The Centers for Disease Control estimate that approximately 1.7 million people per year sustain a traumatic brain injury (TBI), resulting in an estimated 52,000 deaths. The age groups most likely to sustain a TBI are infants (0-4 years old), adolescents (15-19 years old), and the elderly (65 and older). TBI results in the alteration of many signaling pathways and biological processes. These processes contribute to a variety of deleterious pathophysiological sequelae which significantly affect patient morbidity and mortality, including cerebrovascular dysfunctions such as ischemia and increased susceptibility to secondary ischemia, cerebrovascular edema, and increased blood-brain barrier (BBB) permeability. MicroRNAs (miRNAs) are ubiquitous regulatory RNAs that modulate gene expression at the post-transcriptional level by inhibiting protein translation and/or promoting mRNA degradation. MiRNAs play critical roles in regulating many important biological processes, including vascular integrity. We have shown that miRNA expression levels are altered after a controlled cortical impact (CCI) model of TBI, and these altered miRNAs are predicted to regulate the expression of gene products involved in many pathophysiological processes affected by TBI. MiR-223 expression levels in cerebral microvessels are significantly increased 24-72 hr post-TBI, a time period when peak BBB permeability and cerebral edema is observed. Furthermore, preliminary evidence shows that miR-223 directly targets T-lymphoma invasion and metastasis (Tiam1), a central regulator of actin dynamics that impacts endothelial barrier integrity and adherens and tight junction function. We propose to test the hypothesis that increased miR-223 expression in brain microvascular endothelial cells contributes to BBB dysfunction, and blocking miR-223 activity after TBI will improve BBB integrity. The experiments in this grant proposal employ a combination of molecular biology and biochemical approaches using genetic knockout, in vitro, and in vivo model systems to (i) determine the impact of altered miR-223 expression on tight junction protein expression, localization, and barrier function in primary rat brain microvascular endothelial cells, and (ii) evaluate the impact of miR-223 knockout on BBB compromise after TBI. Examining the functional consequences of altered miRNA expression in complex systems is hampered by a lack of methodologies for acutely manipulating miRNAs in vivo. We propose to utilize the rabies virus glycoprotein (RVG) peptide, which selectively transfect cells that express the nicotinic acetylcholine receptor, including vascular endothelial cells, as a novel targeting strategy to transiently manipulate miR-223 in the brain vasculature in vivo. These studies will contribute to our understanding of BBB regulation after CNS trauma, and help characterize a novel method for in vivo manipulation of miRNA levels. Furthermore, if successful these studies will enable future investigations into the effects of transient changes in miRNA expression on protein regulation in vivo, opening up a previously unexplored area of pathophysiological processes initiated after TBI.


Astrocytes are essential for neuronal survival and function. Yet every neurodegenerative disease and every injury to the brain and spinal cord results in "activation" and proliferation of astrocytes, a process termed astrocytosis, which adversely affects neuronal survival and function. Thus, the astrocyte is a two-edged sword, supporting homeostasis in health, but, in pathologic conditions, their activation results in neuronal loss. As an example, the scar that forms in the weeks following stroke is caused by astrocyte proliferation, which further damages neurons, preventing recovery and increasing disability. In chronic neurological diseases, such as Multiple Sclerosis, there is a progressive astrocytosis and a corresponding progressive loss of neurons. GliaMed, Inc., a biotechnology company dedicated to using our proprietary, patent-protected technology to treat a range of neurodegenerative diseases and astrocytoma, has taken the approach that understanding The molecular mechanisms of homeostasis will, by definition, identify important and novel therapies. With this as its scientific cornerstone, and supported by more than a decade of federal and foundation research grants awarded to the Company's scientific founder, the PI on this application, GliaMed has identified both cellular and molecular targets for the effective treatment of a range of conditions that result in loss of CNS homeostasis. In specific, we demonstrated a number of years ago that astrocytes, one of the major celt types in the CNS, are sustained out of the cell cycle by contact with a protein component specific to the neuronal cell-surface. We have recently identified the astrocyte-expressed receptor, termed GMg, and its neuronal ligand, NrS1, that mediate both forward and reverse signaling between these cell types, that results in a number of biologies, including astrocyte cell-cycle arrest. In this application, we provide data elucidating aspects of these interactions, and describe our lead compounds. Further, we provide evidence that these compounds which are based on GM9-NrS1 binding, rescue neurons from programmed cell death and promote axogenesis, both in vitro and in vivo. The overall Specific Aim of this application, based on Preliminary Data provided herein, is to optimize these compounds for in vivo stability and saturation of target sites within the CNS. These data will support the transition of the GliaMed lead compounds from preclinical to clinical development.


Inactivation by hydrolysis of cyclic AMP and cyclic GMP is probably catalyzed by two separate cyclic nucleotide phosphodiesterases. Attempts will be made to separate the two enzymes physically with minimal loss prior to assay or to find means of assaying the two activities in crude extracts by use of selective inhibitors. We will search for hormonal regulation of the quantities or kinetic properties of the enzymes. Brattleboro rats (congenitally lacking vasopressin) will be injected either with vasopressin in oil or the oil alone. The amount and characteristics of the phosphodiesterases in whole rat kidney and various subregions will be determined. Other variables which affect renal concentrating ability will be studied in similar fashion. These include serum levels of Ca ions, body potassium, drugs (especially thiazide diuretics), thyroid status, protein intake.


The purpose of the Mathematical and Statistical Analysis and Modeling Core is to provide an efficient organization for the quantitative support required by all projects in the Center. The range of support needed includes experimental design and data analysis, statistical modeling, mechanistic model formulation, stochastic modeling, and model solution. Specific applications include models of fluid flow and contaminant transport, remediation of contaminated environments, exposure assessment, and statistical and physiologically based pharmacokinetic modeling. The Core comprises a quantitative, interdisciplinary team with backgrounds in deterministic and statistical modeling, biostatistics, applied mathematics, and computer science. Collectively, this group will provide quantitative support through application of known methods and approaches. It will also provide leadership in method development for areas of investigation in the Center with particular need for innovative modeling approaches.


This project involves a continuing effort to develop an understanding of the role of cyclic nucleotides (cyclic AMP and cyclic GMP) in Escherichia coli and the mechanism by which the levels of these nucleotides is regulated within the cells under a variety of conditions. Our findings this year have dealt with the regulation of the activity of adenylate cyclase by the phosphoenolpyruvate linked phosphotransferase system for transport of sugars (PTS). Mutants in one of the proteins of this transport system (Enzyme I) lead to a depression of cellular cyclic AMP levels. Under appropriate conditions, the levels of cyclic AMP in these mutants can be restored to normal by exposure to the phosphorylating agent, phosphoenolpuyrvate. These data have been interpreted in the framework of a model for regulating adenylate cyclase activity by interaction with proteins of the PTS, such that the state of phosphorylation of the PTS proteins determines the activity of the enzyme.


Novel Regulators of Vascular Disease Between 20 and 90 years of age, arterial wall intimal-media thickness increases, promoting arterial stiffness. Increased conduit artery stiffness is a key factor in the pathogenesis of aging-associated diseases such as systolic hypertension, cerebrovascular, cardiovascular, renovascular, and peripheral vascular diseases - all represent a significant burden affecting the Veteran population. This VA-PPA is a unique opportunity to bring together four VA investigators to study the pathobiology of conduit artery stiffness. A series of three inter-related projects and a state-of-the-art animal vascular phenotyping core facility will focus on the common theme of vascular dysfunction associated with matrix protein deposition and medial calcification, two factors known to increase arterial stiffness during aging and chronic kidney disease (CKD). The specific objectives of the three Projects and Core are to: 1. Define the mechanisms by which the aging process reduces endothelial nitric oxide (NO) synthase function and alters vascular production of Transforming Growth Factor-ss (TGF-ss) to facilitate the development of arterial stiffness with aging (Project 1: Molecular Mechanisms of Aging on Vascular Function; PI: Paul Sanders). The working hypothesis of this project is the ratio of active TGF-ss to NO is a critical, modifiable determinant of arterial stiffness of aging. 2. Determine the role of Runx2 in regulating arterial stiffening and elucidate the molecular mechanisms, using a novel smooth muscle-specific Runx2 knockout mouse model (Project 2: Molecular Mechanisms Underlying Arterial Stiffening; PI: Yabing Chen). The working hypothesis of this project is that a high salt intake induces the expression of Runx2 in VSMC, which initiates VSMC calcification and promotes arterial stiffening. 3. Define the role of the heme oxygenase-1 (HO-1)/ferritin system in the prevention of vascular calcification through downregulation of Runx2 and TGF-ss ((Project 3: Role of HO-1/Ferritin in Vascular Calcification During Aging; PI: Anupam Agarwal). Experiments will test the working hypothesis that induction of the HO-1/ferritin system prevents vascular calcification through downregulation of Runx2 and TGF-?. 4. Support a state-of-the-art core facility (Animal Vascular Phenotyping Core; Director: Edgar Jaimes) designed to provide detailed structure-function analyses of arterial stiffness and vascular calcification. The core will consist of a vascular physiology subcore for radiotelemetry, high frequency vascular ultrasound, micro-CT and endothelial function (myography); and a molecular pathology subcore for histology, immunohistochemistry and image analysis. The core will bring to the Birmingham VA Medical Center (BVAMC) new technology to study vascular biology. The long-term goal of this research effort is to lay the essential groundwork necessary to translate the findings to improved cardiovascular outcomes in aging and CKD and further build the research capacity at the BVAMC.


The specific etiology of a majority of cases of keratitis and uveitis, as well as the exact immunologic mechanism of corneal graft rejection is unknown. Yet these entities frequently cause significant visual loss and blindness. The purpose of the proposed project is to elucidate the effector mechanisms involved in the immunopathology of anterior segment ocular inflammation (including graft rejection) and to test experimentally the efficacy and side effects of new therapeutic approaches. These studies will deal with two major subjects: 1) the role of soluble mediators ("lymphokines") of cellular immunologic reactions in ocular inflammatory disease, and 2) the use of blocking antibody serum (BAS) in the prevention of immunologic corneal graft rejections. Lymphokines will be produced by guinea pig lymph node cells and purified and characterized. The lymphokines will then be placed in contact with eye tissue both in vivo and in tissue culture. The ability of lymphokines to cause tissue injury in the presence or absence of inflammatory cells will be investigated. An antibody will be made to a purified lymphokine preparation and its ability to block or suppress lymphokine-mediated tissue damage will be evaluated. Guinea pig anti-rabbit lymphocyte serum will be prepared and modified chemically so that it will not bind complement. The characteristics of modified BAS and the mechanisms by which it protects corneal grafts from rejection will be studied by in vivo and in vitro studies.


The overall goal of this project is to gain a detailed understanding of the molecular function of integrins, a large family of cell surface receptors. Integrins are involved in many developmental processes and also function in numerous fully differentiated cells. With respect to known pathological conditions, most studies of integrins have focused on the circulatory system, the immune system, and the metastasis of tumors, however it is likely that integrins will prove to be important in pathologies in other tissues and diseases as well. The PS integrins of Drosophila are very similar to vertebrate integrins, and provide a unique opportunity to examine integrin function in situ in a complex developmental system. We propose to: (1) Further define the interaction of PS2 integrin with the fly matrix protein tiggrin, using our cell spreading assay and genetically engineered fragments of the tiggrin protein. Also, begin steps to generate mutations in the tiggrin gene, in order to undertake a genetic analysis of tiggrin function during development. (2) Define the requirements for PS l integrin during development, using newly generated mutations in the mew gene, which encodes the alphaPS1 integrin subunit. This study will focus on Comparisons between the myospheroid (betaPS), inflated (alphaPS2) and mew phenotypes, and include analysis of genetic mosaics. (3) Continue to generate and characterize hypomorphic mutations of myospheroid, and combine this with cell culture and phenotypic analyses to define domains of integrins required for specific integrin functions. (4) Develop methods for the regulated expression of PS integrin transgenes in the developing wing of Drosophila, with the goal of using wing as a model system for the fine dissection of integrin function during morphogenesis in situ. This phase of the project will include use of GAL4 enhancer trap technology as well as dissection of the regulatory regions of integrin genes, in order to find enhancers that can provide the required patterns of integrin transgene expression. (5) Use the transgenes in (4) in combination with the integrin mutants to dissect the detailed functions of integrins and integrin "parts" during wing morphogenesis. This will involve the generation of clones of cells mutant for integrin function, and attempts to rescue the mutant phenotypes by expression of normal and genetically altered integrin transgenes.


The goal of the proposed research is to determine the mechanism of electron transfer and thermodynamic properties of the enzyme, assimilatory NADH:nitrate reductase under various environmental conditions. Specific aims are to determine: 1) oxidation-reduction (redox) potentials of the prosthetic groups FAD, heme, and molybdenum; 2) the effect of pH, ionic strength, substrates, and effectors on the redox potentials; 3) the stoichiometry and intermediate redox states in the transfer of electrons from NADH to nitrate; 4) the pathway and transient state kinetics of electron transfer between NADH and nitate; 5) magnetic interactions between prosthetic groups; 6) physical and functional interactions between nitrate reductase and the next enzyme in the pathway of nitrate assimilation, nitrite reductase. For these studies we will utilize EPR spectroscopy, UV/VIS-dual wavelength spectroscopy, microcoulometry, freeze-quench and stopped-flow techniques, and substituted analogues of nitrate reductase. These studies will provide important information on the mechanism of catalysis by nitrate reductase and factors which influence this activity. Since this enzyme catalyzes the rate-limiting step in the process of nitrate assimilation, the proposed research is of importance in terms of plant productivity and the supply of protein. This enzyme also serves as a model system for more complex electron transfer systems.


This project is concerned with the investigation of the mechanism of action of ACTH on the adrenal cortex. Specifically, it relates to the effect of this trophic hormone on the cytochrome P-450 systems of adrenal cortical mitochondria. Methods have been developed for measuring the binding of cholesterol to side chain cleavage cytochrome P-450 in the adrenal cortex and the studies in this project involve measuring how this binding is affected by agents such as ACTH and cyclic AMP. Also under study is the early and late pathway of aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex. Agents to be studied in that project include potassium, ACTH, sodium and angiotensin II. Methods are being developed for the measurement of the late pathway of aldosterone biosynthesis utilizing high pressure liquid chromatography of extracts following incubation of corticosterone with zona glomerulosa mitochondria. In addition, corticosterone binding to the 18-hydroxylase system of zona glomerulosa will be studied by spectrophotometry and electron spin resonance assays. BIBLIOGRAPHIC REFERENCE: Effect of Hypophysectomy on the Volume and Ultrastructure of Zona Glomerulosa in Rat Adrenal. Peter A. Nickerson and Alexander C. Brownie. Endocrin. Exper. 9, 187, 1975.


The goal of this research is to utilize the chiroptical properties of selected chromophoric groups which occur in molecules of biological significance to probe molecular conformation and structure in solution. The general methodology will be to elucidate the relationships which exist between molecular conformation, chromophore environment, and the optical properties of selected model compounds, by calculating the optical properties and the conformational energy as a function of conformation. The method will be evaluated by comparing the calculated results with experimentally determined optical rotatory dispersion, circular dichroism, and absorption sppctra. The model syytems will be chosen to represent various important classes of biological macromolecules, prrmarily proteins. After the technique is proven for a given type of chromophore by appropriate model compound studies, it will be applied to more complex molecules which contain the chromophore of interest. Special attention will be given to proteins such as ribonuclease and lysozyme.


The regulation of promoter activity by nuclear receptors requires the assembly of large multi-subunit complexes that either directly impact the basal transcriptional machinery or modify core histones to affect chromatin structure and remodeling. The mechanisms responsible for maintaining the highly ordered dynamics of protein binding to hormonally responsive promoters have not been definitively established. We have recently developed a novel in situ fluorescence recovery after photobleaching (FRAP) assay that led to the identification of molecular chaperones and their associated co-chaperones as nuclear mobility factors for the glucocorticoid receptor (GR). This assay will be exploited to provide additional mechanistic insights into the role of chaperones in nuclear dynamics of GR. The major hypothesis to be tested in this proposal is that molecular chaperones play a major role in steroid receptor dynamics at target sites within the nucleus via their regulation of receptor nuclear mobility, chromatin exchange and hormone exchange. Specific Aim 1 seeks to identify the role of individual chaperones and the pathway of chaperone complex assembly that mediates their activity as steroid receptor nuclear mobility factors. Chaperone protein assembly will be manipulated in the context of a novel in situ nuclear mobility assay and effects on steroid receptor/chaperone complex formation assessed by co-immunoprecipitation assays. Specific Aim 2 will determine whether molecular chaperones participate in the dynamic exchange of GR and other factors at receptor target sites within the nucleus. The in situ FRAP assay will be used to determine whether specific chaperone complexes are required for the dynamic exchange of GR and various receptor cofactors from a functional target gene. Chromatin immunoprecipitation assays will also be used with permeabilized cells to examine chaperone effects on GR and cofactor recruitment and cycling on the chromatin of target genes. Finally, Specific Aim 3 will determine whether molecular chaperones are required for the exchange of hormone from chromatin- bound GR. The effects of molecular chaperones on hormone exchange on nuclear GRs will be assessed in permeabilized cells using assays that distinguish hormone release from hormone exchange. Chaperone effects on hormone exchange at a specific target site will be revealed using a fluorescent GR ligand. Public Health Relevance: Hsp90 inhibitors such as geldanamycin (GA) are being evaluated for breast cancer therapy due primarily to the their selective action in cancer cells on hsp90 client proteins such as estrogen receptor, p53 and various kinases. However, the development of therapeutic anti-cancer drugs directed against hspQO or other chaperones has not taken into account the newly discovered roles for these proteins in chromatin dynamics or histone modification, which is the subject of this proposal. [unreadable] [unreadable] [unreadable]


Research is focused on studying the events involved in HIV-1 fusion, the development of fusion inhibitors directed against gp41 and the development of monoclonal antibodies with broadly neutralizing activity that are directed against gp41. The HIV-1 Envelope (Env) proteins that mediate membrane fusion represent a major target for the development of new AIDS therapies. Three classes of Env-mediated membrane fusion inhibitors have been described that specifically target the pre-hairpin intermediate conformation of gp41. Class 2 inhibitors bind to the C-terminal heptad repeat (C-HR) of gp41. The single example of a class 3 inhibitor targets the trimeric N-terminal heptad repeat (N-HR) of gp41 and has been postulated to sequestrate the N-HR of the pre-hairpin intermediate through the formation of fusion incompetent heterotrimers. We have now shown that NCCG-gp41, a class 2 inhibitor, and N36Mut(e,g), a class 3 inhibitor, synergistically inhibit Env-mediated membrane fusion for several representative HIV-1 strains (X4 and R5) in both a cell fusion assay (with membrane-bound CD4) and an Env-pseudotyped virus neutralization assay. We have also succeeded in obtaining a monoclonal Fab (Fab 3674) selected from a human non-immune phage library by panning against the chimeric construct NCCG-gp41 (that comprises an exposed coiled-coil trimer of gp41 N-helices fused in helical phase onto the minimal thermostable ectodomain of gp41) that effectively neutralizes diverse laboratory-adapted B-strains of HIV-1 and primary isolates of subtypes A, B and C in an Env-pseudotyped virus neutralization assay, albeit with reduced potency (25x) compared to 2F5 and 4E10. Alanine scanning mutagenesis maps a novel epitope to a shallow groove on the N-helices of gp41 that is exposed between two C-helices in the fusogenic six-helix bundle conformation of gp41. We have shown that bivalent Fab 3674 and the C34 peptide (a potent fusion inhibitor derived from the C-helix of gp41) act at similar stages of the fusion reaction and to neutralize HIV-1 synergistically, providing additional evidence that the epitope of Fab 3674 is new and distinct from the binding site of C34.More recently we have shown that the class 3 inhibitor N36Mut(e,g) prolongs the temporal window during which the virus is susceptible to neutralization by the bivalent Fab-3674, and that bivalent Fab-3674 and N36Mut(e,g) neutralize HXB2 and SF-162 strains of HIV-1, as well as diverse primary B and C clade HIV-1 strains, synergistically in a Env-pseudotyped virus neutralization assay. N36Mut(e,g) also rescues neutralizing activity of the monovalent Fab-3674 against resistant HIV-1 strains and renders a series of related non-neutralizing Fabs neutralizing. Moreover, N36Mut(e,g) exhibits the same effects on the broadly neutralizing 2F5 and 4E10 monoclonal antibodies directed against the membrane proximal extended region of gp41. Very recently, we have subjected Fab 3674 to affinity maturation against the NCCG-gp41 antigen by targeted diversification of the CDR-H2 loop to generate a panel of Fabs with diverse neutralization activity. Three affinity-matured Fabs selected for further study, Fabs 8060, 8066 and 8068, showed significant increases in both potency and breadth of neutralization against HIV-1 pseudotyped with envelopes of primary isolates from the standard subtypes B and C HIV-1 reference panels. The parental Fab 3674 is 10-20 fold less potent in monovalent than bivalent format over the entire B and C panels of HIV-1 pseudotypes. Of note is that the improved neutralization activity of the affinity-matured Fabs relative to the parental Fab 3674 was, on average, significantly greater for the Fabs in monovalent than bivalent format. This suggests that the increased avidity of the Fabs for the target antigen in bivalent format can be partially offset by kinetic and/or steric advantages afforded by the smaller monovalent Fabs. Indeed, the best affinity-matured Fab (8066) in monovalent format (50 kDa) was comparable in HIV-1 neutralization potency to the parental Fab 3674 in bivalent format (120 kDa) across the subtypes B and C reference panels. We have now solved crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5 (developed by Merck), derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop. A key feature of fusion inhibitors that target the N-HR trimer, including antibodies directed against the N-HR trimer, is that deactivation of gp41 in vivo is a slow reversible process that is dependent on chemokine receptor binding to Env, and that the exposed N-HR trimer remains accessible to inhibitors until the final conformational changes in gp41 that lead to the formation of the 6-HB have taken place. This suggests that inhibition of fusion will be most effective when two or more Fabs are bound to the exposed N-HR trimer of the pre-hairpin intermediate of gp41. Since multiple Fabs/antibodies bound to the N-HR trimer are unlikely to dissociate simultaneously, the probability that at least one antibody is bound to the N-HR trimer at all times will be increased. Modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Thus, in the case of the complex with the most potent neutralizing Fab in our series, Fab 8066, the molecules of Fab can readily bind to the N-HR trimer without any steric clashes between adjacent Fab molecules. In contrast, as a consequence of the different mode of binding of the CDR-H2 to the hydrophobic pocket on the surface of the N-HR trimer, binding of three molecules of Fab 8062 to the N-HR trimer may result in steric clash between adjacent Fab molecules involving the CDR-H2, CDR-L1, CDR-L3 loops and loop 71-78. It seems likely in the light of the current structural data, modeling results and neutralization properties of our Fab series, that neutralization is dependent not only on tight binding of a single Fab to the N-HR trimer but also on the ability to bind multiple Fabs to a single N-HR trimer at preliminary step of fusion process.


PROJECT SUMMARY - Regional/National Resource Core for Improving Diabetes Outcomes in Community Health Centers Community health centers (HC) are vanguard providers of health care for vulnerable populations, serving 24 million Americans in 1200 centers in 9000 sites. Forty percent of HC patients are uninsured, 36% have Medicaid coverage, over 60% are racial or ethnic minorities, and 71% are at or below the federal poverty line. Nationally, HCs serve 1 of every 4 people in poverty, 1 of 10 minorities, and 1 of 9 rural Americans. HCs are a critical provider of care to underserved populations, and a vital part of efforts to reduce national disparities. The Chicago Center for Diabetes Translation Research (CCDTR) will serve as a regional and national resource for diabetes translation research in community health centers. We will continue our longstanding collaboration with the MidWest Clinicians' Network (MWCN) of approximately 130 health centers with 300 practice sites in the 10 Midwestern states as well as with partners including the National Association of Community Health Centers (NACHC), Association of Asian Pacific Islander Community Health Organizations (AAPCHO), Migrant Clinicians Network, ACCESS Community Health Network, state Primary Care Associations, America's Essential Hospitals, and the Medicaid Health Plans of America. The CCDTR will have a Regional/National Resource Core for Improving Diabetes Outcomes in Community Health Centers led by MWCN with the collaboration of its partners. MWCN and CCDTR have deep combined research and implementation strengths. In conjunction with other regional and national partners, the collaboration has widespread reach and connections. Tremendous opportunities exist to perform diabetes translation research in health centers and to disseminate findings regionally and nationally. Most MWCN-CCDTR projects capture the heart of translational research - integrating research with implementation science in a collaborative manner that has practical utility to end-users of the information. The Core will improve diabetes translation research and diabetes care and outcomes in safety-net populations. The specific aims of the Improving Diabetes Outcomes in Community Health Centers Core are to: 1) Serve as a regional and national resource to improve diabetes translation research in community health centers and other safety net settings led by the MidWest Clinicians' Network in collaboration with the National Association of Community Health Centers and other partners. 2) Determine best practices for diabetes care in community health centers, identify research voids, and facilitate implementation and evaluation projects.


Autoantibodies to the Golgi complex are found primarily in patients with Sjgren's syndrome and SLE although they are not restricted to these diseases. This competitive renewal application requests funding for the continuation of studies to characterize autoantigens of the Golgi complex and address the mechanisms that lead to the expression of autoantibodies to these intracellular organelle-associated proteins. The P.I.'s laboratory has been responsible for the identification of a family of Golgi antigens known as golgins. Common features of these autoantigens are that they are located on the cytoplasmic face of the Golgi cisternae and they have multiple alpha-helical coiled-coil rod domains flanked by non-coiled-coil and C- terminal domains. The goal of the proposed studies is to determine if the common structure and function for members of this protein family can explain the origin and production of autoantibodies in disease states. Specific Aim 1 will examine common features of golgins that may explain why they are targets of human anti-Golgi autoantibodies. The P.I. will characterize the events associated with the Golgi complex and golgins and their stability during apoptosis and necrosis. Hypotheses on how these autoantibodies may be produced in experimental models will be examined. Specific Aim 2 will examine if Golgi fragments and vesicular structures will induce immune and autoimmune response in experimental mice. Specific Aim 3 will elucidate the target of Golgi complex in lactate dehydrogenase-elevating virus (LDV) infected mice and address mechanism of how autoimmune response to the Golgi complex can be produced in these mice. Earlier studies have shown that LDV infected mice produce anti-Golgi antibodies. Our current data suggest that the autoimmune response to cytoplasmic organelles such as the Golgi complex is uniquely different from other intracellular autoantigens examined to date. It is anticipated that testing of the P.I.'s hypotheses will provide new insights into autoimmunity and autoimmune diseases associated with subcellular organelles.


The research described in this proposal aims to increase our knowledge concerning the fever response in animals other than humans; specifically relating to the occurrence of the fever response in one species of amphibian and two species of mammals living in their natural environment, the generality of the fever response in reptiles, and the energetic cost of the fever response in a free-living reptile. All of the studies concerning the fever response has never been demonstrated or investigated under natural field conditions where the greater heterogeneity of biotic and abiotic factors may lead to a more complex response. Also, although the fever response has been demonstrated in a number of ectothermic vertebrates under laboratory conditions, no systematic study of the fever response has been undertaken with any specific taxonomic group of ectothermic vertebrates to demonstrate the generality of the response and its relationship to life history characteristic of a species. Data on the fever response of the bullfrog, the California Ground squirrel, and the desert cottontail rabbit will first be gathered in the laboratory and than the laboratory baseline data will be applied to experiments on free-living animals. Body temperatures will be determined with the use of temperature sensitive radio transmitters. In the survey of the Class Reptilia for the fever response, II species representing 7 different Families will be given injections of heat-killed Aeromonas hydrophila, a gram negative bacterium known to be pathogenic to reptiles. The reptilian species to be used represent animals from different habitats (aquatic, mesic, desert), with different food habits (herbivorous, insectivorous, carnivorous), and with different activity patterns (diurnal, nocturnal). In the section that pertains to the energetic cost of the fever response, time budget analysis and oxygen consumption measurements will be combined to yield an estimate of the energetic cost of the fever response in a free-living reptile; the chuckwalla.


ABSTRACT c The vasculature endothelium forms a selectively permeable barrier that facilitates transfer of nutrients, oxygen and waste products between the retina and the blood. Therefore, diseases affecting the structure and function of the vascular endothelium, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD), have a devastating effect on the health of the retina and ultimately lead to severe visual impairment. Traditional treatment approaches focus on ameliorating disease symptoms that lead to vision loss, including retinal and choroidal neovascularization. Whilst effective, treatments such as laser photocoagulation are both invasive and destructive, requiring frequent interventions throughout the patient's lifetime, leading to the ablation of neurosensory retina as new blood vessels are cauterized. Moreover, these treatments fail to address the pathologic abnormalities within vascular endothelial cells (VECs) that underlie abnormal blood vessel function in DR. As such, they serve only to temporarily limit progression of the disease. In contrast to existing treatments, gene therapy represents an attractive therapeutic alternative, potentially allowing for the permanent correction of vascular dysfunction prior to the development of sight- threatening complications. The inability to efficiently deliver genetic material to vascular endothelial cells currently prohibits development of any gene therapy treatment aimed at preventing DR. We have recently taken the first step to overcoming this barrier by elucidating a recombinant adeno-associated virus (rAAV) vector mutant with enhanced affinity for VECs. We propose to further develop these vector technologies and optimize their surgical delivery through the following specific aims: 1) Evaluate endothelial cell transduction and maintenance of gene expression in normal and diabetic vasculature; 2) Characterize early stage biomarkers of DR progression and efficacy of endothelial cell gene therapy, and 3) Assess endothelial cell transduction following rAAV administration by selective intra-ophthalmic artery infusion (SIOAI). Utilizing a well-established rat model of type I diabetes (T1D) we anticipate the development of a strategy to effectively deliver genetic material in both normal and dysfunctional VECs. In doing so, we will utilize various advanced imaging modalities to quantin order to maximize the clinical translation of the proposed DR gene therapy, we will optimize key aspects relating to the targeted intravascular delivery of rAAV using a mini-swine model that accurately recapitulates human cardiovascular and ocular anatomy. The Ocular Gene Therapy Laboratory (OGTL) and Advanced Ocular Imaging Program (AOIP) at the Medical College of Wisconsin, together the University of Florida Department of Ophthalmology, provide the perfect collaborative environment to complete the proposed work. Finally, our proposal addresses an emerging need identified in the NEI Publication ?Vision Research: Needs, Gaps, and Opportunities?: ?develop novel, noninvasive imaging techniques for monitoring electrical or metabolic activity of retinal neurons in vivo, ideally at the spatial resolution of photoreceptors or better for early detection of disease and monitoring of therapeutic intervention.?


Previously we have initiated a large cDNA microarray effort in collaboration with the Human Genome Project and the Cancer Genome Anatomy Project (CGAP) to develop a comprehensive and novel molecular classification schema for human gliomas based on a gene expression profile using cDNA microarray technology. We have constructed our own cDNA microarray chips which will be enhanced for new and selective genes thought to be important in glioma biology. This project will include hundreds of tumor specimens and offer an unprecedented opportunity for gene discovery, dissecting signal transduction pathways, and learning this exciting new technology. Glioma stem cell is a tumor subpopulation that can self-renew in culture, perpetuate a tumor in orthotopic transplant in vivo, and generate diversified neuron-like and glia-like postmitotic progeny in vivo and in vitro. Recently, conventional and array-based CGH (aCGH) profiling of human gliomas have shown a significant number of copy number alterations (CNAs) including gain/amplification (1p34-36, 1q32, 3q26-28, 5q, 7q31, 8q24, 11q, 12q13, 13q, 15p15, 17q22- 25,19q, 20p, and 20q), and deletion/loss (3q25-26, 4q, 6q26-27, 9p, 10p, 10q, 11p, 12q22, 13q, 14q13, 14q23-31, 15q13-21, 17p11-13, 18q22-23, 19q, and 22q) (Kotliarov et al., 2006; Nigro et al., 2005; Phillips et al., 2006). The large number of chromosomal aberrations, and the large number of genes contained therein, have to date made it impossible to identify which genes are in part responsible for driving the biology of these tumors. We have analyzed a large number glioma samples for genetic characterization of recurring CNAs using Affymetrix 100K single-nucleotide polymorphism (SNP) array chips and Genechip HumanGenome U133 Plus 2.0 Expression array (Kotliarov et al. 2006). Based on our bioinformatics data from these array and gene expression profiling experiments, we have found novel genes frequently altered in gliomas. Furthermore, we have explored the new biotechnology such as next generation sequencing, for this project. We have generated sequence-verified gene Gateway entry clones of these genes and cloned them into pLenti/UbC/V5 expression vectors for transduction of various target cell lines. With our candidate gene constructs, we will identify whether candidate genes change the biology of these cells in such a way that may be consistent with a role in tumorigenesis (i.e. clonogenecity, proliferation, apoptosis, tumorigenic potential in immunosuppressed animals). The NOB laboratory is using the DHS-seq method to profile genome-wide transcriptional changes in glioma patient samples. As described above, the DHS-seq will reveal dynamic changes in the chromatin, which are important in the development and progression of brain tumors and allow us to identify novel molecular targets to treat this disease. We have tested the DHS-seq protocol on two glioma stem cell lines (827P12 and 923P9) and corresponding xenograft tissues. Preliminary analyses of these data suggest that in combination with gene expression and copy number data, we will obtain novel insights into the genomics underlying brain tumor biology. To this end, the NOB laboratory has begun testing this method on patient samples, using tumor tissues and adjacent normal brain directly from surgical specimens. The plan is to continue processing additional patient samples as they become available with the ultimate goal of incorporating the transcriptome analysis into the comprehensive genomic analysis that is being planned as a component of the molecular tumor board, described in the Clinical Project.


DESCRIPTION: Glaucoma is one of the leading causes of blindness in the Veterans population, estimated to affect 285,000 American Veterans as well as 60 million people worldwide, and is the most common neurodegenerative disease. The exact mechanism by which vision loss occurs in glaucoma is not fully understood, which hinders development of a true cure for this disease. It is thought that excessive deformation of the optic nerve tissues in the posterior eye under the insult of elevated intraocular pressure leads to an unfavorable biomechanical environment for cells of the optic nerve head. These cells include the retinal ganglion cells, whose axons transmit visual information from photoreceptors to the brain. Based upon an increasing body of evidence suggesting that the stiffness of the sclera (the white outer coat of the eye) may greatly influence the degree of deformation of the posterior eye, we hypothesize that stiffening the peripapillary sclera will protect against vision loss in glaucoma. Whereas current therapies only delay the onset of symptoms, stiffening the peripapillary sclera could be a novel treatment for glaucoma. In this study, our approach is to screen the efficacy of potential chemical agents and engineered viral vector approaches that modulate peripapillary scleral stiffness in a rat model of glaucoma. In the first specific aim, we will use an optical tracking technique called digital image correlation to quantitatively assess the magnitude of stiffening these agents have on rat scleras. We will also develop a surgical technique to deliver these agents to the back of the eye under ultrasound guidance without affecting surrounding structures. Once we determine the most effective agents, in the second specific aim, we will deliver these agents to rats with surgically-induced glaucoma. Our experimental paradigm is a 3x2 study, where we will compare rats with and without induction of glaucoma to rats with and without delivery of peripapillary scleral stiffening agents (plus additional sham controls). Over 8 weeks, we will monitor anatomic changes to the eye and quantify the degree of vision loss by measuring the rats' visual acuity and contrast sensitivity. At the conclusion of the experiment, we will analyze the expression of genes and proteins associated with glaucoma and ocular damage to determine the overall health of the eyes. The expected outcome of this study is that rats with stiffened peripapillary scleras will experience less vision loss under glaucomatous conditions than glaucomatous rats with no peripapillary scleral stiffening. This will provide the first in vivo data regarding the efficacy of this novel approach to treating glaucoma. If successful, these findings will motivate development of a human clinical trial to deliver biocompatible stiffening agents to prevent vision loss in glaucoma. Overall, the motivation for this study is the need for a better understanding of ocular biomechanics towards the long-term goal of developing a cure for glaucoma in clinical use for Veterans and others with this disease. This meets the Rehabilitation Research and Development goal of preventing and treating vision loss. This project represents a method to screen potential glaucoma treatments in an animal model, as well as to train the postdoctoral candidate to become a successful and productive independent investigator in the field of ocular biomechanics within the VA research environment.


Three-dimensional coordinate information representing neuronal processes is obtained utilizing a video version of the computer microscope. Usually specimens are prepared by the rapid Golgi method although the measuring system has the capability of accurately reconstructing and analyzing most biological structures which are histologically prepared and preserved on microscope slides. The opportunity is also presented to serially reconstruct electron micrographs. Neurons from the hippocampal and related areas of the human immature brain (18 to 33 weeks gestational age) are measured and analyzed according to their branching characteristics. Statistical parameters representing the branching characteristics of neuronal processes (e.g. branching/fission angles, polar angle and radial separation of endpoints, etc.) are obtained and deviant patterns are correlated with specific forms of profound mental retardation (e.g. Down's Syndrome, chromosomal abnormalities, etc.) Attempts are also in progress to quantitatively analyze and classify dendritic spines in regard to their density, distribution and topological characteristics.


Neoplasms are commonly characterized by abnormal gene expression. Our studies will utilize a unique murine pituitary tumor model (MGH 101A) to investigate altered gene activity. This tumor evolved in our laboratory from a thyrotropic tumor. It is unique in that it has lost completely the ability to produce the beta-subunit of TSH and now only synthesizes the alpha-subunit in a nonregulated and promiscuous fashion. Our preliminary observations suggest that these defects occur at a pretranscriptional level. The projected studies on this pituitary neoplasm will be divided into two parts. First, we will investigate the molecular basis for absent TSH-beta subunit production in MGH 101A. The production of the TSH-beta subunit is normally a highly tissue-specific function in thyrotropic tissue. Its absence could therefore be related to an abnormality in the TSH-beta gene structure or the presence of a transacting factor altering the expression of this gene. Our preliminary experiments have shown that the TSH-beta gene is present and is not grossly abnormal. We will therefore use recombinant DNA techniques and molecular biology to discover the mechanisms for the absent TSH-beta gene expression. A solution to this problem could provide insights into similar phenomena for eutopic and ectopic malignancies, as well as an understanding of those factors which regulate gene expression in eukaryotic cells. Second, we will perform studies to determine the molecular basis for the absent regulation of the alpha-subunit production in this tumor. These experiments will explore those factors of gene structure and cellular environment which are important to the regulation of gent expression. Preliminary information suggests that not only is the alpha-subunit gene present, but that it is transcribed very efficiently. Knowledge gained on the regulation of this tumor's alpha-subunit production may contribute substantive information on pathogenetic mechanisms as well as the treatment of human pituitary and non-pituitary neoplasms.


Studies of the physicochemical properties of epiglycanin will be continued. Its masking role at the cell surface will be further investigated in relation to its immunochemical and immunological properties. The relationship of the masking mechanism to the type and location of the carbohydrate chains on epiglycanin will be further studied by the process of mapping active sites by electron microscopy. The biosynthesis of epiglycanin will be studied by precursor labeling and by the use of the epiglycanin antibody. Investigation of possible inhibitors of the synthesis of this glycoprotein will be pursued.


Continuous, non-invasive methods of measuring arterial PO2/CO2 are clinically desirable; but it has become apparent that the present method of monitoring O2/CO2 transcutaneously is suitable as no more than an indication of trends, as data amassed by us and by other investigators makes clear that transcutaneous O2/CO2 values do not consistently correlate with arterial PO2/CO2. The research proposed here will attempt to provide a clearer understanding of what transcutaneous measurements actually reflect by analyzing the basic physiologic variables and mechanisms reflected in transcutaneous values. We propose to employ--and thereby to validate--new methods of measuring various components of cutaneous blood flow and of determining the index of peripheral O2/CO2 perfusion and evaluating the effect of temperature on these components, as well as on dermal permeability. The ability to distinguish and measure these components should provide a means of correcting transcutaneous values so that they more accurately reflect arterial PO2/CO2. By means of photoplethysmography and measurement of the power output of the heater, in conjunction with an occlusive method of plethysmography, we will be able to measure two major components of cutaneous flow--papillary flow and deep cutaneous flow--and relate their effects under both normal physiologic and pathologic conditions of hypotension, hypoperfusion, anemia, polycythemia, and diminished oxygen unloading. When the validity of our method of corrected transcutaneous measurement has been established with animal subjects, the method will be applied in clinical situations with normal and diseased newborn and adult patients so as to establish its usefulness under all clinical conditions. The knowledge obtained from this study will help us to understand the meaning of transcutaneous O2/CO2 as an independent physiologic variable, which may have significant clinical applications of its own.


The vision of the University of Cincinnati Academic Health Center (AHC) Institutional CTSA is to create an environment to facilitate translating discoveries to clinical application. Our new academic home for clinical and translational research, the Center for Clinical and Translational Science and Training (CCTST), is beginning to transform the research environment within the AHC and our affiliated partners in other areas of the University, the community, and industry. The CCTST will coordinate and plan the overall direction of AHC research infrastructure and training opportunities;serve investigators'needs from project concept to completion;optimize skills and foster career development of both new and experienced investigators;and ensure that community input informs research processes, and that AHC discoveries are translated to the community. The CCTST will coordinate and leverage our existing strengths and develop new initiatives. Through Research Central, researchers will have easy access to centralized study design, biostatistical, bioinformatics, regulatory, and community engagement support. The new Pilot &Collaborative Studies core will expand the highly successful pilot funding program at Cincinnati Children's Hospital Medical Center (CCHMC) to the entire AHC. Biomedical informatics will be coordinated across the AHC. A new K12 program and greatly expanded educational offerings, including a new Certificate in Clinical and Translational Research, will be developed, building on the success of our Dean's Scholars in Clinical Research and K30- funded MS in Clinical and Translational Research programs. Through our community engagement program, we will further bi-directional research linkages with the local community, breaking down bureaucratic barriers by creating IRBs that can coordinate community-based research. Expanding services such as nursing/coordinator support and sample processing provided by the existing General Clinical Research Center (GCRC) at CCHMC and the Cincinnati Veterans Affairs Medical Center will promote patient-oriented research for populations outside the GCRC in the community. New translational technologies, including proteomics. drug discovery, imaging, nanomedicine, gene transfer and stem cell biology and translational and molecular disease modeling, will be made more accessible to researchers. Throughout, we will utilize a quality improvement model to evaluate our progress. These efforts will facilitate transforming clinical and translational research at the AHC. leading to improved health outcomes in the community.


The Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Photography Reading Center (CPRC) provides objective, independent photographic verification of two important components of the CLEK Study: 1.) the presence, absence, and degree of severity of corneal scarring in all study eyes and, 2.) the rigid contact lens fit (fluorescein pattern) in all study eyes that wear rigid contact lenses, as well for the first rigid contact lens base curve that provides clearance of the corneal apex. This proposal provides documentation of the CPRC's ability to perform these functions with high sensitivity and specificity. This proposal also provides a description of the day-to-day organization of work, quality control and data management procedures required to insure the scientific integrity of the CLEK Study and to insure the orderly operation of the CPRC. Documentation of the capabilities of the proposed CPRC Staff for the performance of the Study in accord with the details of the CPRC Operations Manual and with the CLEK Study Operations Manual is provided.


This protocol seeks to determine whether there are specific behavioral changes or a clinically detectable specific pattern of behavioral improvement that occurs within the first week of treatment for major depression. It will also evaluate whether such improvement is due to the drug used in treatment (desipramine or fluoxetine), or rather to the process of recovery, regardless of the treatment modality.


Bacterial DNA replication is carefully controlled at the initiation stage, possible by the regulation of the essential activity of DnaA protein. The cellular membrane has long been hypothesized to be involved in chromosomal replication, with accumulating evidence that indicates membranes have a profound influence on DnaA protein. The long term goal of this research is to elucidate the physiological significance of the influence of membranes on chromosomal replication. The research outlined here uses both biochemical approaches with defined components and genetic and physiological studies to directly test the hypothesis that acidic phospholipids of the cellular membrane participate in the regulation of DnaA protein activity. The Specific Aims are to; 1. Identify the membrane binding site on DnaA protein by chemical crosslinking. Disrupt the membrane binding function by site-directed mutagenesis and isolate the mutant DnaA proteins. Confirm with defined components that the other replication activities of DnaA protein have not been altered. 2. Examine in vivo the replication activity of the mutant Dna A proteins are viable, and if so, if they initiate replication at aberrant times during the cell cycle. Localize mutant forms of DnaA protein within the cell by subcellular fractionation and immuno-gold microscopy to examine the importance of DnaA protein-membrane association for regulated initiations. 3. Generate and screen randomly mutagenized dnaA genes for the ability to suppress the growth arrest of acidic phospholipid-deficient cells. Map the suppressor mutations in dnaA and examine the replication activities of the mutant proteins in vitro. Determine the cellular location of the mutant proteins and analyze their effect on the cell cycle control of chromosomal replication in vivo. This work should provide insight into the regulation of the initiation of DNA replication , which is a key control point in the prokaryotic cell cycle and in the determination of eukaryotic cellular quiescence or proliferation. Furthermore, knowledge gained from the proposed studies of DnaA protein may guide future investigations of how phospholipids may act as regulators of enzymatic activities.


The incidence of obesity is increasing at an alarming rate in Western cultures, leading to its classification as an epidemic. In light of this epidemic, there is great impetus to better elucidate the processes that regulate fat production, storage, and release. The ability to regulate fat storage is a fundamental process required for survival of species ranging from mammals to invertebrates. It has therefore been suggested that diverse model organisms may be exploited in the study of fat metabolism. Very recent studies have demonstrated that the nematode Caenorhabditis elegans provides a useful model for identification of genes affecting fat storage. An RNAi screen to inactivate more than 16,000 genes in C. elegans and screen for alterations in fat storage identified 305 genes that reduced, and 112 genes that increased body fat upon inactivation (Ashrafi et al., 2003). More than 50% of these genes have mammalian homologs not previously implicated in regulating fat storage, and represent excellent candidates for having potential roles in human fat metabolism. In this proposal, we will evaluate several candidate "fat genes" identified in C. elegans by generating and characterizing gene knockout mouse models. To circumvent the traditional time consuming and laborious process of generating gene knockouts via recombination in embryonic stem (ES) cells, we will utilize a resource containing thousands of pre-fabricated ES cell lines containing gene-trap insertions. We have identified 22 existing ES cell lines carrying inactivating insertions within mouse homologs of C. elegans fat genes identified by Ashrafi et al., which are available from the BayGenomics Gene-Trap Consortium. We have confirmed that several of these genes are prominently expressed in mouse adipose tissue, and hypothesize that these genes, which have already been shown to play a role in fat storage in C. elegans, may affect fat development or metabolism in mammals. We will utilize the existing gene-trap ES cell lines to generate mouse knockout strains for several of the putative "fat genes", and characterize them for adipose tissue mass, glucose and energy metabolism. This pilot study will reveal whether "fat genes" identified in the invertebrate C. elegans have a similar role in mammals, and may identify novel genes involved in fat storage and metabolism.


Child maltreatment is a major public health problem. In addition to the risk of physical injury or death, early maltreatment is associated with impairments in social competence, emotional regulation and cognition, as well as to an increased risk for the development of anxiety, mood, and addiction disorders later in life. While it has been assumed that these vulnerabilities reflect impacts on neurodevelopment, practical and ethical concerns prevent studying these associations during early human development. Yet, such data would aid attempts to intervene and reduce the mental health burden associated with these early adverse experiences. The present proposal addresses this problem by using a unique, naturalistic, nonhuman primate model of human childhood maltreatment. Interestingly, maltreatment is not just a human problem; it has been reported in nonhuman primates as well, with rates and types of maltreatment similar to those seen in human populations. This rhesus monkey model provides a means to characterize the long-term impact of infant maltreatment on neurodevelopment using neuroimaging technology. However, in order to accomplish this important goal, preliminary data using in vivo neuroimaging are needed to justify longitudinal analyses comparing abused and non-abused animals, and normative data on early development to determine where perturbations in neural structure and circuits occur as a consequence of early adverse experience. The overall hypothesis of this proposal is based on the argument that infant maltreatment, via a sustained sensitization of the stress response, disrupts normal neurodevelopment, including neural systems underlying the control of socioemotional behavior and stress physiology. There are two aims in this project, both of them applying cutting edge in vivo neuroimaging technology and approaches to study the organization of brain structure, connectivity and function in animals with different rearing experiences. Aim 1 will define the long-term consequences of infant maltreatment on neurodevelopment, characterizing brain differences between monkeys with early exposure to infant abuse and non-abused controls as they transition from adolescence to adulthood. Aim 2 will define normative changes in neurodevelopment in rhesus monkeys, from the early postnatal period until 18 months of age. Neurodevelopmental changes will be assessed for relationship to socioemotional development and physical growth. Significance Understanding the neurodevelopmental consequences of infant maltreatment in a closely related phylogenetic relative will provide the framework for defining the neurodevelopmental impact of human childhood maltreatment, a reference for understanding its role in the development of socioemotional problems and pathophysiology in children and adolescents, and aid the design of treatment plans seeking to limit the adverse consequences of childhood maltreatment. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]


Naturally occurring examples of immune system-mediated HIV control provide encouragement for the ultimate development of effective vaccines and immunotherapies that would limit HIV replication.. Identification of the components, targets, and magnitude of an effective immune response to HIV are important steps toward the development of effective vaccines or immunotherapies. Although patients with normal CD4+ T cell counts and low levels of plasma virus are a heterogeneous group, a small subgroup of patients with truly non-progressive HIV infection and restriction of virus replication likely holds important clues to the basis of an effective immune response to HIV. A small subpopulation (fewer than 0.8% of HIV infected individuals) shows no signs of progression over a 10-year period. We have assembled a stringently defined cohort of such patients, termed long-term nonprogressors (LTNPs) or elite controllers. Many of these patients have been infected for 20 years with no CD4+ T cell decline without taking antiretroviral therapy, and maintain plasma viral RNA below 50 copies per milliliter. Cells from these patients are being used to systematically dissect the mechanisms of immune-mediated restriction of virus replication. HIV-specific T-cell responses of these patients have been studied in extreme detail. Through this project, extraordinary progress has been made in understanding the basis of immunologic control of HIV. Our prior work indicates a dramatic association with the HLA B*5701 allele and that the immune response is highly focused on peptides restricted by this allele. This result established a host genetic and functional link between immunologic control and the CD8+ T-cell response of these patients. More recently we have found that this focus is specific to HIV, and is not found in the response to other pathogens such as Hepatitis C virus or Cytomegalovirus. LTNP patients do not differ in the frequency of HIV-specific T cells or in the ability to recognize the autologous virus, when compared to progressors. The finding of high frequencies of CD8+ T cells specific for the patients virus in both LTNPs and progressors strongly suggests that differences between responses of these patient groups are not quantitative, but rather qualitative in nature. One important qualitative difference in the HIV-specific immune response that distinguishes LTNPs from progressors is the maintenance of HIV-specific CD8+ T cells with a high proliferative capacity. This proliferation parallels perforin expression required for effective killing of HIV-infected CD4+ T cells. In 2009 we established the properties of the HIV-specific CD8+ T-cell response that are tightly associated with the LTNP phenotype. Although the HIV-specific CD8+ T cells of LTNPs have a greater capacity to proliferate and produce molecules responsible for killing HIV infected cells, the mechanism(s) by which these properties translate into effective immunologic control of HIV has remained unknown. Most current assays are not sufficiently powerful to establish if differences in HIV-specific CD8+ T-cell function are determined by frequency, CD8+ T cell proliferation, preferential target or effector cell death, or the mechanism of HIV-infected cell elimination. To better understand the mechanisms of immunologic control, we recently devised a method to measure HIV-infected cell elimination on a per-cell basis. Measured on a per-cell basis, HIV-specific CD8+ T cells of LTNPs efficiently eliminated primary autologous HIV-infected CD4+ T cells. This effective killing was clearly distinguishable from responses of progressors over a very broad range of effectors to HIV-infected targets. Progressors cells did not mediate effective killing even at high effector to target ratios. Defective cytotoxicity of progressor effectors could be restored in vitro. These results establish an effector function and mechanism that clearly segregate with immunologic control of HIV. A deeper understanding of the basis of immunologic control in LTNPs and the loss of immunologic control in progressors is likely to provide information that is critical for development of immunotherapies or prophylactic vaccines for HIV. In future work we seek to understand the molecular basis of the difference in killing capacity between LTNPs and progressors. We are also working to understand the mechanism by which such responses arise in LTNP patients, and determine whether they may be exploited in an HIV vaccine.


Bistability is a striking phenomenon found in various systems in which two very different behaviors can occur for the same external conditions. For example, new-born infants can suddenly stop breathing - for unknown reasons - a condition known as apnea, and, several seconds later, resume normal breathing for reasons which are also unknown. Bistability is also found in simpler systems, including squid giant axons. When the pH of the intracellular medium of the axon is[unreadable] elevated to 7.7, or higher, the axon is either quiescent or spontaneously firing action potentials in a rhythmic manner at a rate of approximately 25 Hz. The spontaneous firing can last for several hours. The ion channel mechanisms underlying this behavior are a persistent, tetrodotoxin-sensitive sodium ion channel, INaP, and a acid-sensing-ion-channel (ASIC) which is also sodium selective. This ASIC differs from the ASIC's found in the mammalian nervous system in that the H+ sensitivity is on the inner surface of the membrane. Alkalinization of the intracellular milleau turns off the ASIC, thereby allowing the INaP channel to destabilize the normal rest state of the axon. We have examined transitions between the two stable states in alkalinized axons, repetitive firing and quiescence, by injecting computer generated noise into the axon. The pattern of on-off switching of the pacemaker depends upon the intensity, spectral properties, and phase angle of the noise stimulus current. Our results reveal a distinct form of bistability in which noise can either silence pacemaker activity, trigger repetitive firing, or induce sporadic burst patterns similar to those recorded in a variety of normal and pathological neurons.


ThermoGen proposes to isolate a library of thermostable glycosidases for use in the synthesis of carbohydrate containing therapeutics. Phase I will identify thermostable glycosidases from ThermoGen's large library of thermophiles and Escherichia coli clone banks of themophilic organisms. Identified glycosidases will be characterized and categorized on the basis of their hydrolytic and synthetic substrate specificity. Phase II will involve the further characterization of these and additional thermostable glycosidases for commercial development. This library of thermostable glycosidases will be available to synthetic chemists for the development and synthesis of carbohydrate containing pharmaceuticals. Phase III will focus on commercialization of identified glycosidases at the industrial scale for use in industrial synthetic pipelines. PROPOSED COMMERCIAL APPLICATION: ThermoGen proposes to isolate and characterize a large set of thermostable glycosidases for use by the synthetic community for the development of new pharmaceutical compounds. These enzymes may also be used to streamline the synthesis of existing commercial processes.


The purpose of the proposed research is to determine how plasma lipoproteins and apolipoproteins bound to lymphocyte membrane receptors - termed proliferation-restraint receptors - inhibit mitogenic recruitment of the cells into and passage through the cell cycle, thereby inhibiting mitogen-induced cell proliferation. The suppressor molecules inhibit biochemical process -e.g., enhanced metabolism of phosphatidylinositol (PI) - which occur within the first hours post-stimulation. Inhibition correlates directly with extent of receptor occupancy. The hypothesis to be tested is that PI metabolism is important in the production of growth factors and/or growth factor receptors which are required for the activated cells to undergo mitosis. The hypothesis will be tested in systems containing defined cellular phenotypes and relying on assays in which events can be assessed in idividual cells by flow cytometry. The three specific aims are: (1) to determine the phenotype of the lymphoid cells required to generate the PI response and to modulate suppression of this response by AP/LP, (2) to determine the influence of AP/LP on IL-2 receptor expression and IL-2 production, paying particular attention to the relationship between the PI response, IL-2 receptor expression and IL-2 production, and (3) To characterize the proliferation-restraint receptor by its specificity for well-characterized ligands and its sensitivity to modification-inactivation. The long term goals of the research are (1) to determine the structure of the lymphocyte proliferation-restraint receptor which recognizes apolipoproteins; (2) to elucidate the mechanism by which it functions to restrict lymphocyte proliferation at the molecular level; (3) to identify the cells, lymphocyte and nonlymphocyte, which express it and are therefore sensitive to its function; and (4) to understand the factors which regulate its expression and its activity. Lipoproteins are natural modulators of the functional capacity of the immune system. Suppression of immune responsiveness by lipoproteins is important in diseases such as cancer in which host immune competence may be a primary defense mechanism. In cell biology/physiology, the proliferation-restraint receptor offers the opportunity to define recruitment of lymphocytes from the nonproliferating state in molecular terms.


Mechanism of dopamine actions in cardiovascular and renal systems will be investigated by delineating the dopamine-dependent cyclic AMP system: the effect of dopamine on intracellular cyclic AMP concentration dopamine binding receptor and adenylate cyclase activity in plasma membrane. The effect of interactions between dopamine and other hormones or catecholamine blocking agents on the cyclic AMP system will also be investigated in cardiovascular and renal systems. Lithium inhibits catecholamine-dependent cyclic AMP, but not all physiological effects of catecholamines. Utilizing this unique effect of lithium, the physiological role of catecholamine-dependent cyclic AMP in cardiovascular and renal system will be further investigated by comparing the inhibitory effect of lithium with that of other adrenergic blocking agents on myocardil contractility, distribution of intra-renal blood flow, and the release of renin. Prostaglandin E2 has vasodilatory effects in inner cortical and outer medullary portions of the kidney. Since vasodilatory effect of the beta adrenergic stimulus in other systems is mediated through cyclic AMP, the possible vasodilatory effect of prostaglandin E2-dependent cyclic AMP will be investigated in various portions of the kidney. The mechanism of interaction between prostaglandin E2 and alpha adrenergic stimulation, angiotensin I or II will also be investigated by studying the effect of these interactions on the cyclic AMP system. These studies are essential for the understanding of mechanism of dopamine action, the release of renin, and prostaglandin E2 in cardiovascular and renal systems.


Molecular signatures collected from intact tissue sections by MALDI imaging mass spectrometry (MALDI IMS) have shown high potential for use as a prognostic or diagnostic pathology tool in the clinical setting. A major obstacle to the widespread deployment of MALDI IMS, however, is the difficulty of identifying the proteins contributing to the signatures. Researchers have tried a number of approaches, including in situ digestion, MALDI TOF/TOF tandem mass spectrometry, and top-down proteomics on specific image regions. In preliminary work, we have obtained promising experimental results using top-down proteomics on intact proteins in the 2 - 20 kDa range. However, the lack of successful algorithms and software to identify the proteins in IMS mass signatures poses a major bottleneck. In particular, available top-down proteomics software relies heavily on high-accuracy mass spectrometry. The requirement for high accuracy precludes the use of some of the most sensitive mass analyzers such as linear ion traps, especially useful for these very small and complex samples. Protein Metrics Inc. is a new software company building on six years of algorithms and software research at Palo Alto Research Center. We plan to extend Byonic, our next- generation proteomics search engine, to intact proteins up to about 20 kDa. For proteins larger than 20 kDa, we will also build software for middle-down proteomics, specifically for assembling large peptides (2 - 20 kDa) produced by limited digestion to recover the identity of the intact proteins observed in IMS. The proposed Phase I feasibility study will allow us to perform controlled studies to determine the best experimental and bioinformatics approaches. Phase II will then build commercial-grade software. The proposed project will advance the state of the art in imaging mass spectrometry. Translation of imaging mass spectrometry to routine clinical pathology use will advance the state-of-the-art in disease diagnosis and treatment, and advance medical imaging and public health. PUBLIC HEALTH RELEVANCE: The project will develop commercial software that will improve our ability to identify the proteins and modifications represented in imaging mass spectrometry molecular signatures. Project success will make imaging mass spectrometry much more useful as a clinical pathology tool.


Modulation of cognitive and emotional responses by the limbic system of the brain is studied in patients undergoing cingulomotomy or amygdalectomy for the relief of intractable pain or epilepsy respectively. Autonomic and electrophysiological changes are studied by 1) stimulation through chronic electrodes situated in various limbic structures and 2) pre- vs. post-operative assessment of the effect of therapeutic brain lesions. Modifications in the appreciation of pain (conditioned avoidance response) and certain psychological functions (memory, perception and learning) are related to mechanisms assigned to the emotional brain of man.


The overall goal is to study the licensing policies and practices of 12 academic institutions regarding their DNA-based patents. The proposed project is conceived as a pilot study that will test the feasibility of conducting a more comprehensive follow-up study of such policies and practices. Specific aims are as follows: (1) To provide a clear, concise definition of the phrase "DNA-based patents"; (2) to analyze DNA-based patents into subtypes, using categories that are useful for understanding the policies and practices under which they are commercialized; (3) to gather and publish precise, up-to-date information on the number of U.S. DNA-based patents held by all U.S. and Canadian academic institutions; (4) to invite the technology transfer offices of all institutions holding at least 25 such patents to participate in a pilot study of their patenting and licensing policies regarding DNA-based patents; (5) with the aid of a project advisory board, to select a representative group of 12 positive respondents for more detailed study of their licensing policies; (6) to provide the technology transfer offices of the 12 participating institutions with categorized lists of their DNA-based patents, and to solicit input on categories; (7) to gather detailed information about the licensing of DNA-based patents at these institutions through a questionnaire and follow-up interviews; (8) to analyze and publish the data that have been gathered, paying special attention to policies and practices regarding the licensing of DNA-based patents that were based, at least in part, on research supported by federal funding; (9) during the second year of the project, to select 10 patents or clusters of patents and to develop case studies that illustrate technology transfer based on DNA-based patents. At least one of these case studies will be focused on a research tool. (10) In light of the results from the pilot study, to consider the feasibility and utility of conducting a more comprehensive follow-up study of licensing policies and practices regarding DNA-based patents at U.S. and Canadian academic institutions. [unreadable] [unreadable]


Thyroid hormone (TH) acts through binding to nuclear protein thyroid hormone receptors (TR), which regulate transcription of specific genes. Clinically significant abnormalities related to TR function are believed present in patients with serious illness, thyroid hormone resistance, and possibly in tumors and obesity. With the availability of cloned cDNAs coding for three forms of human TR (TRalphas and TRbeta), semipurified TR preparations, and cloned TH responsive genes, we are in a position to answer important questions about TH function in human health and disease. While three forms of hTR have been identified, it is possible that other forms are present, and we will screen human cDNA libraries to identify these. We will study the expression of hTR alpha and hTR beta in various human tissues by Northern Blot to determine which forms are expressed in all tissues. The RNAse protection assay will also be used to evaluate the forms of hTR expressed and to quantitate the amount in tissues. To fully evaluate hTR expression, we will develop antibodies to hTRs using synthetic peptides or fusion proteins. With these tools, we can determine hTR mRNA levels, TR protein, and functional TR as (131I)-T3 binding capacity. Function of the hTRs will be determined by co- transfection of hTR and rGH-CAT plasmid constructs into appropriate cell lines. Co-transfection studies will allow us to compare specifically TR alpha and TR beta control of gene transcription, and to evaluate both TRs with different responsive genes. We will clone and sequence portions of the alpha and beta TR genes in order to characterize the genes and to explain the apparent formation of multiple TR isoforms from one primary transcript. mRNA, Southern Blots of genomic DNA, and immunoprecipitable TR protein will be studied in fibroblasts of patients with thyroid hormone resistance. To identify if a specific isoform of TR is related to their illness,-cDNAs will be prepared from their mRNA for specific TRs, amplified, and sequenced. Our studies will identify the forms of functional hTR present in human tissues, their ability to control transcription of specific genes, and the molecular abnormality of TR present in patients with thyroid hormone resistance.


Biomechanical factors, chondrocyte viability, and inflammation are important factors in the pathogenesis of arthritis. Nitric oxide (NO) synthesis is increased in arthritis and is a mediator in the response to mechanical compression, chondrocyte apoptosis and in the inflammatory response to the catabolic cytokine IL-1. The most direct effect of NO is the suppression of energy production, which in many cell types is then associated with decreased oxygen (02) consumption, decreased matrix synthesis and the control of cell death. NO and 02 competitively bind to cytochrome oxidase, which is involved in oxidative mitochondrial respiration (OXPHOS). However, articular chondrocytes are highly glycolytic due to their avascular and hence hypoxic environment and OXPHOS only accounts for up to 25% of total steady state ATP production. It is suggested OXPHOS may contribute to more ATP production under conditions of tissue stress, such as inflammation or mechanical stress, or in arthritis where the oxygen tension of the synovial fluid, and hence cartilage, is further decreased. A steady supply of ATP is essential for articular chondrocyte remodeling of the matrix as an adaptation to biomechanical or inflammatory stresses, but also for the high energy requirements of apoptosis. NO is suggested to have both a protective and destructive role in cartilage. Little is known concerning the effect of NO and oxygen on ATP production in articular cartilage under physiological or ipathological conditions. The putative protective effect of NO might be determined by the intracellular redox state since NO and 02 compete for binding to cytochrome oxidase of the mitochondria. Our primary hypothesis is that oxygen tension determines whether NO inhibits ATP production, and if sufficient ATP is available to respond to inflammatory or mechanical stress. We propose to test whether oxygen tension l affects the ability of NO to inhibit the aerobic OXPHOS pathway and hence the source of ATP required to respond to the inflammatory or mechanical stress. We then propose to investigate how these factors affect matrix turnover and cell death. Since mechanical stress is protective to IL-1 induced matrix breakdown we shall investigate if this phenomenon is influenced by oxygen tension.


The long range goals of this proposal are to better understand how neural activity regulates opioid gene expression, and to understand how these processes contribute to environmental and drug induced stable/adaptive changes in the nervous system. A better understanding of these processes will help to define the adaptive biochemical changes underlying addiction, withdrawal, and drugseeking behaviors. The primary objectives of this research proposal have not changed. The major focus is still to identify and characterize transcription factors which mediate activity-dependent regulation of proenkephalin gene expression via their interaction with a well characterized second messenger inducible DNA enhancer. Studies will focus on defining components the AP-l/ AP-4, and NF-l/ ZFX nucleoprotein complexes which mediate regulation of proenkephalin transcription. Functional and biochemical interactions with the proenkephalin inducible DNA enhancer and signals transmitted through intracellular signaling pathways will be investigated. Recent findings indicate that JunD activates proenkephalin transcription in a fashion which is completely dependent upon the cyclic-AMP dependent protein kinase (PKA), an effect which can be effectively blocked by JunB. We have increased our emphasis on understanding regulation by JunD/JunB in light of the above findings, and the recent discovery that drugs of abuse such as cocaine, amphetamine, and morphine regulate AP-1 (fos/jun complexes) expression in brain. Together, this analysis will define at the molecular level interactions between intracellular signaling pathways, transcription factors, and the DNA elements which regulate endogenous opioid signaling in response to neurotransmitters, drugs, and synaptic inputs.


The immediate objectives of this research proposal are to determine which cellular components are responsible for the behavior of normal and tumor-virus transformed cells in vitro. The longer term goals of this study are to gain new insight into the mechanisms and to elucidate new parameters of the cellular basis of cancer including neoplastic growth and metastasis. Since cytoplasmic fibers are considered to be the major components responsible for many aspects of the behavior of cultured cells, we are emphasizing the study of their structure, function and inter-relationships. Light and electron microscopic studies have revealed the presence of three such fibers in several lines of cultured cells, which have been designated as microtubules, microfilaments and filaments. The localization of all three fibrous components in cells in the various states of physiological activity which occur during the cell cycle, suggests that they play major roles in attachment and spreading, division and locomotion of cultured cells. Furthermore, preliminary observations implicate these fibers in aspects of the social behavior exhibited by cells, including contact-inhibition. The correlation of function with structure is achieved through the coordinated use of light and electron optical techniques. In addition, various drugs such as colchicine, cyclic AMP and Cytochalasin B are used to alter cells in a manner which should yield new information concerning the structures involved in cell behavior. More direct approaches to determining function include the use of heavy meromyosin binding to microfilaments, and fluorescent and ferritin-labelled antibodies made against the major structural and contractile proteins of mammalian cells.


The long-term goal of the research project is to determine the molecular mechanism of the maturation-inducing steroid (MIS) receptor in the regulation of oocyte maturation in vertebrates. Little is known about the physiochemical characteristics of this MIS receptor. Recently, we have isolated and purified the MIS receptor from the plasma membrane of Xenopus laevis oocytes and also produced a polyclonal antiserum against the receptor. The objective of this application is to clone and sequence the MIS receptor and to examine its subcellular localization in Xenopus oocyte. Since the MIS in Xenopus is progesterone, our specific aim 1 of the research is to clone and sequence the cDNA encoding the progesterone receptor. Purified receptor protein will be microsequenced to obtain internal amino acid sequences to design the oligonucleotide probes, cDNA libraries will be prepared from oocytes and screened for positive clones. The cDNA of the receptor will be sequenced and analyzed. Our specific aim 2 is to determine the maturation-inducing activity of the progesterone receptor. The progesterone receptor mRNA will be generated by in vitro transcription of the cDNA and microinjected into immature oocytes. The oocytes will be cultured to allow for the synthesis of the progesterone receptor. Maturation of the oocytes in response to progesterone stimulation will be assayed. Our specific aim 3 is to examine the subcellular localization of the progesterone receptor mRNA and the protein in Xenopus oocytes. Labeled RNA probes will be used for in situ hybridization to determine the distribution of mRNA and the polyclonal antiserum will be used in immunocytochemical localization of the receptor protein. It is expected that the findings will contribute to a better understanding of the mechanism of the MIS receptor in the control of gamete development. The new knowledge will have a great potential for clinic applications such as induction of oocyte maturation in anovulatory patients and development of contraceptive agents for inhibition of oocyte maturation and ovulation in humans and animals.


ABSTRACT: Chronic sequelae of long-term HIV infection and treatment including heart, lung, blood, and sleep (HLBS) co-morbidities are increasingly important causes of morbidity and mortality. Latent HIV and maladaptive inflammation also persist despite successful ART. Therefore, we propose a new training program in HIV-related research, the Pittsburgh HIV Mentored Training for Investigation of Co-morbidities and Cure (HIV MeTrICC), led by Drs. Alison Morris, Dr. John Mellors, and Solomon Ofori-Acquah. They have an outstanding training record for PhDs and MDs, and their research experience spans the spectrum from basic to translational to clinical with expertise in lung, blood, and vascular co-morbidities and HIV cure. HIV MeTrICC will be an interdisciplinary, intensive mentored research training and career development experience focused around two interrelated training tracks ? HLBS Co-morbidities and HIV Cure with the following objectives: 1) To provide intensive mentoring in HIV-associated HLBS Co-morbidities and HIV Cure. Our team of established Mentors will offer unique multidisciplinary mentoring and career guidance to Scholars in the conduct of HIV research projects. Scholars will choose Mentors from the MeTrICC Co-morbidities and HIV Cure tracks. Tracks will be highly interactive. 2) To provide didactic training to foster progression to an independent career in HIV-related research. Formal training will encompass a broad spectrum of research methodologies, technologies, and analytical approaches to basic, clinical, and translational HIV research. Scholars with varying levels of prior research experience will be supported by Individual Development Plans geared to their needs. 3) To provide opportunities for training in Inner-city HIV and International HIV settings. Scholars will be able to choose from two different focus areas if relevant to their career plan. For the Inner-city HIV focus, we partner with the University of Maryland and the NIH to provide experience with the District of Columbia Partnership for AIDS Progress, the nation?s largest urban HIV cohort study. For our International HIV focus, we utilize several sites in Africa with ongoing basic, clinical, and translational research. 4) To provide access to large, multi-center HIV studies: Scholars will have access to large cohort studies including the Multicenter AIDS Cohort Study, the Women?s Interagency HIV Study, the Strategic Timing of Antiretroviral Therapy pulmonary substudy, the Pittsburgh HIV Lung Research Cohort, and the Pharmacokinetic and Clinical Observations in People over 50 sleep Substudy. 5) To support scholars in preparation of an independent research proposal: The program will ensure that Scholars rapidly develop grant-writing skills and submit competitive grant applications to help them secure HIV research funding. We will support five post-doctoral Scholars (MDs and PhDs) for a period of 2 to 3 years based on initial level of experience (i.e. post-docs vs junior faculty). The success of the MeTrICC program will be measured by concrete milestones such first author manuscripts, grant funding, and transition to faculty with the ultimate goal to recruit a new generation of researchers to HIV and HLBS complications.


The N.C.I. Biologic Response Modifer Program has undertaken poly ICLC as a drug to study intensively. In preclinical testing a variety of augmentary actions on several immune functions have been noted, some of which do not appear to be interferon mediated. A Phase I study of biological response modifications in humans is under way in two institutions--N.C.I. and the Naval Hospital in Portsmouth, VA. Upon completion a Phase II study is planned. In addition the N.C.I. has agreed to undertake the monitoring of all cancer protocols with poly ICLC. Six clinical therapy protocols are currently ongoing, involving neuroblastoma, breast cancer, renal cell carcinoma, malignant melanoma, dysimmune peripheral paralytic neuropathies, and multiple sclerosis. There were no beneficial effects in neuroblastoma and breast cancer. There was a partial response in 2 of 5 patients with renal cell carcinoma. It is too early to evaluate, even in a preliminary way the malignant melanoma study. In 14 patients with peripheral neuropathy, there were at least 9 with moderate to marked improvement. In M.S. there are indications of positive effects, but it is too early to have much confidence in the results.


Ribonucleotide reductase (RR) is an important therapeutic target. Therefore, understanding the function and regulation of the RR gene, and development of new RR inhibitors may have a direct impact on cancer treatment. Our long-term goal is to better understand the molecular mechanism of RR gene regulation, and the role of RR in chemotherapeutic drug resistance. We have shown that gemcitabine resistant as well as hydroxyurea resistant clones both overexpress the RR small subunit, hRRM2, mRNA and RR protein. Further studies confirmed that these drug-resistant tumor cell clones are associated with nucleus factor Y (NFY) binding to the RR promoter. Therefore, in Specific Aim I we will investigate whether hRRM2 overexpression in drug resistant cells is directed through transcription factor regulation o: the hRRM2 promoter. Promoter binding of NPY and a new transcription factor, band c, will be examined by gel-shift analysis and specificity will be confirmed by affinity chromatography. The functional consequences of NFY and band c in the regulation of hRRM2 expression will be examined in transfection assays. Since p53R2 is an integral part of the RR gene family, we are also intrigued by the possibility that interactions between different subunits of RR are important. In Specific Aim II, we will examine the interactions of p53R2, hRRM2, and hRRM1 (the RR large subunit), identify interacting domains, and examine the biochemical characteristics of such subunit interactions. A mammalian expression system will be employed to examine in vitro recombination of each subunit. Deletion analysis and mutagenesis will be performed to examine interacting domains and results will be confirmed by affinity chromatography. The plasmid vector containing p53R2 will be transfected into cloned drug-resistant cells for study in vivo. Once we clarify the interaction among RR subunits, we will explore the role of p53 in RR regulation. Our immunoprecipitation results have shown that both p53R2 and hRRM2 can bind to wild-type p53. Our hypothesis is that RR subunit composition may be regulated by p53. In specific aim ifi, we will confirm the protein interaction between p53R2 and wild-type p53 using a yeast two hybrid system. These findings will be confirmed by immunoprecipitation and Western blotting. The effect o wild-type p53 on RR activity and subunit composition will also be examined by transfecting wild-type p53 into cell lines expressing mutant p53. These experiments will help to confirm the interactions between p53R2, hRRM2, and p53. Finally, in Specific Aim IV, we will investigate the molecular pharmacology of a new RR inhibitor, HSC 1 that inhibits human cancer cells through an interaction with hRRM2. Taken together, our proposed studies will provide an integrated evaluation of the molecular control of RR-mediated chemotherapeutic drug resistance.