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This revised application is a clinical trial designed to evaluate crestal bone level changes when implants are placed into extraction sites and immediately provisionalized. Extraction of maxillary incisor teeth is a common procedure as documented in our school clinics. Traditional treatment when a tooth is extracted includes a period of healing followed by placement of an implant. Our preliminary data indicates that the delayed approach results in labial bone loss with resultant horizontal deficiency in over 50% of cases, requiring hard and soft tissue grafting to achieve a functional result. Additional preliminary data indicates that grafting the extraction site and/or immediate implant placement and provisionalization results in a functional restoration, without the need for additional adjunctive grafting procedures. It is unclear how the crestal bone levels change during healing of the extraction site prior to implant placement, in contrast to immediate implant placement, with immediate provisionalization with a crown. We hypothesize that there will be differences in the crestal bone levels (primary endpoint), and differences in soft tissue levels and indices (secondary endpoints) between implants immediately temporized, placed either delayed or immediately after tooth extraction, with more bone loss in the immediate placement group (experimental) compared to the delayed placement group (control). The long-term goal is to determine if the crestal bone remodeling after tooth extraction or the apical position of the implant determines the final level of the crestal bone. Two groups of 45 patients will be evaluated. All subjects will have a single rooted maxillary tooth extracted. One group will have the socket grafted with mineralized bone and allowed four months prior to implant placement. The crestal bone in this group will have remodeled during the four months of healing prior to implant placement. The second group will have the implant placed immediately after tooth extraction, thus the crestal bone will remodel in the presence of the implant and the immediately placed temporary restoration, as a one-stage procedure. For both groups, the implants will be immediately temporized with a crown after implant placement. Standardized hard and soft tissue data will be collected preextraction as baseline, and then prospectively for at least two years in this trial, to compare and contrast the methods.
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{
"pile_set_name": "NIH ExPorter"
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Dramatic increases in computer speed and the development of on-screen keyboards that send text to commercial software programs provided the foundation for this innovative approach to assisted typing. In Phase I, this new approach "Smart Key Technology (TM)" (SKT) was investigated, further developed, and tested. Using "letter prediction, SKT searches currently loaded dictionaries (files containing word, sentence, and abbreviation lists ordinarily used with word prediction) and identifies all letters that follow the currently typed character string. Depending on the option selected, SKT "identifies" the most likely next letter(s) by removing, graying out, or changing the color of unlikely letters presented on the on-screen keyboard. In the experiment conducted, subjects typed significantly more letters during a set time period when using SKT with an on-screen keyboard under both point-and-click (when the keyboard was unfamiliar) and scanning conditions. Most notable was the dramatic improvement under scanning conditions. Hypothesized sources of performance enhancement are 1) reduced visual scanning time, 2) simpler displays, 3) reduced probability of selecting an incorrect key, and 4) decreased key selection time (for scanning keyboards). In the proposed Phase II work, further enhancements of SKT will be made and two important variations of SKT will be developed and tested. PROPOSED COMMERCIAL APPLICATION: Not available
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{
"pile_set_name": "NIH ExPorter"
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It is proposed to study the biosynthesis of phenazine antibiotics. Shikimic acid has been found to be a precursor. The pairing scheme of 2 shikimic acid molecules has been investigated with C14 labeled precursors and is presently studied with C13 labeled precursors. The role of phenazine-1,6-dicarboxylic acid as immediate precursor of phenazine-1-carboxylic acid is studied. Various substituted dihydrobenzenes are synthesized to study their role as intermediates.
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{
"pile_set_name": "NIH ExPorter"
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The computerized anesthesia record has been strongly advocated as a method of increasing the efficiency, accuracy, presentation, and trend identification of patient data, thereby increasing patient safety. Reported problems with the computer-user interface have stopped widespread use. Innovation's over-all objective is to: (1) develop a graphics-based, VISually Interfaced Computer Assisted Anesthesia Record (VISI_CAARE); (2) quantitatively demonstrate that this user interface is successful, and (3) demonstrate VISI_CAARE increases physician vigilance and patient care. In Phase I and simulation of VISI_CAARE increases physician vigilance and patient care. In Phase I a simulation of VISI_CAARE is rendered. A methodology is used to quantitatively demonstrate that the user interface is appropriately designed or identify portions that are not. A plan for the Phase II software development of VISI_CAARE is then established. A model of anesthesiologist vigilance is further developed. Based on the model, preliminary data on anesthesiologist vigilance is obtained. This information will be used in developing the clinical trial of VISI_CAARE is phase 2. Nearly 23,000,000 Americans undergo surgery each year in 28,384 operating room suites. If a computer assisted anesthesia record were used in each of these operating rooms, a market potential of 400 to 500 million dollars would be realized.
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{
"pile_set_name": "NIH ExPorter"
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The objective of this proposal is to determine the extent and mechanism of the mediation of adenosine and adenine nucleotide effects by cyclic AMP in mammalian cells. An attempt will be made to determine whether adenosine and/or the nucleotides are physiological hormone or transmitter like compounds. The adenosine effects will be examined with reference to synergism or inhibition when alone or in combination with other hormines, such as the catecholamines, prostaglandins, and histamine. These effects on cAMP will be studied mainly in cells in tissue culture: to include both established cell lines, and primary cultures of brain cells, peripheral nerve cells, skin epithelia, fibroblasts, liver, capillaries, and heart cells. Effects will also be looked for in non- cultured systems; e.g., brain slices, blocks of peripheral nerve tissue, the isolated perfused liver, and isolated skin epithelia. A detailed investigation of the adenosine action on cAMP turnover and its effects on phosphodiesterase and adenyl cyclase, especially in the cultured brain astrocytoma cells will be undertaken. An effort will be made to determine if adenosine or the nucleotides are released and if so, by what stimuli, and whether once released, if they affect the cAMP system. Purine analogues of the adenine compounds will be examined for their effects on cAMP accumulation, as well as the known inhibitors of adenosine action on cAMP levels such as the methylxanthines.
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{
"pile_set_name": "NIH ExPorter"
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The global epidemic of obesity is alarming, as obesity is a significant risk factor for numerous chronic disorders such as type 2 diabetes, cancer, and cardiovascular disease. Manipulating the adipose lineage may represent a novel therapeutic treatment for obesity and associated diseases; however, any attempts will depend on a deeper understanding of the basic mechanisms controlling adipocyte development. My career goal is to become a leading independent investigator in the burgeoning field of adipose development, with particular focus on the developmental mechanisms controlling the initial specification of different adipose depots in vivo. My postdoctoral research to date in Dr. Bruce Spiegelman's laboratory at the Dana-Farber Cancer Institute has focused on the discovery of transcriptional regulators of preadipocyte determination. Our recent discovery of the transcription factor Zfp423 as a preadipocyte-enriched regulator of preadipocyte commitment crucial for adipocyte development in vitro and in vivo has opened up the possibility of 1) developing tools to localize and isolate committed preadipocytes found in vivo, and 2) elucidate the molecular mechanisms controlling commitment to the adipose lineage. In Specific Aim 1 we will utilize BAC transgenic mice expressing GFP under the control of Zfp423 cis-regulatory elements to localize and isolate preadipocytes in various adult fat depots as well as in developing mouse embryos, and demonstrate their commitment to the adipose lineage. In Specific Aim 2 we will elucidate the molecular mechanisms of preadipocyte determination through biochemical analysis of Zfp423 function and interacting partners. These experiments will reveal the anatomical location of committed preadipocytes during mouse development, establish a novel tool for the purification of adipose precursors in mice, determine the molecular mechanisms by which Zfp423 controls preadipocyte determination, and importantly, identify novel regulators of the preadipose state. A deeper understanding of these unexplored areas of adipose biology will facilitate our attempts to manipulate the adipose lineage as a therapeutic treatment for obesity and metabolic disease. The support of this career development award and the experience derived from carrying out these experiments will facilitate my transition to an independent investigator in the field of adipose biology. PUBLIC HEALTH RELEVANCE: The rising incidence of obesity has increased the urgency of understanding the fundamental systems controlling energy homeostasis, including the formation of fat tissue. The experiments described in this proposal will establish and utilize an important tool for studying fat cell precursors in mice, and reveal the molecular mechanisms controlling fat cell development. This knowledge will facilitate our attempts to manipulate fat tissue biology as a therapeutic treatment for obesity and metabolic disease.
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{
"pile_set_name": "NIH ExPorter"
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Overview of Contract Objectives and Efforts are as follows: 1. Determination of the solubility profile of the supplied bulk drug substance in various physiologically acceptable aqueous vehicles and organic solvents. 2. Development of novel approaches for intravenous delivery of drugs exhibiting inadequate solubility and/or stability in aqueous solution. 3. Development of suitable stability indicating assays to monitor the integrity of the parent compound during formulation studies. Also, study stability of the prototype formulation under accelerated stability conditions at 50 degree C, 37 degree C and 25 degree C. 4. Perform preformulation studies including determination of pKa and partition coefficient. 5. Prepare pilot scale batches of the prototype formulation(s) for preclinical evaluation. 6. Study experimental dosage forms under simulated "end user condiitons", for e.g., compatibility with intravenous fluids, packaging components.
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{
"pile_set_name": "NIH ExPorter"
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The purpose of this project is to study the relationship between the distribution of methyl mercury in the cochlear fluids and the alteration of the cochlear potentials in guinea pigs. Animals are treated with methyl mercury chloride labeled with C14 and/or Hg203. Radioactivity in the endolymph, perilymph, cerebrospinal fluid and whole blood is measured with a liquid scintillation counter. The stimulus-related responses as well as the DC polarization of the endolymph are recorded from the cochlea. The cochlear microphonic and action potential are suppressed by methyl mercury but the endolymphatic potential remains unchanged. No specific accumulation of the methyl mercury is found in either the perilymph or endolymph.
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{
"pile_set_name": "NIH ExPorter"
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Cytochrome P-450 plays a key role in the metabolism, activation, and detoxification of drugs, endogenous steroids, xenobiotics, and environmental carcinogens. Cytochromes P-450 were purified from the livers of rats treated with phenobarbital (PB-P-450), 3-methylcholanthrene (MC-P-450), Beta-naphthoflavone (BNF-P-450), pregnenolone 16-Alpha-carbonitril (PCN-P-450), and the environmentally induced marine fish (Scup-P-450); monoclonal antibody-producing hybridomas were prepared to these enzymes. Among twelve hybrid clones producing MAbs active toward PB-P-450, seven were IgG1 and five were IgM. Three classes of MAbs were produced: one class bound but neither precipitated the PB-P-450 nor inhibited its aryl hydrocarbon hydroxylase (AHH) activity; a second class bound and immunoprecipitated, but did not inhibit enzyme activity; and a third class comprising a single clone, MAb 2-66-3, bound, immunoprecipitated and completely inhibited the AHH of PB-P-450. MAb 2-66-3 did not inhibit the AHH activities of MC-P-450, BNF-P-450, or PCN-P-450. The MAb 2-66-3 inhibited the AHH, ethoxycoumarin deethylase, and benzphetamine demethylase of liver microsomes from PB-treated rats, but did not inhibit these activities in microsomes from control, BNF-, or MC-treated rats. The MAb 2-66-3 showed high cross-reactivity in binding, immunoprecipitation, and inhibition of enzyme activity of PB-induced cytochrome P-450 from rabbit liver. Monoclonal antibodies to PCN-P-450 and Scup-P-450 are in the process of characterization. Monoclonal antibodies to different cytochromes P-450 will be extraordinarily useful for a variety of studies, including phenotyping, genetic analysis, and purification of cytochromes P-450.
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{
"pile_set_name": "NIH ExPorter"
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Understanding the biophysical properties of single neurons and how they process information is fundamental to understanding how brain works. The long-term objective of this proposal is to contribute to understanding the logic of information processing in the dendrites of individual vertebrate neurons. We will utilize new tools, based on a recently developed high-resolution imaging technique with intracellular voltage-sensitive dyes, to examine two notable spatial aspects of dendritic integration; (a) processing of sensory information in two functional compartments of mitral cells of the olfactory bulb and (b) functional subdivisions of CA1 pyramidal neuron dendritic tree. An accurate description of interactions between excitatory and inhibitory components in different dendritic compartments of mitral and tufted cells should facilitate understanding of the process of signal integration in the olfactory bulb. Such an understanding would be an important step toward comprehension of neuronal mechanisms involved in sensory perception. The postulate that synaptic amplification of the backpropagating action potential is restricted to the site of synaptic input is based on computer simulation and indirect experimental evidence. This is an important hypothesis because spatially restricted changes in excitability could functionally subdivide a neuron; multiple functional compartments, in turn, have important implications for synaptic integration and plasticity. The results of this analysis will bear on the basic neuroscience of signaling.
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{
"pile_set_name": "NIH ExPorter"
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The continued research and development of a portable, inexpensive, system for ambient noise attenuation is proposed for use in audiometric testing. The device will be a technologically advanced headset used in conjunction with insert earphones. In combination, the total attenuation of the device will be superior to any current portable alternative. This combination of headset and insert earphones will be designed to attenuate ambient acoustic noise during audiometric testing, and will replace sound isolation test rooms and mobile test facilities under certain ambient noise conditions. The portability of the system will enable it to be used in venues such as schools, nursing homes, prisons, and industrial plants. The result of this research will be tested pre-production units. The ultimate goal of this research and development will be to manufacture a portable audiometric testing system that provides noise-attenuation properties superior to a single-walled room in meeting ANSI S3.1-1 999 standards.
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{
"pile_set_name": "NIH ExPorter"
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[unreadable] [unreadable] The Transfusion Medicine and Hemostasis Clinical Trial Network (TMH CTN) was funded in 2002, in response to a demonstrated need for large multi-institutional clinical trials in transfusion medicine and hemostasis. The TMH Network is a national resource for the advancement of knowledge and understanding in the field of transfusion medicine and hemostasis. The Network will promote the efficient comparison of novel management strategies of potential benefit for children and adults with hemostatic disorders; and will also evaluate novel as well as existing blood therapies and cytokines for the treatment of hematologic disorders. Since its inception, the principal investigators and their collaborators at the 17 funded sites, have worked successfully to develop and implement clinical trials in these areas. Building on the progress to date, over the next the 5 years, the TMH CTN proposes to: 1) Maintain, and further develop, as needed, a structure to support the development, implementation and conduct of clinical trials in the areas of transfusion medicine and hemostasis. This will be done in collaboration with the New England Research Institutes as Data Coordinating Center, and program staff at NHLBI. 2) Utilizing a structure of working and study oversight subcommittees, complete ongoing trials and develop, refine and complete new trials proposed in this application. 3) Develop collaborative relationships and support training opportunities to facilitate the success of the proposed clinical trials and further the fields of transfusion medicine and hemostasis. The University of North Carolina at Chapel Hill, one of 17 funded sites, has been participating actively in the TMH CTN. Dr. Mark Brecher (Department of Pathology and Laboratory) is the lead principal investigator at UNC. Dr. Nigel Key (Department of Medicine- Hematology Division) is the co-principal investigator. Both of them have actively participated in several subgroups of TMH CTN. They also regularly attend the quarterly Steering Committee meetings. Additional TMH study staff includes two full-time research nurses and one part-time medical technologist. Two TMH CTN protocols are ongoing at UNC, and it is the intention of the investigators at this site to support new network protocols in the future. [unreadable] [unreadable] [unreadable] UNIVERSITY OF NORTH CAROLINA CHAPEL HILL CRITIQUES [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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Several lines of evidence suggest that endogenous ciliary neurotrophic factor (CNTF) receptor signaling may promote neuromuscular protection and repair. Thus, proper targeting of CNTF receptor signaling may selectively counteract the devastating effects of neuromuscular disorders by appropriately enhancing these naturally evolved mechanisms. Unfortunately, current understanding of endogenous CNTF receptor function in the adult is extremely limited because blockage of the receptor in vivo (through disruption of the critical CNTF receptor a [CNTFRa] gene) leads to perinatal death. To overcome this problem, we will use Cre/lox techniques to selectively disrupt the CNTFRa gene. Specific Aim 1 will examine the contribution of CNTF receptor signaling to the survival, protection and phenotype maintenance of adult motor neurons. The CNTFRa gene will be disrupted in facial motor neurons of "floxed" CNTFRa mice with: 1) stereotaxic injection of the facial nucleus with an adeno-associated virus that directs Cre recombinase (Cre) expression and 2) a gene construct enabling temporally controlled induction of Cre activity. Preliminary data indicate an essential in vivo role for CNTFRa in adult motor neuron survival. We will quantitatively characterize this function with reporter genes, immunohistochemistry and stereological cell counting and also determine whether insult is required to activate this neuroprotective system (as preliminary data suggest), and if so, what forms of insult. In addition, a reporter gene and immunohistochemistry will be used to determine whether CNTF receptor signaling maintains motor neuron cholinergic phenotype in vivo. Finally, signaling proteins potentially involved in the neuroprotection will be identified through in situ hybridization and immunohistochemistry. Specific Aim 2 will define the role of skeletal muscle CNTFRa in neuromuscular protection and repair. Preliminary data from floxed CNTFRa mice with skeletal muscle specific Cre expression reveal that muscle CNTFRa is required for normal motor recovery following peripheral nerve lesion. We will characterize this functional deficit with "footprint" analysis and identify the underlying cellular mechanisms by quantifying: 1) neuromuscular junction formation, 2) motor neuron survival and cholinergic phenotype, and 3) muscle contractility. In addition, STAT3-based immunohistochemistry will be used to localize cellular sites of muscle-CNTFRa-dependent signaling after nerve lesion. Moreover, microarray analysis of muscle and spinal cord ventral grey matter following nerve lesion will be used to identify novel candidate genes potentially involved in the critical CNTF receptor signaling. Relevance in lay language: We will determine how natural CNTF receptor signaling in mice protects and repairs the adult neuromuscular system. The resulting knowledge should facilitate the design of therapeutics which selectively enhance the appropriate CNTF receptor signaling and thereby effectively treat neuromuscular disease while avoiding side effects. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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The conformations, microdynamic behavior, and interactions with biologically important macromoleculaes of peptide hormones, analogues, derivatives and other biologically important peptides will be examined by a variety of biological activity and the conformations and microdynamic properties of the peptides will be sought, and the results will be used as a basis for preparing better hormone antagonists and more specific agonists, and for examining the chemical-physical basis of hormone action. Specifically labeled (deuterium, carbon-13, carbon-14, fluorine, etc.) derivatives of the neurohypophyseal hormones and biologically important analogues will be prepared. For this purpose, efficient procedures for synthesizing C13 and H2-labeled amino acids be sought. The deuterated and carbon-13 labeled hormones will be used in studies of the conformational and microdynamical behavior of these compounds, and of the interactions of the hormones with their carrier proteins in the neusecretory granules, the neurophysins. Nuclear magnetic resonance spectroscopy, circular dichroism, viscosity studies, binding studies, cross-linking experiments, and other chemical-physical methods will be used to develop a basis for understanding hormone action at the molecular level. The interaction of peptide hormones with their cellular and membrane receptors will be examined. Hormones or hormone glucagon, and oxytocin will be used in these studies. The design of hormone derivatives with properties useful for medical and other applications will be sought.
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{
"pile_set_name": "NIH ExPorter"
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Progression of diabetic foot ulcers is the leading cause of lower limb amputation in the U.S. The high incidence of diabetic foot complications demonstrates aggressive conventional therapy is neither realistically accomplished, nor cost-effective. Furthermore, conventional therapy does not treat the causative microcirculatory pathology. The overall project goal is to evaluate Encelle, Inc.'s proprietary hydrogel matrix (E-Matrix) as a single injection therapy for vascularization of skin lesions. The project will develop characterization assays for E-Matrix to understand the physical and chemical properties of the matrix, measure manufacturing consistency and E-Matrix stability. Efficacy will be evaluated using two wound models resulting from compromised vasculature in pigs. Surgical interruption of the skin vasculature, or phenol chemical burns, will be induced at six separate sites in two cohorts of four pigs each. The effect on wound size and rate of closure following no treatment, a single control injection, or a single E-Matrix injection circumferentially and centrally, will be studied. After 14 days, the animals will be sacrificed, and tissue samples obtained for immunohistochemical preparation and analysis. Tissue analysis using histological and immunohistochemical methods will qualitatively and semi-quantitatively delineate the vascular density at wound sites, differentiation of blood vessel types, the number and type of infiltrating cells, mitotic indices, apoptosis during healing, amount and type of collagen formed and induction of metalloproteinases. Understanding the detailed tissue and cellular response to E-Matrix treatment using reproducibly manufactured E-Matrix with known physical and chemical characteristics in well-defined animal models will provide a strong baseline for the commercial development of this product. PROPOSED COMMERCIAL APPLICATIONS: Potential applications of E-Matrix include the treatment of conditions associated with compromised vascular flow including diabetic ulcers, decubitus ulcers, burns, atherosclerosis of the peripheral vasculature, coronary artery disease, and stroke.
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{
"pile_set_name": "NIH ExPorter"
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Malignant gliomas are common primary human brain tumors, but problems in their pathological classification compromise patient management. These difficulties have sparked considerable interest in molecular genetic approaches. Clinically relevant genetic associations have been discovered, but practical problems have prevented widespread diagnostic application of this knowledge. We hypothesize that custom array comparative genomic hybridization (aCGH) could provide a sensitive, specific, cost-effective and rapid method to assess human malignant gliomas for a variety of clinicopathologically relevant genetic changes and that such first-generation custom CGH arrays will provide the basis for improving diagnostic correlations for future assays. To evaluate this possibility, we propose a two-stage plan that capitalizes on our existing strengths in molecular genetics, pathology, biostatistics and clinical databases. For the R21 component, we will: 1) evaluate custom aCGH sensitivity and specificity in comparison to standard assays; and 2) generate custom BAG arrays for CGH that include targets providing broad genomic coverage as well as focused coverage of chromosomes 1, 7, 9, 10, 19 and X, and other select loci. Once we have met the Milestones from the above R21 Aims, we will proceed in the R33 component to: 1) evaluate whether alterations of particular regions on the assayed chromosomes, as revealed by aCGH, offer improved and/or novel correlations with chemoresponse and survival in two carefully annotated cohorts of malignant glioma patients: a) a retrospective series of anaplastic oligodendroglioma patients; and b) a prospective series of malignant glioma patients. We will then: 2) develop an aCGH-based classification of malignant gliomas using the data from Aim 1 of the R33. The long-term goal of this project is the implementation of a practical molecular assay to detect a variety of clinicopathologically relevant genetic alterations in malignant gliomas. We also anticipate that such information will contribute to identification of key glioma genes as well as to construction of next-generation diagnostic approaches. Given these endpoints, the application is highly responsive to PA-04-102, "Phased application awards in cancer prognosis and prediction."
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{
"pile_set_name": "NIH ExPorter"
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Although malignancy remains a critical health concern, significant medical advances in cancer detection and treatment have improved the survival rates for patients. As patients live longer, the consequences of cancer management have assumed greater importance. Traditionally, cancer patients have been faced with great uncertainty and have had few choices for fertility preservation. However, recent scientific advances employing a three-dimensional scaffold system usingx alginate for the in vitro maturation of ovarian follicles reveals promising new technologies for clinical applications. The mission of the National Physicians Cooperative (NPC) Tissue Core is to collect and distribute human ovarian tissue containing follicles to the basic science components of the Oncofertility Consortium. By doing so, we will catalyze the translation of the basic science knowledge gained in project RO1C and the Biomaterial Core (P30A) to women with a cancer diagnosis. The NPC is comprised of five Core institutions and fifteen Allied centers across the country. The elements of this program include patient screening and selection, collection of clinical and laboratory data, standardization of operative techniques for oophorectomy, post-surgical handling of tissues, tracking and storage of ovarian specimens, and a process of tissue distribution to participating sites. Furthermore, the Core will work closely with the social science section (RO1D) to address the ethical, legal, economic, and social considerations that will emerge from the new discipline of Oncofertility. The primary goals of this proposal are (1) to collect adult human ovaries from women with cancer and distribute them for basic science research;(2) to educate providers, patients, and the community through the Allied centers about fertility options for women with cancer;and (3) to disseminate the technical knowledge of follicle maturation and cryopreservation to the Allied centers. The collective goal is to ultimately provide new options for fertility preservation. The NPC will provide the vehicle through which breakthroughs in basic reproductive physiology will be translated to clinical medicine and, ideally, overcome the infertility and subfertility faced by many women undergoing cancer treatment. Restoration of fertility and other ovarian functions would substantially improve the quality of life for these women.
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{
"pile_set_name": "NIH ExPorter"
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During vertebrate embryogenesis patterning signals emanate from very discrete centers like the Hensen's node, the midbrain-hindbrain junction or, in the case of the limb, the zone of polarizing activity (ZPA) of the apical ectodermal ridge (AER). It has become evident that many of the molecules that control developmental pathways in insects have homologous counterparts in vertebrates. The AER is required for vertebrate limb outgrowth. Formation of the ridge relies on the previous establishment of the dorso-ventral limb axis. The investigator has cloned a gene, r-fng, which is expressed in the dorsal ectoderm and in the ridge. Ectopic expression experiments indicate that r-fng is able to induce additional AERs suggesting that the AER develops from tissue that is expressing high levels of r-fng adjacent to tissue that is not, i.e. a r-fng boundary. A similar mechanism has been proposed in Drosophila, where the fng gene plays a pivotal role in wing development. Identification of the existence of a common signaling process that mediates the formation of the Drosophila wing margin and the vertebrate AER will be quite surprising, even in light of previously demonstrated homologies between the two phylogenetic groups, and it will require the isolation of downstream molecules. In Drosophila, activation of fng induces a cascade of events that results in the activation of the expression of Serrate, Notch, Vestigial, Cut and Wingless at the dorso-ventral boundary. The investigator has cloned chick homologues of Serrate, Notch and Cut and shown they are expressed in the AER. By using wild type and limb mutant embryos, the investigator will study the consequences of disrupting Notch signaling for establishing and positioning the vertebrate AER. The molecules described above are also required during embryogenesis for other processes like neurogenesis and somitogenesis. This suggests that during limb development, somitogenesis, or neurogenesis, a similar combination of genetic building blocks could be used again and again. Understanding the way these genetic bricks interact with each other in order to position and establish the AER will prove to be a rewarding enterprise, not only from the point of view of limb development, but also from a more general perspective of unraveling how evolution has utilized the same combination of molecules to establish other signaling centers during vertebrate development.
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{
"pile_set_name": "NIH ExPorter"
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The overall goal of this project is to identify the neural correlates of recovery from aphasia during the first three months after acute stroke. Aphasia is one of the most common and debilitating consequences of strokes affecting the dominant hemisphere. In the acute and subacute stages, most patients experience some degree of recovery of language function. These first few months after brain damage occurs are critically important because the greatest behavioral gains take place during this time, even when patients do not receive treatment. However the extent of recovery is highly variable: some patients recover most or nearly all language function, while others make few gains and remain chronically aphasic. It is not well understood what neural mechanisms underlie recovery during this period, nor why some patients recover so much better than others. To address these questions, we propose to study individuals with aphasia due to acute left hemisphere stroke. Patients will be examined at four time points after stroke: 24-48 hours, 2 weeks, 1 month, and 3 months. At each time point, a comprehensive yet time-efficient language evaluation will be administered to quantify expressive and receptive language function, and a multimodal brain imaging protocol will be implemented that includes structural imaging (MRI), functional imaging (fMRI), perfusion imaging (arterial spin labeling, ASL) and diffusion tensor imaging (DTI). The proposed research has two specific aims. The first is to identify the neural changes that are associated with successful recovery from aphasia in the acute and subacute periods after a stroke. The second aim is to determine the initial imaging and behavioral variables that are most predictive of the extent of eventual recovery of language function. We hypothesize that the inclusion of fMRI, ASL and DTI in combination with comprehensive language assessments will provide a more complete characterization of the brain's changing state than standard clinical imaging and language measures, and will explain a greater proportion of the variance in recovery patterns. A better understanding of the neural correlates of successful recovery will improve the accuracy of prognoses so as to better plan medical treatments and behavioral interventions.
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{
"pile_set_name": "NIH ExPorter"
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This project represents a comprehensive multidiscipline study of erythema multiforme, a common disorder with significant complications. The primary purpose of this study is to characterize the pathogenesis of erythema multiforme, which we hypothesize involves immunologically-mediated tissue damage initiated by a variety of foreign antigens. Cases of erythema multiforme will be studied for evidence of circulating immune complexes and for evidence of cytotoxicity to epidermal cells. Measurement of activity of the complement system, assays for immune complexes (I125C1q binding and Raji cell), and quantitation of cryoimmunoglobulins will be performed. In order to identify foreign antigens important in the pathogenesis, the components of the cryoprecipitates, both antibodies and antigens, will be specifically characterized. The presence of antibodies which mediate antibody dependent cellular cytotoxicity to epidermal cells will be evaluated. The presence of immunoreactants in skin lesions will be determined by immunofluorescence and electron microscopy. In all cases studied, careful clinical data from history and physical examination, histological data from light microscopic and electron microscopic examinations of skin lesions, and etiological data from history and laboratory evaluation will be collected. By correlating data from the clinical, histological, etiological, and pathogenetic aspects of the study, we will be able to better understand the syndrome. Furthermore, we may be able to identify antigens and antibodies that initiate the process. A much needed double-blind therapeutic study glucocorticoid therapy of erythema multiforme will be performed.
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{
"pile_set_name": "NIH ExPorter"
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This study will compare the risks and complications of the UVC and the PCVC. It is unclear which of these two catheters is safer for babies. This study hypothesizes that the long-erm use of UVC will present fewer infection and other risks than long-term use of PCVC. This incidence of blood stream infection will be compared between grouips of low birthweight infants under 1250 grams at birth randominzed to either long term (8-28 days) umbilical catherization or short term (<7 days) umbilical catheterization followed by percutaneous central venous catheter placement. The primary outcome will be Kaplan-Meier estimates of infection-free line survival. Secondary outcomes will include the incidence of toerh catheter complications, feeding complications and need for additional venous access.
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{
"pile_set_name": "NIH ExPorter"
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DESCRIPTION: The overall goal of this project is to use a subtractive immunization approach, already well-established in the Dr. Quigley's laboratory, to screen for cell surface molecules that may be involved in metastasis. Unlike many other studies in this area, no preconceived bias as to the biochemical nature or function of the molecules is involved. The specific aims are: (1) to use the existing panel of monoclonal antibodies and a simple, quantifiable model to identify cell surface molecules involved in metastasis; (2) to characterize and isolate the cell surface antigens of interest; and (3) to clone and sequence the cDNAs encoding these antigens. The long range goals are to determine molecularly and mechanistically how and where these selected antigens function during metastasis.
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{
"pile_set_name": "NIH ExPorter"
}
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ABSTRACT Although the mechanism of Cd(II)-induced carcinogenesis remains to be investigated, recent studies have indicated that autophagy plays a significant role. Our preliminary studies have shown that exposure of human lung bronchial epithelial BEAS-2B cells to Cd(II) generates reactive oxygen species (ROS), which are responsible for Cd(II)-induced malignant cell transformation. We have also shown that Cd(II) is able to induce autophagy in normal BEAS-2B cells. The activation of autophagy by Cd(II) is likely to be a cell self- defense mechanism against Cd(II)-induced oxidative damage. Our preliminary studies have also shown that Cd(II)-transformed BEAS-2B cells exhibit autophagy deficiency (autophagy incompetence), resulting in accumulation of autophagosomes and increased level of p62 protein. The increased p62 causes a constitutive activation of Nrf2, which in turn upregulates its target antioxidant proteins, superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2), and anti-apoptotic proteins, Bcl-2 and Bcl-xL, most likely due to increased binding of Nrf2 to antioxidant response element (ARE) sites of these target proteins. The upregulations of these antioxidants (decrease ROS) and anti-apoptotic proteins result in development of apoptosis resistance of Cd(II)-transformed cells. The constitutively elevated p62 and Nrf2 are responsible for survival advantage of Cd(II)-transformed cells. Natural compound sulforaphane increased autophagy in normal cells exposed to Cd(II) and restored autophagy competence in Cd(II)-transformed cells. The central hypothesis of this application is that autophagy is a cell defense mechanism in Cd(II)-induced malignant cell transformation and that autophagy deficiency is responsible for tumorigenesis of Cd(II)-transformed cells. Aim 1 will investigate the protective role of autophagy against Cd(II)-induced malignant cell transformation. The hypothesis of this aim is that autophagy protects against Cd(II)-induced malignant cell transformation by decreasing ROS through facilitating mitochondrial turnover in normal cells and that sulforaphane enhances autophagy and decreases the cell transformation. Aim 2 will demonstrate that autophagy deficiency in Cd(II)- transformed cells increases cell survival through constitutive activation of p62/Nrf2 signaling. The hypothesis of this aim is that Cd(II)-transformed cells are autophagy deficient, resulting in accumulation of autophagosomes, constitutive activations of p62/Nrf2, elevated levels of antioxidants, Bcl-2, and Bcl-xL, decreased levels of ROS, and acquisition of apoptosis resistance. Aim 3 will investigate roles of autophagy deficiency, elevation of p62, constitutive activation of Nrf2, and apoptosis resistance in tumorigenesis of Cd(II)-transformed cells and protection by sulforaphane in vivo. The hypothesis of this aim is that autophagy deficiency in Cd(II)-transformed cells promotes tumorigenesis through elevation of p62, constitutive activation of Nrf2, and acquisition of apoptosis resistance and that sulforaphane restores autophagy competence and inhibits tumorigenesis.
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{
"pile_set_name": "NIH ExPorter"
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This application seeks support for a post-doctoral training program in urology based in the Scott Department of Urology at Baylor College of Medicine. The SDU has major ongoing clinical activities, as well as basic research with an emphasis in Prostate Biology, Male Reproductive Biology and Pediatric Urology/Developmental Biology. There are 20 training faculty, 8 of whom have primary appointments in Urology. The training faculty includes 7 M.D.s, 12 Ph.D.s, and 1 M.D./Ph.D. Dr. Lamb will serve as Principal Investigator of the T3 proposal, Dr. Morton will be PI of the K12 proposal, and Dr. Lipshultz will serve as the Director of the Fellowship Training Program. The major research areas in the program include: prostate biology, cell biology, steroid hormone receptors, male reproductive biology, genetics, cell cycle control, aging, human benign prostatic hyperplasia, pediatric urology/developmental biology, gene therapy, and cloning genes. The trainees will be Ph.D. candidates, most likely joining the program directly from completion of graduate school. A focus of our program will be the attraction of highly qualified trainees who seek and intensive substantial research training in Prostate biology, Male Reproductive Biology and Pediatric urology/Developmental Biology, with an emphasis on the clinical translation of the basic research. We have developed a strong recruitment plan, emphasizing the career development of women and minorities in the field. Strengths of the training environment include highly respected, experienced faculty, a high national ranking for research funding, a structured mentoring program and required coursework, a cell biology graduate student program with relevant course work and overall depth in cell biology. Our long-term goals are to train these young investigators to compete successfully for peer-reviewed funding and eventually to increase basic research efforts in Urology.
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{
"pile_set_name": "NIH ExPorter"
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UDP-glucuronosyltransferases (UDPGTs) are a group of glycoprotein enzymes concentrated in the endoplasmic reticulum of liver cells, which are essential for the detoxification of internal metabolites and foreign substances, e.g. drugs, carcinogens and environmental toxins. UDPGTs exist as several isoforms; inherited defects of specific forms of UDPGT result in life-long jaundice in man and mutant (Gunn) rats. In preliminary studies we have shown that Gunn rats have approximately normal amounts of two functionally defective UDPGT isoforms. However, the biochemical basis of functional heterogeneity and inherited functional disorders of UDPGTs is not known. The purpose of the proposed project is to determine the possible role of glycosylation and other types of co-translational and post- translational changes in the generation of functional heterogeneity and inherited functional defects of UDPGTs. We plan to develop monoclonal and polyclonal antibodies that are specific for individual UDPGT isoforms and to use these to study the expression of UDPGT genes in inbred normal rats and congeneic Gunn rats. Precursors of individual UDPGT isoforms will be identified by cell-free translation of liver mRNA from normal rats; these precursors will be compared with those derived by translation of Gunn rat liver mRNA. Biosynthetic steps of synthesis of UDPGT precursors and processing of their protein and carbohydrate components will be studied by metabolic labeling of hepatocytes isolated from livers of Gunn rats and congeneic normal rats. Since UDPGTs are integral endoplasmic reticulum proteins, in addition to elucidation of the biochemical basis of inherited disorders of this essential enzyme system, these studies may help us understand the mechanism of assembly of liver endoplasmic reticulum proteins in general.
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{
"pile_set_name": "NIH ExPorter"
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ABSTRACT Radiotherapy and chemoradiation for head and neck cancer treatment exposes normal tissues to radiation, which has many devastating effects and often results in difficulty with communication and swallowing. While muscle weakness and fibrosis are possible etiologies for disruptions in critical communicative and deglutition functions following radiation, very little research has been performed on underlying biological changes within muscles of the head and neck following radiation, or possible treatments for these lasting negative effects. Skeletal muscles can adapt at multiple levels of structure and function to changing demands. Exercise training of the tongue, or enhanced muscle contraction via neuromuscular electrical stimulation (NMES) may be beneficial for preventing or reversing muscle tissue damage. However, controlled research examining these putative benefits has not been performed and optimal treatment modalities have not been established. Our hypothesis is that radiotherapy and chemoradiation-induced decline in speech and swallowing function is largely due to alterations in tongue muscle structure and function. Further, we hypothesize that tongue exercise or NMES will result in phenotypic changes in extrinsic tongue muscles that will improve tongue muscle function. To examine these clinically-relevant issues, we will use a rat model to test two different tongue treatment paradigms (tongue exercise; NMES) for remediation of radiotherapy and chemoradiation- induced muscle damage. The proposed research has two specific aims: 1) To determine how treatment modality affects morphological, biochemical, and physiological changes in radiotherapy and chemoradiation- induced muscle damage of the tongue, 2) To discover how tongue exercise and/or NMES treatment affects functional measures of deglutition following radiotherapy/chemoradiation. This work is innovative and significant because the mechanisms by which tongue exercise or NMES can prevent or treat the effects of radiation- or chemoradiation-induced communication and swallowing dysfunction is largely unexplored. Our animal model and treatments are analogs to treatments used in human patients and follow the Institute of Medicine guidelines for increasing probability of translation. Further, this work is highly significant in providing a basis for understanding the mechanisms underlying the potential of therapeutic interventions for radiation- and chemoradiation-induced cranial impairments. Translation of findings will assist with increasing the effectiveness of treatments for radiation- and chemoradiation-induced tongue muscle impairments that are so prevalent in patients with head and neck cancer.
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{
"pile_set_name": "NIH ExPorter"
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Our overall objective is to understand how processing of damaged DNA relates to human genetic disease, cancer and aging. Having pioneered the discovery of nucleotide excision repair (NER), we are elucidating the sub-pathways of global genomic repair (GGR) and transcription-coupled repair (TCR). The TCR-deficient diseases, Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS), present indistinguishable biochemical responses to UV; UVSS patients have only superficial consequences of sunburn while those with CS suffer severe neurological/developmental defects, segmental progeria and early death. Notably, no cancers of any type have been reported for patients with these syndromes. We hypothesize that the severe features of CS are due to apoptosis triggered by prolonged transcription arrest as a consequence of defective TCR of oxidative base damage or to defective transcriptional bypass of such damage generated by endogenous reactive oxygen species, while UVSS cells are normal with respect to processing base damage in expressed genes. In support of this model we find that CS cells are hypersensitive to oxidants, and that UVSS but not CS cells are proficient in host cell reactivation of plasmids containing oxidized bases. However, definitive biochemical evidence for TCR of oxidized bases is lacking. Our model for TCR postulates that an arrested RNA polymerase (RNAP) recruits repair enzymes to transcription- blocking lesions. Reported studies with an oxidized base positioned at a unique site in the DNA template strand indicate that RNAP can bypass, transiently pause or arrest at these lesions. We propose to use a novel transcription assay with multiple randomly-positioned lesions induced in the template, to let the transcription system tell us which lesions and which sequence contexts are most relevant for further analysis. After determining the types and positions of the lesions that cause arrest in vitro, we will construct single-lesion vectors for transfection into human cells to measure in vivo transcription rates upstream and downstream of the lesion; sequencing the transcripts will reveal transcriptional mutagenesis. We propose to develop the sensitive Comet-FISH approach with gene-specific probes to comparatively quantify low levels of particular oxidative lesions and their removal from transcribed or silent sequences and from the genome overall. Cells with missing or reduced base excision repair or NER activities will be employed to investigate processing of oxidative lesions. Specific enhancement of 8oxoG in DNA will be achieved by interference RNA-mediated MTH1 knockdown, to eliminate complications of other lesions and other oxidative effects. We will focus on differences between CS and UVSS as a model system to elucidate the role of processing of oxidative base damage in aging, disease and neurological degeneration, as well as the underlying cause of the cancer-resistance of these syndromes. PUBLIC HEALTH RELEVANCE: Free radicals from endogenous and environmental sources are a constant threat to genomic integrity. The induced damage can arrest DNA and RNA polymerases, events that can unleash irreversible apoptotic pathways or mutagenicity. We propose novel approaches for elucidation of the effects of oxidative DNA lesions on transcription, and for the analysis of repair of physiologically relevant levels of these lesions in transcriptionally active or silent genomic domains and in the genome overall, using the Comet- FISH assay. Results from the project will advance our understanding of cellular processes leading to carcinogenesis, aging, and other pathologies. They will also further the development of effective strategies for therapeutic intervention in human disease.
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{
"pile_set_name": "NIH ExPorter"
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OBJECTIVE: To determine whether the gestational age at the time of prenatal androgen exposure contributes to the altered glucose/insulin dynamics in female rhesus monkeys, as part of a study investigating the etiology of polycystic ovarian syndrome (PCOS). RESULTS Female rhesus monkeysexposed to androgen early in gestation had a significantly lower percentile for the relationship between insulin sensitivity and acute insulin response (10.3 q 7.0 %; p < 0.01) compared to the late treated androgenized females and control females (40.5 q 7.2 % and 31.3 q 6.0 % respectively). This result indicated that early exposed androgenized females have reduced pancreatic b-cell response relative to their insulin sensitivity. Overall, these findings suggest that prenatal androgen excess during early gestation exerts a significant adverse effect on pancreatic b-cell function in adulthood. Two of these androgenized females have since become glucose intolerant. Prenatal androgen excess in females may, therefore, pose a risk of early development of diabetes in adulthood. FUTURE DIRECTIONS We plan to explore the role of prenatal androgen excess in causing pancreatic impairment in female rhesus monkeys and examine potential mechanisms. KEY WORDS testosterone, diabetes, pancreas, polycystic ovarian syndrome, insulin, glucose. UW Graduate School Research Committee, UW Medical School Committee
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{
"pile_set_name": "NIH ExPorter"
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Obesity is now established as a potential cause for colon cancer. While the underlying mechanisms mediating the obesity-colon cancer link are not well understood, increasing evidence supports that Insulin resistance resulting from long-term energy imbalance and subsequent perturbation of metabolic homeostasis and insulin signaling pathways form the core of obesity-related colon carcinogenesis. Genetic variations within genes in key insulin and growth factor signaling pathways may, or in combination with obesity, drive the development of colon cancer, but have been little studied. The fact that obesity is escalating as an epidemic worldwide makes the exploration of the mechanistic connections between obesity and colon cancer a pressing public health and research priority. Therefore, we propose a genetic epidemiologic study of the relation of colon cancer with obesity and candidate genes in four critical insulin and related growth factor signaling pathways: 1) phosphatidylinositol-3 Kinase/protein kinase B (PI3K/AKT) signaling cascade;2) AMP-activated protein kinase (AMPK) pathway;3) mitogen-activated protein (MAP) kinase pathway;and 4) peroxisome proliferators-activated receptors (PPARs). Each of these pathways plays an important role linking increased adiposity to colon carcinogenesis and model systems indicate that crosstalk occurs among them. We will use both conventional statistical approaches and a novel hierarchical model to comprehensively evaluate obesity and adult weight gain, candidate gene polymorphisms and haplotypes, and their potential joint and interactive effects on colon cancer. The unifying theme of this proposal is that obesity and candidate gene variants within these pathways may act alone or jointly to drive colon carcinogenesis. This study builds upon an ongoing population-based case-control study where epidemiologic data and DNA samples from 1,250 incident colon cancer cases and 1,500 population controls has already being collected. This relatively large study population will be readily available and allow us to dissect complex gene-gene and gene-obesity interactions to gain in- depth understanding of mechanistic link between obesity and colon carcinogenesis. PUBLIC HEALTH RELEVANCE: This study builds upon an ongoing population-based case-control study where epidemiologic data and DNA samples from 1,250 incident colon cancer cases and 1,500 population controls have already being collected. This relatively large study population will be readily available and allow us to dissect complex gene-gene and gene-obesity interactions to gain in-depth understanding of mechanistic link between obesity and colon carcinogenesis.
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{
"pile_set_name": "NIH ExPorter"
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Many environmental mutagens and carcinogens are known to interact with cellular DNA and RNA in vivo. In these studies synthetic oligo-and polynucleotides will be used as models for nucleic acids to determine the significance of such interactions. This approach uses simple, well- defined systems to yield detailed information on the chemistry of base substitution, and on the accompanying changes in physical properties of the polymers. Biological effects will be assayed in biosynthetic systems which utilize the reacted polynucleotides as templates for replicating and transcribing enzymes. Investigations will proceed along two lines: a) the study of certain known substitutions, e.g. on the O6 position of guanine, which are believed to have special biological significance and b) the development of a screening method for detecting the biological effects of more complicated chemical interactions with suspected carcinogens. Since many carcinogens are known to be metabolically activated, model compounds will be incubated with microsomes in the presence of nucleosides. In this way, reactive species can be detected and elucidated.
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{
"pile_set_name": "NIH ExPorter"
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Fatty acids (FA) and monoacylglycerol (MG), the hydrolytic products of triacylglycerol (TG), provide a major source of dietary energy. Nevertheless, substantial gaps remain in understanding the basic mechanisms of MG and FA assimilation. These include a molecular level understanding of the individual functions of two highly expressed enterocyte fatty acid-binding proteins (FABP), intestinal FABP and liver FABP. In addition, the mechanisms of differential trafficking of MG and FA added to the apical (AP) vs. basolateral (BL) poles of the enterocyte, where AP lipid is more highly incorporated into TG, and BL lipid into phospholipid, are not understood. In this proposal, we will use an integrated approach to address the cellular and molecular mechanisms that underlie the polarity of intestinal FA and MG transport and metabolism, and the specific roles of the two enterocyte FABPs in FA transport, lipid metabolism, and lipoportein secretion. Further, we will explore the regulation and function of monoacylglycerol lipase (MGL), which we have recently shown to be expressed in rat intestine. The specific aims are 1) To determine the functions of IFABP and LFABP in cellular transport and metabolism of FA and MG: Structure-function studies in model systems, analysis of the apo- and holo-LFABP tertiary structures, and direct protein transfer of wild type and specific FABP mutant forms into cultured Caco-2 enterocytes, will allow us to determine the individual functions of IFABP and LFABP, and the structural basis for these functional properties; 2) To determine the cellular basis for metabolic compartmentation of FA and MG in the entcrocyte. Studies in rats, mice null for specific enzymes and transport proteins, and Caco-2 cells, will allow us to examine the role of plasma membrane transporters, intracellular FABPs, and specific intracellular enzymes, in the underlying mechanisms of this metabolic divergence; and 3) To explore the role of intestinal monoacylglycerol lipase, using studies in rodents and cultured cells, MGL is higher in neonatal intestine and declines to lower levels in the adult animal, thus we will examine the mechanisms that underlie changes in MGL expression. The ultimate aim of this research program is to understand how to control the rate and extent of dietray lipid assimilation by modulating specific transport and metabolic processes. Such modulation has important implications for the treatment of obesity, diabetes, and hyperlipidemias. PERFORMANCE SI-_E(S) (organization, city, state) 1. Rutgers University, Department of Nutritional Sciences, New Brunswick, NJ 2. City University of New York, College of Staten Island, Department of Chemistry, NY 3. Rutgers University, Department of Chemistry & Biochemistry, Newark, NJ KEY PERSONNEL. See instructions. Start with Principal Investigator. List Name Judith Storch, Ph.D. Sunita Cheruka, Ph.D. Jesse Gabriel, B.S. Sarala Kodukula, B.S. Yin Ziu Zhou Ruth E. Stark, Ph.D. Richard Mendelsohn, Ph.D. Mahmood Hussein, Ph.D. Nada Abumrad, Ph.D. Luis Agellon, Ph.D. Susan K. Fried Disclosure Permission Statement. [unreadable] PHS 398 (Rev. 05/01) Use continuation pages as needed to provide the required information in all other key personnel in alphabetical order, last name first. Organization Rutgers University Rutgers University Rutgers University Rutgers University Rutgers University City University of New York Rutgers University State University of New York, Downstate State University of New York, Stony Brook University of Alberta, Canada Rutgers University the format shown below. Role on Project Principal Investigator Postdoc. Associate Graduate Res. Assistant Sr. Research Technician Research Technician Collaborator/subcontract Collaborator Collaborator Collaborator Collaborator Collaborator Applicable to SBtR/STTR Only. See instructions. [] Yes [] No Page 2__ Form Page 2 [unreadable] [unreadable] PrincipInavlestigator/PDroirgercat(omLrasfitr,stm, iddle): Storoh, Judith The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page, RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page .................................................................................................................................................. 1 Description,
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{
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This proposal is in response to RFA-NS-02-010 Parkinson's Disease Neuroprotective Clinical Trials: Clinical Center. Parkinson's disease is a progressive and debilitating neurodegenerative disorder affecting one-percent of the population over the age of 55 years. The primary pathological features of PD are the loss of nigrostriatal dopaminergic neurons and subsequent depletion of striatal dopamine. The cause of PD is currently unknown but both environmental and genetic factors may contribute to the etiology of the disease. Since PD is a progressive disease, pharmacological and/or non-pharmacological modalities may be developed to provide neuroprotection through either protecting/rescuing surviving neurons and slowing disease progression. The primary goal of this proposal is to identify and recruit patients and conduct clinical trials of neuroprotection in early stage PD. An alternative approach to slowing disease progression is by promoting neuroplasticity in surviving nigrostriatal neurons. One possible non-pharmacological intervention is the process of activity-dependent neuroplasticity that may be achieved through long-term exercise (such as treadmill training). The secondary goal of this proposal is to determine if long-term treadmill training attenuates disease progression in PD through activity dependent neuroplasticity.
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{
"pile_set_name": "NIH ExPorter"
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The program development core (B), which is led by the Center Director, vrill provide modest support for the time of faculty, staff, and research assistants engaged in innovative pilot research projects that are likely to lead to major NIA support. As noted above, these funds wdll be allocated by the Director vrith the advice and consent ofthe Steering Committee. There is an annual competition for these funds, held each Spring in order to complement the Fall competition for research funding by the Graduate School ofthe UW-Madison. A small fraction of these funds vrill be held back in each cycle to provide flexible support for emergencies and emerging opportunities. Budgeted funds vrill be supplemented by the Graduate School wdth a Research Assistant position, in addition to substantial research funds for CDHA members that are provided by the regular Graduate School competition.
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{
"pile_set_name": "NIH ExPorter"
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For over five decades, a large group of productive investigators at VA Commonwealth University (VCU) has made significant contributions to our understanding of drugs of abuse. The interests of these scientists encompass synthesis, pharmacokinetic, neurochemical, molecular, behavioral and pharmacological characterization of most classes of abused drugs. While productive collaborations have always existed among these investigators, the drug abuse research effort at VCU has been enhanced by the creation of this NIDA Center. The primary objective of the Center is to foster multifaceted basic science research. The Center also will continue to provide a mechanism for uniting scientists from many other disciplines in order to share information and new research ideas regarding abused drugs. In this regard, the Center serves a coordinating role for all drug abuse grants at VCU, including numerous NIDA R01 grants, contracts, a training grant, several individual training awards and a program project. Another major contribution continues to be the training and development of young scientists in drug abuse research. The role of the Core is to provide program management and resources, and to facilitate interaction and cooperation through its administration, drug supply, shared instrumentation, and mouse knock-out facility sub-cores. In addition, a Small Grants Program provides a mechanism to attract established scientists in other fields to drug abuse research and to allow for pilot investigations in response to new drug abuse problems by all junior and senior scientists. The Center serves as a national resource through extensive collaborations and shared information with scientists at the national and international level. In addition, the Center maintains an inventory of cannabinoid analogs that we distribute to scientists at VCU and to NIDA-sponsored researchers throughout the US and the world. Of course, basic research will continue to be the primary focus of the Center. The five proposed projects address the mechanisms through which endocannabinoids produce a blockade of morphine tolerance, decrease seizure threshold following chronic cannabinoid administration, and affect the reward and withdrawal of chronic nicotine. These efforts are complemented by studies to understand the plasticity of cannabinoid receptors and characterization of cannabinoid properties of orphan G-protein coupled receptors. Coordination among the projects is directed toward the nature of ligand receptor interactions, roles of receptor-associated proteins, receptor phosphorylation states, characterization of new receptor subtypes, and signal transduction mechanisms. We are also investigating the interactions between the endocannabinoid system and opioids and nicotine. The roles of the nicotinic receptor subtypes in the development of reward and dependence will also be addressed. This center has much significance on human health since additional knowledge of this endogenous system will improve our ability to treat disease. [unreadable] [unreadable] CENTERS CHARACTERISTICS [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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The American Health Foundation, with its well established and functional interdisciplinary team, expects to have continued impact on the solution of major environmental cancer problems ranging from specific items with widespread usage such as tobacco and cosmetics, and food products such as saccharin, to hazards affecting limited groups like shipyard workers. We also hope to continue to make fundamental contributions to the understanding on the mechanisms by which various environmental forces relate to human cancers through in vitro and in vivo studies. Our major efforts will continue to be a comprehensive program of research leading to the prevention of major human cancers.
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{
"pile_set_name": "NIH ExPorter"
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Angiotensin II (Ang II) signaling, including matrix metalloproteinase type II (MMP2) activation, has been linked to an age-associated increase in migration capacity of vascular smooth muscle cells (VSMC), and to other proinflammatory features of arterial aging. Calpain-1 activation is required for MMP2 expression in fibroblasts and is induced in cardiomyocytes by Ang II. The consequences of engagement of calpain-1 with its substrates, however, in governing the age-associated proinflammatory status within the arterial wall, remain unknown. 1. The present findings demonstrate that transcription, translation, and activity of calpain-1 are significantly up-regulated in rat aortae or early-passage aortic VSMC from old (30-mo) rats compared to young (8-mo). 2. Dual immunolabeling of the arterial wall indicates that colocalization of calpain-1 and Ang II increases within the aged arterial wall. To further explore the relationship of calpain-1 to Ang II, we chronically infused Ang II into young rats, and treated cultured aortic rings or VSMC with Ang II. We also constructed adenoviruses harboring calpain-1 (CANP1) or its endogenous inhibitor calpastatin (CAST) and infected these into VSMC. Ang II induces calpain-1 expression in the aortic walls in vivo and ex vivo and VSMC in vitro. The Ang II mediated, age-associated increased MMP2 activity and migration in VSMC are both blocked by calpain inhibitor 1 or CAST. 3. Over-expression of calpain-1 in young VSMC results in cleavage of intact vimentin, and an increased migratory capacity mimicking that of old VSMC, which is blocked by the MMP inhibitor, GM6001. Furthermore, age-associated central arterial wall stiffness is linked to extracellular matrix (ECM) remodeling, including fibrosis and vascular calcification. Angiotensin II induces both matrix metalloproteinase type 2 (MMP2) and calpain-1 expression and activity in the arterial wall. But the role of calpain-1 in MMP2 activation and ECM remodeling remains unknown. 4. Dual histo-immunolabeling demonstrates co-localization of calpain-1 and MMP2 within old rat vascular smooth muscle cells. Over-expression of calpain-1 induces MMP2 transcripts, protein levels and activity, in part, by increasing the ratio of membrane-type 1 MMPs to tissue inhibitor of metalloproteinases 2. 5. The effect of calpain-1 over-expression-induced MMP2 activation is linked to increased collagen I and III production and vascular calcification. 6. In addition, over-expression of calpain-1 also induces transforming growth factor-beta1/Smad signaling, elastin degradation, alkaline phosphatase activation and total calcium content, but reduces the expression of calcification inhibitors, osteopontin and osteonectin, in cultured vascular smooth muscle cells in vitro and in carotid artery rings ex vivo. 7. Interestingly, both calpain-1 and collagen II increase with aging within human aortic intima. In aged human aortic wall, both calpain-1 and collagen II are highly expressed in arteriosclerotic plaque areas compared to grossly normal areas. Cross-talk of two proteases, calpain-1 and MMP2, leads to secretion of active MMP2, which modulates ECM remodeling via enhancing collagen production and facilitating vascular calcification. These results establish calpain-1 as a novel molecular candidate to retard age-associated ECM remodeling and its attendant risk for hypertension and atherosclerosis.
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{
"pile_set_name": "NIH ExPorter"
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Fifteen years after the first description of monoclonal antibodies, we are seeing their successful application as drugs to treat a variety of diseases. The insights and technologies of protein chemistry and molecular biology are having a significant impact on improved design of these agents. Results of recent clinical trials have identified new problems which are being rapidly addressed in the laboratory to provide new solutions. Therefore, at this juncture, there is a critical need to bring together basic and clinical scientists as well as individuals involved in process design and regulatory affairs to design strategies for the future. Along these lines, sessions at this conference will address issues in the areas of binding sites, effector mechanisms, surface receptor molecules, immunoconjugates, protein design, clinical trials, animal models, and other issues that impact on these strategies.
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{
"pile_set_name": "NIH ExPorter"
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Flavopiridol is a synthetic flavone that inhibits cyclin-dependent kinases. It has been shown to enhance apoptosis when administered after taxanes in human breast cancer cell lines. We are conducting a phase I/II clinical trial of flavopiridol and docetaxel in patients with locally advanced and metastatic breast cancer. We propose that flavopiridol is synergistic with docetaxel and will increase its efficacy. Incorporated in this study is the serial examination of molecular parameters, cyclin D1, p53, bc12, and mib1, in accessible tumor biopsies and from the buccal mucosa, as a potential surrogate tissue, to see if desired biochemical effects are achieved with this combination. The development of target-based anticancer drugs, such as small molecule inhibitors of epidermal growth factor tyrosine kinase (EGFRTK), is becoming an attractive therapeutic strategy in the oncology field. The examination of the effects of these mechanism-based drugs at the cellular level is a logical approach to test these compounds and may in the future replace the traditional way of determining the appropriate dose of new agents. The development of reproducible assays and tools to assess the effect of a drug on targets, such as inhibition of enzyme activity, the validation of surrogate endpoints to check the desired effect of the drug, and the implementation of novel clinical trial designs that allow the testing of biologic endpoints as a primary aim, will become a challenge to both clinicians and basic scientists in the new era of targeted therapy. We are conducting a pilot study of an EGFRTK inhibitor to address these issues. Adjuvant therapy of breast cancer has clearly provided a significant survival benefit for women in all age groups. Though screening mammography has resulted in earlier detection, still 50 percent of patients with early breast cancer present with node-positive disease. There is a significant recurrence rate in these patients, even with doxorubicin-based chemotherapy. Approaches for increasing the survival include the addition of taxanes. A multicenter trial, NSABP B30, for which Dr. Swain is the protocol chair, is a three-arm trial that will explore the efficacy of docetaxel on survival and quality of life. The three arms include a combination of docetaxel and doxorubicin versus a combination of docetaxel, doxorubicin, and cyclophosphamide, versus doxorubicin and cyclophosphamide followed sequentially by docetaxel. This trial will include 4,000 patients with node-positive breast cancer and has currently accrued 2,100 patients. A quality of life question is being asked in all patients and also specifically in premenopausal women. It has been shown in a meta-analysis of ovarian ablation that this approach increases survival to the same degree as chemotherapy. Therefore, the trial will evaluate ovarian suppression induced by chemotherapy and determine whether this suppression leads to an increased survival. The other quality of life issues relate to the toxicity of the three combinations. Other research areas with NSABP include membership in the Cardiology Advisory Panel for the adjuvant Herceptin trial (NSABP B31) and Protocol Chair for the IressaTM trial. Iressa is an epidermal growth factor tyrosine kinase inhibitor and will be evaluated with docetaxel in metastatic breast cancer.
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{
"pile_set_name": "NIH ExPorter"
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The proposed research will study normal and pathological human auditory nerve action potentials (APs) recorded from external auditory meatus and brainstem evoked response (BERs) recorded from vertex. The project includes investigations relevant to the problems of utilizing these responses to quantify auditory system pathology (by predicting the pure-tone audiogram) and to localize it. Cochlear excitation patterns generated by filtered clicks will be determined with the derived AP technique and supplemented by computer modeling. The relationship between filtered-click AP and BER waveforms and thresholds and the underlying cochlear excitation patterns will then be determined. The ability filtered-click AP and BER thresholds and waveforms to predict pathological cochlear excitation patterns and thence pathological pure-tone audiograms will then be tested in patients with hearing loss. The project also includes research designed to refine and expand previous results which suggest that "square-wave" click AP and BER Latency-Intensity (L-I) functions provide a clinically useful means of separating cochlear from retrocochlear pathology. Data will be collected from a larger series of patients than presently available to confirm the validity and improve the accuracy of the retrocochlear signs derived from the L-I functions and also to test hypothetical AP/BER L-I patterns indicative of conductive hearing loss.
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{
"pile_set_name": "NIH ExPorter"
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PROJECT SUMMARY Analytic treatment interruption (ATI) studies remain the ultimate test of efficacy for HIV curative strategies, which will need to demonstrate a significant delay of viral rebound for clinical approval. ATI trials will become increasingly important as new HIV curative strategies are discovered and advance in clinical trials. However, there is a current knowledge gap in our understanding of factors that affect the timing of viral rebound and the sources of rebounding virus. This has led to significant challenges in designing novel therapeutics for HIV remission and evaluating their efficacy in pre-clinical and early phase clinical trials. In this application, we propose a collaborative effort by a multidisciplinary team of virologists, immunologists, and mathematicians to tackle these challenges by studying a rare cohort of participants from prior AIDS Clinical Trials Group ATI trials. We plan to define virologic characteristics associated with viral rebound timing and rebounding viral variants. This includes assessing the impact of immune escape mutations, quantifying viral RNA species within cellular subsets, and determining the integration site characteristics of rebounding virus. Concurrently, we plan to perform an in-depth evaluation of both cellular, antibody, and inflammatory markers associated with HIV rebound timing. Finally, we will use the results of this study to enhance a mathematical model of post- treatment HIV control. The knowledge gained will inform the design and evaluation of novel strategies for drug- free HIV remission.
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{
"pile_set_name": "NIH ExPorter"
}
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Research Involving Outpatient Settings Network (RIOS Net) is a practice-based research network of primary care clinicians practicing in community health centers, Indian Health Service and tribal health centers, as well as academic sites serving low-income persons. Formerly known as the New Mexico Practice Based Resource and Research Network, RIOS Net is devoted to the study and improvement of primary care of medically underserved, low-income and ethnically diverse communities. Member clinicians serve predominantly Native American and Hispanic and communities, with many practices located in rural areas. RIOS Net proposes to join other AHRQ-supported PBRNs in conducting the NAMCS-based, networkdefining survey of patients seen by member clinicians. Of 67 members in the network at the time of application, 54 have volunteered to participate in the survey. A design utilizing regionally-located research assistants as "detailers" to assure participation and high quality of data recording is planned. The data obtained will be of value to RIOS Net not only in clarifying the spectrum of health concerns seen by members, but also in advancing the network's process for deciding on future research topics. RIOS Net also proposes to explore the impacts of ethnicity and rurality on patient/consumer perspectives about health data privacy and confidentiality. The composition of the communities served by R10S Net members, being predominantly Native American and Hispanic, offers a unique opportunity to better understand the similarities and differences in views about privacy and confidentiality, particularly regarding PBRNs, in these important populations. A qualitative study, based on focus groups conducted among representative segments of the RIOS Net communities, will be conducted. The result will be a report that will guide both future researchers and others with need to use health data originating in similar communities. An equally important outcome will be the beginning of a partnership with the communities that will operationalize participatory community involvement in research.
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{
"pile_set_name": "NIH ExPorter"
}
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Alloimmunization to antigens on platelets is one of the most severe limiting factors in platelet transfusion. In this application to become a member of a cooperative trial for the prevention of alloimmunization, we propose two strategies which might limit alloimmunization from this source in patients treated for acute non-lymphocytic leukemia: 1. selection of platelet donors by matching HLA antigens, particularly the so-called "public" antigens; 2. removal of leukocytes from platelet products by filtration. In a cooperative study, we would be able to implement other strategies if they were selected. We also propose methods for following the development of alloimmunization to both HLA and platelet-specific antigens and for determining the epidemiology of alloimmunization.
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{
"pile_set_name": "NIH ExPorter"
}
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Research suggests that children's behavioral trajectories are influenced by the behavioral climates of their elementary schools, and that the quality of school and classroom environments may have long-lasting effects on behavioral outcomes in adolescence and adulthood. Moreover, several interventions to improve the behavioral climate of schools have demonstrated efficacy, while other promising approaches are currently being evaluated. Additional information about the effects of school behavioral climate in naturalistic settings is needed, however, in order to evaluate the potential demand for school intervention programs nationwide. In particular, there have been few studies of the current quality of elementary school environments in the U.S., and few attempts to estimate the effects of school behavioral climate on child behavior using non-experimental data. Also, several limitations in study design and scope apply to the existing literature. This proposed study will begin to address these limitations by estimating models of the quality of elementary school's behavioral climates, and models of the effects of behavioral climate on childhood behavior problems using data from a nationally-representative, non-experimental, longitudinal study of kindergartners. The specific aims of this study are: 1) to estimate the proportion of elementary schools in the U.S. with disruptive learning environments; 2) to identify elementary school and teacher characteristics that are significantly associated with the disruptiveness of classrooms and with school climate; and 3) to estimate the effects of children's school environments on the persistence of behavior problems from kindergarten through third grade. The data will come from the Early Childhood Longitudinal Study -- Kindergarten Cohort (ECLS-K), a longitudinal study of approximately 22,000 children attending kindergarten at baseline. Effects of school climate on child behavior will be estimated using multilevel models that allow for interactions between school climate and behavior problems at baseline. The study will provide a more comprehensive picture of the status of schools and their effects on child behavior than has been available previously.
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{
"pile_set_name": "NIH ExPorter"
}
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In 2007, the University of Alabama at Birmingham (UAB) and its affiliates completed three important, large- scale strategic planning efforts. Together, these processes generated a "blueprint for our research future" that lays the foundation to transform our substantial clinical and translational research enterprise through integration and innovation across all disciplines, Schools, and our health care system. This transformation will be driven by the UAB Center for Clinical and Translational Science (CCTS). The CCTS vision is to transform the UAB environment by building productive and efficient interdisciplinary research teams through educational ingenuity, regulatory reorganization, resource coordination, and methodological innovation. The mission is to develop a transformative infrastructure that spans the spectrum from preclinical research to bench-to-bedside translation (T1 research) to community implementation (T2 research), and will meet 5 goals: 1) transform the Investigator;2) transform the Training Environment;3) transform the Resource Infrastructure;4) transform the Approach to Interdisciplinary Research via an emphasis on outcomes research and health disparities research;and 5) establish a new Research Model through the Self-Monitoring and Improvement Program. To meet these goals, the CCTS will rely on: 1) a re-organized reporting structure that assures the PI full institutional support in implementing the CCTS roadmap;2) a revised CCTS leadership that includes faculty from the Schools of Medicine, Public Health, Nursing, Health Professions, and Optometry;and 3) innovative partnerships involving UAB, Southern Research Institute (our affiliated, not-for-profit research organization for preclinical drug discovery/development), the Children's Health System, the HudsonAlpha Institute for Biotechnology, the greater Birmingham community, and a long-standing collaborative network that involves Historically Black Colleges and Universities (HBCUs) and underprivileged communities in our region. UAB, the Health System, Health Services Foundation, Research Foundation, and Southern Research Institute have made substantial commitments to the CCTS including: 1) significant new funding for the clinical and translational research infrastructure (~$150 million, of which over $18 million are directly for CCTS activities and programs);2) a redesigned and enhanced biomedical informatics infrastructure;and 3) over 20,000 sf of prime clinical and administrative space. When combined, the re-organized^ CCTS governance and leadership, the substantial commitment of funds and resources, the interdisciplinary culture of UAB, and the new CCTS programs, will create a transformed environment for clinical and translational research that will benefit our trainees, investigators, patients, community, and the national CTSA effort.
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{
"pile_set_name": "NIH ExPorter"
}
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Progressive kidney disease is characterized by the accumulation of fibrotic mediators in the kidney. Glomerulonephritis, diabetic nephropathy, pyelonephritis, and renovascular disease together account for over 75% of patients requiring renal transplants. In these conditions it is chronic progressive renal fibrosis with associated loss of functioning nephrons that results in irreversible renal injury. While the renin angiotensin system (RAS) has traditionally been viewed as a circulating axis, evidence is accumulating that an active intrarenal RAS plays an important role in chronic kidney disease and fibrosis. Based on recent evidence reported from our lab that mast cells express and release active renin, we hypothesize that renin released from mast cells infiltrating the kidney triggers intra-renal RAS and local angiotensin (ANG II) formation. The overall objective of this proposal is addressing the important issue of whether infiltrating mast cells and mast-cell renin play a significant role in ischemic organ damage and fibrosis. Using a model of progressive renal fibrosis (unilateral ureteral obstruction (DUO)) we will study the role of mast cell renin and local ANG II production in this process as well as investigate the role of adenosine in regulating mast cell renin gene expression. Our preliminary results demonstrate: 1. Human and rodent kidney mast cells express active renin. 2. Mast cell deficient mice do not develop renal fibrosis with UUO 3. Stabilizing mast cells in rat UUO kidney prevents renal fibrosis. 4. Inhibiting the renin released from mast cells reduces vasoconstriction in isolated and perfused kidney. 5. Stimulating the adenosine A2b receptor increases mast cell renin gene expression. In Specific Aim I, we will characterize the molecular identity of mast cell renin and examine the effects of mast cell renin and local ANG II formation on fibrosis and vasoconstriction in UUO. These experiments will be done with mast cells isolated from human and rodent kidney and with HMC-1 cells, a cultured cell model of human mast cells. The UUO animal studies will be carried out in rat and mast cell- deficient c-Kit knockout mice and their congenic controls. Specific Aim II will examine regulation of mast cell renin expression, synthesis, and release by adenosine. These experiments will be done with isolated human kidney mast cells and with HMC-1 cells. If our hypothesis is proven correct then we will have identified a novel therapeutic target (mast cell renin) for ameliorating renal function in chronic kidney disease. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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The purpose of this work is to synthesize and evaluate compounds which interact with the stimulant binding site(s) on the dopamine transporter, with the aim of identifying compounds which are antagonists for the reinforcing and/or psychotogenic properties of cocaine. The current program to synthesize and evaluate compounds which bind irreversibly to sites on or related to the dopamine transporter will be continued. This program involves substitution of phencyclidine, GBR-1 2783 ("GBR"), dopamine, methylphenidate and mazindol, with electrophilic groups such as isothiocyanate, maleimide, bromoacetyl, and fluorosulfonyl. This work has been successful in identifying a phencyclidine (Fourphit) which antagonizes the stimulant effects of cocaine. Completion of this program will benefit drug research by expanding the available data base on which to search for features which distinguish compounds which act as effective cocaine antagonists. In addition, similar derivatives of LR5182, a compound with some structural similarities to cocaine, but higher selectivity and affinity for the dopamine transporter, will be synthesized. Several lines of evidence suggest that there may be more than one substrate and/or stimulant binding site per dopamine transport complex. Based on such a model, the effects of linking two dopamine molecules together and varying the distance of separation will be explored. As a variation of the above theme, a GBR derivative with two reactive groups in the same molecule will be synthesized and the tritiated form will be used in protein isolation and characterization studies. The mutual inhibition of GBR, methylphenidate and cocaine binding suggests that derivatives of these stimulants may serve as antagonists or partial agonists for cocaine. Methylphenidate has structural analogies with cocaine, but appears to have a much lower abuse potential; methylphenidate derivatives may have promise as "partial agonists". The SAR that has been gained about cocaine will be applied to methylphenidate, with the aim of producing more metabolically stable compounds with higher affinity. The need for more soluble and less lipophilic GBR-derivatives has become apparent; these type of derivatives might have improved potential as therapeutic agents. The use of epoxides as electrophiles specific for sulfhydryl groups will be tested with the synthesis of the 3-epoxide derivative of GBR-12783. The effect of the newly synthesized compounds on the dopamine transport complex will be determined by examining their ability to inhibit [3H]-CFT binding and [3H]-dopamine transport in vitro. Compounds which prove to be irreversible inhibitors of stimulant binding, exhibit increased affinity for the transporter, or possess novel pharmacological properties in the in vitro screening above will then be submitted to NIDA for both in vitro and in vivo analysis using the NIDA/SRI protocol. Selected compounds from those identified above will be tested for their ability to antagonize the behavioral effects of cocaine and other stimulants in our established rat models; [3H]-CFT binding will be determined ex vivo in striatal tissue from the same animal. Two different behavioral paradigms will be used,in order to better correlate binding and behavior.
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{
"pile_set_name": "NIH ExPorter"
}
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Development of a safe, effective and affordable vaccine for HIV-1 is potentially the most efficient means of controlling HIV-1 infection worldwide. However, the genetic and biological variability of HIV-1 represents significant obstacles for vaccine development. Recent evidence indicates that CTLs may play a role in clearing viremia during primary infection and maintaining a disease-free state in HIV-1-infected patients. Thus, efforts to develop an HIV vaccine should focus on eliciting a broad cross-reactive CTL response as one major component. One promising approach to generating an effective CTL response in vivo is through the use of DNA vaccination. Current efforts using HIV-1 env and gag/pol DNA constructs as immunogens suggest that additional vaccine components are needed to confer broad protection. Therefore, a putative HIV-1 vaccine might benefit by inclusion of additional immunogenic targets. In addition to the structural and enzymatic proteins, HIV-1 also contains regulatory and accessory genes. These genes are highly conserved in vivo and may provide additional targets for CTL responses. We hypothesize that such targets could induce a broad virus-specific CTL response that could help to limit viral escape and confer protection against viral challenge. To use the accessory genes as part of a multicomponent vaccine, we have engineered a novel construct that expresses HIV-1 accessory genes vif, vpu, and nef under the control of a single promoter. To test the "proof of concept" we propose the following aims: (1) We will immunize human HLA-A2 transgenic mouse and evaluate the cellular immune responses using HIV-1 infected human targets; (2) we will use a non-human primate model to test the ability of this vaccine construct to confer protection either alone or in combination with env and gag/pol vaccine constructs. We hypothesize that cell-mediated responses induced by the accessory genes may include broader recognition of divergent HIV-1 clades and should be useful in both prophylactic as well as therapeutic vaccination schemes against HIV-1.
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{
"pile_set_name": "NIH ExPorter"
}
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Alcohol use disorder commonly occurs among patients with schizophrenia and contributes greatly to the morbidity of schizophrenia. Patients with schizophrenia tend to consume modest quantities of alcohol on a regular basis and are less likely to develop alcohol dependence than alcohol abuse, but even this modest use of alcohol dramatically worsens their symptoms and decreases their overall functioning. Green (co- investigator) and colleagues have suggested that such moderate alcohol use may, however, transiently ameliorate a brain reward circuit deficiency that underlies alcohol use disorder in these patients. Unfortunately, available treatments for co-occurring alcohol use disorder in schizophrenia are very limited. This revised R03 proposal seeks to begin a line of research toward the development of such an animal model of alcohol use disorder in schizophrenia, an animal that exhibits characteristics of schizophrenia, and like patients with schizophrenia, also drinks at least moderate amounts of alcohol. To develop this animal model, we propose to use, as a base, a rat with a neonatal ventral hippocampal lesion (the NVHL rat), a well-established animal model of schizophrenia, a rodent that as an adult exhibits requisite characteristics of schizophrenia, demonstrates abnormalities in its brain reward circuit, and, interestingly, has recently been shown to exhibit increased cocaine self-administration. Our preliminary data in a small group of adult NVHL rats also suggest that this rat will voluntarily drink at least moderate amounts of alcohol. This revised research proposal seeks to further probe the potential role of the NVHL rat as an animal model of schizophrenia and comorbid alcohol use disorder. Using free-access conditions, we will: (1) compare the amount and preference of alcohol drinking [and blood alcohol level] in NVHL rats versus sham-operated rats; and (2) compare the size, frequency and temporal distribution of alcohol drinking bouts in NVHL rats versus sham-operated rats. If NVHL rats can be differentiated from sham rats according to these measures, we plan to continue research with NVHL rats in subsequent studies to: (1) explore mechanisms mediating alcohol drinking in these animals (e.g., to address the question of whether alcohol use serves to transiently ameliorate a deficit in brain reward functioning); and (2) screen medications that might be able to decrease alcohol drinking in this rat. Ultimately, we expect to translate the findings from our studies with the NVHL rat into studies involving human subjects, with the long- term goal of this research to find novel medications to treat patients with schizophrenia and alcohol use disorder, and thus to improve the outcome of these patients. Alcohol use disorder occurs commonly among patients with schizophrenia and greatly worsens the overall functioning of these patients. This research seeks to develop an animal model of alcohol use disorder in schizophrenia, an animal model that exhibits schizophrenia-like characteristics as well as increased alcohol drinking. This animal model, when developed, will be used: (1) to elucidate the underlying basis of alcohol use disorder in patients with schizophrenia; and, (2) to develop novel medications to limit alcohol use in these patients. [unreadable] [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
}
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This project is directed toward the development of computer- based mathematical and statistical analyses, pattern recognition, and image processing of data, principally X-ray micrography and electron energy loss spectra, or from the electron/light microscopy images of biological specimens.
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{
"pile_set_name": "NIH ExPorter"
}
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Adaptation to persistent neural input is a fundamental component of neural computation. Persistent neural activity in neurons within a sensory system can be caused both by a constant sensory stimulus and by intrinsic cellular factors, which are independent of sensory stimuli. While adapting to a persistent sensory stimulus could help a neuron adapt to its sensory environment, adapting to intrinsic properties could substantially limit the sensitivity of the sensory system to weak stimuli. Thus, understanding how neurons adapt to both stimulus-induced and intrinsic persistent activity is critical to understanding how sensory systems process information across a large range of sensory environments and delineating the factors that limit neural processing. Few studies have addressed these issues clearly because it requires access to multiple levels of an intact neural circuit and ability to manipulate both stimulus-induced and intrinsic persistent activity. The Drosophila olfactory system provides a unique opportunity to resolve these issues. This model organism provides experimental access to the first two stages of the olfactory system, the olfactory receptor neurons (ORNs) and the projection neurons (PNs), as well as the ability to control persistent activity in the ORNs. Stimulus-induced persistent ORN activity can be driven by a constant odor stimulus and control of the intrinsic ORN activity is provided by the inherent structure of the Drosophila olfactory system. ORNs span a large range of intrinsic activity, with some only generating 1 spike/sec while others generate 32 spikes/sec, and a specific PN only receive input from ORNs with the same level of intrinsic activity. Further, established fly genetic allow even more precise control of intrinsic activity. Activity of ORNs and PNs can be measured using electrophysiology and specific cells can be targeted using known maps of fly anatomy and genetic labeling of neurons with green fluorescent protein. In Specific Aim 1 we will compare how PNs signal brief ORN odor responses during increasing odor-induced persistent activity from the ORN. In Specific Aim 2 we will compare how PNs signal brief ORN odor responses across PNs that receive input from ORNs with a range of intrinsic activity. Additionally, in Aim 2 we will evaluate if PNs adapt to odor-induced persistent activity differently than intrinsic persistent activity. In Specific Aim 3 we will test how the adaptation observed in Aims 1 and 2 depend on known biological mechanisms. Insight gained from these studies into how downstream neurons adapt to changes in their persistent input and how that adaptation depends on the source of the persistent activity will help generate accurate models of how the neural system processes sensory stimuli and identify the factors that can limit sensory processing.
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{
"pile_set_name": "NIH ExPorter"
}
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We hypothesize that the basement membrane (BM) receptor dystroglycan (DG) mediates critical signals in epithelial cell development, homeostasis and tissue-specific functions. DG has been studied most extensively in muscle tissues, where loss of DG function has been implicated in muscular dystrophies, but investigation of DG functions in epithelia has lagged. Information emerging from recent studies in epithelial cells has implicated DG in a wide range of cell responses to the BM, including cell polarization, cell survival, BM organization, and tissue-specific gene expression. But inconsistencies exist between results from different experimental systems, and DG's roles in epithelial tissues remain poorly defined, as do the signaling mechanisms by which DG mediates its functions. Importantly, alterations in the cell-BM interactions play important roles in cancer progression, and loss of DG function is evident in carcinoma cells. Therefore, DG functions in epithelial cell also have potential high relevance to human pathology. To assess the roles of DG in epithelial cells, we have established a unique system for the genetic manipulation of DG function in cultured mammary epithelial cells using cells derived from the "floxed DG" (flanking-Lox DG) transgenic mouse line. These cells allow selective deletion of DG gene expression in functionally normal cells through "Cre-/Lox" recombination, permitting direct comparisons of DG +/+ and DG-/- cells in assays of normal cell behavior and signal transduction. Using this experimental system we will: 1) determine the cell responses induced by DG signaling in cultured mammary epithelial cells; 2) determine the consequence of loss of DG signaling in cells exposed to the in vivo microenvironment; and 3) dissect the signaling pathways emanating from DG that are responsible for identified receptor functions. A thorough evaluation of DG roles in mammary epithelial cells will advance our basic understanding of how cell-BM interactions regulate normal epithelial cell behavior. A thorough assessment of DG signaling in epithelial cells is also necessary to understand the signaling imbalances that result from the frequent loss of this BM receptor in cancer progression.
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{
"pile_set_name": "NIH ExPorter"
}
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The goal of this project is to isolate and characterize genes that are important to the development and function of the Drosophila visual system and to use them to identify human homologs. Two new directions have emerged. One is to use mutant analysis to understand the roles of glia in the visual system and the other is to explore the nature of the interphotoreceptor space in Drosophila. The applicant will continue to use genetic screens and monoclonal antibodies to initiate these studies.
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{
"pile_set_name": "NIH ExPorter"
}
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Retinopathy of prematurity (ROP) is a potentially blinding disease of premature infants in which retinal blood vessels fail to develop properly. A multicentered clinical trial (CRYO-ROP Study) sponsored by the National Eye Institute demonstrated that peripheral retinal ablation at a defined stage of the disease reduced the risk of unfavorable structural outcome (i.e., retinal detachment); however, over 47.1 percent of treated eyes had vision greater than 20/200 at 5 1/2 years. The CRYO-ROP Study also defined a number of risk factors for unfavorable visual and structural outcome in ROP. Despite these advances, ROP remains a leading cause of blindness in children. More effective treatment is required in order to further reduce the rate of blindness. It is hypothesized that earlier treatment of ROP will improve the visual and structural outcome in infants with ROP. Peripheral retinal ablation will be administered to infants who develop prethreshold ROP (ROP that is severe, but not as advanced as threshold ROP as defined by the CRYO-ROP Study). To minimize the risk of treating eyes unnecessarily, since ROP may regress spontaneously in some cases, we will apply a risk management analysis program (RM-ROP2) to eyes with prethreshold disease and enroll infants with a 15 percent or greater risk of an unfavorable structural outcome for treatment. The RM-ROP2 utilizes a number of risk factors to calculate the probability of an eye having an unfavorable structural outcome. Prethreshold infants with a 15 percent or higher risk of an unfavorable outcome will be randomized. One eye will receive earlier treatment and the other will receive conventionally-timed treatment, as defined by the CRYO-ROP Study if required. Outcome will be determined at a 6 month post-term ophthalmologic examination and at a 9 month post-term visual assessment examination.
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{
"pile_set_name": "NIH ExPorter"
}
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Project 4 focuses on the ability of circadian genes in the ventral tegmental area (VTA)-nucleus accumbens (NAc) circuit to regulate mood and motivational state. This is related to the knowledge that abnormal mood and other symptoms in many patients with depression show prominent circadian oscillations. We have demonstrated that NPAS2 (neuronal PAS domain protein 2), a transcription factor highly homologous to Clock, regulates an animal's responsiveness to emotional stimuli, including their activity in animal models of depression. Interestingly, NPAS2 is not expressed in the suprachiasmatic nucleus (SCN), a hypothalamic region important for circadian oscillations and their entrainment by environmental lighting. Rather, the highest expression of NPAS2 is seen in the NAc. Our hypothesis is that NPAS2[unreadable]acting within the NAc[unreadable] contributes to circadian variations in mood, locomotor activity, and motivation. In parallel, we have established a powerful influence of Clock itself on mood: mice lacking functional Clock protein exhibit a striking array of behavioral symptoms reminiscent of mania. This phenotype is reversed by lithium, and we have growing evidence that Clock action in the VTA per se is an important mediator of this behavioral phenotype. The goal of the proposed studies is to carry out a systematic evaluation of the role played by NPAS2, Clock, and related circadian gene products expressed in the VTA and NAc in the regulation of mood and motivation. This will be accomplished by use of mice with mutations in these various genes and of viral vectors that selectively manipulate the activity of the genes within the VTA-NAc. In addition, we will further establish the regulation of circadian gene expression in the VTA and NAc in response to chronic exposure to stress and antidepressant treatments. As well, we will identify and characterize the target genes through which NPAS2, Clock, and other circadian genes, acting as transcription factors, regulate the VTA-NAc circuit. We are also interested in cross talk between these circadian genes and CREB. CREB is known to regulate certain circadian genes in SCN, and we have found similar regulation in the VTA-NAc. Moreover, CREB, and circadian transcription factors, share some of the same target genes (e.g., cholecystokinin) in these brain reward regions.
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{
"pile_set_name": "NIH ExPorter"
}
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The incidence of malignant melanoma and malignant melanoma deaths have greatly increased in the United States. By the year of 2000, the life time risk for developing cutaneous malignant melanoma is predicted to be at about one in 90. There is an urgent need to further understand key factors in the pathogenesis of malignant melanomas. Malignant melanoma is deadly, because of the high tendency to spread to other organs. Unfortunately, similar to all other cancers the molecular mechanism of metastasis of melanomas remains elusive. The goal of this project is to begin to understand how a localized melanoma spreads to other organs and cause death. The malignant form of human skin melanoma expresses a high amount of a surface protein, called MUC18. This protein is normally present only in smooth muscle cells and in the cells of the inner linings of blood vessels. The protein appears to play a role in cell to cell communication and attachment. It has been suggested that MUC18 is involved in the progression of human melanomas. Definite proof for the direct contribution of the MUC18 protein in potentiating metastatic ability of melanomas however remains obscure. The immediate goal of this project is to find out whether this protein actually facilitates spreading of melanoma cells to other tissues in a mouse model. Eventually, new knowledge learned from this work should be applicable for developing an effective immuno-therapeutic cure for this fatal human skin cancer.
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{
"pile_set_name": "NIH ExPorter"
}
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The long-term objective of this program is a commercially viable system that will play a key role in restoring and preserving vision for millions of patients in this country and abroad. The system will couple high-frequency ultrasound (40 MHz) with novel signal processing to provide accurate, precise biometry of the cornea and anterior chamber. Measurements will be used by ophthalmologists to: 1) plan refractive laser surgery of the cornea (for vision correction and restoration); 2) select the appropriate type and size of recently developed intra-ocular lenses (IOL) to correct refractive errors or restore vision following cataract surgery; and, 3) document causes of undesired side-effects following laser and IOL procedures, so that corrective actions can be initiated promptly. The system will incorporate advanced, patented methodology developed by the Weill Medical College of Cornell University (WMC) and Riverside Research Institute (RRI). Ultralink, Inc. has already implemented an initial "proof-of-concept" system that successfully demonstrated the practicality of these objectives. This innovative system functioned extremely well, but it also identified several practical issues that will be addressed in this program so that these techniques can realize their full clinical and commercial potentials. The program will integrate system design (Phase I), and development and testing (Phase II) at Ultralink, WMC, and RRI. PROPOSED COMMERCIAL APPLICATION: The research will provide advanced ultrasonic systems to provide precise biometric data that is essential for optimal laser?refractive surgery and intraocular lens implants.
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{
"pile_set_name": "NIH ExPorter"
}
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Several drugs, popularly used by adolescents at dance parties, raves, and clubs, have been shown to be a serious health concern to medical researchers. MDMA (ecstasy), Lysergic Acid Diethylamide (LSD), Rohypnol, Methamphetamine, Ketamine, and Gammahydroxybutyrate (GBH) are substances generally labeled "club drugs." Such drugs, while not necessarily new, have gained recent popularity though use at raves. The impact of these drugs on the health of youth has been researched in recent decades. While results are mixed, studies do suggest that ecstasy in particular may be associated with convulsions, cardiac and pulmonary problems, hypothermia, and in rare instances, death. However, ecstasy and other club drugs are most dangerous when used in combination. According to a recent SAMHSA Dawn Report, most medical emergency incidents involving club drugs also involved the use of alcohol, marijuana, cocaine, and other club drugs. Additional work also suggests that the use of polydrugs is common in the rave/club scene. This study proposes to explore the relationship between the Internet and the transmission of information among youth about "club drugs." The Internet has figured prominently in the "rave scene" since the early years of the movement. The Internet has been a primary site of communication about rave parties, DJs, and artists. Moreover, it provides space for youth to connect with others involved in the rave scene through chat rooms and email subscription lists. While raves, house and bush parties change locale often, the setting of web-based interaction remains static and constant. The role of Internet interaction in the use of club drugs has been unexplored. The current research proposes an Internet-based qualitative study of youth involved in Internet chat rooms and discussion boards. The goal of the research is to solicit new and pertinent information related to the context and setting of club drug use (e.g., when, where, and with whom are drugs primarily used; under what circumstances are some drugs chosen over others; etc.). Moreover, it will explore the role of the Internet in transferring and mediating information regarding drug use across settings (e.g., is the Internet a source of new information about drugs among users, how is Internet use related to the local club scene; do drug use trends disseminate through a "virtual community" or through local, geographically tied networks, how can the Internet be used a prevention and education tool among "club drug" users; etc.). For these reasons, the methodology for this project will involve the extension and adaptation of traditional ethnographic methods to the practice of cyber-ethnography. The project team will combine, as well as adapt field methods for what will at first be a largely unseen group of subjects. We will engage in a two-pronged approach to capture both the online and off-line (so-called 'real life') interactions and networks of the subjects. Primarily, field notes will be generated by monitoring online sites and participant observation in site-communications and other Internet-based communications with members of these communities (e.g. email and America Online Instant Messaging). In addition, we will construct an online survey administered to interested members.
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{
"pile_set_name": "NIH ExPorter"
}
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Human African trypanosomiasis (HAT) is an infectious disease with a large global health burden occurring primarily in Central and Eastern Africa. HAT is 100% fatal if untreated and the current drug therapies suffer from poor safety profiles, difficult treatment regimens, limited effectiveness, and high costs. Furthermore, drugs that cross the blood brain barrier (BBB) are notoriously difficult to develop and have a lower probability of reaching the market place. Here, we propose to apply a quantum-similarity approach to discover novel T. Brucei inhibitors. The approach allows for simultaneous interrogation of multiple targets and pathways, and for incorporation of various pharmacological requirements early in the discovery process, thus increasing the chances of finding novel therapeutics with great selectivity, potency and pharmacokinetic properties. Instead of screening millions of compounds for novel modulators of the target of interest, our focused testing of 10-20 commercially available compounds per target (pathway, protein etc.) with predicted (inhibitory) activity by the modeling effort, allows us to quickly explore novel chemical spaces for therapeutic applications. The modeling, virtual search, identification and rank ordering of novel classes T. brucei inhibitors, as well as the de novo design of novel anti-trypanosome drug candidates will be done by Gradient Biomodeling. The experimental evaluation of the compounds will be done in the laboratory of Dr. Kojo Mensa-Wilmot at the University of Georgia.
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{
"pile_set_name": "NIH ExPorter"
}
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PROJECT SUMMARY The central goal of our proposed research is to obtain a functional understanding of the native and toxin-exposed gut microbiome in xenometabolism from phylum to taxa to specific enzymes. The genomes of the microorganisms within the gut microbiome encode an extensive capacity for the metabolism of dietary compounds, pharmaceutical drugs, environmental chemicals, persistent organic pollutants, and other xenobiotics. Gut microbiota-xenobiotic interactions are also reciprocal, as exposure to xenobiotics can perturb the metabolic function of the microbiome. Our current understanding of microbiome functions are largely inferred from corollary measurements that can ascribe the potential for a function, but can not actually confirm that the function is present and active. We will make a significant advance in the understanding of microbial metabolic functions and how they influence human health by applying an activity-based protein profiling (ABPP) platform for in situ analysis of taxa and enzyme functional activities. By being able to identify the functionally active cells and enzymes within the microbiome, meaningful conclusions can be drawn about the connection between the gut microbiome and xenobiotic metabolism. Our ABPP platform will deploy chemical probes that target only catalytically active enzymes involved in xenometabolism in the gut microcbiome. The probes target the functional enzymes, but will enable isolation, characterization, and quantification of both the cells expressing those functions, and the specific enzymes catalyzing xenobiotic metabolism. With our ABPP platform we will address the central hypothesis that xenometabolism in the gut microbiome can be performed with phylogenetically disparate populations and different enzymes. We will initially develop a suite of new activity-based probes for five central xenobiotic metabolizing enzyme families in the gut microbiome. We will apply these to mouse gut and fecal microbiomes, and use flow cytometry, imaging, and proteomics to characterize functional cells and enzymes. We will also evaluate how exposure to a toxin, which is also a strong agonist of the aryl hydrocarbon receptor, impacts the compositional and functional environment of the microbiome. Completion of this project will provide a rich new functional understanding of microbial xenobiotic metabolism at the level of specific cells and enzymes. Our research will also yield a platform for future microbiome studies to evaluate individual variability and susceptibility to diseases, to understand consequences associated with xenobiotic exposures in adults and developing children, as well as for the future of precision medicine.
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{
"pile_set_name": "NIH ExPorter"
}
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There are several important reasons for studying the cellular and molecular mechanisms underlying the behavioral effects of repeated psychostimulant administration. First, abuse of psychostimulants such as cocaine is a major medical and social problem. Studies suggest that the neurophysiological adaptations associated with repeated psychostimulant administration contribute to drug craving. Second, the cellular changes underlying repeated psychostimulant administration are a form of neuronal plasticity. Studying the psychostimulant-induced neuronal adaptations will shed more light on the mechanisms of neuronal plasticity in general. Third, studying the molecular adaptations associated with psychostimulant exposure will help us to understand the neural mechanisms that lead to subjective experiences such as pleasure and pain, one of the most fascinating problems in the field of neuroscience. Growing evidence indicates that the neurotrophin family of growth factors plays an important role in the effects of psychostimulants. BDNF and NT-3 microinjections into the VTA have been shown to enhance the behavioral activating effects of cocaine. The goal of the research outlined in this proposal is to expand on these findings by studying the role of BDNF and NT-3 receptors, trkB and trkC respectively, in cocaine-induced behavioral sensitization. Specifically, this will be accomplished in the following two stages. First, rats will be injected with acute and repeated systemic injections of cocaine and levels of trkB and trkC protein in the ventral tegmental area (VTA) will be measured. Second, trkB and trkC receptors will be up-regulated and down-regulated in the VTA using sense and antisense recombinant adenovirus vectors. Following virus treatment, rats will be given acute and repeated systemic cocaine injections. Subsequent behavioral activity will be measured and the VTA will be excised to test for changes in tyrosine hydroxylase activity as a measure of cocaine-induced neuronal plasticity.
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{
"pile_set_name": "NIH ExPorter"
}
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Nosocomial bacterial sepsis is a frequent and serious complication of the care of very low birth weight (VLBW) premature infants. Nosocomial sepsis is associated with higher mortality, as well as prolonged and more expensive hospital stays. The increased susceptibility of these extremely small babies to nosocomial bacterial sepsis is related to impaired immune function (Wilson, 1986), and to the invasive nature of the necessary supportive care. Recent evidence has been reported suggesting that immaturity of the skin may be a significant additional risk factor for developing nosocomial sepsis. Nopper and co-workers (1996) studied whether application of an emollient ointment (Aquaphor Original) would improve skin integrity and decrease transepidermal water loss in extremely premature babies. This study had an unanticipated additional finding of a lower incidence of nosocomial sepsis and/or meningitis in the group treated with an emollient ointment. The Neonatal Skin Care Study (NSCS) is designed to determine whether prophylactic application of Aquaphor reduces the incidence of nosocomial sepsis in babies of birth weight 501-1000 grams. The study will be conducted at participating centers in the Vermont Oxford Network, a non profit voluntary collaboration of NICU's. Infants meeting all eligibility criteria will be randomized to the Prophylactic Aquaphor Group or the Routine Skin Care Group. The Prophylactic Aquaphor Group will receive routine twice daily application of Aquaphor during the first 14 days of life. Both the Prophylactic Aquaphor Group and the Routine Skin Care Group may receive prn treatment with Aquaphor ointment for dermatitis or evidence of local skin injury. The primary outcome measure will be the incidence of mortality and/or nosocomial infection at 28 days of age.
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{
"pile_set_name": "NIH ExPorter"
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The specific aims are to determine whether the hormonal responses to leuprolide acetate (Lupron) will distinguish among the causes of hypogonadism in pre-teen children with hypogonadism, among the causes of precocious puberty, and among the causes of inappropriate puberty. In the latter regard, girls with be compared to adult women with polycystic ovary syndrome to determine whether they have similar ovarian dysfunction, and boys with gynecomastia will be evaluated to test the hypothesis that gynecomastia occurs in boys who produce higher levels of estradiol relative to testosterone than normal. (This is an investigator-initiated study using the probe brandname Lupron)
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{
"pile_set_name": "NIH ExPorter"
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This Phase II STTR application is a continuation of a previous Phase I award between the Wright group at Dartmouth College and Promiliad Biopharma. The goal of this application is to continue our efforts to develop novel chemical libraries with the diversity and complexity common to natural products as a marketable product to the pharmaceutical industry. Key to our strategy is the use of fermentation derived natural products and recombinant non-natural variants as high complexity feedstock materials for diversity oriented synthesis. Unlike analog generation, these recombinant natural products are specifically designed to allow chemical diversification. Our approach differs substantially from combinatorial biosynthesis in that only a few, well-defined compounds are needed from fermentation sources. By only requiring a limited set of recombinant compounds from the organism, careful optimization can be conducted to ensure high yields of material. While this strategy relies on nature to construct a high degree of the complex features of the library, diversity-oriented strategies are utilized to generate large libraries that cover vast areas of structural space. Under the Phase I award, we have developed a number of direct and efficient strategies for converting a recombinant building block (nonactate) into several unique molecular architectures. This Phase award will allow our efforts to transition from the methods stage to Phase III, large-scale production and marketing of compound libraries. Under this award we will (1) prepare moderate sized libraries of highly diverse and complex molecules from nonactate, (2) develop new methods for scaffolds generation from the polyketides methymycin, pikromycin and spinosyn and (3) develop two new recombinant natural feedstocks, pikrodiene and spinosene. Through these efforts, Promiliad will be in a position to become a major supplier of new chemical entities to the pharmaceutical industry. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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STUDY OF TOXICITY OF FLUIDS FLOWING THROUGH DIALYZERS
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{
"pile_set_name": "NIH ExPorter"
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Compared to their male counterparts, women in American society who abuse or become dependent upon alcohol have been consistently overlooked as subjects for research investigation related to development of addictive behaviors and appropriate treatment approaches. Only recently have scientific inquiries been directed toward study of potential sex-specific differences in the effects of alcohol on emotions and behavior, an little is known about the effects of alcohol on functioning in women or about the interaction of the unique aspects of female endocrinology and response to or self-selection of beverage alcohol. A series of nine studies is designed to begin to address these gaps in existant knowledge which make use of intra- and intersex comparisons (i.e., normally cycling women at high versus low estrogen points of the menstrual cycle; women maintained on oral contraceptives versus nonoral contraceptive women; women versus men). These studies will yield normative data on the effects of acute alcohol intoxication on the cognitive processes of learning and memory (acquisition, storage, and retrieval), on fluctuating mood states, and on self and observer ratings of intoxication levels. Relationships between self-selection of beverage alcohol and such potentially influential but possibly independent variables as estrogen levels (preovulatory-ovulatory versus premenstrual-menstrual levels), mood state changes occuring concomitant with or independent of such changes, and more enduring mood dispositions of depression and anxiety will be explored with intra- and intersex comparisons performed as appropriate. Finally, the tension-reduction hypothesis will be tested using an expectancy format to describe the effects of acute alcohol intoxication or the expectancy of intoxication on self-reported states of anxiety and depression, electrodermal and heart rate activity, and self and observer reported intoxication ratings with appropriate intra- and intersex comparisons. Essentially, the range of studies will reflect in part the variety of research work accomplished with samples of male nonalcoholics alone.
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{
"pile_set_name": "NIH ExPorter"
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A landmark therapeutic breakthrough was the preemptive use of antiviral drugs including ganciclovir (GCV) and foscarnet (FOS) to control cytomegalovirus (CMV) viremia in hematopoietic cell transplant (HCT) recipients. These drugs have remarkable properties to limit progression from viremia to disease but also have significant side effects, including exacerbating neutropenia, delay of immune reconstitution, and renal toxicity in the case of FOS. To overcome using these antiviral agents, we proposed a peptide vaccine to stimulate the host immune response to CMV. In the last funding period we conducted a Phase 1 trial of the peptide vaccine with or without Pfizer PF03512676 adjuvant that confirmed the safety of 2 different formulations, with evidence of immunologic activity based upon flow cytometry analysis of CMV-pp65- specific T cells. A limitation of the peptide vaccine is its restriction to individuals with the HLA-A2 haplotype which narrows the target population. While we are seeking to confirm a therapeutic benefit of the peptide vaccine in HCT recipients, broadening the population that would benefit from a vaccination strategy is best approached by employing a delivery vector that expresses full length CMV antigens. We have used modified vaccinia Ankara (MVA) virus for that purpose in collaboration with NCI-NExT to manufacture a candidate for clinical testing. In this revised application, we propose 3 clinical trials that will comprehensively evaluate our clinical vaccine candidate. In Aim 1, a safety study (Trial 1) will be conducted in healthy volunteers stratified by age and poxvirus exposure who are equivalent health-wise to immunocompetent HCT donors as required by the FDA and will establish the safety and dose level for a 2nd trial in HCT. In Aim 2, a double-blind Phase 2 trial is described in which 150 HLA matched related HCT donors (MRD) will be randomized equally to receive either vaccine or placebo. Immune responses and clinical correlates of vaccine-stimulated protection will be followed in recipients whose donors are enrolled in Trial 2. In Aim 3 (Trial 3), we propose to directly immunize 150 HCT recipients undergoing unrelated (8/8 HLA matches) donor (URD) HCT after gaining safety experience by following the clinical course of 30 recipients in Trial 2. This approach is justified since these recipients are at high-risk for CMV reactivation and do not have consistently accessible donors. Trials 2 and 3 will each enroll 150 HCT subjects, randomized equally to vaccine or placebo and both will have 90% power to detect a 50% reduction in viremia (60% to 30%) in the vaccine arm. Safety monitoring employing defined stopping rules will guard against adverse consequences for recipients in both trials. Combined results from carrying out both Trials 2 and 3 will provide a precise immunoprophylaxis strategy for control of CMV disease in the HCT setting. We will discover the optimal formula for immunizing both donors and recipients to prevent CMV viremia and minimize usage of potentially toxic antiviral agents. Success with this CMV vaccine would have a significant clinical and cost-saving effect on recipient management after MRD or URD HCT.
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{
"pile_set_name": "NIH ExPorter"
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[unreadable] This Lung Cancer SPORE application is a competing renewal application submitted by the Vanderbilt-lngram Cancer Center and its affiliated institutions. Cancer is now the number one killer of Americans, and lung cancer is the number one cause of cancer death, causing the deaths of more people than breast, colon, and prostate cancers combined. [unreadable] [unreadable] The Vanderbilt-lngram Cancer Center SPORE application in lung cancer is composed of investigators with expertise in proteomics, cellular and molecular biology, epidemiology, pathology, pulmonary medicine and medical, radiation, and surgical oncology committed to translational research with the goal of reducing the incidence, morbidity and mortality of lung cancer. We are applying a broad range of state-of-the-art technologies, including leading edge protein mass spectrometry, expertise in signal transduction, and in eicosanoid biology, as well as innovative bioinformatics and statistical analyses to this problem. We have assembled a multidisciplinary team of internationally recognized basic and clinical investigators into a cohesive program with a history of progress in the translation of laboratory findings into the clinic and back, as well as a track record of extensive collaborations with the other lung SPOREs, multiple cooperative groups, as well as the two other SPOREs at Vanderbilt. We have demonstrated flexibility and responsiveness in our program to optimize achieving the broader goals of our SPORE. [unreadable] [unreadable] The overall goal of our program is to define complex pathways and important intermediates in these pathways to help in the early detection and optimal treatment of patients with lung cancer. Our program is focused on elements of these pathways of potential utility both as clinically useful biomarkers for early detection, prediction and prognostication, but also as potential direct therapeutic targets. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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The long-term goal of research proposed in the present application is to delineate neuronal systems and processes involved in simple forms of learning and memory. More specifically, this research seeks to advance our understanding of the involvement of the cerebellum in a simple motor learning task, classical eyelid conditioning in rabbits. Three experiments are proposed that use anesthetized classical conditioning preparations. These preparations involve the pairing of brain microstimulation in place of peripheral conditioning stimuli and recording unit activity from brain stem nuclei known to be involved in generating conditioning responses in place of monitoring overt behavioral responses. The proposed experiments include: (1) An analysis of extracellular single unit cerebellar activity during and after conditioning, (2) Manipulations of the "behavioral" training regimen used during the anesthetized conditioning, and (3) Intracellular recordings coupled with HRP labeling after conditioning. These experiments have been designed to explore similarities and differences in conditioning between awake and anesthetized rabbits, to provide information about changes in the activity of cerebellar neurons induced by the anesthetized conditioning procedures, and to provide some insight into the connectivity between cerebellar and brain stem neurons involved in the conditioning. The further development of this anesthetized conditioning preparation should facilitate the definition and study of cerebellar and brain stem circuits activated during this form of learning. Intracellular analyses of neuronal activity should provide initial information about cellular processes and synaptic mechanisms associated with classical eyelid conditioning. We anticipate that these mechanisms may be generalizable to other motor learning and memory situations and therefore advance our understanding of basic cerebellar and brainstem neuronal function. Results from these experiments should also prove useful for understanding loss of normal function associated with cerebellar pathologies caused by injury or disease. Moreover, advancing our knowledge of cellular and synaptic processes involved in simple motor learning and memory should prove useful for understanding more complex learning and memory processes and the cellular bases of learning impairments and memory loss associated with disease and injury.
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{
"pile_set_name": "NIH ExPorter"
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The specific objectives of this project include 1) to determine the analog and dose of retinoid that can prevent/delay mammary tumor development, 2) to determine the retinoid and tamoxifen combination effect on delaying mammary tumor development and 3) to determine the influence of type of diet and dietary ingredients in delaying the development of mammary tumors. For this purpose female transgenic (TG.NK) mice with human breast cancer oncogene "neu" (erbB2) that develop 50% incidence of mammary tumors by 26 weeks of age were a) fed different diets to delay the development of tumors, b) treated with selected doses of retinoids to establish the most effective retinoid and dose and c) the most effective diet and retinoid analog will be further evaluated in combination with tamoxifen to determine the synergism between the diet and drugs. Retinyl acetate in diet delayed the development of mammry tumors in a dose response manner but may be toxic at the doses causing delay in tumor development. The experimental retinoid analog 4-hydroxyphenyl retinamide (4-HPR) when given in diet delayed the development of mammary tumors and showed a dose response at nontoxic doses. Another experimental retinoid analog Arotinoid Ro 40-8757 (Roche) caused a dose-related delay in development of mammry tumors but also caused dose-related toxicity. These results indicate that the transgenic TG.NK mouse model may be appropriate to evaluate intervention strategies to delay/prevent the development of breast cancer. About 50% of the TG.NK mice fed a purified diet AIN-76A containing 5% cellulose as fiber developed mammary tumors by 26-weeks of age. A Nonpurified diet NIH-07 with -3.5% active fiber (fiber that can bind estrogen and other substances) delayed the development of mammary tumors as compared to the purified diet. Another nonpurified diet NTP-2000 with lower protein and higher active fiber than NIH-07 diet markedly delayed the development of mammary tumors. These results indicate that increased fiber consumption appears to delay the development of mammary tumors in the TG.NK mouse model and the delaying effect of fiber was comparable to nontoxic doses of some retinoid analogs. This finding is in agreement with human epidemiological observations that higher fiber consumption may decrease the risk of breast cancer in women. The TG.NK model appears to be suitable for evaluation of intervention strategies such as diet, retinoid and tamoxifen combinations to prevent/delay breast cancer.
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{
"pile_set_name": "NIH ExPorter"
}
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Novel strategies are needed to prevent and treat sexually transmitted infections. The alpha and beta defensins are a family of small, cysteine-rich peptides that are expressed in mammalian neutrophils and epithelial cells, and which exhibit antimicrobial activity against a wide range of bacteria, fungi and enveloped viruses. Human defensin-5 (HD-5) is a novel member of the a-defensin family which to date has only been found in the Paneth cells of the small intestine. The investigators recently detected a HD-5 homologue in the epithelial cells of the human female genital tract. They have demonstrated that HD-5 is differentially expressed in different regions of the tract and that expression is altered by inflammatory processes and by the stage of the menstrual cycle. The experiments outlined in this proposal will test the following hypotheses: (a) expression of HD-5 in female genital tract epithelia is upregulated by sexually transmitted pathogens, proinflammatory cytokines, and female reproductive hormones (b) HD-5 is secreted into the genital tract lumen, (c) HD-5 has effective microbicidal properties against common sexually transmitted organisms, and (d) HD-5 is a chemoattractant for leukocytes. This research will help elucidate the role of defensins at genital tract mucosal surfaces, and may facilitate the development of novel prophylactic and therapeutic agents against sexually transmitted pathogens which are based on these endogenous antibiotic peptides.
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{
"pile_set_name": "NIH ExPorter"
}
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The ability to explore, detect and modify the protein features of cell surfaces is a key in the long-term development of many diagnostic and therapeutic strategies. A major obstacle is the fact that the fundamental nature of protein-membrane interactions is largely unknown. Two model systems have been developed for using molecular genetics to determine the principles regulating such interactions: the lysis control genes S of bacteriophage lambda and E of bacteriophage PhiX174. These genes are small (S = 107 codons, E = 91 codons) and lethal, and thus are well-suited for mutational analysis. In vitro mRNA and protein synthesis will be sued to characterize both individually and in pairs representatives from a collection of about 50 missense S mutants in terms of membrane interaction and protease accessibility. Intragenic suppressors of many of these S mutants will be isolated and sequenced to determine interaction domains within the polypeptide. The novel translational control system of S will be probed by site-directed mutagenesis, to determine the parameters by which the two different protein products are synthesized in regulated proportion. The E gene will be subjected to fine structure lac fusion analysis and reversion studies to determine the minimum requirements for membrane insertion of the polypeptide. A missense analysis of the E gene will be carried out in the same way done successfully for the S cistron.
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{
"pile_set_name": "NIH ExPorter"
}
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The aims of this proposal are designed to uncover biological functions of the human Y chromosome beyond reproduction, which will have significant implications for human health and disease. Until now, Y chromosome research has focused solely on two functions: sex determination and sperm production. Our ongoing genomic studies of multiple mammalian Y chromosomes have revealed a set of broadly expressed, dosage sensitive regulatory genes that have been conserved on the Y chromosome. Because many of these broadly expressed genes likely have important and widespread biological functions, we hypothesize that the disruption of these genes may contribute to disease, especially Turner syndrome and diseases with sex-biased prevalence, susceptibility, or severity. Historically, deletion-mapping studies have focused solely on the Y chromosome's testis-specific genes, which are involved in sex determination and sperm production. We will perform the first systematic examination of phenotypes associated with disruption of the Y chromosome's broadly expressed genes by studying ~75 patients with Y-chromosome anomalies affecting one or more of these genes. We will evaluate phenotypes of patients using a combination of approaches: 1. At the NIH Clinical Center, patients will undergo a thorough examination of all major organ systems, including physical, reproductive, endocrinologic, and radiologic tests. We will characterize phenotypic abnormalities in patients, detecting patterns that correlate with particular Y-linked gene defects. Bringing patients to the Clinical Center ensures that all testing will be conducted in the most rigorous and systematic manner possible, thus allowing the identification of previously overlooked phenotypes. 2. In the Page lab, the cellular and molecular phenotypes of the same set of patients will be characterized using a combination of whole-genome approaches: RNA-seq and ChIP-seq. We will perform similar analyses in controls with intact Y chromosomes to identify perturbations in chromatin modification, transcript level, or splicing efficiently that may be linked to Y-chromosome defects. Through comprehensive phenotypic characterization, at molecular, cellular, anatomical, and physiological levels, of individuals with Y-chromosome anomalies, we can begin to understand the contribution of Y-linked genes to basic biological processes and fundamental differences between the sexes. Ultimately, these insights could lead to a greater appreciation of the etiology of Turner syndrome phenotypes and the underlying causes of sex biases in disease.
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{
"pile_set_name": "NIH ExPorter"
}
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Like other enveloped viruses, paramyxoviruses spread infections from cell to cell and from host to host in the form of particles which are formed by budding from infected cell membranes. Paramyxovirus matrix (M) proteins organize the assembly process, linking together the viral glycoproteins and the viral ribonucleoproteins (vRNPs). Interactions between M proteins and nucleocapsid (NP) proteins are the driving force for active incorporation of vRNPs into virions, and hence are fundamentally important for the infectivity of nsRNA virus particles. Here, we propose to investigate paramyxovirus M protein interactions that drive the packaging of vRNPs into particles. Our first aim is to manipulate and inhibit paramyxovirus genome packaging interactions by targeting the nucleocapsid proteins. We will attempt to achieve competitive inhibition of M-NP interactions using foreign proteins and peptides that incorporate into budding particles using the same interactions that normally direct vRNPs into virions. We also propose a series of experiments to define parameters that govern M-NP interactions among the paramyxoviruses. Our second aim is to define and manipulate paramyxovirus genome packaging interactions by targeting the matrix proteins. Guided by second-site mutations and the recently-determined M protein structure, we have defined PIV5 M mutations that enhance interactions with NP. Consequences of enhanced M-NP interaction will be assessed both in transfected cells using mini-genome assays and in the context of recombinant virus. We hypothesize that M-NP interaction strength in wt virus is carefully balanced. M-NP interaction that is too strong will benefit virus assembly, but will also lead to inappropriate generation of M- bound vRNPs during the early phases of infection, thereby impairing viral transcription and/or genome replication. We will also define individual roles for two distinct clusters of second-site mutations on the M protein surface, with the hypothesis that the C-terminal surface indirectly influences virus assembly and NP binding through interactions with host factors.
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{
"pile_set_name": "NIH ExPorter"
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This is a phase III randomized, blinded study of nevirapine vs. nevirapine placebo for the prevention of maternal to fetal transmission in pregnant women who present during the third trimester of pregnancy. Participating mothers will be given a single blinded dose during active labor. Neonates will be given a single dose of nevirapine elixir or placebo elixir by mouth at 2-3 days of life. The primary endpoint is incidence of transmission of HIV infection from mother to fetus. The secondary endpoints are monitoring of maternal and neonatal toxicities, determination of nevirapoine pharmacokinetics in neonates, determinine the relationship between maternal plasma viremia and risk of vertical transmission and the effect on neonate plasma viremia.
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{
"pile_set_name": "NIH ExPorter"
}
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Coronaviruses are positive-strand RNA viruses that cause respiratory infections in humans and important gastrointestinal and neurologic disease in other animal species. Mouse hepatitis virus (MHV) is a model for studies of coronavirus replication and pathogenesis. The central goal of the proposed research is to define interactions of mouse hepatitis virus with host cells during formation and function of viral replication complexes. MHV directs viral RNA synthesis on membranous replication complexes that are distributed thoughout the cytoplasm. These complexes are distinct from sites of virion assembly in the intermediate compartment of the Golgi (ERGIC). Proteins processed from the replicase gene polyprotein localize with the nucleocapsid (N) protein at sites of viral RNA synthesis in two closely associated protein complexes, the p22/p65 complex and the hel/N complex. At late times of infection, the hel/N complexes separate from the p22/p65 complexes and translocate to sites of virion assembly in the ERGIC. However, the precise composition of replication complexes is not known, and the mechanism of hel/N complex translocation has not been determined. This proposal describes an integrated research program to define the organization and translocation of MHV replication complexes. In Specific Aim 1, the protein and membrane composition of MHV replication complexes will be determined. In Specific Aim 2, the mechanism of hel/N complex translocation to the ERGIC will be defined. In Specific Aim 3, the functions of viral proteins in replication complex translocation will be determined. These studies will identify fundamental mechanisms by which MHV directs replication complex formation and function and will define virus-cell interactions that are essential for successful virus replication.
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{
"pile_set_name": "NIH ExPorter"
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Opiates are commonly abused substances, and forensic urine drug-testing for them involves an immunoassay screen and gas chromatographic/mass spectrometric (GC/MS) confirmation. There are also medical reasons to test urine for opiates, and confirmation procedures other than GC/MS are often used for medical drug-testing which are more compatible with the demands of clinical services and which identify a wider range of opiates than those in standard forensic batteries. One such procedure involves thin-layer chromatographic (TLC) analysis of opiate derivatives and can distinguish eight clinically encountered opiates, including morphine, acetylmorphine, hydromorphone, oxymorphone, codeine, dihydrocodeine, hydrocodone, and oxycodone. Medical drug-testing results are sometimes challenged by patients, causing physicians to request additional confirmation of the identified opiates. To our knowledge, no previous report examines all opiates specified above in a single GC/MS pr ocedure, but we find that they can be distinguished by GC/MS analyses of trimethylsilyl (TMS) ether derivatives, the mass spectra of which contain prominent molecular ions. Inclusion of deuterium-labeled internal standards permits quantitation of each of the eight opiates in urine. The GC/MS assay is linear over a concentration range which spans the TLC cutoff level, and coefficients of variation of 10% or less at concentrations below the TLC cutoff are achieved by for all opiates specified above except for oxymorphone and oxycodone, which exhibit coefficients of variation of 18-19%. This procedure has proved useful as a third-stage identification step for medical drug-testing specimens in which results from prior immunoassay and TLC analyses were challenged.
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{
"pile_set_name": "NIH ExPorter"
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The proposed project is a continuation of research on the effects of marihuana (delta-9-tetrahydrocannabinol) on the reproductive system. Sexually mature female Rhesus monkeys are administered tetrahydrocannabinol (THC) and gonadotrophic hormones and sex steroids are measured. The initial phase of this study has shown that THC can alter reproductive hormones in both male and female monkeys. The proposed continuation of this research will place these changes in perspective by comparing the effects to other drugs that alter reproductive hormones; by comparing the effects produced in females with effects in males; by evaluating the effects on fertility in the female.
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{
"pile_set_name": "NIH ExPorter"
}
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An experimental model for myocardial dysfunction has been developed in sheep. Nineteen animals were placed on cardiopulmonary bypass and subjected to 13 to 19 minutes of myocardial ischemia at 37 C. Measurements of cardiac output, stroke volume, and left ventricular stroke work pre and postoperatively, showed that these indices of hemodynamic function were significantly depressed up to one week after ischemic injury. Analysis of Starling curves, confirmed the presence of ischemia induced myocardial depression. Histologic studies demonstrated primarily subendocardial necrosis.
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{
"pile_set_name": "NIH ExPorter"
}
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This proposal is to perform preliminary studies that will ultimately result in antibiotics with synergistic activity against the HTV opportunistic pathogen, Mycobacterium tuberculosis (TB). TB is the leading cause of death among HTV-infected patients in the developing world. This bacterium predictably becomes resistant to each drug soon after it is introduced. A new approach is required that specifically prevents the emergence of drug resistance and its transmission between patients. We have discovered that mutations in codon 306 of the TB embB gene predisposes strains to acquire progressively more drug resistance and increases their potential for transmission, although the mutation does not cause resistance to any given drug on its own. Here, we propose to determine the mechanisms underlying these observations, with the ultimate goal of designing drugs that inhibit this activity. We hypothesize that the drug ethambutol (EMB) normally acts synergistically with other antibiotics to prevent the emergence of drug resistance, and that this synergy is lost in embB306 mutants. This synergy appears to be effective below the minimal inhibitory concentration of EMB. To better understand this process, we will investigate whether EMB-associated inhibition of lipoarabinomannam (LAM) biosynthesis increases the permeability of the TB cell wall to antibiotics and results in synergy. We will also investigate whether increased intracellular antibiotics can delay the development of drug resistance. Finally we will study whether EMB-inhibition of LAM modulates the early interactions between TB and the immune system, potentially explaining the increased transmissibility seen in the embB306 mutants. Experiments with wild type and embB3Q6 mutant strains will be performed in parallel to determine if embB306 mutants are resistant to these novel effects of EMB. Our specific aims are to determine in vitro: 1. if EMB causes synergistic with other antibiotics by increasing antibiotic permeability, and if embB306 mutants are resistant to this synergy; 2. if embB306 mutants have altered phagocytosis, growth and cytokine induction in macrophages and dendritic cells with and without EMB treatment; 3. if embB306 mutants are more likely than wild type strains to become resistant to other drugs in vitro; 4. if embB306 mutations affect biosynthesis of LAM, capped LAM, arabinogalactan (AG) or other cell wall components in the presence and absence of EMB.
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{
"pile_set_name": "NIH ExPorter"
}
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Insulin resistance is a marker for increased risk of Non-insulin dependent diabetes mellitus, hypertension, and cardiovascular disease. The degree to which insulin sensitivity is determined by genetic and/or environmental factors is unknown. In order to define the heritability of insulin resistance, maximally insulin-stimulated glucose uptake will be measured using the glucose clamp technique in MZ and DZ twin pairs who are free from overt disease.
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{
"pile_set_name": "NIH ExPorter"
}
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EXCEED THE SPACE PROVIDED. This application requests funding for the Tulane National Primate Research Center (TNPRC) for the five year period beginning May 1, 2003 through April 30, 2008. Funds will be used to support the administration, operations, veterinary resources, and scientific research resources of the Center. Additional funds to support pilot research projects and colony-health-related research resource projects are also requested. The research program at the TNPRC has been focused on infectious disease research for close to three decades. Although the majority of the Center' s research efforts are devoted to infectious disease, there are also rapidly expanding programs in gene therapy and assisted reproductive technologies. The infectious disease program currently focuses on AIDS, Malaria Microsporidial diseases and Lyme disease. These are multidisciplinary studies involving investigators in- numerous Divisions at the TNPRC and collaborators outside the Center. The studies cover the spectrum from transmission, diagnosis and pathogenesis to development of vaccine strategies and chemotherapeutic treatments, and from pathologic lesions to gene sequences. In addition, the development of the gene therapy program allows for novel approaches to the treatment of many types of disease and preclinical modeling of a variety of gene therapy modalities. The last five years and particularly the last year have seen significant changes at the Center, following the retirement of Dr. Peter Gerone and the naming of Dr. Andrew Lackner as the new Director. Since October 1, 2001 when Dr. Lackner became Director, leadership in several of the divisions has changed, new divisions have been formed and the business office (now Administrative Services) has begun reorganization. The result is a Center that now places more emphasis on research programs and which has experienced a 50% growth in its grant portfolio. These changes have been facilitated by a very helpful and collegial host institution. The new organizational structure and leadership team at the TNPRC and recent successes in obtaining grant funding and attracting outside collaboration suggest that the Center is poised for a period of significant growth and improvement.
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{
"pile_set_name": "NIH ExPorter"
}
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To visualize binding of the glucocorticoid receptor to its DNA target in living cells, we are using a specially constructed cell line, 3617 cells, characterized by two key features. First, 3617 cells are stably transformed with a GFP-tagged glucocorticoid receptor (GFP-GR), and second the 3617 genome contains an ~200-fold tandem array of the mouse mammary tumor virus (MMTV) promoter. Since GR binds to the MMTV, we should be able to use fluorescence light microscopy to visualize GFP-GR binding to the MMTV tandem array in the 3617 cells. We have now demonstrated that this can be done. When 3617 cells are stimulated with hormone, GFP-GR moves into the nucleus. In addition to a punctate staining pattern seen in control cells lacking an array, a significant percentage of 3617 cells also exhibit a GFP-GR staining pattern indicative of the array. The array appears as either a large, amorphous spot or a more elongated linear structure. Such structures are in fact the array, as demonstrated by fluorescence in-situ hybridization (FISH). This technique reveals RNA transcript accumulation. In 3617 cells, FISH demonstrates that the RNA transcripts known to be regulated by the MMTV promoter colocalize uniquely with the array structures visualized by GFP-GR. The ability to visualize GR binding in living cells now paves the way for an in vivo analysis of steroid receptor function by a variety of light microscopy methods. Two aspects of the GFP-GR array are being studied. First, we are using FRAP microscopy (fluorescence recovery after photobleaching) to study exchange rates of molecules at the MMTV template. We have shown that GFP-GR exchanges rapidly at this site in live cells, and future studies will examine exchange of other factors known to be present at this template. Second, we are examining structural changes in the array over time. We find by time lapse microscopy that hormone induces decondensation followed by recondensation of the array. We have also shown using RNA FISH that decondensed arrays produce more transcript. Thus, the observed opening and closing of the array likely reflect the activation and subsequent down regulation of the MMTV known to occur from previous run on transcription studies.
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{
"pile_set_name": "NIH ExPorter"
}
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In animals or humans suffering the consequences of hemorrhagic shock (HS), polymorphonuclear neutrophilic granulocytes (PMN) are the major cellular element that mediates the inflammatory process. We propose that PMN activation is an early event in HS initiated by the consequences of ischemia and resuscitation which includes the production and action of granulocyte colony-stimulating factor (G-CSF). We have demonstrated elevated G-CSF mRNA levels in critical organs including the lung, liver, and bowel in our rat model of HS. Levels of G-CSF mRNA increased with increasing severity of HS. We have identified bronchial epithelial cells as the major cellular source of G-CSF production in the lung in HS. In addition to our findings in rats, we have observed a 55-fold increase in G-CSF mRNA production in the livers of patients with HS. To establish that G-CSF production in the organs of animals and patients with HS is deleterious to the host, we demonstrated that instillation of G-CSF alone into the lungs of rats results in PMN recruitment and lung damage. In related work, we have shown that G-CSF specifically activates a distinct member of the signal transduction and activator of transcription (STAT) family, Stat3(, in PMN. PMN of rats subjected to HS revealed greater Stat3( activity than sham animals. In addition, PMN from patients suffering from HS showed clear evidence of G-CSF-induced Stat3( activation. Taken together, our findings to date strongly support the hypothesis that G-CSF is produced in critical organs in HS where it binds to PMN resulting in organ inflammation. The overall goal of this proposal is determine how G-CSF production occurs in HS and how its production leads to PMN recruitment and activation in HS and its morbid sequellae. Towards this end, the two Specific Aims of this project are: AIM I: To determine the consequences of G-CSF production on PMN recruitment, Stat3( activation, and organ injury in HS and to examine the effect of blocking the G-CSF signaling pathway in PMN on morbidity and mortality of HS. AIM II: To determine the factors contributing to G-CSF production at critical sites of PMN recruitment and organ injury.
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{
"pile_set_name": "NIH ExPorter"
}
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The Wake Forest University MFMU Center is reapplying for participation in the MFMU Network. The strengths of this Center include: (1) The Center has a large stable base of ethnically diverse obstetrical patients and deliveries by virtue of the fact that the hospital at which the Center is based, Forsyth Medical Center, is the only hospital delivering in-patient obstetric care for a four county area. A high proportion of these patients are high-risk patients since the unit is the only tertiary perinatal center for a 17 county area in Northwest North Carolina; (2) The Wake Forest University Center has had a strong record of participation in the Network for the past nine years. It has a solid record in design of protocols (it is the lead Center for an active MFMU Network protocol), in recruitment of subjects, and in accuracy and reliability of data collection. The Center?s investigators appear as authors on 33 Network publications; (3) The Center?s Principal Investigator, Dr. Paul Meis, has played an important role in the activities of the Network as Chair of the Publications Committee and of the Progesterone Subcommittee. He has worked closely with the Program Officer and the Biostatistical Center to facilitate high quality publications of the work of the Network; and (4) The Wake Forest University Center has unusual potential for epidemiologic research. The fact that it is the only tertiary perinatal center for seventeen counties facilitates the performance of geographically based studies of maternal and infant epidemiology. Dr. Meis and Dr. T. Michael O?Shea, the neonatal consultant, have special interest in this area and have collaborated on studies in the past using a combined perinatal-neonatal database. Drs. Meis and O Shea have been active in the leadership of the Society for Pediatric and Perinatal Epidemiologic Research. Dr. O?Shea is submitting an application for participation of Wake Forest University in the NICHD Neonatal Network.
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{
"pile_set_name": "NIH ExPorter"
}
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Molecular Probes proposes to develop applications in basic and applied research and in medical diagnostics for the fluorescent latex microspheres that it has prepared. The superiority of these products in terms of their range of size, wavelength and fluorescence intensity will enhance their application in areas of research and industry that have sensitivity requirements that currently available fluorescent latex and other fluorescent reagents cannot meet. The new microspheres will be prepared from oil-soluble fluorescent dyes recently developed by Molecular Probes research. The specific applications the investigators propose to develop are: 1) new fluorescent latex-based hybridization reagents; 2) labeling reagents with improved fluorescence intensity, greater Stokes Shifts and multiple excitation/emission for better detectability and multi-color applications in imaging and flow cytometry; and 3) new, ultra-sensitive fluorescent probes for low-density cellular receptors. Most of the work envisioned in this proposal will be carried out by Molecular Probes research personnel, with assistance from outside collaborators.
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{
"pile_set_name": "NIH ExPorter"
}
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The overall objectives of this proposal are to use a combination of traditional medicinal chemistry and computational, structure based drug design to develop novel small molecule inhibitors of 1-deoxy-D-xylulose-5- phosphate reductoisomerase (DXR) and test their in vitro biological activities on pathogenic bacteria and parasites. Isoprene biosynthesis is essential to all organisms. Humans use the mevalonate pathway to produce isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), two common precursors for all isoprenoid biosynthesis;however, in most pathogenic bacteria, such as P. aeruginosa and M. tuberculosis, as well as apicomplexan parasites, such as P. falciparum and T. gondii, the non-mevalonate pathway is used to make IPP and DMAPP. Since humans lack all the 7 enzymes in the non-mevalonate pathway, it has become an attractive target for anti-infective drug discovery. Fosmidomycin has been found to be the only potent inhibitor of this pathway, blocking DXR, the 2nd enzyme, and has antibacterial activity against many Gram- negative bacteria and antimalarial activity in recent clinical trials. However, Gram-positive bacteria (e.g., M. tuberculosis) and some Gram-negative bacteria as well as certain pathogenic parasites (e.g., T. gondii) are resistant to fosmidomycin. In addition, it has a poor pharmacokinetic profile with a half-life in plasma of 0.5-1.5 h. Given the current devastating situation facing quickly rising drug resistance as well as shortage of new anti- infective drugs, there is a pressing need to find new weaponry for infectious diseases. The first Specific Aim is to use a combination of medicinal chemistry and computational, structure based drug design to develop novel inhibitors of DXR. Based on rational, structure based design, we have found novel, drug-like lead inhibitors with Kis as low as 310 nM against a recombinant E. coli DXR enzyme. Our docking studies showed that they could bind to DXR in different modes from that of fosmidomycin. These drug-like compounds should have great potential for further development. We propose 1) to use medicinal chemistry to make several series of compound libraries based on the scaffolds of the lead inhibitors, in order to find compounds with improved activity;2) to carry out QSAR studies of these compounds;3) to obtain x-ray crystal structures of DXR in complex with our novel inhibitors;and 4) to use the results from the computational and crystallographic studies to guide our further drug design and synthesis. The second Specific Aim is to test in vitro biological activity of our inhibitors on a broad range of bacteria and apicomplexan parasites as well as their recombinant DXR enzymes. Finally, we will also test the cytotoxicity of our potent DXR inhibitors on human cell lines to evaluate their potential toxicity. PUBLIC HEALTH RELEVANCE: The research proposed is designed to lead to new potential therapeutics to treat drug-resistant infectious diseases. We will focus on the discovery and development of novel compounds that block essential biological targets that are exclusively found in bacteria and malaria parasites.
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{
"pile_set_name": "NIH ExPorter"
}
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New computational approaches to the simulation of nucleic acid interactions will be developed, including improved molecular mechanical force fields, new methods to calculate free energies and new methods to enhance conformational sampling. These new approaches will be applied to DNA and RNA the structures and the interactions of DNA and RNA with proteins and small molecule Iigands. The goal is to accurately reproduce experimental structures and free energies of binding in these systems when these are known. Applications include interactions of DNA with anticancer drug daunonycin and its analogs, minor groove topoisomerase I inhibitors which have anti-cancer activity, and polyamides which are among the most promising therapeutic approaches to target specific DNA sequences, as well as interactions of DNA with both binding proteins (the zinc finger Zif 268) and proteins that catalyze specific site cleavage of the DNA (Eco RV). Dr. Kollman will also study RNA-ligand interactions which are relevant in anti-viral and anti-AIDS chemotherapy, as well as elucidating the mechanism of catalysis of the ribosome, certainly the most important ribozyme yet discovered.
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{
"pile_set_name": "NIH ExPorter"
}
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DESCRIPTION: Only a tiny % of tumor cells within the tumor tissue is motile and capable of escaping and initiating metastasis. The gene signature of these motile tumor-initiating cells predicts clinical outcome in breast cancer patients. Patients exhibit heterogeneous responses to drugs targeting the cytoskeleton, which is a common target for therapies aiming to inhibit tumor cell growth, motility and invasion. To date, there is no clinical assay to 1) isolate the highly motile cell subpopulations from patient biopsies, and 2) investigate their responses to cytoskeleton-targeting drugs. A clinically relevant method for identifying optimal individualized therapies is to screen the effects of drugs on patient-derived tumor specimens transplanted in mice (tumorgrafts). However, this method suffers from four major drawbacks: 1) only 28-37% of patient-derived tumorgrafts grow in mice, 2) this method, if successful, requires 4-12 months, 3) it requires large tissue specimens, and 4) it examines the effects of drugs on the bulk tumor cell population, and not specifically on the motile tumor-initiating cells. It is thus urgent 1) to deveop a clinically relevant technology that will enable physicians to accurately and rapidly assess the relative abundance of motile cell subpopulations in primary tumors at the time of initial diagnosis, and 2) to understand how cytoskeletal-targeting drugs affect the properties and function of these motile cells within a heterogeneous tumor in order to develop personalized therapies. Such technology should be high- throughput, rapid with superior detection sensitivity, and capable of studying the responses of motile single cells isolated from a small number of tumor cells harvested from patient biopsies. The microfluidic technology meets all these requirements. In Aim 1, we will develop, test and validate a microfluidic assay that predicts the metastatic propensity of human breast cancer cell lines and patient-derived breast cancer cells. Because of the heterogeneous responses of cancer patients to cytoskeleton-targeting drugs, we will use our microfluidic assay to rapidly assess the responses of 8 human breast cancer tumorgrafts as well as patient-derived breast cancer cells to different cytoskeletal modulators to identify optimal personalized therapies (Aim 2). Most of the drugs to be tested are FDA-approved or in clinical trials, ensuring that this study could rapidly impact the clinical treatmen of breast cancer. Since cell properties regulate migration, invasion and metastasis, we will use a novel high-throughput, single-cell imaging technology to define the morphological, and molecular signatures of migratory vs. non-migratory breast cancer cells isolated by the -fluidic assay (Aim 3). In view of compelling data showing the involvement of giant obscurins in cancer metastasis, we will evaluate giant obscurins as a novel biomarker for breast cancer (Aim 4). We will also examine how the expression profile of giant obscurins: 1) is altered in migratory versus non-migratory cells; 2) regulates the responses of breast cancer cells to cytoskeletal drugs; 3) affects the morphological and molecular signatures of breast cancer cells; and 4) how ectopic expression of an obscurin signaling cassette can suppress tumorigenicity and metastasis.
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{
"pile_set_name": "NIH ExPorter"
}
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Cardiac myosin plays a key role in cardiac contractility. Mutations in the cardiac myosin heavy chain gene is linked in some families to familial hypertrophic cardiomyopathy (HCM), the most common inherited cardiac disease. To date, over 40 mutations in the S-1 and proximal S-2 regions of the heavy chain have been detected. Recently, mutations in the regulatory and essential light chain of cardiac myosin have also been found. Interestingly, two mutations in the essential light chain map quite close to a number of heavy chain mutations. We have studied a heavy chain mutant, R719Q, and an essential light chain mutant, M149V, which lie close to each other in space using the crystal structure of chicken fast skeletal muscle as a guide. In contrast to other mutations in the heavy chain that we have studied, these two mutations are associated with a higher rate of movement of actin filaments in an in vitro motility assay. The location of these two mutations suggests that they lie in what may be a hinge region between the heavy chain and light chain where two domains slide past one another during force transduction. The increase in rate of actin filament translocation in vitro may reflect an increase in the crossbridge cycling rate in vivo. Such an increase in crossbridge cycling rate would require a higher energy consumption, contributing to hypoxia of cardiac muscle in HCM patients, who usually have ventricular hypertrophy, which could augment the effects of hypoxia by increasing the distance for oxygen exchange between muscle cells and capillaries.
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{
"pile_set_name": "NIH ExPorter"
}
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Along with chemotherapy and surgery, radiation therapy (RT) is a leading treatment option for cancer. It is an element in the treatment plan of over 50 million cancer survivors worldwide. Even with advances in conformal image guided therapies, RT is still limited by damage to normal tissues. Both short and long term side effects have been associated with RT, including anemia, gastrointestinal distress, skin irritation, tissue fibrosis, and even secondary cancer. These compromise the quality of life for many patients during RT. RT kills cells through free radical generation that overwhelms cells' radical scavenging protective mechanisms, leading to DNA damage. As a preventive measure, antioxidant drugs have been discovered that can supplement cells' radical scavenging and reduce RT side effects. These drugs have their own side effects and whether they also protect tumor tissue is unclear, leading to controversy over their use. Cerium oxide nanoparticles (CONPs), made from an oxide of the rare-earth element cerium, may be a novel potential therapeutic for their application as protectors of normal tissue from RT. CONPs have been used as industrial catalysts, are well known for their unique redox chemistry, and have recently been investigated for their biomedical applications. CONPs have demonstrated protection of cells from free radicals produced by inflammatory, oxidative, and radiation damage, with minimal dose related toxicity. It has been shown that CONPs do not protect cancer cells from radiation damage, though the mechanisms of this loss of radio-protection are poorly understood. This project attempts to significantly increase understanding of these mechanisms by attempting to discover the effects of the tumor microenvironment (TME), specifically low pH and hypoxia, on CONPs' radio-protective properties. pH and hypoxia are the focus of this study because CONPs lose their redox properties at lower pHs, and the hypoxic conditions of the TME are critical to radical production and tumor radio-resistance and may also affect how CONPs react in tumor tissue. The effects of CONPs have been studied in vivo, but there is a lack of mechanistic studies under clinically simulating treatment conditions. This proposal will utilize multi-modality molecular imaging and conformal image guided RT to better understand the underlying mechanisms of CONPs' radio- protection in a spontaneously induced colon cancer mouse model. Molecular imaging will consist of SPECT- /PET-CT imaging of radiolabeled CONPs' (rCONPs) to measure uptake and pharmacokinetics, while MRI and tomographic photoacoustic imaging (TPA) will be used to determine in vivo pH and oxygen gradients. [18F]FDG PET imaging will be used to monitor tumor growth and tumor/normal tissue response to RT. Comparing the normal/tumor tissue response to the rCONPs' uptake and the pH/O2 conditions in vivo, with in vitro cross validation, will reveal how they are interrelated. The methods of this project are comparable to those available in the clinic and information from these studies may help accelerate CONP translation into RT clinical trials.
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{
"pile_set_name": "NIH ExPorter"
}
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Brief preconditioning ischemia produces tolerance to subsequent prolonged ischemia that would otherwise cause brain injury. The genomic signature of the tolerant brain is transcriptional suppression. The development of tolerance, however, requires new protein synthesis, indicating that changes in protein expression contribute significantly to the mechanism of tolerance. To understand how, we characterized the proteome of the tolerant brain (Stapels et al. Sci Signaling, 2010). It is enriched in histone proteins and, remarkably, in polycomb group (PcG) proteins, which function as transcriptional suppressors. Thus, we may have discovered the mechanism that induces transcriptional suppression in tolerance. Our results implicate epigenetic regulation mediated by PcG proteins. Further, our results show that PcG proteins, previously known as regulators of segmentation during development in Drosophila, have a novel neuroprotective function in the brain. Our preliminary data on ischemic tolerance in vivo and in vitro show that the development of ischemic tolerance is dependent upon the expression of PcG proteins: knockdown ablates tolerance, and over- expression produces tolerance. Accordingly, we offer the following aims to establish and define PcG proteins role as actuators of tolerance. Aim 1. To identify early, differential changes in PcG protein abundance and activity during the induction of tolerance. We will characterize changes in the expression of PcG proteins within different polycomb protein repressive complexes (PRCs). We will also characterize PcG protein- mediated histone modifications during the development of tolerance modeled in mice, over time. The results will define which PcG proteins and complexes participate in tolerance; demonstrate a rapid increase in PcG protein abundance at the initiation of tolerance; and establish epigenetic regulation through histone modi- fication as a mechanism underlying ischemic tolerance. Aim 2. To establish an essential role for PcG proteins in the development of ischemic tolerance. We will evaluate the effect of PcG protein expression on the outcome of ischemia using loss-of-function and gain-of-function approaches. Knockdown or over'expression of PcG proteins will be achieved by using small hairpin RNA (shRNA) or recombinant cDNA, respectively, both in vivo and in vitro. The results will demonstrate that the abundance of PcG proteins profoundly affects the outcome of ischemia. Aim 3. To demonstrate that PcG proteins control the expression of genes that are suppressed in ischemic tolerance. We will investigate the interaction of PcG proteins with the promoters of genes downregulated in tolerance using ChIP assays. When genes encode channels, electrophysiological analyses of cultured neurons, over- or under-expressing PcG proteins, will be performed to establish the effect of PcG proteins on the activity of tolerance effectors. We will also manipulate PcG proteins and potassium channels simultaneously and examine the effect on tolerance induction in mice in vivo. The results will show that tolerance effector genes and gene products can be modulated by alterations in PcG protein abundance. PUBLIC HEALTH RELEVANCE: The brain can be made tolerant to stroke, which is often caused by brain ischemia, when the brain is exposed to a small, non-lethal ischemia prior to a prolonged, otherwise injurious ischemia. Understanding the molecular mechanisms that underlie brain ischemic tolerance is extremely important in our efforts to develop new treatment for stroke. In the proposed study, we will investigate a novel mechanism that may be able to induce ischemic tolerance in the brain, a mechanism that involves a special group of proteins called epigenetic regulators (proteins that control the way genes in the brain respond to ischemia), by using various advanced neuroanatomical, biochemical, cell biology, molecular biology and physiological techniques.
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{
"pile_set_name": "NIH ExPorter"
}
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The purpose of this project is to study effects of cytokines upon host defense and upon phagocytic cell function. Interferon gamma (IFN-g) corrected abnormal oxidative metabolism and bactericidal and fungicidal activity of neutrophils and monocytes from patients with chronic granulomatous diseases of childhood (CGD) whether administered in vivo or in vitro. 127 CGD patients (27 at NIH) were entered into a double blinded study of the efficacy of every other day prophylactic IFN-g (0.05 mg/square-meter) in preventing infection. IFN-g administration decreased the incidence of infection by 70% and decreased hospital days for those infections which did occur. Normal monocytes treated in vitro with IFN-g increased mRNA transcripts and protein levels of membrane and cytoplasmic NADPH oxidase components, while Interleukin 4 (IL-4) markedly decreased these components. The p47-phox oxidase component increased or decreased more than other components, indicating that this component is most critical in cytokine regulation of oxidase activity. IFN-g can induce differentiation of HL-60 myeloid leukemia cells with acquisition of oxidase capacity. During such differentiation the most dramatic increase was seen with p47-phox, while p67-phox appeared most slowly. In other studies B-cells from patients with Hyperimmunoglobulin E and Recurrent Infections (Job's) Syndrome (HIE) were shown to have abnormally high spontaneous in vitro synthesis of IgE which was decreased by administration of IFN-g in vitro or in vivo. Production of IgG1,3, and 4, but not IgG2 or IgM was also decreased by IFN-g. In other studies CGD monocytes in vitro formed granulomas and these granulomas could be dispersed with steroids.
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{
"pile_set_name": "NIH ExPorter"
}
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Certain cationic, amphiphilic drugs induce a phospholipid storage disorder when administered to animals and humans. The alveolar macrophage (AM) is particularly sensitive to this disorder. Following the induction of phospholipidosis in animals, the AMs accumulate in the alveoli, become markedly enlarged and show unusual ultrastructural changes. Because the AM plays such an important role in host pulmonary defense, it is the objective of this project to determine if changes occur in AM function as a result of the induction of this disorder. The proposed research will focus on two aspects of this problem: (a) We will examine whether the AMs from rats treated with the phospholipidosis-inducing drug, chlorphentermine are more susceptible to lipid peroxidation than normal AMs. Lipid peroxidation will be induced by cumeme hydroperoxide and cell viability will be assessed. (b) We have shown previously that AMs from chlorphentermine-treated rats release less reactive oxygen species than normal AMs when challenged with zymosan particles. We will now examine whether the adminstration of iprindole, another drug which causes phospholipidosis, results in the same phenomenon in AMs. Since the release of oxidizing species from AMs may be important in the microbicidal activity of these cells, this study will provide important information on this aspect of AM function.
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{
"pile_set_name": "NIH ExPorter"
}
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This project brings to completion data access, organization, documentation, coding, entry, analysis and dissemination of results from an existing prospective longitudinal data set. The data focuses on a cohort of children born in the greater Helsinki area between July l, 1975 and June 30, 1976. Through the use of Finnish national registries, and temperament assessment probes, it allows for the exploration of relations between maternal pregnancy stress, maternal depression, and neonatal delivery status on the temperamental characteristics of the child. Further, it allows for the exploration of relations between maternal ratings of infant temperament (6-8 months) and preschool temperament (5 years), and teacher ratings of temperament in early adolescence (age 12), and behavior problems/mental illness during childhood and adolescence. Behavior problem data are available from mothers, school nurses, and teachers. Mental illness is assessed through physician diagnosis at hospitalization, with records available over the life-span of the child. Important mediating variables also studied include family stress (financial and medical, psychiatric problems of siblings, parents, and grandparents), developmental rate of the child during the preschool years, somatic illness of the child from age 1 through 16, and educational outcomes in preadolescence. Major longitudinal questions will be analyzed using the partial least squares approach to model testing. Due to large sample sizes (800 to 2100 for various measures) and the range of data available from infancy through age 16, this data set allows for the most extensive analysis of long-term temperament/mental illness relations currently available.
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{
"pile_set_name": "NIH ExPorter"
}
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Germline and somatic mutations in the Apc (Adenomatous polyposis coli ) gene are thought to be one of the seminal genetic events in the etiology of human and murine colorectal cancer. ApcMin mice carry a germline mutation in the Apc gene and experience reduced lifespan due to adenocarcinoma burden. Wild type, but not truncated, APC binds to and regulates -catenin, the mammalian homolog of armadillo required for cadherin-mediated cell adhesion. Apc heterozygosity caused by the MIN mutation may contribute to the deregulation of -catenin protein levels and result in overexpression of an inducible, proinflammatory enzyme cyclooxygenase-2 (COX-2). Evidence for the regulation of COX-2 expression by APC and possible interaction between NO and COX-2 prompted us to examine the expression of inducible nitric oxide synthase (NOSII) and COX-2 in cells with ApcMIN mutation. Using two conditionally immortal murine intestinal epithelial cell lines contrasting in Apc genotype ("Immortomouse"/Min Colonic Epithelia, Apc +/-; Young Adult Mouse Colon epithelia, Apc +/+), we have demonstrated that IMCE cells had significantly higher expression of both NOSII and COX-2 in response to lipopolysaccharide (LPS) and interferon-gamma (IFN-g) as compared to YAMC cells. Levels of the metabolic products of these two enzymes, NO and PGE2, respectively, were also elevated markedly. Interestingly, the expression of COX-2 can be also induced by NO-releasing compounds, such as SNAP and NOR-1. These drugs increased PGE2 generation in IMCE and YAMC cells as well. IMCE cells consistently expressed higher COX-2 mRNA and protein and produced more PGE2 in comparison to YAMC cells in response to the same treatment. Preliminary data indicates that NO may induce COX-2 expression and PGE2 generation by increasing the nuclear translocation of the transcription factor -catenin, possibly through affecting the degradation of -catenin by APC protein. Therefore, over- production of NO, observed in IMCE cells in our study, may further potentiate COX-2 overexpression and PGE2 generation affected by Apc genotype.
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{
"pile_set_name": "NIH ExPorter"
}
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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have developed a microfluidic platform to generate stable gradients across 3D hydrogels and require confocal microscopy to image the cells as they migrate via chemotaxis toward EGF and SDF-1 gradients (FITC-dextran with a similar molecular weight). We will be using HT1080 (fibrosarcoma), MDA-MB-231 (metastatic breast cancer), and RAW (mouse macrophage) cell lines with GFP and RFP. In addition we will be conducting co-culture studies in our device in which the cells will be suspended in a 3D matrix (collagen, matrigel). We would like to also image the cell-cell interactions as well as the cell-matrix interactions (cell migration, degradation, proliferation, spreading, etc.) .
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{
"pile_set_name": "NIH ExPorter"
}
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Platelets undergo the initial reactions which lead to hemostasis and to thrombosis. The nature of these reactions, i.e. the mechanisms whereby a specific stimulus interacts with a platelet membrane site, and those whereby the signal that a stimulation has occurred triggers an eventual platelet response are not yet fully elucidated. We seek to approach this problem by (a) isolating and characterizing the membrane binding site (i.e. receptor), utilizing photoreactive derivatives to obtain a covalently linked stimulus-receptor complex, (b) using the techniques for evaluation of changes in the platelet membrane potential and in intraplatelet pH which we have developed to probe this stimulus response, its transmembrane monovalent cation (Na+, K+, H+) gradient dependence, and its metabolic requirements, using specific indicators and/or specific antagonists, (c) evaluating the role of Ca++ in these responses to stimulation, utilizing arsenazo III, a sensitive Ca++ indicator, (d) determining the effect of certain membrane perturbants as well as some drugs on the platelet and on its response to thrombin stimulation. The results of these studies shold help us to understand the platelet reaction in response to stimuli, to delineate these mechanisms, and to design prevention or treatments for platelet related disorders of the circulatory system such as occur in thrombosis, atherosclerosis, thrombastenia, myelo-proliferative disorders and certain immune reactions.
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{
"pile_set_name": "NIH ExPorter"
}
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The overall objective of this proposal is to examine the factors which regulate the expression and cellular sorting of the insulin-like growth factor II/cation-independent mannose 6-phosphate receptor (IGF-II/CI-MPR) and the cation-dependent mannose 6-phosphate receptor (CD-MPR) in developing rat intestine and polarized epithelial cells. Quantitative Western blot analysis and Northern blot analysis will characterize and quantify the protein and mRNA levels, respectively, of the IGF-II/CI-MPR and CD-MPR. In addition, IGF-II binding studies will be performed to determine the functional activity of the IGF-II/CI-MPR in intestinal membranes. These studies will be carried out using a) different anatomical regions of the intestine, b) villus and crypt cells, and c) intestine from different developmental stages of the rat. Similar studies as above will be carried out on a human colon adenocarcinoma cell line, Caco-2, which spontaneously undergoes enterocyte-like differentiation in culture. The biosynthesis and processing of the MPRs will be characterized in these cells as will the effect IGF-II and other growth factors have on this cell's rate of differentiation. The cell surface distribution of the MPRs in polarized cells grown on filters will be determined by cell surface labeling techniques and uptake studies using 125I-IGF-II and [3H]lysosomal enzymes. In addition, the kinetics of intracellular movement of the MPRs will be analyzed in these cells. The role that the MPRs' covalent modifications (i.e., acylation, phosphorylation, glycosylation) have as regulators of the receptors' intracellular targeting will be addressed using metabolic labeling studies in conjunction with processing inhibitors as well as site-directed mutagenesis of the MPRs' cDNA. Once the targeting pathway of the MPRs has been established, the structural elements of the receptors' cytoplasmic domain and/or extracytoplasmic domain important for their intracellular targeting will be examined by site-directed mutagenesis of the MPRs' cDNA. These mutant MPR cDNAs will be transfected into Caco-2 and/or opossum kidney cells in order to study the effect these mutations have on the intracellular targeting of the MPRs in a polarized cell. the studies outlined in this proposal will examine at the cellular and molecular levels the possible roles the MPRs have in epithelial differentiation and in regulating the functional capacity of enterocytic lysosomes, and thus will provide a basis for a clearer understanding of the role of this organelle in protein metabolism in the gut.
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{
"pile_set_name": "NIH ExPorter"
}
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The goal of these studies is to examine interactions between arachidonic acid pathways and nitric oxide pathways in renal immune injury. Previous studies by the Principal Investigator, as well as others, have demonstrated that in experimental models of renal immune injury, both of these pathways are activated and result in synthesis of proinflammatory eicosanoids and generation of nitric oxide in isolated glomeruli. Furthermore, there is evidence that infiltrating activated macrophages are a key site where both are generated. Eicosanoid species produced include both cyclooxygenase and 5-lipoxygenase metabolides. It is postulated that the vasoconstrictive eicosanoids produced may be important contributors to renal vasoconstriction and structural damage to the glomerulus. The Principal Investigator also postulates that nitric oxide may play a detrimental role in glomerular and inflammatory injury due to generation of peroxynitrite secondary to interactions of oxygen free radical species with nitric oxide, and hypothesizes that the induction of iNOS may have deleterious consequences due to this generation of nitric oxide in inflammatory renal injury. These studies are also motivated by evidence that nitric oxide may activate cyclooxygenase in vivo and in vitro. Therefore, the hypotheses are: 1) In glomerular immune-injury NO generated as a result of iNOS activation regulates synthesis of pro-inflammatory eicosanoids, and these eicosanoids modulate expression and activity of iNOS, and 2) In glomerular immune-injury, NO preserves renal hemodynamic function and activation of iNOS promotes glomerular cell injury. There are four specific aims in this proposal. The first specific aim will examine whether nitric oxide derived from iNOS activation by glomerular inflammatory injury will regulate cyclooxygenase activity and also whether there is an effect to activate lipoxygenase activity. The second specific aim will determine whether eicosanoids will modulate iNOS expression and generation of iNOS derived nitric oxide. The third specific aim will examine whether in glomerular immune-injury NO serves a beneficial function to preserve renal blood flow in glomerular filtration, and the fourth specific aim will determine whether in glomerular immune-injury NO may serve a harmful function via interaction of NO with superoxide in formation of peroxynitrite or similar oxidants. It is anticipated that the observations will provide new insights in the pathobiology of glomerulonephritis.
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{
"pile_set_name": "NIH ExPorter"
}
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DESCRIPTION (adapted from the Abstract): The long-term objective of this project is to create and distribute a resource of photo-realistic three-dimensional virtual anatomic specimens. The specific aims of this application are: 1) to create a set of QuickTime VR objects of normal anatomical specimens; and 2) to make this resource freely accessible to users via the Internet. Virtual objects will allow examination and study of anatomical features and relationships in situations where the availability of real specimens is impractical or inappropriate. It is anticipated that users in the area of medical and allied health education, patient education, etc., will utilize these QTVR objects in electronic presentations or will produce derivative works such as interactive learning program. QTVR technology creates the illusion of handling an object (turning it over and viewing it from any angle) by playing a series of digital movie frames in the proper sequence determined by the user?s cursor movement. This technology has proven effective in representing gross anatomical specimens of many types. It has also proven possible to change the metaphor of the QTVR objects to show movement of anatomical specimens (such as a knee joint) or to demonstrate a sequential dissection of a specimen. The QTVR format is inherently capable of creating interactive applications or QTVR files can be incorporated into authored programs, including web-based applications. To achieve the aims of the project, 75 to 100 anatomical specimens will be prepared, photographed and processed into QTVR object movies. For each specimen, several object movies at varied size and resolution will be produced to accommodate multiple end-user purposes. The resulting 500+ movie files will be placed on an Internet server at this institution and mirrored on at least one other server. The complete resource will be made available to registered users via a password protected, searchable database. This resource will be maintained indefinitely and will likely be expanded by continued production in our laboratory and by external contributions to include anomalous and pathologic specimens.
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{
"pile_set_name": "NIH ExPorter"
}
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