-bound and oligomerization-prone, to soluble proteins upon which the powerful tools of structural biology can be brought to bear. An equally important second long-term goal is to elucidate and understand the characteristics of protein structure leading to aggregate and membrane bound states. Determining the structures of previously unattainable targets will expedite the development of therapeutics for a host of diseases, such as disorders resulting from amyloid fibril formation, a specific type of protein aggregation. Specifically, the experiments proposed here focus on the caveolin-1, a key regulator of signal transduction. Caveolin-1 binds to a large number of different cellular proteins, and can inhibit key enzymes, including protein kinase A (PKA) and endothelial nitric oxide synthase (eNOS). Such activities allow selections for functional, yet more soluble, caveolin-1 variants. As both an aggregation-prone and membrane-associated protein, caveolin-1 provides an ideal system for the planned experiments. In essence, one series of experiments will uncover molecular determinants for both aggregation and membrane binding. In the first specific aim, the determinants of solubility, aggregation, and membrane-binding will be investigated during experiments aimed at engineering soluble variants of caveolin. The structure of the soluble variant will be determined by solution phase NMR in the second specific aim. This structure will be compared to a structure determined by solid-state NMR of an aggregated variant of caveolin. Structural insight will be then guide mutagenesis experiments aimed at testing the mechanistic basis for protein aggregation and membrane-binding. [unreadable] [unreadable] [unreadable]


Abstract. According to the CDC, at least 2 million people per year in the U.S. develop illness arising from antibiotic-resistant bacteria, and there are at least 23,000 resultant deaths annually. Certain patient populations, such as surgery and trauma patients, are especially susceptible to infections with opportunistic microorganisms and receive aggressive antibiotic therapy. The development of new antibacterial therapies - to be used either in combination with current antibiotics or as an alternative to antibiotics - would reduce complications associated with a wide range of diseases and infections. This project will explore a novel approach to antibacterial therapy through inhibition of hydrogen sulfide (H2S) production. Over the last decade, H2S emerged as an endogenous gasotransmitter produced by mammalian cells. Recent work shows that H2S is also produced by bacteria. Importantly, bacterial H2S has been identified as a novel mechanism that protects the bacteria against antibiotic induced, oxidant-mediated killing in vitro. Whether or not bacteria produce H2S as a defense against the immune system is not known. Our preliminary data show that inhibition of either host or bacterial H2S improves bacterial clearance in vivo. Therefore, H2S-producing enzymes may be novel targets for antibacterial therapy. Since H2S also has multiple physiological roles, it is critical to understand the regulation and relevance of specific host and bacterial H2S-synthesizing enzymes during host- pathogen interactions. This project will address these questions through the following Specific Aims: Aim 1. To identify the contributions of specific H2S-synthesizing enzymes to the production of H2S that mediates bacterial and host responses during infection. Tissue H2S levels, expression and activity of the various H2S-synthesizing enzymes will be measured before and during infection. Mice deficient in H2S-synthesizing enzymes will be compared to wild type mice. Infections with wild type versus H2S-deficient strains of bacteria will elucidate the relative contributions of bacterial- versus host-derived H2S. Patterns and potential enzymatic regulators of constitutive and infection-associated H2S levels will be identified. Aim 2. To determine the roles of H2S in host susceptibility to infection and bacterial resistance to antibiotic therapy. The functional relevance of specific enzymes to infection responses will be determined in two models of infection: S. aureus pneumonia and E. coli abdominal sepsis. H2S-deficient knockout mice will be infected and survival, bacterial clearance, local and systemic inflammation, organ dysfunction, and immune cell activation will be measured and compared to that in WT mice. The same outcomes will be measured after infection with H2S-deficient strains of bacteria. Resistance of H2S-deficient and wild type bacteria to oxidative killing by the host will be examined. Finally, the efficacy of antibiotic treatment in combination with host or bacterial H2S-deficiency will be evaluated. This project will identify specific sources of bacterial or host-derived H2S to be pharmacologically targeted in the future as a fundamentally novel approach to treat bacterial infection.


Many clinical and pathological studies have suggested that the hypertrophied left ventricle may have an inadequate coronary circulation in the absence of disease of the epicardial coronary vessels. Thus, this question has obvious clinical implications for patients with many forms of heart disease. The primary objective of this proposal was to study the effects of left ventricular hypertrophy induced by a pressure load (Goldblatt hypertension) on the coronary circulation. The four major investigations proposed were: 1) study maximal coronary vasodilation; 2) response of the coronary circulation to treadmill exercise; 3) transmural lactate levels in the left ventricle; and 4) reactive hyperemia response in the coronary circulation. In all studies, dogs with pressure induced left ventricular hypertrophy were to be compared with controls. The effects of left ventricular hypertrophy and maximal coronary vasodilitation have been evaluated. Our general conclusion has been that the maximal dilator capacity of the coronary circulation is decreased by about 40-50% in the hypertrophied left ventricle. This is not the result of an increase in systolic compressive forces secondary to systemic hypertension since a similar abnormality was found in dogs with left ventricular hypertrophy who had been relieved of their hypertension. This suggests that left ventricular hypertrophy is associated with an architectural abnormality in the coronary circulation.


Four large multi-generational Tourette's Syndrome families are being studied. Identification of a biological marker or demonstration of a genetic linkage to a marker locus is being pursued. Data is being collected for documentation of both genetic and environmental factors important for the expression of TS and OCD.


The overall objectives for the proposed MBRS program at North Carolina Central University are: 1)to substantially improve and strengthen the research capabilities and competitiveness of science faculty; and, 2) to increase the number of underrepresented minorities seeking careers in biomedical research. To fulfill the first objective, NCCU's MBRS program seeks to build upon existing and emerging research strengths of the University. In this renewal, five subprojects are submitted from the Department of Biology, and one subproject is submitted from the Department of Chemistry. The request includes support for 6 Principal Investigators, 17 students, 8 research technicians, and 1 secretary. Among these projects are common threads of health related research problems which will foster collaborations and sharing of biomedical research ideas and technologies. The cover areas of cancer biology, molecular biology, microbiology, mycology, environmental health and bio organic chemistry. Four of the six subprojects are new MBRS projects and two are competitive continuations of existing MBRS projects. This program continues to be enriched by interdepartmental and interinstitutional collaborations that foster research productivity and assist in alleviating isolation often felt by researchers at minority and/or non research-intensive institutions. Improved productivity will be made evident y measurable increases in faculty/student publications, presentations at seminars and meetings, in patents, faculty research honors, and the acquisition of additional non-MBRS funding to support research and research training activities. This program will be enriched by the proposed MBRS seminar series. The second broad objective involves enhancing the student participation program. By insuring appropriate mentoring and guidance, students should improve their productivity, visibility, and accountability. Students will be involved in state-of-the-are research projects, in presenting their data in seminars and at meetings, in co-authoring publications, and in establishing linkages with students and faculty involved in other research programs at NCCU and at collaborating institutions and expected outcome is that this program will continue to motivate more minority students to pursue terminal degrees and careers in biomedical research.


Project Summary/Abstract During meiotic prophase, homology recognition between partner chromosomes is coordinated with the assembly of a proteinaceous scaffold, the synaptonemal complex (SC), along the length of the chromosome pair (synapsis). Interhomolog recombination progresses within the context of assembled SC, and many interhomolog recombination events rely on SC for their proper distribution and/or completion. Our lab investigates SC structure and how SC assembly is coordinated with meiotic prophase chromosomal events. Recently we demonstrated that In S. cerevisiae, at least some components of the SC remain dynamic during meiotic prophase, even after chromosomes are fully synapsed. Moreover we found that recombination sites maintain a different SC dynamic as compared to other sites on the SC. Furthermore, our preliminary studies show that SUMO, which localizes to SC structures, has a mutually dependent relationship with the coiled-coil Zip1 protein to assemble SC on meiotic chromosomes. With pioneering imaging studies using super-resolution light microscopy, we have also shown that SUMO localizes to Zip1 N termini, at the center of SC structure. Finally, the lab has isolated zip1 alleles that assemble on chromosomes independent of canonical regulators. Each zip1 allele represents a single amino acid substitution at the extreme C terminus of Zip1 and may reveal aspects of the mechanism underlying Zip1 assembly. The purpose of this grant proposal is to 1) use our established genetic and microscopy tools to define the molecular architecture and dynamics of all known synapsis proteins within the SC, and 2) to isolate point mutant alleles of synapsis initiation proteins and Zip1 in order to identif molecular features of each protein responsible for their function in synapsis and other meiotic prophase chromosomal events.


The Structural Basis of APOBEC Functions The APOBEC (Apolioprotein B mRNA-editing Enzyme Catalytic polypeptide) family of cytidine deaminases is a family of enzymes that deaminate cytidine residues of DNA/RNA. APOBEC proteins possess significant cellular functions and anti-viral activity, which partially accounts for the intense attention to this the field of research in recent years. APOBEC proteins are found only in vertebrates and APOBEC3 (Apo3) proteins are found only in primates. By binding and deaminating DNA/RNA, APOBEC enzymes achieve remarkably diverse cellular functions. For example, APOBEC1 (Apo1) edits the mRNA of a protein involved in lipid metabolism; AID plays a key role in somatic hypermutation for antibody maturation; APOBEC2 (Apo2) may play a regulatory role for heart muscle development; and Apo3 proteins, especially Apo3G, can restrict important viral pathogens, including Human Immunodeficiency Virus (HIV) and Hepatitis B Virus (HBV), and retro-element mobility. As a result, a novel approach to HIV therapy focuses on utilizing the potent anti-viral activity of Apo3G and Apo3F. Our long-term goals are to understand the structural/functional relationship for APOBEC cellular function and their anti-viral activity. Our specific aims are to extend our prior success in the structural characterization of APOBEC proteins to the studies of the structural basis of APOBEC's functional mechanisms, including their antiviral activity, with particular focuses on Apo3G and Apo3F. The research will provide valuable information for understanding the molecular details of the APOBEC enzyme family and for the potential drug development to provide therapy for HIV, immune diseases and other diseases related to APOBEC function or malfunction. PUBLIC HEALTH RELEVANCE: Understanding The Structural Basis of APOBEC Functions The Apolioprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC) family of cytidine deaminases are found exclusively in verterbrates. APOBEC nucleic acid deaminases modify genes by deaminating cytosines in mRNA coding sequences and in ssDNA. Their critical biological roles include lipid metabolism, humoral immune response, and potential regulations of developmental process of certain human organs and reproductive system. Additionally, these enzymes can inhibit the replication of retroviruses, such as the human immunodeficiency virus (HIV) and hepatitis B virus (HBV), and retrotransposons. The important beneficial mutational ability of APOBEC proteins can become detrimental to the stability of genome if their activity is not tightly regulated. The understanding of the structural basis of the molecular mechanisms of APOBEC function, which is still poorly understood, bears scientific significance and direct health relevance. We propose to study the structure/function of this important APOBEC deaminase family, with focuses on APOBEC3G and 3F (Apo3G and 3F) proteins and their interactions with cellular and viral ligands, using mainly structural biology, assisted by biophysics, molecular biology, and functional biochemistry.


Develop an in vitro test for sarcoidosis.


ABSTRACT Pancreatic cancer (PC) is one of the deadliest cancers with a 5-year survival rate of 7%. Notable racial/ethnic differences in PC incidence have been observed, with U.S. blacks having 30% higher rates compared to whites. The rates of PC in Native Hawaiians and Japanese Americans have been rising in past two decades and have surpassed those of whites, but no study has been conducted to identify the cause(s) of this rising incidence. In the large prospective Multiethnic Cohort Study (MEC; N>200,000) we observed highly significant differences in PC incidence across racial/ethnic populations, with African Americans having 39% higher rates compared to whites. Native Hawaiians are observed to have the highest rates in the cohort that are 74% higher than whites while rates in Japanese Americans are 32% higher and rates in Latinos are similar to whites. We expect that inter-ethnic differences in risk factor prevalence are likely to explain the observed ethnic differences in PC incidence. However, to date, few studies have included African Americans and no studies have included Japanese Americans, Native Hawaiians, or Latinos; thus, the factors underlying PC disparities remain undefined. The goal of this study is to identify factors that explain racial/ethnic disparities in PC incidence, particularly the excess risks observed in African Americans, Japanese Americans, and Native Hawaiians. We hypothesize that in addition to known risk factors, host genetic factors and unknown non-genetic factors contribute to the observed racial/ethnic differences in PC incidence. To test our hypothesis, we will leverage the well-characterized lifestyle and genetic data of the MEC, a long-standing ethnically diverse prospective cohort of >200,000 African American, Latino, Native Hawaiian, Japanese and white participants established in the early 1990's in California and Hawaii. The MEC is uniquely positioned to address racial/ethnic disparities in PC incidence, with >2,100 incident cases of PC diagnosed over >20-years of follow-up and with detailed lifestyle and exposure data collected in a consistent fashion amongst all populations to permit valid ethnic comparisons. Our specific aims are: 1) To quantify racial/ethnic-specific associations of known and potential/suspected risk factors with PC incidence in African Americans, Japanese Americans, Latinos, Native Hawaiians, and whites; 2) To determine whether the risk factors confirmed or discovered in Aim 1 contribute to the observed ethnic differences in PC incidence; 3) To characterize the association of known common genetic variants with PC risk in African Americans, Japanese Americans, Latinos, and Native Hawaiians and build a quantitative risk model to compare the distribution of genetic risks across populations associated with these marker alleles. By leveraging the existing resources/infrastructure, this study will efficiently and cost-effectively examine multiple factors that may contribute to racial/ethnic disparities in PC. Because of the lack of effective treatment and low survivorship, identifying modifiable risk factors is critical in reducing PC burden; this is even more crucial for minority populations who are at greater risk and are likely to have limited access to care.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The 90-kDa ribosomal S6 kinase 2 (RSK2) is important serine-threonine kinase broadly expressed in response to various growth factors. RSK pathway is a key regulator of cancer cell proliferation. In humans, RSK2 gene mutations are manifested in Coffin-Lowry syndrome characterized by severe psychomotor retardation. Mechanism of activation of RSK2 is still unclear, and many contradictive reports are published. Elucidating the molecular structure of RSK2 is essential for understanding its function. RSK2 belongs to the family of unusual serine-threonine kinases that contain two distinct kinase domains connected by a linker region. First, we focused on the regulatory C-terminal domain of RSK2 (CTD), activation of which resulted in activation of full length protein. Recently we determined the X-Ray structure of the isolated CTD RSK2 at 2.0 [unreadable] resolution and successfully published it (Nature Structural &Molecular Biology, 2008). The structure revealed a C-terminal autoinhibitory [unreadable]L-helix which was embedded in kinase scaffold and pre-determined kinase inactive conformation. We suggested a mechanism of activation by interaction with up-stream kinase, ERK, which would displace the autoinhibitory helix from its position resulting in the re-arrangement of conserved Glu500 residue.


The protocols involved in this project are as follows: 02-M-0120, 03-M-0175, 88-M-0131, 92-M-0174. This report includes work arising from the following protocols: NCT00030147, NCT00060736, NCT00001231, and NCT00001322. In addition to the established increased risk of first onset depression during the perimenopause, there is preliminary evidence of estradiols antidepressant efficacy in perimenopausal depression (PMD). Indeed, observational studies report the emergence of depressive symptoms after the discontinuation of estradiol therapy (ET) in 5-10% of women. The role of estradiol -either declining or low levels - in the precipitation of PMD is unknown, largely due to the associational and indirect nature of the evidence linking ovarian function and depression. Correlative studies with plasma FSH or response to ET do provide indirect evidence of the relevance of changes in reproductive hormones to mood disturbances. However, even prospective epidemiological studies cannot test the estradiol withdrawal hypothesis since perimenopausal changes in the secretion of several hormonal and metabolic factors could confound the effects of estradiol withdrawal. In other studies of the role of ovarian steroids in affective disturbance, changes in ovarian steroids (in the context of otherwise normal levels) have been shown to directly trigger depression, but only in a subset of women with histories of mood disorders linked to reproductive function. In this study, we directly examined the hypothesis that declining ovarian function estradiol withdrawal - underpins depression occurring during the perimenopause. We further sought to examine whether the response to estradiol withdrawal differed in women with a past PMD compared with those without any past depression. In this second study, we determined if sudden, blinded withdrawal of ET would precipitate depressive symptoms and if it would do so differentially in those with a history of PMD. Our findings support both predictions. Estradiol withdrawal precipitated depression in women with a history of PMD, but no depressive symptoms were seen after estradiol withdrawal in women with no past history of PMD. Further, depressive symptoms did not emerge in those women with past PMD who were maintained on ET. Importantly, the recurrence of depressive symptoms in women with past PMD occurred in the absence of differences in several measures that could influence mood, including baseline clinical characteristics (other than PMD), the severity of daytime/nighttime hot-flushes, and plasma hormone levels after withdrawal. The lack of depressive symptoms in the control women despite identical hormone manipulation (and similar levels of hot-flushes and plasma estradiol levels) demonstrates that estradiol withdrawal differentially impacts CNS function in some women so as to render them susceptible to depression. This suggests that perimenopausal changes in estradiol can trigger depression, but only in the susceptible subgroup. These observations, in the context of similar plasma reproductive hormone levels, suggest that normal changes in ovarian estradiol secretion can trigger an abnormal behavioral state in susceptible women. There are several mechanisms by which changes in estradiol might mediate the observed effects on mood. First, estradiol modulates the activity of virtually every system implicated in the pathogenesis of depression, including regulation of neurotransmitter synthesis and metabolism, stress axis activation, neuroplasticity (including regulation of BDNF), epigenesis, and immune system activation. Second, estradiol signaling through estrogen receptor (ER) beta reverses depressive-like behavior in animal studies. Third, reward responsiveness, which is disturbed in depression, is modulated by estradiol in both rodents and humans. Finally, of particular note, discontinuation of long term ET in postmenopausal women was accompanied by decreases in medial frontal and temporo-occipital metabolism. Thus, through local signaling or network-level dysfunction, particularly in fronto-limbic regions, estradiol withdrawal could precipitate affective dysregulation. What remains unclear is the reason for the differential susceptibility to the mood destabilizing effects of estradiol withdrawal. Of interest in this regard is the recent identification of increased sensitivity to estrogen regulation among transcripts that were differentially expressed in women who developed postpartum depression. Alternatively, basic science studies report the de novo production of estradiol from androgens in brain regions involved in mood regulation (e.g., hippocampus). Thus, it is possible that women who did not develop depressive symptoms after ET withdrawal have sufficient CNS aromatase activity to prevent the development of depressive symptom during declining estradiol secretion. The investigation of these possible mechanisms will be the focus of future studies. One possible source of the susceptibility to developing perimenopasual depression is an abnormal hypothalamic pituitary-adrenal (HPA) axis. Dysfunction of the HPA axis is a frequent accompaniment of depression, and HPA axis function is modulated by both aging and ovarian steroid secretion. To date, only a few studies have evaluated basal (but not stimulated) HPA axis function in women with perimenopause-related depression, with findings suggesting differences in adrenal androgen secretion but not basal cortisol secretion in PMD compared with controls. We examined HPA axis function in depressed and asymptomatic perimenopausal women using the combined dexamethasone-corticotropin releasing hormone (Dex/CRH) test. In contrast to recent suggestions that declining ovarian estradiol secretion (and in turn alterations in neurosteroid synthesis), preliminary results suggest that abnormalities of HPA axis function do not distinguish perimenopausal women with depression from those without depression. Thus, unlike depression in premenopausal women, HPA axis dysfunction is not a frequent accompaniment of depression during the perimenopause. These findings suggest that reproductive endocrine-related mood disorders are not uniformly associated with the HPA dysregulation and could reflect underlying pathophysiologic processes that are distinct from those reported in women with non-reproductive-related depressions. Finally, we continue our longitudinal investigation of the onset of depression during the perimenopause. Over sixty women have completed this study that involved measuring early follicular phase blood samples, daily and weekly symptom self-ratings, and formal in-person interviews every six months during the transition from regular menstrual cycle function (premenopausal) until one year postmenopause. Women participate in this study for an average of five years (range = 3-10 years). Preliminary results confirm previous observations of a clustering of depressive episodes during the late menopause transition a stage of reproductive aging associated with estradiol withdrawal. Moreover, we have observed that depression occurring during the perimenopause,( but not other events related to the perimenopause including menstrual cycle irregularity, aging, or vasomotor symptoms) is accompanied by significant declines in measures of quality of life, social support, and personal disability.


Multiple Sclerosis (MS) is an inflammatory, demyelinating disease of the Central Nervous System (CNS) which likely involves an autoimmune mechanism directed against self-myelin associated antigens. There is substantial evidence suggesting that B cells are involved in at least one mechanism of MS pathogenesis. However, both functional and molecular analysis of the immunoglobulins (Ig's) produced by clonally expanded CSF B cells has been limited. We hypothesize that a subset of clonally expanded B cells in the CSF of MS patients is involved in at least one mechanism of MS pathogenesis by producing antibodies that bind to myelin associated antigens. In order to identify complete Ig rearrangements that may play a role in the autoimmune activities evidenced in MS patients and test for their antigenic specificity, we intend to define the Ig repertoires in the CSF of MS patients in order to identify Ig's from clonally expanded B cells. We then plan to determine the antigenic specificity of these unique Ig rearrangements by cloning both the heavy and light chain segments into an expression vector, isolating the resultant Fab fragments, and testing them for their antigenic specificity. We can also use PCR to track the persistence of these clones in the original MS patients. Moreover, we can determine if other MS patients have the same clonally expanded B cells in their CSF. These studies provide the necessary foundation to initiate and assess the potential role of B cells in at least one mechanism of MS pathogenesis, and ultimately will allow us to explore whether generating specific immunotherapies directed against these clonally expanded B cells in the future is warranted. [unreadable] [unreadable]


The purpose of this proposal is to work collaboratively with the CDC and other SB clinics to build a disease specific national registry to improve care for individuals with SB. Proposed Aims: As part of the national network of participating SB clinics, the Greater San Francisco Bay Area SB Partnership will: a) Recruit and collect standard data on at least 125 SB patients for each of three years of the project using standard forms;b) Submit the de-identified data to a central repository using a web based electronic medical record (EMR) system;c) Adopt the EMR for ongoing clinical use;d) Participate in the national Coordinating Committee to promote improvements to the registry, propose research questions, develop an analytical plan, and promulgate changes to clinical practice standards based on data findings. Background: SB is the most common permanently disabling birth defect, with an estimated 70,000 individuals living with the more significant forms of this neural tube defect. Relatively little is known about how SB care varies by location, and currently, not much research is occurring related to SB. Consequently, the evidence based on which the current treatment of SB occurs is weak. It is anticipated that the establishment of a National SB Patient Registry (spearheaded by CDC, the SB Association, and Agency for Healthcare Research and Quality) will result in improved quality of care for all individuals with SB, and will identify and support opportunities for collaborative research. The proposed Greater San Francisco Bay Area SB Partnership is a collaboration between two high quality SB programs with very diverse patient populations: one at Children's Hospital &Research Center Oakland and the other at the University of California at San Francisco Benicoff Children's Hospital. Together, the sites follow 255 diverse patients in their clinics from newborn to age 21;about one half of these patients are Latino. Methods: Following protocol established by the CDC, the Greater San Francisco Bay Area SB Partnership will attempt to recruit all newborns, children, adolescents, and young adults to participate in the registry during their visits to SB clinic. Data will be collected on each patient for a minimum of three years using standard forms. Non-identifiable data will be entered into a web based EMR maintained by CDC. The project's Coordinating Committee will determine research topics and data analysis.


Arsenic is a carcinogen, a Superfund toxic compound, and a common drinking-water contaminant. The Environmental Protection Agency (EPA) has identified 1,300 sites on its National Priorities List (NPL) and arsenic has been found in at least 781 of these sites in the USA. Exposure may occur by a variety of pathways including inhalation of dusts in air, ingestion of contaminated soil or water, or through the food chain. The primary mode of exposure in non-occupationally exposed populations is drinking-water contamination from either alluvial deposits or industrial contamination. The precise relation of arsenic to cancer has not been established at low exposure levels. In addition, arsenic toxicokinetics in humans remains poorly understood. Thus, we propose to assess exposure and skin-lesion risk, as well as arsenic dosimetrics in a population with a wide range of arsenic exposure via drinking-water contamination. The proposal builds upon a small study in which a case-control study has been initiated. This proposal will use the necessary resources to analyze biomarkers of exposure and susceptibility and for statistical analyses of both a case-control study of skin lesions and a repeated-measures biomarker dosimetry study. The proposed studies in Bangladesh will assess this risk in a population with a wide range of exposures - from low to very high. Together, these data will add substantially to the existing risk assessment information by elucidating both early and late outcomes alter arsenic exposure, and the influence of susceptibility traits at all levels of exposure. This project is relevant to the overall aims of NIEHS in several ways: First, we will examine a range of health effects of an important environmental carcinogen. Second, we will define human biomarkers of exposure, early effects, and genetic susceptibility to arsenic exposure. Third, we will examine exposure-response relationships for arsenic-induced non-malignant skin lesions as well as for skin cancers.


Nonsteroidal anti-inflammatory drugs (NSAIDs) display striking antineoplastic activity, although toxicity resulting from cyclooxygenase (COX) inhibition and incomplete protection from disease progression limits their use for cancer chemoprevention. Preliminary studies suggest that the mechanism for the chemopreventive activity of a highly efficacious NSAID, sulindac, does not require the inhibition of COX-1 or -2 and that cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) inhibition is responsible for its tumor cell growth inhibitory activity. We hypothesize that it is feasible to chemically modify sulindac to block COX inhibitory activity, while enhancing tumor cell growth inhibitory activity by increasing selectivity for cGMP PDE. In support of this hypothesis, we have synthesized a prototypic amide derivative of sulindac sulfide that does not inhibit COX-1 or -2, yet displays high potency to inhibit colon tumor cell growth and cGMP PDE in vitro. Sulindac sulfide amide (SSA) displayed adequate oral bioavailability and appeared to be less toxic than sulindac in mice. Using a xenograft model involving human colon tumor cells, SSA displayed comparable in vivo antitumor efficacy as sulindac, albeit at high dosages. These studies demonstrate the feasibility of uncoupling the COX inhibitory activity from the growth inhibitory activity of sulindac and supports further research to develop additional derivatives that are more potent and/or bioavailable than SSA. The following specific aims are proposed: 1) prepare a series of sulindac analogs to optimize for potency, selectivity, and oral bioavailability, 2) evaluate in vitro activity, 3) determine the mechanism of action and identify the target cGMP PDE isozyme, and 4) evaluate in vivo chemopreventive efficacy. Our goals are to identify a safe and efficacious derivative of sulindac for cancer chemoprevention and to critically test the hypothesis that cGMP PDE is a target for cancer chemoprevention. This multidisciplinary research proposal is anticipated to result in a new drug candidate for cancer chemoprevention, and in particular, patients with familial or sporadic adenomatous polyposis who are at high risk of disease progression. Nonsteroidal anti-inflammatory drugs (NSAIDs) display striking cancer chemopreventive activity, although toxicity resulting from cyclooxygenase (COX) inhibition and incomplete efficacy limits their clinical use for patients at moderate to high risk of disease progression. Preliminary studies suggest that the mechanism for the antineoplastic activity of sulindac does not require COX inhibition and that cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) inhibition is responsible for its tumor cell growth inhibitory activity. We hypothesize that it is feasible to chemically modify sulindac to block COX inhibitory activity, while enhancing tumor cell growth inhibitory activity by increasing selectivity for cGMP PDE. Our primary goal is to identify a novel sulindac derivative as a clinical candidate for patients with either familial or sporadic adenomatous polyps who are at high risk of disease progression. A secondary goal is to critically test the hypothesis that cGMP PDE is a target for cancer chemoprevention and involved in carcinogenesis. This multidisciplinary research proposal may lead to a novel strategy for colon cancer chemoprevention.


The primary goal of the conference for which funding is being requested will promote the marketing of safe, fresh and accurately labeled shell eggs and egg products through the development of uniform, science based and coordinated state and federal regulations. The conference is designed to achieve this goal by providing educational segments related to current egg safety and quality issues and the opportunity for state, federal and industry representatives to interact and share information and ideas. The conference will also provide a forum to present specific issues which will be referred to the NERO committees comprised of specialists in that area. The committee will review the information and provide specific recommendations for the most effective and cost efficient method to achieve the desired results. The conference will address new rules for S. enteritidis, label requirements for egg, scientific information related to campylobacter in eggs as well as other presentations related to food safety. The conference will be held at the Florida Mall Hotel in Orlando, Florida from March 13 - 16, 2005.


Connexin 32 (Cx32), is a gap junction protein found in the paranodal loops and Schmidt-Lantermann incisures of myelinating Schwann cells. Mutations in the gene encoding the human gap junction protein lead to CMTX, the X-linked form of the clinical condition known as Charcot-Marie - Tooth disease, a group of inherited peripheral neuropathies. Some mutations in Cx32 appear to allow for appropriate membrane targeting and formation of functional channels, some may lead to altered cellular processing, and others prevent Cx32 synthesis entirely. Mutations in Cx32 may be important in determining the processing and biophysical behavior of the Cx32 ion-channel. I hypothesize that mutations in Cx32 found in patients with CMTX, lead to the loss of ability of Cx32 to provide a vital cellular communication pathway. Various mutations may lead to this loss-of-function by altering the biophysical properties of the pore, or by affecting cellular processing of the protein or its ability to organize into functional channels. To evaluate these possibilities: 1) I will determine the biophysical properties of the channels formed by connexins containing mutations corresponding to those found in patients with CMTX; channels will be expressed in Xenopus oocytes or transfected cells; and 2) I will observe the cellular trafficking of wild-type and mutant forms of Cx32 in real time, in living cells, using an EGFP-tagged form of the molecule. The work described in this proposal will provide the applicant with an opportunity to acquire the necessary intellectual and technical skills to be an independent investigator in translational neuroscience research.


Functional nerve stimulation (FNS) is a means to restore lost function to patients with spinal cord injury. By directly stimulating the nerve, it is possible to maximize muscle contraction and minimize injected charge. An effective neural electrode must meet the following criteria: 1) The electrode must be able to activate any subset of the neural axon population, either deep or surface, selectively and independently and; 2) The electrode must not cause significant damage to the nerve. Current nerve cuff designs place electrodes around the nerve. These exhibit some degree of selectivity, but, cannot provide adequate functional control since it is not possible to selectively activate portions deep in the nerve without stimulation of the surface of the nerve. Intraneural electrodes have been developed to provide access to the deep portions of the nerve. Two of the current intraneural approaches are regeneration electrodes and wire electrodes. The regeneration design places a silicon substrate penetrated by holes between two severed ends of a nerve. The axons then regenerate through the holes. Wire electrodes are electrical wires threaded through the neural fascicles, penetrating the epineurium and a second protective layer, the perineurium. Both the threaded wire and regeneration designs provide selective stimulation of neural regions, but produce significant damage to the fascicles and axons. We propose a new design which combines the advantages of the standard nerve cuff and intraneural electrodes. This new device slowly places electrodes inside the nerve, but between the fascicles without perforation of the perineurium. The interaction takes place over the course of several days by taking advantage of the mechanical energy stored in the cuff prior to installation. Preliminary experiments have shown that the slow rate of penetration causes minimal damage and trauma to the nerve while still placing electrodes deep in the nerve. The aims of this proposal are 1) to show that chronic damage from the electrode is minimal, 2) to determine the selectivity of the design for electrical stimulation of the sciatic nerve in the cat, 3) develop finite element model of fields generated within the nerve and use this model to optimize the electrode design, and 4) to develop a electrode that utilizes currently available silicon technology to maximize the selectivity and function of the electrode. The end-product of this project will be an electrode capable of producing selective activation of neural regions by placing multiple contact points within the nerve with minimal axon damage. The same electrode could then be used for recording neuronal activity such as sensory signals and this Slowly Penetrating Interfascicular Nerve Electrode (SPINE) could, therefore, become a general purpose neural cuff design capable of both selective activation and recording.


The long-range goal of this research is to isolate and characterize all of the components necessary for reconstitution of hormonally regulated adenylyl cyclase system. Using the liver glucagon-sensitive adenylyl cyclase systems as a model system and by monitoring principally specific binding of labelled glucagon and adenylyl cyclase activity, we specifically propose during the next 5 years: (1) Purify the catalytic unit of the liver adenylyl cyclase system in a form that is either sensitive or insensitive to stimulation by nucleotide, and study its subunit composition. (2) Develop the necessary methodology to purify the glucagon receptor from liver and initiate its purification. (3) Identify and purify key regulatory components of the system: a Guanyl nucleotide regulatory component associated with the catalytic subunit using Gilman's reconstitution assays with membranes from AC- S49 lymphoma cells as means of identifying the component once separated from the catalytic subunit of the system; and b. Brain stimulatory factor(s). (4) Continue exploring the kinetics of this system with respect to nucleotide and hormone stimulation with the aim to further delineate its regulatory aspects and to identify possible additional components involved in normal functioning of adenylyl cyclases.


Attended NCRR/NIH session to edit a workshop report on ~Technologies for the Future~, May 15, 1996. Consulting with various personnel on the progress/nuances of 2~PE scanning microscopy.


Description: (Applicant's Description) Since its origin in Fall 1996, the Pharmacology Core has steadily grown into being an important CCC resource for several investigators. Its mission is providing expertise in pharmacological sciences, including kinetics, dynamics, biopharmaceutics and physical pharmacy. Pharmacokinetic support includes drug absorption, metabolism, distribution, elimination, bioactivation and drug-drug interactions. Pharmacodynamic support includes molecular effects of drug exposure, and in vitro toxicity testing. Biopharmaceutic support includes the influence of dose and dosage form on pharmacokinetics. Physical pharmacy support includes production of (approximate dose) prototype forms of drugs for preclinical studies and clinical forms for IND studies. Enhancements of research by the Core take the form of study design, data acquisition, data analysis, and interpretation of results, including technical writing for manuscripts and grant proposals written by CCC investigators. This core also developed and oversees the Quality Assurance Plan for tracking and handling clinical pharmacology specimens throughout the Institute. Key features of this Core are the scope of services provided, its integral role in clinical specimen handling, existing collegial relationships, direct consultation for requesting investigators, and the interests and commitment of its staff. Pharmacological contributions benefited 5 Programs, including Developmental Therapeutics, Breast Cancer, Molecular Biology and Genetics, Prostate Cancer and Protease Programs.


Description: (Applicant's Description) This facility was previously part of the joint Transgenic/Histology facility. It was designed for the efficient processing, sectioning and examination of tissue from mouse and other species, and provides expert advice and assistance in slide preparation. It serves all groups using mutant mouse strains and is available to all other groups. A consultant veterinary pathologist provides expert review of slides. Support comes from this CCSG (equipment and one full-time technician) and from individual users (personnel and full cost supplies). Professor Tyler Jacks has supervisory responsibility for the facility. His research program centers on the generation of mouse models of human cancer, and he and his laboratory have performed extensive histological analysis of tumor models as well as developmental phenotypes in the mouse.


The mechanism of muscle wasting in AIDS is not known. It is the hypothesis of this proposal that wasting in AIDS is associated with increased production of glutamine by muscle. Glucocorticoids, by stimulating muscle proteolysis and glutamine synthetase activity, are a key contributing factor to wasting. Therefore, pharmacological blockade of glucocorticoid receptors might prevent or alleviate a significant component of the wasting syndrome. The proposed studies will employ muscle biopsy specimens from human AIDS patients, as well as mice with a persistent retrovirus infection (LP-BM5), the manifestations of which simulate many features of human AIDS. The specific objectives are: (1) to characterize muscle wasting in AIDS; (2) to evaluate the mechanism of changes in glutamine synthetase and glutaminase activities and glutamine production in muscle from human AIDS patients and LP-BM5- infected mice; (3) to determine alterations in glucocorticoid receptors in muscle from human AIDS patients and from LP-BM5- infected mice and the mechanisms underlying such changes; and, (4) to assess antiglucocorticoid therapy for muscle wasting in LP-BM5- infected mice. The proposed studies are expected to contribute to a better understanding of biochemical mechanisms underlying muscle wasting in AIDS, provide useful biochemical markers for clinical assessment of early wasting, and lead to the development of specific treatments for this devastating conditions.


The Epithelial Na+ Channel or ENaC is rate limiting to sodium absorption in renal epithelia. Channel activity sets the final urinary sodium concentration and determines in part sodium excretion. Channel activity affects body sodium and water balance and therefore blood pressure. This channel exists at the plasma membrane largely in an electrically inactive pool. Recently, it was demonstrated that this inactive pool can be activated by cleavage of two of the channel subunits by serine proteases. Thus, this mode of channel regulation affords ENaC expressing renal epithelia an enormous capacity to rapidly respond to changes of sodium load, renal filtration, and body sodium balance. Activation of silent channels by proteases is proposed to involve the removal of short inhibitory domains from the channel's alpha and gamma subunits. However, our data clearly demonstrate that non-cleaved channels can be active, while some cleaved channel subunits do not form active channels. We demonstrate that Po can be regulated by multiple mechanisms that include channel processing, membrane lipid rigidity, channel partitioning into lipid rafts, and interaction between inhibitory domains of the ENaC subunits. We propose that multiple interactions between the ENaC subunits determine channel Po. Cleavage of the channel subunits provides a mean of removing or altering some of these interactions and leads to channel activation. We also propose that channel subunit interactions affect subunit processing, membrane partitioning and stability. The unifying hypothesis is that unprocessed channels are stable at the membrane but inherently inactive unless conditions exist to reduce subunit-subunit inhibition, and that moreover, proteolysis activates or primes the channels for activation by permanently removing such inter-subunit interactions. Proteolysis then predisposes the channel subunits for internalization unless processed subunits are protected in raft membrane domains. To address the mechanism of control of channel activity, we develop new tools that utilize 1) engineered cleavage sites 2) a recently identified constitutively ENaC subunit (epsilon) 3) homology mapping of the channel to the crystal structure of ASIC1 and 4) analysis of channel partitioning into membrane domains. Our data will pave the way for understanding channel Po regulation and every downstream process that further modify this Po. PUBLIC HEALTH RELEVANCE: We examine the mechanisms that control the spontaneous activity of the renal epithelial sodium channel. The activity of this protein is important in determining kidney salt excretion and consequently salt retention by the body and blood volume and pressure. Understanding the spontaneous activity of this protein would help understand the basis of salt sensitivity of blood pressure and is important in designing therapy for individuals with high blood pressure.


Cytochrome P-450IIE1 is a constitutive, hepatic enzyme that has been implicated in the activation of pro-toxins and pro-carcinogens. Studies with animals have shown that P-450IIE1 can be significantly induced by a class of compounds that include ethanol, acetone, and isoniazid, as well as by abnormal metabolic states such as fasting, obesity, and diabetes. The major hypothesis to be tested in this proposal is that humans exposed to the same class of chemicals, or with a similar altered metabolic state, will have elevated levels of P-450IIE1 enzyme and can be identified with an in vivo catalytic probe. Four compounds (enflurane, chlorzoxazone, 4-methylpyrazole, and acetone) have been identified as potential probes based on literature reports and preliminary results. The utility of probes based on literature reports and preliminary results. The utility of probe candidate(s) will be validated by: a) determining the in vitro catalytic specificity of the probe towards human liver P-450IIE1, b) identifying an in vivo metabolic clearance parameter that reflects intrinsic P-450IIE1 catalytic activity, and c) determining whether the in vivo clearance parameter predicts direct measurements of hepatic P-450IIE1 levels in normal and isoniazid-treated populations. Once developed, the in vivo probe will be used to study the inducibility of functional P-450IIE1 enzyme in people with Type I and Type II diabetes, the morbidity obese, and obese people experiencing rapid weight loss. If P-450IIE1 levels are significantly elevated in selected human populations, those persons will be at greater risk for developing the carcinogenicities and cytotoxicities thought to be related to P-450IIE1 catalytic activity. This proposal will provide a safe and facile method for their identification.


In earlier work, hydrogen enchange methods developed in this laboratory were used to study structure and dynamics in a number of necleic acid systems. More recently we have adapted the method of D. Cross (absorbance-detected H-D exchange in stopped-flow) to the study of large nucleic acids and have used this to study base-pair opening reactions in synthetic double helices and also the ethidium-DNA interaction. The stopped-flow approach promises a remarkable advance in such investigations; data collection is very much faster, extension of time resolution to msec makes accessible the important early-time exchange region, and it appears possible now to study nucleic acids while complexed to major amounts of protein. We want to bring these capabilities to fruition. This will involve: completing the calibration of the various necleotide parameters for UV-detected H-D exchange, extending our previous studies of opening reactions to other synthetic duplexes and DNA, and attempting to gain information on the structure of the open form. In addition we want to see what can be learned about DNA in chromatin and RNA in ribosomes.


Increased susceptibility to infections including tuberculosis (TB) is a major cause of morbidity and mortality in diabetes. Despite its clinical importance, this phenomenon has received little basic research attention. We will investigate TB resistance in mice using models of type 1 and type 2 diabetes. Diabetic and non-diabetic control mice will be challenged by low-dose aerosol infection with Mycobacterium tuberculosis (Mtb). We will characterize TB susceptibility with parameters of survival, bacterial load, and lung leukocyte recruitment. The basis of susceptibility will be evaluated by characterizing cytokine expression and by testing macrophage, dendritic cell, and T cell functions. We will investigate whether advanced glycation end products (AGE), which have been linked to diverse complications of diabetes, are responsible for impaired host defense. We will also evaluate the potential role of other biochemical mechanisms implicated in diabetes complications including polyol pathway flux, hexosamine pathway flux, and over-production of diacylglycerol with activation of protein kinase C. Hyperlipidemia is a common co-morbidity in diabetes that exacerbates diabetic vasculopathy. In preliminary studies we found that hypercholesterolemia also increases TB susceptibility. We will explore similarities and differences in the effects of diabetes and hyperlipidemia on protective immunity, and we will characterize TB susceptibility of mice with combined hyperlipidemia and diabetes. This project will identify specific deficits in protective immunity caused by hyperglycemia and hyperlipidemia, and may provide new insights to critical parameters of host defense against TB. Understanding the mechanisms of susceptibility will inform the development of treatments to reverse this diabetes-related complication. At the same time our model will be used to test the impact on protective immunity of novel treatments for diabetes co-morbidities such as statins, aminoguanidine and pyridoxamine. While our focus is on TB, the new knowledge generated by this project will have broad relevance to other infections associated with diabetes and will further basic understanding of the interplay between immunity and metabolism. This project studies how diabetes weakens the body's ability to fight tuberculosis. Learning more about the harmful effects of diabetes on the immune system will suggest new ways to prevent and treat infections that are a major problem in people with diabetes.


[unreadable] [unreadable] This application is the competing renewal for the ongoing postdoctoral training program at Drexel University College of Medicine focused on the biological bases of nervous system disorders. The objective of the program is to provide trainees with the perspectives and skills needed to prepare them for independent careers in neurobiological research. We request support for four fellows, who will have either the Ph.D. or M.D. degree. The program will provide opportunities for multidisciplinary research projects in the laboratories of both basic and clinical scientists and exposure to a wide variety of experimental and clinical issues. The program is broadly-based, and consists of four focal groups that are highly interactive with one another. The first group focuses on cellular and developmental neurobiology; the second group on systems and behavioral neurobiology, the third group on spinal cord and regeneration; and the fourth group on neuroengineering. There are a total of 10 senior level faculty members who will act as the primary training faculty. There are an additional 8 junior level faculty members who will also act as trainers, under the supervision of the senior faculty. The entire participating faculty, both senior and junior, is funded by the NIH, well published, and active in both research and training. In addition, there is an abundance of clinical researchers at three different hospitals within our university system who will participate in the training of the fellows. In addition to the primary relationship between a trainee and the mentor, there are a number of activities that ensure a broad exposure to the issues and techniques of modern neurobiology. These include courses developed around the four focal groups, journal clubs and seminars, research conferences and shared laboratory facilities. The progress of each trainee will be formally reviewed regularly and monitored constantly, through research meetings and seminars. This process assures full access to the various disciplines and perspectives available in the program. [unreadable] [unreadable]