This proposal is for a conference grant for the Twelfth Microbial Genomes Conference (formerly the E. coli and Small Genomes Conference) to be held September 26-30, 2004, at the UCLA conference center at Lake Arrowhead, California. The genomics landscape has changed now that numerous genomes are available and that microarrays and bioinformatics methods are advancing rapidly. It is a critical time to foster multi-disciplinary approaches to making sense out of all the genomic sequence information. Considerable integration of different aspects of microbiology will undoubtedly take place, including new perspectives on evolution and the requirements for life. To achieve these goals, particularly of integration and synergy, scientists from diverse disciplines must interact. Thus, this conference will include active genome researchers, such as sequencing experts and informatics specialists working in database design, sequence analysis, or simulation; geneticists developing new methods to take advantage of genomic information, e.g. for functional analysis; and cell biologists and biochemists who have specialized in areas now ripe for genomics such as metabolism, chromosome structure, or gene regulation. One focus of this conference will also be on microbial communities and biodiversity. An additional focus will be on modeling and constructing circuits.and pathways. This meeting has become established as a major annual microbial genomics conference during the last twelve years and has assured status and quality. [unreadable] [unreadable] [unreadable]


PROJECT SUMMARY The available evidence suggests that intrauterine and/or maternal bacterial infection is a major cause of early and late pregnancy complications. Current antibacterial and anti-inflammatory drugs used to treat bacterial infection during pregnancy are considered harmful to maternal and fetal health. Thus, continued research to look for better treatment options to avoid bacterial infection-induced pregnancy complications is alluring. The decidua and placenta are uniquely positioned at the maternal-embryonic interface to serve as a first line of defense against bacterial toxins, but their defensive mechanisms against bacterial toxins are poorly understood. Lipopolysaccharide (LPS) is an endotoxin of Gram-negative E. coli that is associated with infection-induced pregnancy defects. LPS recognition by cell surface proteins is an important step required for the initiation of its inflammatory signaling or inactivation by cell surface molecules. Our preliminary results establish that: 1) the uterine luminal epithelium, decidua and placenta express the LPS sensing and signaling TLR4/CD14/MD2 complex; 2) LPS injection on day 5 of pregnancy terminates pregnancy by day 8 via selective-activation of the MyD88-dependent TLR4 signaling pathway at the embryo implantation site (EIS); 3) LPS induces expression of proinflammatory cytokine genes such as Tnf-? & Il-1?, chemokine genes such as Cxcl1&2, and neutrophil recruitment to the EIS; 4) cell surfaces of the decidua and placenta express tissue- nonspecific alkaline phosphatase (TNAP) isozyme that is capable of dephosphorylating LPS; 5) dephosphorylated LPS is non-inflammatory and non-toxic to murine pregnancy; and 6) AP isozyme treatment alleviates LPS-induced early pregnancy loss in mice. These preliminary findings have led to the hypothesis that LPS-induced microenvironment disruption at the early EIS is a result of concerted action of resident decidual cells and recruited neutrophils, and LPS detoxification by supplementation and/or induction of TNAP production/activity may abrogate LPS-mediated early pregnancy defects/loss. To test our hypothesis, we have proposed three mechanistic aims. In Aim 1, we will use Cre-lox and antibody- mediated neutralization technologies to establish that an important step in the development of LPS-induced unwanted inflammation at the early EIS is decidual cell-type-dependent recruitment of inflammatory neutrophils. In Aim 2, we will generate novel female mice with uterine deletion of TNAP gene Alpl to determine whether endogenous TNAP deficiency in the uterus augments the response to LPS. In Aim 3, we will examine the potential of TNAP and its activator or inducer in mitigating LPS- or E. coli-induced early pregnancy defects/loss. Upon completion of these aims, we hope to gain: 1) insights into the mechanisms of infection-induced inflammation at the early EIS; and 2) develop a novel LPS-detoxification therapeutic strategy to avoid bacteria- induced early pregnancy complications.


The objectives of this project are to determine the effects of 2450 MHz microwave radiation on biological material at the cellular and macromolecular level and relate the amount of microwave energy absorbed to the effects. Various cellular and macromolecular systems will be tested by exposure to microwaves under carefully controlled temperature and dose conditions. Parameters such as viability, cell membrane permeability and denaturation will be used as indicators of microwave effects.


Clonal evolution is a key feature of cancer progression and relapse. Our recent study, which utilized a newly developed pipeline that estimates the fraction of cancer cells harboring each somatic mutation within a tumor through integration of whole-exome sequencing and local copy number data, linked the presence of subclones harboring putative driver mutations with adverse clinical outcome in chronic lymphocytic leukemia (CLL) and suggested that CLL therapy may accelerate the process of clonal evolution (Landau et al., Cell 2013). We propose that presence of subclonal mutations that are putative drivers are indicative of an active evolutionary process. We now seek to definitively establish the impact of subclonal mutations on CLL biology, the development of disease relapse and clinical outcome. This will be achieved by longitudinal analysis of clonal structure of serial samples collected from patients enrolled on phase II and phase III clinical trials (and hence uniformly treated) that address the treatment landscape of CLL. In particular, we will perform detailed genetic analysis of samples from patients receiving standard-of-care first line fludarabine-based chemotherapy (Aim 1). In parallel, we will examine patient samples exposed to ibrutinib, a highly promising irreversible inhibitor of Bruton's tyrosine kinase which i anticipated to be a cornerstone of future CLL therapy (Aim 2). Analysis of samples exposed to both these types of therapies will include characterization of subclonal structure as well as assessment of the dynamic phenotypic changes (detected by single cell RNA-sequencing) to validate mutation analysis and determine the transcriptional networks of drug resistant cells in order to reveal potential novel and effective treatment combinations. To causally link the impact of putative drivers and therapy on CLL clonal evolution, we will generate an in vivo model to study interclonal dynamics in the setting of therapy (Aim 3). We will use transformative genome-editing techniques to generate cell lines that model leukemic subpopulations bearing representative CLL driver mutations and thereby mechanistically dissect the contribution of individual genetic lesions to the evolutionary landscape. By creating an animal model of clonal evolution, we will have the potential to more effectively evaluate preclinically the impact of nove therapeutics on clonal selection. In total, these studies are designed to establish a framework for understanding the role of the dynamic evolutionary landscape of CLL on the diagnosis, prognosis and treatment of this currently incurable disease.


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. Aging is associated with increased risk for heart disease, diabetes and physical disability. In women, the incidence of these age-related conditions increases dramatically after menopause. This has led to the hypothesis that ovarian hormone deficiency contributes to these adverse health outcomes. However, the effect of ovarian hormone deficiency, per se, on risk factors for disease and disability has not been clearly defined. Thus, the primary goal of the proposed studies is to characterize the effect of ovarian hormone deficiency on glucose, insulin, fat and protein metabolism. Our overall hypothesis is that ovarian hormone deficiency alters substrate turnover and utilization in a manner that increases the risk for developing chronic disease and disability. Specifically, alterations in glucose, insulin and fat metabolism increase the risk for developing heart disease and diabetes and changes in protein metabolism contribute to reduced lean tissue mass which, in turn, promotes disability. To address our hypothesis, we will measure substrate metabolism using stable isotope tracer methodology in healthy, premenopausal women before and after pharmacological ovarian suppression. Women will be randomized to receive the gonadotropin-releasing hormone agonist leuprolide acetate or placebo. Measurements of substrate metabolism will be performed during both the follicular and luteal phases of the menstrual cycle prior to treatment and 2 months after the initiation of treatment. Experiment 1 will investigate the effect of ovarian hormone deficiency on the glucose and insulin response to hyperglycemia. Experiment 2 will examine the role of ovarian hormone deficiency in the regulation of whole-body lipolysis under postabsorptive and epinephrine-stimulated conditions. Experiment 3 will examine the effect of ovarian hormone deficiency on whole-body protein metabolism under postabsorptive and simulated-postprandial conditions.


Two strains of mice were used in this project. Female C3H and CBA mice were exposed to a total body dose of radiation of 3 Gy with or without Tempol supplementation in the animal's food. Immediately following the radiation exposure, animals were be placed on either control or TP-containing food. The groups include: a) no radiation, control food, b) 3 Gy, control food, c) no radiation, TP food, and d) 3 Gy, TP food. For C3H mice, one additional group was added receiving 3 Gy total body irradiation where the administration of the TP containing food was delayed one month post-irradiation. Preliminary data show TP food supplementation after radiation: a) did not alter food consumption compared to animals on a control food diet, b) compared to animals on control food diet, the TP diet resulted in decreased weights in both mouse strains (40% for C3H and 20% for CBA), and c) TP food supplementation post-radiation significantly enhanced the survival of both mouse stains. Median survival values for 0 Gy, 3 Gy, 0 Gy TP, and 3 Gy TP for C3H mice was 706, 434, 764, and 670 days, respectively. For CBA mice median survival values for 0 Gy, 3 Gy, 0 Gy TP, and 3 Gy TP for C3H mice was 901, 660, 939, and 782 days, respectively. The incidence of hematopoietic neoplasms (predominantly lymphomas) was significantly reduced in both mouse strains by TP treatment and both the onset and incidence of solid neoplasms was significantly reduced in CBA mice treated with TP. These preliminary data would encourage further research and development of TP as a chemopreventive agent. The second hypothesis is also being tested that mice protected from lethal total body irradiation by administration of a radioprotector immediately before radiation exposure will experience an elevated risk of cancer induction. Mice were exposed to a total body radiation dose of 10.8 Gy, a radiation dose that results in 100% lethality. Ten minutes prior to the 10.8 Gy exposure the animals will be injected with a radioprotector. The control for this group, another set of animals was exposed to 5.4 Gy total body irradiation. This radiation dose was derived from the radiation dose modification factor (2) when the radioprotector is administered 10 min before total body irradiation. These animals will also be followed for their entire lifespan for tumor induction as outlined above. Preliminary data show that the median survival for mice receiving 0, 5.4 or 10.8 Gy were 706, 460, and 491 days, respectively. There was no difference between the 5.4 and 10.8 Gy groups (p = 0.42);however, the median survival of both irradiated groups was significantly shorter compared to unirradiated mice (p <0.0001). Cancer incidence (hematopoietic plus solid tumors) was similar between the 5.4 and 10.8 Gy groups and was significantly greater than for unirradiated controls. However, the ratio of hematopoietic to solid tumors differed between the two groups, with the 5.4 Gy group having a higher incidence of hematopoietic neoplasms compared to the 10.8 Gy group (1.8 fold). A greater incidence of solid tumors was observed in the 10.8 Gy group. These preliminary results suggest that mice protected from lethal whole body radiation have a shortened lifespan, due in large part, to cancer induction post-radiation compared to unirradiated controls. Lastly studies have been initiated to determine if metabolites in the urine of mice receiving whole body radiation can predict for radiation-induced cancer induction prior to the observation of tumor mass.


Older adults often struggle to communicate in adverse listening situations, especially when they must understand one talker when there are other voices in the background. Current rehabilitation of older adults with hearing loss focuses primarily on restoring audibility via hearing aids. Unfortunately, this is of limited benefit in dificult listening environments because both speech and background noise are amplified. Restoring audibility is not enough to ensure successful communication in situations where listeners must ignore the distracting speech of other talkers. The long-term goal of this research is to develop evaluation and treatment methods that focus on both the sensory and cognitive age-related changes that mediate comprehension in these situations. The proposed experiments are designed to provide vital information about why older adults experience problems in the presence of more than one talker. Past research has yielded conflicting conclusions about the relative importance of age-related peripheral/sensory factors and age-related cognitive changes in explaining deficits in speech understanding. The studies in this proposal will help to clarify tis issue by examining how listener-related factors (specifically, working memory, processing speed, attention-switching, inhibitory control, and hearing loss) interact with stimulus-related factors to create problems understanding speech in multi-talker situations. Two specific aims will be addressed: 1.) To understand why competing speech signals are so disruptive for older listeners, and how hearing loss and cognitive processing affect their ability to cope with this interference; and 2.) To determine the degree to which hearing loss and cognitive function impact speech recognition in more realistic communication situations. The proposed experiments use both established tasks (e.g., simultaneous speech-on-speech masking, ratings of effort) and innovative techniques (temporally interleaved speech, eye tracking, limitations on response time) to address these questions. The performance of middle-aged as well as older listeners will be measured in order to provide information about which functional abilities begin to decline early vs. later in the aging process. The end result of this project will be an enhanced understanding of the factors that limit older adults from fully participating in conversations in everyday listening conditions. This, in turn, may drive improvements in rehabilitative protocols that incorporate treatment of both bottom-up and top-down contributors to age-related speech understanding problems. PUBLIC HEALTH RELEVANCE: Many older adults struggle to understand speech in adverse listening situations. The purpose of this project is to identify why they experience these problems. The long-term goal of this research is to develop effective and comprehensive rehabilitation programs aimed at improving older adults' functional communication ability in difficult listening environments.


It has been shown that T lymphocytes are primarily responsible for the cell-mediated immunity of the host and the most important cells in the body to eliminate the neoplastic cells. The development of these cells depends upon the thymus gland. We have shown that there is a decrease of T cells in patients whose thymus glands were irradiated during early infancy. This population of patients also has a higher incidence of cancer. It is possible the decreased T lymphocytes may account for the increased incidence of cancer. Our present proposal is to extend our studies to include other populations of thymus-irradiated patients, especially the prenatal fetus, the premature infants and adult patients treated with radiation to their lung or breast. We hope these studies will determine the contributions of the thymus gland upon the T cell development during the different periods of life and the radiation effect to this gland at various thymus gland maturation stages as expressed by the proportion of T cells in the peripheral blood. Also, we will determine the radiation-induced chromosomal abnormalities in the peripheral blood lymphocytes in these patients.


Current studies have indicated that antibodies directed against the stalk region of CD23 cause enhancement of IgE synthesis in both the human in vitro and mouse in vivo systems. CD23 transgenic mice, which overexpress CD23 on all lymphocytes and FDCs, exhibit drastically reduced IgE production in both helminth and alum/ag models. The data suggest a model where the role of CD23 is initially to serve as a component of innate immunity to signal for IgE production by becoming destabilized and cleaved and later by overexpressing at the cell surface and modulating IgE production. This continuation application proposes to investigate the mechanism of these effects. Aim#1 examines the mouse system where the destabilizing mab 19G5 gives enhanced IgE synthesis in vivo. In the current funding, the metalloprotease, ADAM10 has been identified as the primary CD23 sheddase in mouse and humans. The role of ADAM10 in allergic disease will be modeled by making transgenic mouse that overexpress ADAM10 or dominant negative ADAM10. In addition, we will examine the mechanism for the 19G5-induced IgE production by investigating the association of CD23 with another negative signaling molecule, LAX, which has recently been shown to both modulate CD23 expression and regulate IgE levels. Aim#2 will investigate the affect of CD23 overexpression and CD23 destabilization on the mouse asthma model with respect to both modulation and exacerbation of disease. We will utilize both IgE and the new ADAM10 transgenics in order to evaluate the mechanism of the suppression of eosinophilia as well as the capacity of CD23 to modulate the asthma phenotype. Aim#3 will investigate the human in vitro IgE synthesis models with respect to the mechanisms involved in IgE synthesis enhancement, seen with anti-stalk antibodies and synthesis suppression, seen with certain anti-lectin mabs. The importance of ADAM10 in human CD23 cleavage and IgE production will also be explored as will the involvement of LAX. Finally, we will determine if IgE production by B cells obtained from normal and allergic subjects is affected differently by destabilization or stabilization of CD23. In summary, these studies examine the mechanism of action of a natural regulator of IgE production, CD23, with the objective of developing protocols to enhance CD23 expression and thereby regulate IgE, and by analogy, allergic disease in which IgE plays a dominant role.Project Narrative: This project examines mechanisms involved in control of IgE synthesis by a natural regulator. The latter is CD23, a low affinity receptor for IgE. Accumulated evidence indicates that cleavage of CD23 by the metalloprotease ADAM10 increases IgE production in both mouse and humans. This application proposes to study mechanisms involved in this regulation in order to develop new protocols to control allergic disease.


The proposed research plan seeks to determine the morphological changes in cells comprising the Japanese quail oviducts and testes during and after ingestion of various levels of the estrogenic insecticide Kepone. The morphological observations will be accomplished by gross, microscopic and ultramicroscopic (transmission and scanning electron microscopy) means. The first study will determine whether the damages recorded in cytoplasmic organelles of magnum and shell gland regions of the quail oviduct are reversible after ingestion of different concentrations of Kepone was discontinued. This study will involve the examination of cytoplasmic organelles in cells of different oviduct regions during their progressive atrophy after Kepone stimulation ceases. The second study will determine the detrimental effects of various concentrations of the estrogenic pesticide Kepone on the internal organization and ultrastructure of cells in quail testes. This study will determine the severity of low, moderate and heavy Kepone concentrations on spermatogenic process, sperm maturation and structure of interstitial cells. The third study will determine whether the damages in the testicular cells of the quail can be reversible and whether recovery of normal testicular morphology is possible after Kepone ingestion has been discontinued. This study will determine whether recovery of testicular morphology is complete after ingestion of mild Kepone dosage as well as whether damage is severe and permanent after ingestion of heavy Kepone concentration is discontinued. The data from this project will provide some important clinical and ecological information concerning the fate of reproductive capabilities of organisms, including man, exposed to various Kepone concentrations.


FOXO transcription factors extend lifespan and delay age-related disease in animals, and many studies have now linked FOXO3A DNA variants to exceptional longevity in humans. Thus, the time seems right to look for human genes that are likely to regulate FOXO-dependent, or other, longevity pathways. FOXO proteins can be activated in many ways to extend animal lifespan. For example, C. elegans FOXO can promote longevity in response to reduced insulin/IGF-1 signaling, altered serotonin signaling, and elevated AMP kinase activity, elevated heat-shock factor activity, elevated lin-4 microRNA activity, elevated Jun kinase activity and other inputs. Thus, there could be many gene perturbations that can extend healthy lifespan in humans; and some of these perturbations may be safer and more effective than others. Because it is not possible to do genetic screens for long-lived humans, we are doing genetic screens in human cells instead. Our experimental strategy is based on the observation that all FOXO- dependent life-extending pathways tested so far (as well as many other life-extension pathways) increase resistance to oxidative stress. Although the role of oxidative stress resistance in life extension is not clear, the correlation is tight enough that in many model organisms, screens for oxidative stress resistance have yielded long-lived mutants. Therefore, to obtain a set of potential human longevity genes, the Kenyon lab has carried out a genome-wide siRNA screen for oxidative stress resistance in a human primary cell line. The gene hits include known C. elegans FOXO regulators, regulators of other longevity proteins such as TOR and NRF2, and new genes as well. From this set, the Kenyon lab will identify good candidates for new human longevity and healthspan genes. To do this, they will determine which knockdowns trigger other correlates of longevity, such as xenobiotic resistance or autophagy. In addition, they will ask which knockdowns perturb the activities of FOXO3A, TOR or NRF2. Finally, to link these genes to longevity, they will test for their ability to influence lifespan in C. elegans and for their altered expression in centenarian families. This fresh approach will define new potential drug targets for extending the youthful and productive years of human life, and for delaying age-related diseases such as cancer, heart disease and/or neurodegenerative disease. PUBLIC HEALTH RELEVANCE: The wonderful finding that changing specific genes can extend healthy lifespan and counteract age- related diseases in animals makes a search for human longevity genes imperative. This study takes a fresh approach, by looking for genes that, when altered, give cultured cells from human's properties characteristic of cultured cells from healthy, long-lived animal mutants and from very long-lived animal species. This study will define new entry points for extending the youthful and productive years of human life, and for delaying age-related diseases such as cancer, heart disease and neurodegenerative disease.


We are requesting the continuation of a training program in theoretical neuroscience with funds to support 4 predoctoral trainees. Our goal is to train students who combine exceptional skills in mathematics, statistics, modeling and machine learning with a deep understanding of neurobiology. The complexity of neural systems and of the data that we can now obtain demands researchers with these skills if we are to realize the neuroscience community's goals of achieving a mechanistic understanding of nervous system function and making significant progress in the treatment of neural disorders and mental illness. Training will occur at the Center for Theoretical Neuroscience at Columbia University, supported by the 6 faculty at the Center, faculty visitors to the Center, and 26 other faculty from the Departments of Neuroscience, Psychology, Biology, Biochemistry, Biomedical Engineering, and Statistics at Columbia. The Theory Center provides an exceptional environment in which pre-doctoral, post-doctoral and faculty researchers interact and collaborate extensively both within the Center and with researchers in experimental laboratories. Most trainees are members of the Columbia graduate program in Neurobiology and Behavior (with a small number drawn from other graduate programs) and take the courses that satisfy the requirements of that program. The required courses are augmented by a large selection of electives, including courses in theoretical neuroscience, statistics and machine learning. The goal of this training program is twofold: 1) To produce theoreticians who combine outstanding skills in analysis and model-building with a deep understanding and sense of biological neuroscience; and 2) To train experimentalist who are skilled at applying theoretical and computational methods in their research. This will be accomplished through extensive collaborations with outstanding experimental laboratories both at Columbia and elsewhere combined with training in state-of-the art theoretical methods. Whenever possible, students will be co-advised by both a member of the Theory Center faculty and a researcher from our associated experimental faculty, and they will be given desk space both in the Theory Center and in the laboratory of their experimental co-advisor. The opportunity to be involved in the operations of an outstanding laboratory and in the activities and intellectual atmosphere of a world-class theory center provides an exceptional training experience.


The maternal-fetal interface is critically important since is at this level that uteroplacental and fetoplacental blood flows are regulated and dysfunctions are seen in diseases of pregnancy, e.g. preeclampsia. Vasodilation is controlled by PGI2 and Nitric Oxide (NO) of endothelial origin and locally shows obligatory requirements for cell-cell communication via Gap junctions, a common theme in this PO-1. Uterine Artery Endothelial Cells in Pregnancy (P-UAEC) and HUVECs undergo Programmed Adaptation to maintain NO production via a Connexin (Cx) 43 mediated mechanisms. HYPOTHESIS Aim 1: Endothelial Programmed Adaptation is modulated by the Gap junction protein Cx43 that is elevated directly by Shear Stress via cAMP and/or cGMP to maintain vasodilator (PGI2 and NO) production. Purpose Aim 1: To determine in P-UAECs, HUVECs and HUAECs the effects of: 1) acute vs. prolonged Laminar Shear Stress on levels of phosphorylated and total Cx43 & eNOS and vasodilator production (PGI2 and NO) relative to cyclic nucleotide (cAMP and cGMP) adaptation; 2) inhibition of PKA, PKG, Adenylate Cyclase and sol Guanylate Cyclase; and 3) Indomethacin and L-NAME on these shear stress responses. HYPOTHESIS Aim 2: These important shear stress-mediated vasodilator mechanisms are abrogated by patho-physiologic levels of representative cytokines elevated in preeclampsia [VEGF and Tumor Necrosis Factor] via functional Gap junction disruption. We hypothesize that shear stress elevates cAMP /cGMP which is protective for maintaining vasodilator production via Gap junction assembly. Purpose Aim 2: To determine in P-UAECs, HUVECs and HUAECs if these cytokines disrupt the shear stress-associated cyclic nucleotide Gap junction-mediated vasodilator mechanisms we will evaluate: 1) effects of treatment with VEGF and TNF on the acute and prolonged laminar shear stress responses; 2) the therapeutic protective effects 8-Br-cAMP and 8-Br-cGMP on VEGF and TNF disruption of shear stress responses; and 3) the therapeutic protective effects cAMP phosphodiesterase (PDE)2, cAMP PDES, cAMP PDE4, or cGMP PDE5 inhibitors on VEGF and TNF disruption of shear stress responses. These data provide the first mechanistic framework for understanding the interactions between shear stress-mediated cyclic nucleotides for therapeutic protective actions and Gap junctions to regulate endothelial vasodilator (PGI2 & NO) production and it's abrogation via cytokine (VEGF & TNF) mediated disruption of cell-cell communication.


Antimacrophage serum and indomethacin are each capable of blocking the ability of endotoxin to inhibit the hormonal induction of certain liver enzymes of short half-life such as tryptophan pyrrolase and phosphoenol pyruvate carboxykinase. These studies will continue in an effort to identify the role macrophages play in the process. The ability of endotoxin to interfere with the binding of glucocorticoids to liver cells will also be investigated. These experiments will be carried out first in intact mice and subsequently in hepatoma cell cultures. The contribution of endotoxin to the pathogenesis of experimental murine typhoid will be evaluated by determining changes in infected mice that parallel alterations characteristically induced by endotoxin.


Fibrinogen (340 kDa) is a multifunctional plasma protein that after activation spontaneously polymerizes to form a fibrin gel. Fibrin prevents the loss of blood upon vascular injury and serves subsequently as a provisional matrix on which various cell types adhere, migrate and proliferate during wound healing and other physiological and pathological processes. The ability of fibrinogen to polymerize and degrade in a controllable fashion and to adhere to different cells makes it an ideal bioadhesive (fibrin sealant) that has been used increasingly in numerous surgical application for arrest of bleeding, gluing of tissues, delivery of bioactive substances, etc. Better understanding of the mechanisms underlying fibrin-dependent processes and more effective application of fibrin sealant require comprehensive knowledge of the structure and interactions of this complex molecule. Recent studies established a high resolution structure of about two-thirds of the molecule. The first aim of this application is to complete the high-resolution structure of the remaining regions and to further clarify conformational changes upon conversion of fibrinogen to fibrin. Our recent discoveries that fibrinogen alphaC-domains interact with high affinity with plasminogen, tPA and apo(a) suggest their involvement in fibrinolysis and atherogenesis. The second aim is to characterize these interactions and to clarify their role in regulation of these processes. Interactions of the other fibrin(ogen) regions, gamma-modules and the betaN-domains, with receptors on leukocytes and endothelial cells was shown to stimulate inflammation and angiogenesis. The third and fourth aims focus on further establishing the mechanisms of these interactions. These aims will be accomplished by preparation of various fibrin(ogen) domains and combinations thereof by limited proteolysis and recombinant techniques, and studying their structures and interactions by biochemical and biophysical methods. These studies will generate basic knowledge that will have an impact on the understanding of and ability to control the above mentioned fibrin(ogen)-dependent processes. They will also contribute to the development of improved fibrin sealants with desirable properties.


The overall goals of this grant proposal is to determine role(s) of lens proteinases, their cognate inhibitors and proteolytic products of crystallins in the formation of high mo. wt. (HMW) proteins during the development of senile cataract. In this study, human lenses of different ages and cataractous lenses will be utilized. The following four specific aims will be pursued: (1) Regulation of proteolysis in lens: The regulation of proteolysis in the lens at the enzyme and protein substrate levels will be determined, i.e. by searching for zymogens and/or proteinase-inhibitor complexes of two lens proteinases, i.e.a 25 kDa and a membrane proteinase, and examining the effect of oxidation on endogenous proteinase inhibitors and the preferential proteolysis of native vs. oxidatively modified crystallins by endogenous proteinases. (2) Origin of degraded polypeptides from crystallins: The identity of "parent crystallins" from which the three in vivo found degraded polypeptides of human lenses, i.e. 5.5 kDa, 9 kDa and 15 kDa, originated will be established by comparing their amino acid sequences. Lens crystallins will be proteolyzed in vitro with the two lens proteinases (25 kDa and a membrane proteinase) and proteolysis products compared with similar polypeptides found in vivo for their Mr's and partial N-terminal amino acid sequences. In addition, the origin of the degraded polypeptides from crystallins by a non-enzymatic reaction (Fenton reaction) will also be examined. (3) Cross-linking of degraded polypeptides to form HMW proteins: The mechanism of cross-linking to form HMW proteins in vitro by degraded polypeptides per se or between these polypeptides and individual crystallins will be determined. (4) Search for and quantitation of cross-linked products of degraded polypeptides in HMW and water insoluble proteins of normal aging and cataractous lenses. Antibodies raised against the specific cross-linked degraded polypeptides will be utilized to identify and quantitate the levels of aggregates of these polypeptides by Western blot analysis and a radioimmunoassay method respectively. The above studies will show: (i) how the lens proteolytic process is regulated in vivo; (ii) whether the degradation of crystallins in vivo is enzymatic or non-enzymatic, and (iii) if the degraded polypeptides cross-link per se and with individual crystallins to form the HMW proteins that exist in increasing levels in aging and cataractous lenses.


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. This project focuses upon Infrastructure development in Computational Biology and Bioinformatics as part of the Core Resources at the Center for Biomedical Research (CBR) at Tuskegee University. Research involving advanced information technologies, the Internet and databases is crucial in examining and analyzing patterns of disease occurrence, genetic traits and molecular level dynamics including AIDS research.


As a national resource for molecular biology information, NCBI's mission is to develop new information technologies to aid in the understanding of fundamental molecular and genetic processes that control health and disease. More specifically, the NCBI has been charged with creating automated systems for storing and analyzing knowledge about molecular biology, biochemistry, and genetics; facilitating the use of such databases and software by the research and medical community; coordinating efforts to gather biotechnology information both nationally and internationally; and performing research into advanced methods of computer-based information processing for analyzing the structure and function of biologically important molecules. To carry out its diverse responsibilities, NCBI: conducts research on fundamental biomedical problems at the molecular level using mathematical and computational methods maintains collaborations with several NIH institutes, academia, industry, and other governmental agencies fosters scientific communication by sponsoring meetings, workshops, and lecture series supports training on basic and applied research in computational biology for postdoctoral fellows through the NIH Intramural Research Program engages members of the international scientific community in informatics research and training through the Scientific Visitors Program develops, distributes, supports, and coordinates access to a variety of databases and software for the scientific and medical communities develops and promotes standards for databases, data deposition and exchange, and biological nomenclature


Numerous cell surface receptors transduce signals through heterotrimeric GTP binding proteins (G proteins). The alpha subunit of these proteins is a molecular switch, cycling between GDP-bound (inactive) and GTP-bound (active) forms. The purpose of this study is to characterize the intracellular regulation of G-protein-mediated signal transduction. GTPase activity of the alpha subunit is enhanced by a novel family of regulators of G protein signaling (RGS proteins), resulting in inhibition of Gi and Gq-coupled signaling. This project studies specifically the interaction between RGS proteins and G proteins and the resultant control of G protein function. RGS proteins demonstrate little specificity for Gi and Gq subunits in cell-free systems, yet they may discriminate between G-protein-coupled receptors (GPCRs) linked to the same G-alpha. To address the issue of receptor specificity directly, fusion proteins consisting of different GPCRs fused to a single G-alpha subunit were constructed and expressed in cell lines, and receptor-stimulated GTPase activity in the presence of RGS proteins is studied. Although no GTPase accelerating (GAP) activity of any RGS protein for Gs-alpha has been detected, RGS16 interacted unexpectedly with Gs-alpha and inhibited Gs-induced cyclic AMP (cAMP) generation in mammalian cells. Regulation of RGS16 activity was studied by examining whether the protein underwent post-translational modification. RGS16 shares amino terminal cysteine residues with RGS4 and RGS5 that were hypothesized to be sites of palmitoylation. RGS16 underwent palmitoylation, and palmitoylation-defective RGS16 mutants demonstrated impaired ability to inhibit both Gi- and Gq-coupled signaling in mammalian cells. RGS16 also underwent tyrosine phosphorylation in response to both GPCR or receptor tyrosine kinase (Epidermal Growth Factor, EGF) stimulation. Mutation of a conserved tyrosine residue blocked tyrosine phosphorylation of RGS16, and the tyrosine mutant was quantitatively less able to inhibit Gi and Gq- induced mitogen activated protein (MAP) kinase activation. RGS16 co- immunoprecipitated with the EGF receptor complex and inhibited EGF- induced MAP kinase activation. A novel RGS protein with a divergent RGS box was cloned, D-AKAP2, which also contains a protein kinase A anchoring (AKAP) domain. Expression of D-AKAP2 in human embryonic kidney (HEK)293 cells resulted in inhibition of signaling induced by Gs-coupled GPCRs but not those coupled to Gi or Gq. - GTP, G proteins, RGS proteins, signal transduction


Funding is requested in partial support of the 7th Biennial Conference of the American Society for Matrix Biology (ASMB), which is to be held from Oct. 12-15, 2014, in Cleveland, OH (ASMB2014). ASMB is a national society whose mission is to promote basic, translational, and clinical research on the extracellular matrix (ECM), cell-ECM interactions, and ECM-based therapies and devices, and to support professional development of the matrix biology research community. Its biennial conference provides an in-depth forum integrating new information on the genetics, molecular biology, biochemistry, pathobiology, and translational relevance of all aspects of ECM. Additionally, this conference provides a continuum with cell biology and intracellular signaling via an emphasis on cell-ECM interactions and ECM-driven signaling. This all-encompassing, matrix-immersion aspect of the ASMB conference is lacking in large society conferences and is distinct from the single molecule- or process-focused conferences available to the ECM community, including Gordon, Keystone and FASEB conferences. ECM and matrix receptor gene mutations lead to inherited connective tissue disorders, skeletal dysplasias, muscular dystrophies and blistering skin diseases, and ECM dysregulation is universally a major factor in acquired diseases, both acute and chronic, affecting all organ systems. ECM is especially relevant to skin and the musculoskeletal system, where it is a primary component of bone, cartilage and tendons. Among acquired conditions, the pathology of osteoarthritis, osteoporosis, aneurysms, cancer and metastasis involves ECM destruction, and all fibrotic conditions and aberrant wound healing feature an excess or dysregulated ECM. ASMB2014 has considerable translational relevance, especially to tissue repair and engineering, and provides the basic science foundation upon which to advance these fields. The ASMB Conference brings together scientists, clinician-scientists, post-doctoral trainees, and graduate students from diverse research fields and medical specialties to discuss the most recent advances in ECM biology and their relevance to human disease. The exciting 3-day program comprises a keynote address on protein phosphorylation by Dr. Jack Dixon, five plenary sessions and 18 concurrent sessions on focused topics that provide many opportunities for short talks by trainees. In addition to advancement of the matrix biology field, ASMB2014 demonstrates a commitment to providing opportunities for in-training and women matrix biologists and has specific mentoring components. These goals and activities of ASMB2014 are fully consistent with the mission of NIH and the intent of the R13 mechanism.


Despite recent progress in medical therapy, heart failure is one of the most common causes of death in western countries. Understanding the molecular mechanism mediating growth and death of cardiac muscle is fundamentally important and can potentially lead to better medical treatment for heart failure. This laboratory has been working on an enzyme termed mammalian sterile 20-like kinase 1 (Mst1) which plays an essential role in regulating growth and death of cardiac myocytes. Although this enzyme is activated in the heart in response to high blood pressure and during heart failure, the molecular mechanism by which Mst1 is activated in the heart is not well understood. The aims of this project are to investigate the role of putative regulators of Mst1, termed Ras-association domain family 1, isoform A (RassfIA) and K-Ras, in mediating the activation of Mst1, growth and death of cardiac myocytes, and the resulting impact on heart function. Genetically altered RassfIA and K-Ras mice will be subjected to transverse aortic constriction and used to study the role of RassfIA in the development of cardiac hypertrophy and heart failure. Echocardiography, hemodynamic analysis, post-mortem organ measurements, histology and biochemical analysis will all be used to determine and compare resulting cardiac phenotypes. The possibility that RassfIA can modulate cardiac fibrosis will also be addressed with a focus on NF-KB as a potential mediator. Cultured cardiac myocytes and fibroblasts will serve as tractable systems to allow for the examination of signaling pathways involving RassfIA and Mst1. This work will also seek to evaluate K-Ras as an upstream activator of this signaling pathway in the heart. The knowledge obtained from this investigation will further elucidate the mechanism of Mst1 regulation in vivo and should be useful for the development of better treatment for heart failure.


Project Summary: This application seeks to support the career development of Dr John H. Stewart, IV. His long-term goal is to attain the necessary skills to become a well-trained clinician-scientist with a focus in the treatment of metastatic colorectal cancer and eventually become competitive for independent funding support. This goal will be met by a structured career development plan as well as a well-designed research project. Dr. Stewart's career development strategy will involve continued engagement with his present mentoring committee and participation in a structured didactic course of study to fill gaps in his scientific background. Dr. Stewart will present his research findings at national meetings and publish his work in peer reviewed journals during the proposed career development period. In addition to the aforementioned career development plan, Dr. Stewart and his mentoring committee have devised a research project titled "Oncolytic Vesicular Stomatitis Virus (VSV) for the Treatment of Colorectal Cancer". The overall hypothesis of this research project is that local delivery of oncolytic VSV can effectively treat properly selected, metastatic colorectal tumors of the liver and peritoneum. This hypothesis will be tested by three specific aims. In Specific Aim 1, he will seek to identify the molecular determinants of host-cell permissiveness to VSV in colorectal cancer cells. In Specific Aim 2 he will define the apoptotic pathways activated by VSV in colorectal cancer cells. In Specific Aim 3 he will delineate the efficacy of loco-regional delivery of VSV in small animal models of metastatic colorectal cancer. The research proposed in this application will set the stage for further translational research by advancing the novel paradigm of local delivery of oncolytic VSV for the treatment of metastatic colorectal cancer. The Comprehensive Cancer Center of Wake Forest University provides an excellent environment for a mentored training program. This center incorporates expertise from a broad variety of disciplines and has extensive resources. Dr. Stewart has laboratory space that is fully funded by the Department of Surgery. He has the full support of his Division Chief to devote at least 75% of his time to the career development plan outlined in this proposal. Relevance: It is projected that 153,760 individuals will be diagnosed with colorectal cancer in 2007. Despite advances in early diagnosis, surgery and systemic therapy, an estimated 52,180 patients will die of metastatic disease this year. Clearly, new therapies are needed to treat advanced colorectal cancer. This project will attempt to address the need for novel treatments of metastatic colorectal cancer by exploring the efficacy of VSV in the treatment of this disease.


A set of comprehensive neuropsychological test batteries is used to provide a complete assessment of various cognitive and sensory function that can be related to damage or dysfunction in different regions of the e brain. The adult battery comprises tests designed to tap the following aspects of behavior: attention, executive function, language, memory, motor functions, orientation, selected sensory and perceptual functions, vigilance and visual-spatial functions. In addition, adults are given a test of general intelligence and. In some studies, subjects are administered a structured psychiatric interview. Modified batteries have been developed for the assessment of infants, preschool children, and children ages 6-16. The data provided by these batteries are being used to construct a neuropsychological theory of the elements of attention that may be applied to the neurological and psychiatric diagnostic groups under study in the LPP. The LPP has as its major focus disorders involving impaired attention, including schizophrenia, epilepsy, eating disorders, affective disorders, head injuries, and AIDs dementia complex. Comparisons are being carries out between the neuropsychological profiles of various groups of psychiatric patients and those of patients with known cerebral lesions in specified brain regions. Our data are also being used to delineate neurobehaviorally-defined subgroups within diagnostic categories, an undertaking aimed at reducing variability in psychiatric diagnosis, treatment, and outcome. The data provided by this protocol provide a complete behavioral assessment that may be integrated with concurrently gathered electrophysiological, neuroradiological, and biochemical information.


Some aspects of the karyotype are highly conserved in certain mammalian groups, while in other groups the karyotype can undergo extensive repatterning in a short period of time. While it is clear that chromosomal mutation represents a reorganization of the genetic material, the molecular basis and effects of such change are largely a matter of speculation. The suggestion has frequently been made that mobile genetic elements play a role in chromosomal rearrangement. This proposal investigates the role of repetitive sequences in karyotypic repatterning by studying the relationship between hypervariable repetitive sequences and karyotypic plasticity in different mammalian groups. Three mammalain groups will be investigated: cetaceans, which are karyotypically conservative; deer mice, which have undergone karyotypic orthoselection for pericentric inversions and heterochromatic additions; and equids, which have shown an extremely rapid and varied repatterning of the karyotype. Hypervariable DNA will be isolated from one species from each group by phylogenetic screening, and approach for identifying rapidly diverging repetitive sequences. The correlation between the amount and nature of hypervariable DNA and the karyotypic stability of each group will be investigated. Because the equids are so karyotypically unstable, the most derived equid genome will be further investigated. Repetitive elements from this group will be more extensively characterized. Equid DNA will be factionated by density gradient centrifugation, and these fractions will be used to determine whether repetitive families from the Hartmann's mountain zebra are non-randomly distributed in the genome. A clear perception of genome organization may be central to understanding development and the regulation of gene expression.


Hepatitis B Virus (HBV)is a hepadnavirus that is a major cause of acute and chronic hepatitis in humans. Three hundred million people globally are chronically infected with HBV, making HBV one of the most common human pathogens. HBV replication is not cytopathic, and evidence has shown that hepatic pathology is immune-mediated. Thus, understanding the pathogenesis of acute and chronic hepatitis B virus infection mandates understanding the immune responses underlying these processes. Natural hepadnaviral infections occur only in humans and other outbred species whose immune systems are poorly characterized and difficult to study. This proposal seeks to use a new transgenic mouse model of hepatitis B virus infection to identify mechanisms involved in immunopathogenesis of acute and chronic hepatitis B virus infection, including understanding the role of the innate immune response in these disease processes. It is our hope that in identifying mechanisms underlying these disease processes, we will be able to design specific immunotherapies to prevent and treat HBV-related liver disease. Our specific aims are: 1. To determine the mechanism by which non-classical NKT cells mediate acute experimental hepatitis in our transgenic mouse model of primary HBV infection; 2. To test the hypothesis that the early activation of non-classical NKT cells and/or NK Cells in our model of primary HBV infection can significantly influence the chronic phase of hepatitis in our disease model; and 3. To develop an in vitro model of non-classical NKT cell activation in response to Hepatitis B virus, which will establish a fundamental experimental system for identifying the mechanisms of non-classical NKT cell activation.


The overall goal of this proposal is to define new molecular and cellular mechanisms that enable vertebrate photoreceptors to adapt to constantly changing conditions of ambient illumination. We will focus on the massive light-driven translocation of major signaling proteins between the subcellular compartments of the photoreceptor cell, a phenomenon which has attracted overwhelming interest throughout the visual community over the past few years. We will continue to explore the mechanistic aspects and the physiological role of protein translocation and will extend our efforts to address the potential involvement of protein translocation in photoreceptor neuroprotection. The first two Aims are devoted to the translocation of arrestin. Our recent findings indicate that arrestin translocation is triggered at a critical threshold of light intensity by a specific signaling mechanism. The nature of this mechanism will be elucidated in Aim 1. The fact that arrestin is a key player in inactivation of the visual signaling cascade suggests that its light-driven translocation to the light- sensitive compartment of the photoreceptor cell, the outer segment, contributes to light adaptation by making light responses less sensitive and more rapid. This hypothesis will be directly addressed in Aim 2. The other two Aims are focused on the translocation of transducin. In Aim 3 we will follow up on our recent finding that a protein called phosducin serves as a trafficking chaperone for transducin translocation. We will test the hypothesis that the light-dark cycle of transducin translocation is regulated through the concurrent cycle of phosducin phosphorylation and dephosphorylation. Finally, in Aim 4, we will use the mechanistic information learned during the previous grant cycle to generate a mouse model in which transducin translocation is impaired but other transducin functions are preserved. We will utilize this model to explore the putative role of transducin translocation in the neuroprotection of the rod from metabolic and oxidative stress imposed on rods during the many decades of their regular exposure to light. The proposed experiments are relevant to understanding the molecular and cellular mechanisms that regulate normal photoreceptor activity, mechanisms that may be perturbed in several degenerative retinal diseases, both inherited and associated with aging. The studies proposed in this application address the molecular and cellular mechanisms that allow our visual system to efficiently adapt to the drastic variations in light intensity encountered during a normal day. We will explore the role of these mechanisms in the neuroprotection of the photoreceptor cells from the metabolic and oxidative stresses they experience during the lifetime of light exposure. Because these mechanisms are likely to be perturbed in a variety of degenerative diseases of the retina, both inherited and associated with aging, addressing these questions on a molecular level opens doors for developing strategies for disease prevention and future therapeutic interventions.


This study examines whether reductions in air pollution are associated with reductions in the prevailing mortality rates. The areas considered are census tracts that have "high" residential stability; this restriction reduces the effect of migration into and out of the area. Analysis of data from Buffalo will be completed by the end of the first year of the project. Next year, data from Detroit, Seattle, and the major cities of Ohio will be analyzed. Results to date from Buffalo support the hypothesis of an association between reduced air pollution levels and a reduction in the prevailing mortality rate; however, these results are not statistically significant. Consideration of the larger data sets will clarify this point. In addition to time trend analyses, the study is examining whether there are any unusual geographical clusterings of mortality rate changes and performing other exploratory analyses. The deaths are analyzed by cause of death to specify the nature of the putative air pollution and mortality association. Primary attention, of course, is given to lung and chronic heart disease.


Perfluorochemicals attracted widespread attention in 1966, when Clark and Gollan showed that mice could survive for prolonged periods when completely immersed in liquid perfluorochemicals equilibriated with O2 at atmospheric pressure. The exceptional O2-carrying capacity of perfluorochemicals has provided the basis for using physiolologic emulsions of perfluorochemicals as O2-transport fluids after hemorrhage or during surgery when blood transfusions are impossible or refused. Treatment with perfluorochemical emulsions (PFCE's) and O2-breathing also reduces myocardial damage after coronary artery occlusion, protects against acute focal cerebral ischemia after ligation of the cerebral artery, and minimizes injury after spinal cord compression, reflecting improving O2 delivery into ischemic tissues. Solid tumors have abnormal, insufficient vasculatures and, as a result, contain areas which are hypoxic because of transient or chronic deficiencies in blood flow. Hypoxic cells are very resistant to the cytotoxic effects of ionizing radiation and limit the efficacy of radiotherapy in achieving local control of some solid tumors. We found that administration of a perfluorochemical emulsion and a high-oxygen atmosphere increases the response of BAlll2 rat rhabdomyosarcomas and EMT6 mouse mammary tumors to radiation. This was shown to reflect a decrease in the proportion of hypoxic tumor cells, rather than a direct toxic or radiosensitizing effect of the PFCE. This project will examine further the clinical potential of this effect, using solid EMT6, KHJJ, and BAlll2 tumors to test the effects of PFCE's and O2-breathing on tumor cell survival curves, tumor growth, tumor cure, and tumor oxygenation. The effect of treatment with PFCE's on the efficacy of fractionated radiotherapy and chronic low dose rate irradiation will also be examined. The effects of exchange transfusions, producing high flurocrits and low hematocrits, will be tested. The toxicities and physiological effects of the PFCE/O2 treatments and the effects of the treatments on tumor growth and metastasis will also be examined. The effects of PFCE's on the radiation responses of several normal tissues (including lung, skin, liver, and the hematopoetic system) will also be studied. These data will be used to determine whether therapeutic gain can be obtained by adjunctive treatment with PFCE's and oxygen, to assess further the clinical potential of PFCE's as adjuncts to radiotherapy, and to identify any problems which may be encountered in the clinical use of these adjuvants.


The specific aims of the study are to recruit 200 obese individuals with type II diabetes and 200 obese individuals who have a positive family history of diabetes. These subjects will be randomly assigned to usual care or to behavioral weight control programs which include either structured diet, structured exercise or the combination. All subjects will be assessed at baseline, 6 months, 1 year and 2 years on measures of weight, glycemic control, coronary heart disease (CHD) risk factors, and eating and exercise behaviors.


The Division of Cancer Treatment and Diagnosis (DCTD) supports the development of novel diagnostic and therapeutic approaches for cancer by expediting the initial and subsequent large-scale testing of new agents, biomarkers, imaging tests, and other diagnostic and therapeutic interventions (radiation, surgery, immunotherapy) in patients. Within DCTD, eight major programs and a patient clinic work together to bring unique molecules, diagnostic tests, and therapeutic interventions from the laboratory bench to the patient bedside. Activities supported under this contract include: isolation and evaluation of natural products; molecular pharmacology; high-throughput screening of compound libraries; pharmacodynamic marker and pharmacokinetic assay development and utilization in clinical trials; drug toxicology research; information systems, technologies, and data science; drug discovery; radiochemistry and radioactive drug production; archival of clinical trial images, in vitro and in vivo translational research; repository support for natural products, patient-derived xenografts, tumors and biologic anti-cancer agents; a biopharmaceutical production facility; medical writers; nursing support services for clinical trials performed in the NIH Clinical Center, correlative studies in collaboration with biopharmaceutical companies; support for precision medicine trials; advanced genomics capabilities supporting tumor evaluation in pre-clinical models and clinical trials; patient biorepositories for NCI-funded clinical trials; nanotechnology characterization and evaluation.


The overall goals of this proposal are to determine the dynamic properties of the intermediate filament (IF) cytoskeletal networks in fibroblasts and epithelial cells as well as to determine the nature of the interaction between IF and the cell surface. IF dynamics will be studied in mitotic cells in which the IF system is depolymerized and in daughter cells in which IF are repolymerized. Specifically, studies of the molecular basis of the extensive remodeling of Type III IF networks containing vimentin or vimentin/desmin will be undertaken. The explosive depolymerization which typifies early-mid prometaphase and the rapid reassembly of IF which takes place during anaphase-telophase can be explained, for the most part, by the hyperphosphorylation of IF structural proteins. Two mitotic kinases have been found in BHK cells which are involved in the phosphorylation reaction. We are undertaking an extensive analysis of one of these kinases termed vimentin protein kinase (VPK). VPK is a complex of three proteins: a 65kD component, a 110kD component, and p34cdc2 which is the catalytic subunit of maturation promoting factor (MPF) and is known to play a major role in triggering mitosis. We plan to carry out experiments aimed at determining the functional significance of the specific site(s) phosphorylated by VPK and other kinases which act on Type HI IF proteins using a variety of cell physiological (e.g., microinjection), morphological (immunofluorescence, confocal, and electron microscopy), biochemical and molecular biological (the use of bacterially expressed proteins and transient transfection of cultured mammalian cells) techniques. We will also attempt to study the interphase dynamic properties of IF. For these studies we will utilize the microinjection of derivatized IF proteins such as biotinylated vimentin and keratin into live cells and to determine their fate in situ using confocal and electron microscopy. Similar approaches using x-rhodamine labelled IF proteins will be used to determine whether or not a steady state or dynamic equilibrium exists in interphase cells through the use of fluorescence recovery after photobleaching (FRAP) experiments. Studies are also described which are aimed at determining the biochemical basis of the binding of IF to cell surface associated desmosomes in epithelial cells. These studies involve the isolation and biochemical characterization of bovine tongue desmosome components and the determination of how keratin polypeptides bind to them. The information derived from our investigations should shed new light on the properties and functions of IF in mammalian cells. A better understanding of IF structure and function is basic to understanding the pathogenesis of a variety of diseases in which changes in IF occur, such as Alzheimer's disease, Parkinson's disease, various cancers, and alcoholic cirrhosis.


Oral clefts (including cleft lip, CL; cleft palate, CP; and cleft lip and palate, CLP) represent a complex and heterogeneous group of crainofacial defects that constitute a major public health burden due to their high prevalence. We propose an international collaborative study to test specific candidate genes and their potential interaction with environmental risk factors using the case-parent trio design. Since this case-parent design avoids the problem of confounding due to population stratification, it is most suitable for international collaborative studies where samples from genetically distinct populations are collected. We feel it is necessary to extend genetic epidemiologic studies of oral clefts into international collaborative efforts to accumulate adequate samples sizes in the shortest time possible. This will require extensive planning to insure similarity in ascertainment of cases, comparability of exposure data collected by each center, and high levels of quality control in the processing of DNA and typing of genetic markers. We are proposing to develop an international study of genetic epidemiology of oral clefts based on case-parent trios (plus sib controls, where available) at 5 centers: Johns Hopkins (USA), Singapore, Taiwan, Hong Kong and Japan. During the 2 year planning phase proposed here, we will hold two meetings among co- investigators to develop standardized research protocols to collect DNA and environmental exposure data from cases and their parent (plus available sib controls). The first meeting will be held in Baltimore, where the Hopkins group will share their experience in the genetic epidemiology of oral clefts. The second meeting will be held in Singapore to review how research protocols should be modified for maximum effectiveness in the Asian centers. After this planning phase, this group of collaborators will prepare and submit a grant to fund a full scale international study of oral clefts.


Prostate cancer (PCa) is the second most common cause of cancer-related death in men in the United States. While multiple molecular events contribute to prostate cancer progression, it has become increasingly evident that epigenetic changes play pivotal roles in regulating cancer development. Polycomb repressive complex (PRC) members maintain the gene expression status of a cell by epigenetically modifying histone proteins. Histone methyltransferase EZH2, an oncogenic PRC2 member, initiates the transcriptional repression by trimethylating histone H3 at lysine 27. We have shown that EZH2 is overexpressed in aggressive prostate and breast cancers, predicts disease outcome, and is required for cancer cell survival. In addition, our studies have shown that EZH2 down-regulates multiple tumor suppressors and a genomic loss of microRNA-101 results in unregulated expression of EZH2 in aggressive tumors. While the role of EZH2 in regulating protein-coding genes is known, its role in regulating microRNA (miR) expression has not been studied. MiRs are critical regulators of cellular functions and are commonly altered in cancers including down regulation of several tumor suppressor miRs. Thus, we hypothesize that transcriptional repressor EZH2 plays a key role in regulating microRNA expression by epigenetic silencing and EZH2-regulated miRs play critical roles in PCa progression. Our preliminary data suggest that EZH2 down regulates multiple miRs, including miR-203 and the miR-200a, miR-200bc family. MiR-203 and mir-200a, bc in turn regulate PRC1 members, BMI1 and RING2. Thus the aims of this proposal are to extend these findings and gain further insights into the regulation of miRs by EZH2 and the role of these miRs in PCa development. In order to accomplish these goals, in Specific Aim 1, we will investigate the role of EZH2 in regulating miR expression in multiple cell types. We will first perform miR profiling using RNA from prostate cell lines in which EZH2 expression is modulated. We will validate the EZH2 regulated miR expression in prostate tumor tissues and correlate them with EZH2 expression. In Specific Aim 2, we will investigate the role of EZH2-regulated miRs in PCa and their role in targeting the PRC1 members. In Specific Aim 3, we will investigate the role of EZH2-regulated miRs in prostate tumorigenesis. We will use select EZH2-regulated miRs that are directly repressed by EZH2 and characterize the consequences of their modulation using both cell line and in vivo models of PCa. Relevance to Public Health: Successful completion of the proposed work will provide evidence for an intricate network of miR and epigenetic regulators in PCa development and identify potential diagnostic and prognostic markers, which in turn may improve PCa therapy through better diagnosis and disease monitoring. Our results may ultimately provide credence for therapeutic "re-introduction" of EZH2-repressed tumor suppressor miRs in cancer. PUBLIC HEALTH RELEVANCE: Prostate cancer is one of the most common causes of cancer related death of men in the United States, yet the etiology of this disease is relatively unknown and current therapies have a significant failure rate for advanced disease. Oncogenic histone methyltransferase EZH2 which is overexpressed in aggressive prostate cancer is known to play essential roles in aggressive prostate cancer and our preliminary studies have found that it regulates critical tumor suppressive microRNAs in prostate cancer cells. Our proposed investigations will provide evidence for the role of EZH2 in regulating critical tumor associated microRNAs, suggest biological role of EZH2 regulated microRNAs and will identify potential prostate cancer biomarkers and valuable therapeutic targets for prostate cancer.


This proposal will determine the intracellular signaling pathways that are important for the replication of SMC after arterial injury. The first group of experiments will document the activation of the MAP kinase pathways, namely p42/44erk in the ballooned injured rat carotid arteries. Activation of this pathway will be inhibited in vivo with a topically applied MEK1 inhibitor and the effect on SMC replication quantitated. Furthermore, the effect of activation of the p46sapk on SMC growth by transfecting cells with the upstream activator of p46sapk and measuring their ability to replicate in response to known mitogens. The second specific aim will determine the role of mitogen-activated protein kinase phosphatases (MKP-1) on SMC replication in rat carotid arteries. Initially MKP1/2 expression will be documented at various times after injury. MKP-1 will be inhibited with pervanadate and its expression blocked with an antisense oligonucleotide. The replication of carotid artery SMC to known agonists will then be quantitated. The third aim will determine if the MAP kinases, p42/44erk are activated by FGF2 and PDGF. Rat arteries will be denuded of endothelium in a manner known to induce a minimal SMC replication, and then FGF2 and PDGF will be administered. The activity of p42/44erk and the expression of MKP-1 will be measured in the SMC of these arteries. The final group of experiments will document the time course of cyclin D and E activity and the presence of cyclin inhibitors, p21 and p27 at times after carotid injury. Further studies will evaluate the effect of p27 on SMC replication in vivo by inhibiting its expression with an antisense oligonucleotide and then measuring the ability of intimal SMC to respond to known mitogens. In other studies the inhibitor, p27 will be over expressed in SMC and its role in suppressing SMC replication, when challenged with a mitogenic stimulus, quantitated. These studies will provide important data on the signaling pathways thought to be active in SMC and determine if their activation is critical for SMC replication.


DESCRIPTION (provided by applicant: Diabetes is a disease of multiple organs responding to complex genetic and environmental factors. A complete understanding of insulin resistance and type 2 diabetes mellitus (T2DM) requires an integrative approach that asks how different cell types influence each other through hormonal, neural, and metabolic signals all in the context of extra-organismal stresses including overnutrition and disruptions in normal circadian rhythms. A goal of all studies proposed in this application is to explain normal and pathological metabolism in molecular terms, emphasizing both cell autonomous processes as well as those that depend on organismal integration. The Program Project brings together five outstanding investigators, each with considerable past success as an independent investigator, but each also with a genuine belief in the value of scientific collaboration. Each PI focuses on a specific organ system and how it interacts with other tissues and external stresses, and works in close communication other PIs who study related problems. In Project 1, Lazar addresses how resistin coordinates the multi-organ response to nutritional overload, in which an inflammatory response leads to adverse consequences in insulin target tissues and the cardiovascular system. In Project 2, Stoffers focuses on the response of the beta cell to the stress of peripheral insulin resistance, testing an intriguing model that connects transcriptional regulation to endoplasmic reticulum biosynthesis and cell growth. In Project 3, Ahima uses a mouse feeding entrainment model that mimics circadian disruption in humans to examine how the central nervous system influences hepatic metabolism. In Project 4, Kaestner also studies the response to a perturbed central clock, but in the context of how the hormonal milieu influences the transcriptional control of liver glucose metabolism. Lastly, in Project 5, Birnbaum also considers how hepatic metabolism responds to the stress of obesity, but also asks how it is normalized by the antidiabetic drug metformin. The projects are supported by three Cores that provide histochemical analysis, generation of genetically modified mice, and their metabolic phenotyping.


The periaqueductal gray (FAG) and the adjoining dorsal raphe nucleus (DRN) are mesencephalic cell groups that can act to control pain perception. A consensus has arisen that the PAG functions in initiating and implementing behavioral coping strategies to situations involving stress, fear or pain. The DRN is a major source of forebrain serotonin, which modulates many behaviors and has been implicated in the pathophysiology of depression. Control of pain by these two nuclei is likely a single element of a multimodal response pattern to stressful situations. Substance P (SP) is a neuropeptide well known for playing a role in pain transmission. When released, SP binds the neurokinin 1 (NK1) receptor and precipitates receptor activation and internalization. The NK1 receptor is enriched within the dorsal and ventrolateral FAG as well as the DRN. In this region, focal application of SF is antinociceptive, eliciting the local release of endogenous opioids. In addition, SP neurotransmission is associated with anxiety, cardiovascular adjustments and grooming behavior. Therefore the PAG and DRN represent potential sites where SP may influence several individual components of behavioral coping strategies. The proposed experiments will examine internalization of the NK1 receptor produced by exogenous and endogenous SP using immunohistochemical methods to gain insight into the role of SP neurotransmission in the FAG and DRN. The topography of NK1 internalization by these stimuli will reveal the potential overlap of neural circuits used in coping with these stimuli. In addition, the proposed AIMS will broadly establish the neural circuitry that SP engages to modulate these areas. That is, the hypothesis that enkephalin- or serotonin-containing neurons have the NK1 receptor will be tested using light and electron microscopic analysis. The results of these studies will-yield insight into how distinct modes of stressful stimuli impact SP neurotransmission within neural circuits that coordinate defensive coping strategies.


This proposal is an application for the continuation of Public Health Service Grant HL-11119. Its purpose is to make available a common amino acid analyzer and sequencer operation for eight research groups in the Departments of Biochemistry and Molecular Biology and Biological Sciences, by providing support for an operator and the supplies and maintenance for the available equipment. The present research projects dependent on these facilities include the mechanisms of enzyme catalysis (M. Bender); the isolation of antigenic peptides from lactate dehydrogenase C4 (E. Goldberg); the comparative biochemistry of oxygen-carrying proteins (I.M. Klotz); the structure and function of photoreceptor complexes in phogosynthesis (P. Loach); enzymes and proteins of coagulative processes (L. Lorand); the mitochondrial metabolic role of cytochrome c (E. Margoliash); the antigenicity of globular proteins (E. Margoliash); the biosynthesis of bacterial cell wall components (F.C. Neuhaus); the structure and function of prophobilinogen synthase (D. Shemin); and the mechanism of action and design of antitumor agents (R.B. Silverman). These studies are supported by the Public Health Service and the continued existence of a joint amino acid analysis and sequencer operation is the most economical way of providing for their prosecution while obviating the duplication of efforts and its resultant inefficiency.


The Cell Deposition System (CDS) deposits biological cells on predetermined sites on a microscope slide. With this improvement in flow microfluorometry (FMF) and cell sorting technology, it will be possible to associate an arbitrary cell on the microscope slide with its FMF data. Such correlation is possible by using a computer to store a cell's FMF data and concomitantly determine a cell's location on the slide. An initial study (Tyrer et al., Proc. IEEE 67:10, 1979) demonstrated the feasibility of this device. This proposal seeks to develop an improved CDS prototype which can be used in our laboratory for slide preparation of diagnostic samples. This requires a straight-forward instrumentation development task for a laboratory prototype; studies of the interaction between slide substrate, cell fixative, cell stain and cellular morphology; and studies of the interaction between substrate, cell nutrients and cells when dealing with viable cell systems. This will result in the reliable preparation of slides so that human visualization as well as automated cell localization and analysis can be accomplished with confidence. The prototype will be used in two separate applications to determine a wide range of utility in fixed or living cells. Applications in cancer detection include preparation of microscope slides by placement of cells in a highly ordered fashion for human or machine analysis. The slides will have only cells of interest and be relatively free of debris. The other application involves depositing viable cells in a mechanically stable cell culture medium. Individual cells can then be assessed for their tissue culture character. The CDS will permit true single cell analysis since the computerized determination of cell address and storage of FMF values can be used to drive a Scanning Transmission Microscope (STOM). The STOM, a computerized image analysis microscope system, can use the CDS-derived data and microscope slide preparations to locate and further analyze interesting cells.


We propose to develop an integrated microchip platform for rapid, reliable and comprehensive diagnosis of cancer by examining the pathophysiological mechanism of tumor-immune interaction. This will be done at both molecular and cellular level. By applying this platform to analyze a large number of clinical samples, we expect to gain new insights about cancer immunobiology that point to new approaches for cancer prevention and treatment. During the K99 phase, I have demonstrated an Integrated barcode platform that can measure a large number of protein markers from small quantities of whole blood or from single cells. A panel of signaling proteins that have important implication in tumor inflammation has been integrated Into the microchip platform. In the ROO phase, I will further utilize this technology to Investigate the fundamental biology of tumor-immune interaction via measuring the paracrine signaling pathways between tumor and immune cells. I will also study the heterogeneity of tumor microenvironment by combining population dynamics modeling and the microchip-based molecular analysis. Once this model is trained with experimental or clinical data, it will become a useful tool to predict the outcomes or therapeutic responses of cancer patients. In addition, an on-chip culture of tumor cells and immune cells will be developed to emulate tumor microenvironment and then used for rapid, effective anti-cancer drug screening. To accomplish these goals, I will (1) use the Integrated barcode chip platform to study chronic Inflammation as a common mechanism in various human diseases and to reveal their correlations; (2) apply this tool to monitor the Immunological responses of cancer treatment; and (3) design a Tumor-on-a-Chip to re-engineer the tumorimmune Interactions ex vivo and use this platform for effective anti-cancer drug screening with rich feedback.


The long term objective is to study the chemistry of the prosthetic group of visual pigments. For, although the spectroscopy of retinal, N-retinylidene n-butylamine (NRBA) and related polyenes has been examined in some detail, knowledge of the relationship between the chemistry and spectroscopy is not clearly understood. In order to correctly deduce the chemical role of the prosthetic group within the environment of the pigment protein, the chemistry of the prosthetic group in chemically defined experimental systems must first be probed. In this work, the hydrogen bonding (H-bond) and proton transferring reactions of CH3(CH=CH)5C=NC4H9 (compound I) and NRBA are to be studied both qualitatively and quantitatively. Some of the experimental variables to be studied are: i) donor reagents such as phenol, substituted phenols, acetic acid and chloro substituted acetic acids, ii) selected solvents such as hydrocarbons and chlorohydrocarbons, and iii) changing temperature. It was learned in prior studies that, in hydrocarbon solvents, when I is treated with a weak acid such as phenol, and when the temperature is progressively lowered, three separate spectra sequentially appear. These spectra belong to respectively: compound I, H-bonded I, and proton transferred I. This entire process can be placed in the formal framework of acid-base equilibria studies. The degree of conversion from one species to another will be obtained from changes in absorbance that will be used to find equilibrium constants. The resulting values will be used to determine the temperature dependent thermodynamic constants deltaH, deltaS. Compound I and NRBA undergo proton transfer with certain weak acids, and these reactions will be examined quantitatively in the same manner. The obtained thermodynamic constants for both reaction steps and the three excitation energies will link the three species together in a relative energy state diagram. From these results, it will be possible to measure the relative stability contributed to each ground and excited state by the selected donors and solvents.


The overall objectives of this grant are to determine the effect of cell surface alterations on the immunologic behavior of cells. Certain substances all appear to increase cell immunogenicity in appropriate doses so that many studies have involved the design of model systems for tumor immunotherapy using such altered tumor cells. In recent years, attention has turned less and less to the design of model systems for tumor immunotherapy than to the investigation of the mechanisms of the beneficial effects of tumor immunotherapy using active specific methods of immunization in vitro. Attention has particularly turned toward investigation of how these and other substances interact with cells and alter cellular behavior in vitro.


The purpose of this study is to evaluate the benefits of combined Glucotrol XL and acarbose therapy in patients with type II diabetes mellitus who cannot achieve adequate glycemic control with Glucotrol alone. The comparison of treatments will include evaluation of fasting plasma glucose, post-prandial glucose excursions, incidence and frequency of hypoglycemia, free insulin, lipids, and overall metabolic control. This is a randomized, double-blinded, parallel controlled trial involving 26 patients with type II diabetes mellitus who are hyperglycemic on the maximum dose of a commercially-available sulfonylurea. Patients will undergo screening laboratory evaluations (including CBC with differential, UA, OP16, stimulated C-peptide in response to a mixed meal, lipoprotein profile, total insulin, HBA1c, EKG, fasting plasma glucose, and Beta HCG for females of reproductive age) and a brief interview to determine if they qualify for study participation. Patients who qualify will proceed to visit 2 at which time a history and physical examination will be conducted and the patients will be instructed in a 60% carbohydrate diet. At that time, patients will be switched to Glucotrol XL who are not already receiving it. Following a lead in period of 6 weeks, patients must have a fasting plasma glucose >140mg/dl on the maximum dose of Glucotrol XL (20mg/dl) and a 60% carbohydrate diet in order to enter the study. The next visit (visit 3) will consist of an admission to the General Clinical Research Center (GCRC) at Duke University Medical Center at which time the following laboratory tests will be performed: fasting plasma glucose, HBA1c, C-peptide, total insulin, 1 hr post prandial insulin and glucose levels, 2 hr post prandial insulin and glucose levels, and lipoprotein profile. During this admission, patients will be randomized to one of two treatment arms. One treatment will consists of therapy with Glucotrol XL and placebo for 4 months. The other treatment consists of 4 months of Glucotrol XL combined with acarbose therapy. The initial dose of acarbose will be 25 mg tid, increasing to 50mg tid after 2 weeks and, if necessary, to 100 mg tid after an additional 2 weeks. Our end point will be a fasting plasma glucose under 120 mg/dl or maximum dose acarbose. Approximately 2 months following the admission, patients will return for visit 4 as an outpatient. The purpose of this visit is to assess the level of glucose control and to monitor for drug toxicity. Assessment will include examination of home blood glucose monitoring and the following labs: LFT's and HbA1c. Approximately 2 months after visit 4, patients will be readmitted to repeat the laboratory evaluations of the first admission. The study is ongoing, and results are not available to date. SIGNIFICANCE: Type II diabetes mellitus is a syndrome characterized by resistance to insulin and relative insulin deficiency. While some patients can be managed and euglycemia achieved using oral hypoglycemic agents alone, others require insulin (frequently in substantial doses). Correction of fasting hyperglycemia is a main concern, since it is often impossible to correct subsequent daily hyperglycemia without improving the fasting blood glucose. However, the use of insulin therapy in patients with type II diabetes is associated with several problems, including invariable weight gain and facilitation of atherogenesis. It is therefore desirable to develop other forms of therapy which might minimize insulin use in this patient population.


DESCRIPTION: (Adapted from the Applicant's Abstract.) The objective of the proposed research continues to be a comprehensive, systematic and parametric evaluation of the roles that spectral locus and temporal variation play in selected binaural experiments. By producing interaural temporal and intensive stimuli, we hope to extend greatly knowledge of how the human's ability to lateralize, to discriminate and to detect is affected by the spectral region and spectral compositions of sounds that are processed via binaural interactions. One subset of experiments concerns lateralization and will evaluate, as a function of spectral locus and temporal variations, how extent of laterality is affected by combinations of interaural time-delays, phase-shifts, and intensive differences. These data will be used to extend a new model, the "weighted-image" model to incorporate interaural intensive differences across spectral regions. Other lateralization experiments will assess the relative potency of onset/offset and ongoing interaural delays in combination with interaural intensive differences and how each affects the intracranial locus of acoustic images. Another subset concerns the detection and discrimination of interaural delay when a narrow spectral region containing the delay is known vs. when the listener is uncertain with respect to its spectral location. Another group is designed to assess discrimination of interaural delay when the base or initial delay is varied. Narrow bands of noise will serve as targets and these experiments are crucial for determining how well relative discriminations can be performed for high-frequency stimuli. The potential health benefits are a better understanding of how the ear and brain process information and a potential for "better" diagnostic procedures that may, eventually, have clinical significance.


Purkinje neurons and granule neurons are two major cell populations in the cerebellar cortex. Purkinje neurons are well recognized by the size and complexity of their dentritic arbors. The numerous spines on the arbors serve as postsynaptic sites for reception of afferent input from the parallel fiber axons of granule neurons. The presynaptic axonal membranes, associated vesicles containing neurotransmitters and the thickened postsynaptic spinous membranes form structural complexes called synapses. The total number of synapses on Purkinje dendritic arbors of normally aging rats was known to be significantly reduced after chronic ethanol treatment, an effect that was reversed after recovery from the ethanol treatment. There were no ethanol-induced decreases in the number of parallel fibers because the number of granule neurons did not change. The overall size of the dendritic arbors was unchanged, but dendritic segments within the arbors were longer than normal following ethanol treatme nt, suggesting that some segments and their branch points had been deleted. The purpose of this study was to determine whether the loss of synapses represented a direct effect of ethanol on the density of spines (spines/nm) on the dendritic segments or an indirect effect of segment deletion. Spine densities on terminal dentrites were determined in Purkinje neurons from 40 old male Fischer 344 rats that were assigned randomly to one of three treatments groups, a chow-fed control group (n=8), an isocaloric and isovolumetric (pair-fed) control group (n=16), or an ethanol-fed group (n=16). Brain tissues were prepared by the Golgi-Cox procedure for light microscopy and coded to avoid investigator bias. 14 Purkinje neurons from each rat (a total of 560 cells) were randomly selected for quantitation of spines. In each neuron, spine counts and terminal segment lengths were determined for 14 paired and 14 umpaired terminal dendritic segments (a total of 15,680 measurements). Group averages tested by ANOVA showed no statistically significant decreases in spine density on spiny branchlets of Purkinje ne urons after chronic ethanol consumption. It was concluded that ethanol did not reduce the density of spines on dendritic segments of Purkinje neurons directly and that the decrease in synaptic density after ethanol treatment represented an indirect effect of segment deletion. (Funded by NIH/NIAAA grant AA05592) Keywords: Cerebellar Cortex, Ethanol, Golgi Cox, Purkinje Neurons, Synaptic Spine


The long range goal of this project is to use functional magnetic resonance imaging (FMRI) to elucidate the brain mechanisms responsible for normal vision and for brain-related visual pathologies. As a new technology for studying brain function, FMRI is potentially revolutionary because it can produce high resolution pictures and movies of dynamic brain activation during sensory input, task performance, or cognitive activity. Since it is non-invasive and has no known health risks, hundreds of images can be obtained from a single subject, thus permitting detailed and thorough studies of brain function in both human and animal subjects. This project seeks to develop and test this technology further and then apply it to the study of visual perception. Fortunately, the neural basis of vision in monkeys is sufficiently well understood to provide a solid background for interpreting the results of FMRI experiments and for comparing and interrelating visual mechanisms in human and simian species. This will permit the knowledge gained in previous animal experiments to be applied more directly to the study of the human nervous system. To this end, the specific aims of the project are: 1) to determine the parametric relationships between the FMRI signal and visual stimulation, 2) to use FMRI to study the functional specialization of cerebral cortex by creating maps of topographic and functionally selective activation within and among different cortical visual areas in both humans and monkeys, 3) to use FMRI to record and analyze the temporal and spatial evolution of cortical activity during prolonged visual tasks, and 4) to explore the potential application of FMRI in the assessment of visual deficits resulting from cortical pathology. To accomplish these aims, a high magnetic field strength MR scanner (Bruker 3 Tesla) will be used to image cortical activation during computer controlled visual stimulation delivered via a custom designed optical system. Using novel computer algorithms, the 3-dimensional patterns of activation will be displayed on unfolded, two-dimensional maps of the cortical surface. Ultimately, these techniques will be used to study abnormal brain activation in a small number of patients with well characterized visual deficits. The goal will be to explore the potential use of FMRI in the assessment of brain pathology. The results of this project will provide new insight into the relationships between physical brain mechanisms and both normal and impaired perceptual experience. In addition, the testing and refinement of the FMRI technology will contribute significantly to the development of a powerful new tool for medical science.


Moderate alcohol intake is thought to be cardioprotective. The postulated mechanism has always been the elevated high-density lipoprotein cholesterol concentration seen in alcohol consumers compared to nondrinkers. Recent studies have raised the possibility that insulin sensitivity may be mediating the effect between moderate alcohol consumption and lowered cardiovascular disease (CVD) risk. This study therefore has the following aims: 1.To assess whether the daily consumption of a moderate amount of alcohol can improve insulin sensitivity in a volunteer group of nondrinkers predefined as insulin resistant. 2. To determine if moderate alcohol intake by improving insulin sensitivity will have the beneficial metabolic outcomes in HDL, insulin and glucose to lower CVD risk. 3. To investigate if measures of endothelial function (asymmetric dimethylarginine and soluble cellular adhesion molecules) as risk factors for CVD are affected by moderate alcohol consumption and insulin sensitivity. Insulin sensitivity/resistance will be measured by the insulin suppression test. Given the morbidity and mortality of insulin resistance and CVD, it becomes important to clarify the role of moderate alcohol consumption to both.


Group A Streptococcus (GAS, also known as Streptococcus pyogenes) is a common human pathogen, most frequently causing pharyngitis or "strep throat." However, severe invasive infections like necrotizing fasciitis and toxic shock syndrome are also caused by this organism, leading to its nickname in the lay press - "flesh-eating bacteria." This work will help explain the process by which a microbe in the throat can invade and cause life-threatening illness. Previous studies found that GAS alter transcription of multiple virulence factors in response to the human cathelicidin, LL-37, an antimicrobial peptide. A membrane-bound sensor kinase (CsrS) and its cytoplasmic response regulator (CsrR) appear to drive this reaction. This application aims to examine the role of LL-37 in stimulating an invasive phenotype in GAS. We will focus on the interaction of GAS with two host cell types commonly encountered early in infection: keratinocytes and macrophages. We will examine the role that LL-37 and CsrRS play in GAS's ability to avoid internalization and killing. Overall, these studies will examine a fascinating evolutionary interaction between a human pathogen and the innate immune system, offering new insights into the pathogenesis of GAS infections. PUBLIC HEALTH RELEVANCE: The public health burden of GAS infection remains high. Sequelae of infection, including rheumatic heart disease, continue to plague especially developing areas, where prompt antibiotic therapy is unavailable. Additionally, there has been a rise in invasive GAS infections as a new, more virulent strain has spread around the globe. For these reasons, improving our understanding of the earliest stages of human infection will be essential to battling this common yet evolving pathogen.