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Our studies are focused in three areas. The first involves characterization of the role of T cell antigen receptor (TCR) signals, and in particular, individual TCR signal transducing subunits and signal transducing motifs in T cell development. Second, we have extended our studies to include analysis of signal transducing molecules that function downstream of the TCR or that inhibit TCR signaling. The aim of these studies is to understand how these molecules participate in TCR mediated signaling and to determine what roles they and the signaling pathways they regulate play in T cell maturation and T cell activation. Finally, the lab has initiated a new area of investigation of the genes controlling the generation and maintenance of Hematopoietic Stem Cells. Role of T cell antigen receptor (TCR) signaling in thymocyte development. Signal transduction sequences (termed Immunoreceptor Tyrosine-based Activation Motifs;ITAMs) are contained within four distinct subunits of the multimeric TCR complex (zeta, CD3-gamma, -delta, -epsilon). Di-tyrosine residues within ITAMs are phosphorylated upon TCR engagement and function to recruit signaling molecules, such as protein tyrosine kinases, to the TCR complex, thereby initiating the T cell activation cascade. To determine if TCR signal transducing subunits perform distinct or analogous functions in development, we previously generated zeta deficient and CD3-epsilon deficient mice by gene targeting, genetically reconstituted these mice with transgenes encoding wild-type or signaling-deficient (ITAM-mutant) forms of zeta and CD3-epsilon, and characterized the developmental and functional consequences of these alterations on TCR signaling. The results of these studies demonstrated that TCR-ITAMs are functionally equivalent but act in concert to amplify TCR signals. TCR signal amplification was found to be critical for thymocyte selection, the process by which potentially useful immature T cells are instructed to survive and differentiate further-(positive selection), and potentially auto-reactive cells that may cause auto-immune disease are deleted in the thymus (negative selection). Thus, the multi-subunit structure of the TCR may have evolved to enable complex organisms to develop a broad, self-restricted yet auto-tolerant T cell repertoire. In current studies we are using conditional gene expression systems to analyze the importance of TCR signaling at specific stages of development. In addition, we are using microarray and subtractive cloning to identify genes involved in T cell signaling and T cell development. Signaling molecules that function downstream of the TCR or that function to "fine-tune" the TCR signal. Our results with TCR-ITAM mutant mice suggested that other signaling molecules can compensate for the reduction in TCR signal strength. An initial FACS-based search for candidate compensatory molecules led us to CD5, a TCR associated trans-membrane protein that inhibits TCR signaling. Importantly, we found that CD5 surface expression is regulated by and parallels TCR signal intensity. Thus, rather than simply functioning as a co-receptor, CD5 acts to fine-tune TCR signals during thymocyte selection since its level of surface expression depends upon the intensity of TCR signaling. An obvious benefit of such fine-tuning of the TCR signaling response would be to enable the generation of a T cell repertoire with the maximum possible diversity since it would allow a broader range of TCRs to pass through the signaling window of positive selection. Since little was known about how CD5 regulates TCR signaling, we initiated a project to characterize CD5 function, both genetically and biochemically. The results of these experiments suggest a mechanism for CD5 mediated TCR signal inhibition that we are currently testing experimentally. We have also begun a search for additional tuning molecules using a microarray based screen. The identification of such molecules may have importance for the diagnosis and treatment of human autoimmune diseases. In another study, we identified a novel T-lineage restricted putative adaptor protein, designated Themis. Biochemical studies indicate that Themis functions at a distal point in the TCR signaling pathway and may have an important role is helping to sustain TCR signaling. Themis-/- mice have been generated and their phenotype reveals an important role for this protein in late thymocyte development and selection. Current and projected experiments are directed at elucidating the mechanism by which Themis functions in T cell signaling and development. Genes controlling Hematopoietic Stem cell specification and maintenance. The hematopoietic system is composed of a functionally diverse group of cells that originate from a common hematopoietic stem cell (HSC) capable of long-term self-renewal and multi-lineage differentiation. Self-renewal ensures that a pool of HSCs persists throughout life, whereas differentiation leads to the continuous generation of all circulating blood cells including lymphocytes, myeloid cells, erythrocytes and platelets. We initiated experiments aimed at identifying genes important for HSC generation and maintenance. Our initial studies focused on the role of LIM domain binding protein-1 (Ldb1) in hematopoiesis. The results of these experiments revealed a critical function for Ldb1 in regulating the self-renewal/differentiation cell fate decision in hematopoietic stem cells and suggest that Ldb1 nucleated transcription complexes may control maintenance of lineage specific stem cells. Consistent with this, a genome-wide ChIP-seq screen identified Ldb1 binding sites within the promoter/gene body of a high percentage of genes known to be essential for HSC maintenance. Deletion of Ldb1 also resulted in decreased expression of these genes suggesting that Ldb1 complexes function as "master regulators" of the transcriptional program regulating HSC maintenance/self-renewal.
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The objective of the proposed research is to describe the cholesterol esterification systems in rat intestine, mucosal cholesterol esterase and acyl CoA:cholesterol acyltransferase (ACAT) with respect to the following: Cholesterol esterase: a. To degine the mechanism of entry of pancreatic cholesterol esterase into mucosal cells were it functions as mucosal cholesterol esterase. b. To purify mucosal cholesterol esterase by affinity chromatography and determine directly its properties relative to pancreatic cholesterol esterase. c. To determine the physiological substrate(s), i.e., luminal and/or biosynthetized cholesterol, for cholesterol esterase. ACAT: a. To localize ACAT in the intestine (in the absence of cholesterol esterase) with respect to proximal and distal, villous tips and crypts and subcellular organelles. b. To determine the substrate specificity of ACAT in vitro, i.e., cholesterol specific or non-specific. c. To determine the physiological substrate(s) for ACAT, i.e., luminal and/or biosynthesized cholesterol. We also plan to describe the intestinal transport forms for luminal and biosynthesized cholesterol.
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{
"pile_set_name": "NIH ExPorter"
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The purpose of the project is to examine the short-term and long-term consequence of developmental exposure of oxytocin (OT). The time parameters, doses and specificity of OT will be examined, in experiments that will be used to define animal models for the more fine-grained analyses of Projects II-IV. Animals from this project will be used in Project IV in an analysis of cFos changes as an immediate result of treatment with OT during development. Basic functions, including patterns of growth, reproduction and basal endocrine parameters will be examined in rats and voles. The behaviors and endocrine measures to be studied are functions which have been shown to be sensitive to OT in adulthood. Among the behaviors to be measured are birth, maternal behavior and mating and natural estrus, along with pain thresholds. Among the endocrine parameters to be measured are steroid and peptide hormones (including OT and AVP). We also will examine the growth patterns of these animals and metabolic hormones including CCK, insulin, as well as glucose.
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{
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In the past year, we have completed several studies related to cardiovascular biomarkers and have published the findings (see below). We have assessed the commutability of frozen samples for the determination of HDL-C and LDL-C by direct assays. Results show that fresh frozen samples may not be fully commutable for some direct lipoprotein assays, thus potentially limiting the use of these tests for evaluation frozen samples for large epidemilogic studies of cardiovascular biomarkers. In two different studies on samples from MESA, we showed that a novel assay for small dense LDL and a test based on the ratio of LDL-P to HDL-P are superior to other conventional markers for predicting cardiovascular risk. In another study, we showed in a cohort of CGD patients that they have increased cardiovascular risk markers but are seemingly protected against the development of cardiovascular disease possibly because of reduced levels of NADPH oxidase activity. In terms of work in progress, we are currently developing a new lipoprotein phenotype classification, using NMR for the measurement of lipoprotein particles distribution, and are examining its potential utility in predicting CVD events in MESA.
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{
"pile_set_name": "NIH ExPorter"
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Borrelia burgdorferi sensu lato, the etiologic agent of Lyme disease, causes a multi-system disorder that may progress into a persistent infection characterized by significant morbidity. The overall goal of this proposal is to identify and characterize virulent strain associated (VSA) outer membrane (OM) proteins, both those that are outer membrane- spanning (Oms) and lipoproteins, that function as protective immunogens. The investigators' long-term goal is to link protective immunity elicited by these proteins to pathogenic mechanisms. Specifically, this study proposes to: (1) Purify and characterize a 28 and 35 kilodalton (kDa) virulent strain associated (VSA) OM proteins of B. burgdorferi that are specific to infectious isolates. The native forms of the 28 and 35 kDa OM proteins will be purified from OM vesicles derived from B. burgdorferi and their amino terminal or internal peptide sequences determined. These sequences will then be used to identify the genes encoding these proteins by screening the B. burgdorferi genomic database; (2) Characterize four virulent strain associated (VSA) antigens of B. burgdorferi identified from a phage lambda expression library. We will determine the extent of differential expression of the VSA antigen in virulent versus avirulent B. burgdorferi and evaluate whether these antigens are either expressed in infected animals or temperature regulated; and (3) Test these OM VSA antigens identified for their ability to confer protective immunity. Recombinant VSA proteins will be overproduced, purified, and used to immunize mice. The native VSA OM 28 and 35 kDa proteins will be incorporated into liposomes and the resulting proteoliposomes used to immunize mice. Immunized animals will then be challenged with infectious B. burgdorferi. Serum specific for protective immunogens will be tested to determine whether these antibodies confer passive protection and whether antibodies mediate adherence inhibition or preferentially kill in vitro cultivated virulent B. burgdorferi in a complement dependent manner. Determination of the protective abilities of different B. burgdorferi antigens expressed in the host relative to those expressed during in vitro cultivation, will facilitate in the development of a vaccine cocktail to protect against non-clonal, environmental isolates of B. burgdorferi. Furthermore, these studies will provide insight into pathogenic mechanisms mediated by VSA antigens of B. burgdorferi.
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"pile_set_name": "NIH ExPorter"
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For many cocaine addicts, drug use is a stress-driven behavior. Previous research has shown that stressor- induced cocaine seeking is mediated in part by corticotropin-releasing factor (CRF) stimulation of a neurobiological pathway involving dopamine (DA) neurons in the ventral tegmental area (VTA) that likely project to nucleus accumbens (NA). The regulation of this pathway by stressors and CRF appears to emerge as a consequence of prior cocaine use and therefore may be relevant to the onset of addiction. We have demonstrated that rats provided long access to cocaine for self-administration (SA) each day (LgA rats) display greater reinstatement in response to a stressor (electric footshock; EFS) or administration of CRF directly into the VTA, compared to rats provided shorter daily drug access (ShA rats). Furthermore, we have found that that the establishment of heightened stressor-induced reinstatement appears to require elevation of glucocorticoids (GCs) at the time of earlier LgA SA, suggesting that the induction of addiction-related neuroplasticity leading to heightened stressor-induced drug seeking following excessive patterns of cocaine use is a GC-dependent process. The goal of this proposal is to test the hypothesis that cocaine addiction is associated with an emergent or augmented CRF regulation of dopaminergic neurons projecting from the VTA to a subregion of the NA, the shell, that is attributable to increased VTA CRF receptor (CRF-R) expression or function and leads to a heightened susceptibility to stressor-induced craving and relapse. Furthermore, we hypothesize that the establishment of heightened CRF regulation is dependent upon elevated GCs and activation of GC receptors (GR) at the time of earlier drug exposure. These hypotheses will be tested in this proposal in three specific aims. In the first aim we will further investigate the relationship between augmented VTA CRF sensitivity and stressor-induced reinstatement through a series of experiments that examine the relative time-courses of altered CRF- and EFS-induced reinstatement as they relate to changes in CRF-R expression and trafficking and determine the involvement of CRF-R subtypes in the VTA in stressor-induced cocaine seeking through antagonist administration and receptor knockdown by RNA-interference. In the second aim we will examine the role of altered CRF actions in the VTA in the augmented stressor-induced regulation of NA DA and its involvement in stressor-induced cocaine seeking using in vivo microdialysis in and administration of DA receptor antagonists into the NA core and shell. In the final aim, we will examine the GC- dependence of the effects of LgA SA on stressor-induced cocaine seeking and NA DA neurotransmission and CRF-R expression/trafficking through a surgical adrenalectomy and diurnal GC replacement approach that eliminates evoked GC secretion while maintaining normal diurnal patterns of plasma GCs and through central infusion of the GR antagonist, RU-486 via osmotic minipump. Understanding the neurobiological processes through which stressor-induced regulation of cocaine use is established in cocaine addicts should facilitate the development of new and more effective treatment approaches, particularly for subpopulations of cocaine addicts whose drug use is stress-driven. PUBLIC HEALTH RELEVANCE: This project examines the neurobiological mechanisms through which susceptibility to drug relapse during periods of stress is heightened in cocaine addiction. The project focuses on neuroplasticity involving the regulation of neural pathways underlying drug use by corticotropin-releasing factor (CRF), a neuropeptide previously implicated in stress and anxiety. The ability of a rat model of excessive drug use to enhance CRF regulation of drug-seeking behavior in a manner that depends on secretion of glucocorticoid hormones will be tested.
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{
"pile_set_name": "NIH ExPorter"
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This project provides statistical, operational, and coordinating support for the design, conduct, analyses, and reporting of clinical trials in the therapy of malignant diseases conducted by the Cancer and Leukemia Group B (formerly Acute Leukemia Group B). It services the cooperative research of 450 scientists in over 75 international hospitals and clinical centers. About 10 new investigations are undertaken each year, while an average of 30 studies are accruing nearly 2000 patients with a variety of cancers and leukemias each year. These studies range in character from the investigation of potential therapeutic effects of new chemotherapeutic agents in advanced cancer patients to the systematic search for curative combinations of surgery, radiotherapy, chemotherapy, and immunotherapy in newly diagnosed cancer patients. Uniform data are centrally collected, processed, analysed, and stored from these experiments, and interim results are regularly reported to participating physicians. These data now form a library of nearly 20,000 cancer patients treated in accordance with carefully designed experimentally therapeutic trials over two decades of Group research.
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{
"pile_set_name": "NIH ExPorter"
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Mechanistically, how do drugs and dyes intercalate into DNA? Do they intercalate directly into the A, B and Z structures -- or is there some other structural form for DNA into which drugs and dyes intercalate? We have proposed the existence of another DNA structure to be involved in the intercalation process. This structure -- called Beta DNA or Beta premelted DNA -- forms spontaneously in the centers of soliton-antisoliton (i.e., kink-antikink) bound states in DNA structure. Such structural solitons contain a modulated Beta alternation in sugar puckering about the central Beta premelted core region which gradually merges into B (or A) DNA on either side. We have called such composite structures -- Beta premeltons. We are uncertain, as yet, whether the centers of Beta premeltons are open enough to accomodate an intercalator directly -- however, structures such as these are known to have an intrinsic ability to undergo low frequency breather motions, and it is possible that such motions facilitate the intercalation process. We would like to provide direct X-ray crystallographic evidence for the existence of the Beta premelted structural form. This could be accomplished by crystallizing a series of suitable drug-oligonucleotide complexes, and then determining their three-dimensional structures by X-ray crystallography. Such structures could contain the Beta alternation in sugar puckering and demonstrate neighbor exclusion intercalative drug binding. In addition, we would like to crystallize the Beta premelton. Such a structure could be stabilized by binding actinomycin D to the center of a self-complementary double-stranded long chain oligonucleotide. If this can be crystallized, X-ray analysis could reveal the modulated Beta alternation in sugar puckering around the central Beta premelted core region merging into B (or A) DNA on either side. This would provide evidence supporting the model that has been proposed.
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"pile_set_name": "NIH ExPorter"
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The Ly49 and KIR families are comprised of both inhibitory and activating receptors; the latter interacting and signaling through DAP12. We have continued our dissection of the proximal events associated with DAP12 signaling over the review period. The result of this work includes the definition of an apparent defect in DAP12 signaling in mice of the 129/Sv background that can profoundly effect the analysis of gene targeted mice, delineation of two levels of regulation of DAP12 signaling by CD45 (signal initiation and dephosphorylation of DAP-12 itself), and most recently, dissection of the overlapping use of the non-catalytic adaptor proteins Linker for Activation of T cells (LAT) and the Linker for Activation of B cells (LAB). TREM-1 regulates sepsis whereas TREM-2 can control the development of DC, microglia and osteoclasts. Our studies of the TREM cluster have led to publications regarding; 1) the identification of TREM-like transcript-1 (TLT-1), a protein that does not couple to DAP-12 but instead interacts with SHP-1; 2) characterization of TLT-1 as a platelet specific receptor sequestered in alpha granules; and 3) identification of soluble TLT-1 in serum, and solution of the crystal structure of the extracellular domain. We have also targeted TLT-1 in mice, shown its role in platelet aggregation in vitro, and defined some proteins that interact with it. Although platelets and thrombosis can regulate inflammation, cancer growth and metastasis, and adaptive immunity, we have chosen not to propose further TLT-1 studies under our base allocation here. If however, sufficient resources were available, we would continue biochemical study of TLT-1. We are currently, evaluating our TLT-1-/- mice in collaboration with a former fellow from our lab.
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{
"pile_set_name": "NIH ExPorter"
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Epidermal stem cells are of great importance because they play a central role in tissue homeostasis, wound repair (both delayed in aging), as well as in tumor initiation, and for gene therapy. Studies of epidermal stem cells have been hampered by a lack of markers by which to identify them. Studies of putative markers have in turn been limited by lack of adequate functional stem cell assays. Studies of epidermal stem cells performed to date rely on relatively short-term in vitro colony forming units as a tool to assess ?stemness?. However, studies in other tissues have shown that in vitro colony forming units do not represent the true long-term repopulating cell. Borrowing from well-established functional assays in hemopoietic cells, we have designed a novel in vivo competitive repopulating assay to study stem cell function in epidermal cells. Our assay utilizes green fluorescent protein (GFP) to distinguish epidermally derived test cells from non-green competitor cells. These keratinocytes produce an epidermis when seeded in a chamber on the back of a non-GFP mouse. A limiting dilution technique allows the absolute number of stem cells in a keratinocyte population to be determined using this assay. This assay has two key features. The first is the use of a long-term competitive assay that ensures not only the detection of the most primitive epidermal stem cells but also the survival of the transplanted keratinocytes, even when seeded with very low numbers of test cells. The second is the use of a limiting dilution design to allow stem cell quantification. Our long-term objective is to elucidate the effects of aging on epidermal stem cells, and determine the mechanism(s) by which the delayed epidermal homeostasis and wound healing may be improved. The specific aims include, first, to fully optimize our assay, to enable us to assess aged and young murine epidermal stem cells. We will then compare the absolute number of stem cells in aged vs. young epidermis, and determine the proliferative potential of an equal number of stem cells from young vs. aged epidermis. This work will allow us to begin to understand the changes in epidermal stem cells with age, and may point to further studies to improve epidermal cell function and/or wound healing in the aged. These studies will also provide the first functional in vivo assay for epidermal stem cells, an important step for analvzing (T putative markers for this elusive epidermal cell, for cutaneous stem cell-targeted gene therapy, and for more basic studies of epidermal stem cell regulation and differentiation.
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To establish the relationship between eeg sleep, sympathetic tone and immune function, to characterize the nocturnal secretory profile of lymphokines, neuroendocrine hormones and neurotransmitters, to examine nocturnal secretion of lymphokines, hormones and catecholamines in psychiatric patients with sleep disturbance, and to test link between sleep, neuroendocrine and sympathetic measures and immunologic responses using partial sleep deprivation.
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{
"pile_set_name": "NIH ExPorter"
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The ability to image and quantify nicotinic receptors in human brain may be crucial to better understanding the neurobiology of smoking addiction. Analogs of A-85380 and A-84543 have been evaluated as imaging agents for nicotinic acetylcholine receptors (nAChRs) with positron emission tomography (PET) and single photon emission computed tomography (SPECT). In collaboration with Yale University, the SPECT studies with 5IA revealed that cerebral nAChRs can be quantified in human non-smoking volunteers. Toxicological studies in animals demonstrated the safety of 2-[F-18]fluoro-A-85380 (2FA). The first human PET studies in non-smokers with 2-[F-18]fluoro-A-85380 (2FA) demonstrated the feasibility and safety quantitatively imaging nAChRs in the thalamus and visualizing these receptors in brain regions containing low to moderate receptor densities and that multiple studies on a single volunteer are possible within dosimetry limits. Preliminary analysis of PET studies in smokers with 2FA demonstrate increased densities of nicotinic receptors in many brain regions compared to the brains of non-smokers. Mice studies showed that the administration of the non-selective inhibitor of cytochrome P450, cimetidine, substantially slowed the rapid in vivo metabolism of 5IA, suggesting that the use of this or similar compounds could reduce the dose of radioactivity needed to successfully image nAChRs in human volunteers. In collaboration with researchers from the University of Michigan, the loss of nAChRs in the striatum of unilaterally MPTP-lesioned NHPs with 2FA and PET was demonstrated. Kinetic studies with 2FA and PET in NHPs revealed that 2FA accumulates relatively slowly in brain, partially because its low lipophilicity slows its blood-brain-barrier penetration. A series of 5-heteryl-6-halogeno-A-85380 derivatives with binding affinity (Ki) at the nicotinic acetylcholine receptor (nAChR) ranging from 3 to 150 pM and a lipophilicity (logD) range of -1.6 to +1.5 has been synthesized as potential PET ligands. Most ligands of the series exhibited a higher binding affinity at the alpha4beta2* subtype of nAChRs than epibatidine. Molecular modeling studies revealed an important role of the orientation of the external heterocyclic ring on the binding affinity of the ligands with nAChRs. Two compounds of the series were radiolabeled with 18F. Recent PET studies with one of these compounds ([18F]NIDA52189) demonstrated that its binding potential values in Rhesus monkey brain was ca. 2.5 times that of 2-[F-18]F-A-85380, the only available PET radiotracer for imaging cerebral nAChR in humans. Preliminary toxicology and dosimetry studies suggest that [F-18]NIDA52189 is suitable for quantitative imaging of extrathalamic nAChRs. Imaging of the central CB1 cannabinoid receptors by PET will improve our understanding of the roles of these receptors in the brain. Existing PET radiotracers for imaging CB1 are very lipophilic compounds with high non-specific to specific binding ratios and as a result are not adequate for quantitative studies. A 14 member series of analogs of 1-[(N-methyl-piperidin-2-yl)methyl]-3-naphthoylindole, a CB1 agonist with high binding affinity, with reduced lipophilicities has been synthesized. The lead compound shows a Ki of 0.9 nM, which represents one of the highest affinities observed for CB1 receptor ligands; further, it has a cLogD value, which should reduce non specific binding, a problem observed with all previously tested ligands. Our study showed that substitutions to the lead compound at the 4 position on the naphthyl ring gave similar Ki values that allowed us to vary the lipophilicity of the ligand, giving cLogD values in the range of 2.7 to 4.5. Substituting a fluorine atom for hydrogen gave a derivative with the lowest Ki value, 0.7 nM, and an unexpectedly low experimental LogD of 2.6. A 4-nitro precursor for nucleophic radio-fluorination of this compound has been used giving good radiochemiccal yields and specific activity. When the entomerically purified tracer was used in an ex-vivo study on mice the target to non-target ratios were about 1.7-1.8. for measurements from the hippocampus to brain stem regions. Other tracers are planned including the 4-(2-hydroxyethyl)naphthoyl derivative which has a better combination of lipophilicity and binding. Corticotropin-Releasing Hormone (CRH) acts as a major regulator of the hypothalamic-pituitary-adrenal (HPA) axis coordinating neuroendocrine, autonomic, immune, and behavioral responses to stress. It is prevalent in the central nervous system (CNS) where it acts as a neurotransmitter. A high-affinity, nonpeptide radioligand for the CRHR1 was synthesized and showed distribution in rat brain consistent with CRHR1 receptor subtype using in vitro autoradiography. This is the first nonpeptide radiotracer combining high affinity and appropriate lipophilicity that penetrates the blood-brain barrier and hence has the potential to be used for PET imaging studies. In vivo visualization of changes in the CRH1 receptor or its occupancy would further the understanding of the role of stress in drug abuse.
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I am studying unnatural hydrophobic amino acids and their effect on the stability of T4 lysozyme using free energy perturbation (FEP) methods. The effect of mutating hydrophobic amino acids on protein stability has been an area of great interest since these residues are responsible for two important forces which stabilize proteins: packing in the protein core and the hydrophobic effect. The use of unnatural amino acids allows us to create mutations that specifically vary the amount of nonpolar surface area buried. The project consists of three parts: (1) Design and analysis of unnatural amino acids that replace Leu 133 in the hydrophobic core of T4 lysozyme using computer graphics and molecular dynamics. This part of the project has been completed, and several unnatural amino acid mutants of T4 lysozyme have been created and their stabilities relative to the wild type determined by collaborators at UC Berkeley. (2) FEP studies of two mutations of isolated unnatural amino acids in water: CPE -> ETH and NVL -> MSE. The purpose of these calculations is to determine the methodology required to simulate these mutations accurately. (3) Calculation of relative protein stabilities of the mutants CPE versus ETH and MSE versus NVL. FEP techniques should enable us to better understand how hydrophobic residues stabilize proteins since the relative stability of different mutants can be determined in the native and denatured states. Thus, contributions due to interactions in the native state such as packing can be separated from solvation effects in the denatured state. I use the CGL for viewing protein structures and making slides and photographs for posters, presentations, and publications. These slides and photographs were made by myself by taking pictures of images displayed on the screen.
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The long-term goal of this project is to develop a new technology to improve the screening for and diagnosis of breast cancer. Non-invasive electrical impedance measurements that are made using a hand-held probe have been shown to improve the specificity and sensitivity of mammography for breast tumor diagnosis in patients with ambiguous mammograms. This non-invasive technology poses no known risks to the subject, and provides a new diagnostic parameter to assess suspicious anomalies. This new technology, called Electrical Impedance Tomography (EIT), makes images of the interior of the body by applying electrical signals to electrodes applied to the skin. These signals cause small electric currents to flow in the body and the voltage and current values at the electrodes are used by mathematical algorithms to reconstruct the values of the electrical conductivity and permittivity inside the body. These reconstructed conductivity and permittivity values can be presented as EIT images. When this is done using several different signal frequencies, the technique is called Electrical Impedance Spectroscopy (EIS), and further diagnostic information may be elucidated. We propose to determine the feasibility of improving the diagnostic ability of mammography by combining a 3-D tomosynthesis mammography system with an EIT/EIS system in order to make simultaneous, in-registration images of breast electrical properties using data collected from two parallel arrays of radiolucent electrodes applied to the mammogram compression plates. We have implemented an electronic instrument, built radiolucent electrode arrays, and written algorithms to reconstruct EIT images for a region of interest within the breast that lies between electrode arrays attached to the compression plates of an x-ray mammography unit. The system operates at frequencies between 3 kHz and 1 MHz, has 60 electrodes, and forms and displays images at 2.5 frames/second. It is being applied to patients undergoing biopsy for breast cancer diagnosis. We have identified a parameter that is calculated from the EIS data that has a markedly different value at the cancer sites in four of four breasts with carcinoma within the ROI, when compared to its value in 44 non-malignant breasts. These findings are confirmed by biopsy and 3-D tomosynthesis mammography. There is nearly no overlap between the values of this parameter with cancer and values in normal tissue or the benign lesions studied so far. We have also developed a display method that superimposes a semi-transparent color over a mammogram image to show the magnitude of this malignancy measure. This is a proposal to continue these studies in more patients with improved hardware, reconstruction algorithms and analysis and display methods. If this combined EIT/EIS/Tomosynthesis system can improve the sensitivity and specificity of mammography for breast cancer screening, it will have a major impact on the ability to detect and treat this major cause of mortality. PUBLIC HEALTH RELEVANCE This project seeks to develop a means to improve the diagnosis of breast cancer by improving the sensitivity and specificity of mammography. Mammography screening reduces breast cancer mortality, but it remains a technology that could be improved. This project seeks to refine Electrical Impedance Tomography as a diagnostic modality, and to develop a scientific basis to justify a large-scale clinical trial of the technique. Success of such a trial would reduce the need for biopsy and increase the detection of tumors at an earlier stage, which would reduce the morbidity and mortality of breast cancer.
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The aim of this project is to elucidate the neuronal (CNS) mechanisms that mediate the effects of several hallucinogens (psychotomimetics) and other, related compounds. To this end, a sensitive, specific, robust and reliable "behavioral asset" in which rats or pigeons are trained to detect (discriminate) drug-induced alterations in their own internal environments (states), will be used to study several clinically-important ergot alkaloids including lergotrile and dihydro-ergotoxine (Hydergine). These compounds have been used to treat a variety of cardiovascular, neuroendocrine and neuropsychiatric disorders ranging from migraine headache to Parkinsonism and senile dementia. Special attention will be paid to (1) the extent to which the discriminable effects of therapeutic ergots resemble those of their close structural congener lysergic acid diethylamide (LSD) and therefore might predict hallucinogenic or psychotominetic liability; (2) the neuronal mechanisms underlying ergot cues; (3) possible correlations between the discriminable and (other) physiological and neurochemical effects of ergots.
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{
"pile_set_name": "NIH ExPorter"
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The proposed research into the income and life styles of middle class Black Americans is the first stage of more extensive research into the economic conditions, attitudes, values, and mobility of middle class Blacks. The present study is a preliminary investigation into (1) the factors related to the achievement of middle and upper income among black and white families, (2) life styles among middle and upper income black and white families, and (3) a profile of Black wives in middle and upper income families. While future research will involve the collection of original data, the present project will make use of the Bureau of Census' Public Use Samples of the 1970 decennial census. Both contingency analysis using chi square and regression analysis will be the principle technique, and will involve two regression equations (one for the Black sample and one for the white sample) using the same independent variables. The second problem will be investigated with the aid of both contingency analysis and partial correlation using dummy variables, and the third primarily with contingency analysis.
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{
"pile_set_name": "NIH ExPorter"
}
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Alzheimer Disease (AD) and dementia affect an estimated 5.4 million people in the US with a new case diagnosed every 68 seconds. Obesity is a proposed modifiable risk factor for dementia and cognitive dysfunction. Obesity and dementia disproportionately affect African Americans (AA), yet AAs are underrepresented in studies of adiposity and cognitive function and mechanisms through which obesity increases dementia risk are poorly understood. In addition, risk scores to aid clinicians in identifying AA at risk of cognitive dysfunction are lacking. We will examine vascular and non-vascular mechanisms through which adiposity may affect brain structure, cognitive function (cross-sectional) and cognitive decline (longitudinal) using the GENOA study, a cohort of AA sibships. Adiposity measures, traditional and novel biomarkers, and vascular risk factors will be used to develop a cognitive dysfunction clinical risk tool for AA. We hypothesize a cascade of adiposity effects occurring through inflammation, adipokine, and vascular risk factor pathways which contribute deleteriously to cognitive function/decline. The specific aims of this study are: (1) Quantify cross-sectional adiposity and brain structure relationships, and cross-sectional and longitudinal relationships between adiposity and cognitive function, accounting for brain structure. We hypothesize that: 1.1) Greater central and overall adiposity will be associated with more brain atrophy, white matter hyperintensities, increased ventricular and decreased total brain volume across age. We also hypothesize that, accounting for brain structure, cardiovascular risk factors and clinical disease: 1.2) Greater adiposity will be associated with poorer cross-sectional cognitive function; and 1.3) Greater increases in adiposity will be associated with greater cognitive decline; and 1.4) Cross-sectional associations of CT-imaged abdominal adiposity will have stronger associations with cognitive function than waist circumference or body mass index. (2) Contrast the mediating effects of brain structure, inflammation, adipokines and vascular disease/risk factors in explaining relationships of adiposity to cognitive function/decline. We hypothesize that: Inflammation, adipokines and vascular contributors will explain 2.1) cross-sectional associations of adiposity to cognitive function and 2.2) associations of adiposity to cognitive decline, more so than brain structure. (3) Develop a clinical risk score to predict 3.1) cognitive dysfunction and 3.2) decline in AA. We hypothesize that an inflammation biomarker profile and adiposity measures will improve existing cardiovascular risk scores to predict 3.1) cognitive dysfunction and 3.2) cognitive decline. The proposed analyses will fill salient gaps in knowledge regarding prevalent and modifiable risk factors for cognitive impairment and will improve clinicians' ability to identify AA at high risk of cognitive dysfunction and decline for targeted interventions.
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{
"pile_set_name": "NIH ExPorter"
}
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Recent experiments indicate that enzymatic DNA methylation participates in the control of gene expression in higher eukaryotes. Presently, the mechanics of regulation and specificity of the enzyme, DNA methyltransferase (DMase) to methylate DNA are unclear. Studies have demonstrated aberrations in tissue-specific patterns of genomic methylation during carcinogenesis, tumor hypomethylation, and recently carcinogen interference in the transmethylation reaction catalyzed by DMase. Since cancer is charaterized by abnormalities in gene expression and persistant DNA replication, changes in genomic methylation by carcinogen binding may be causally related to carcinogenic initiation and/or malignant evolution. The proposed research focuses on regulation and enzymology of DMase in normal liver and during hepatocarcinogenesis; the elucidation of factors for faithful transmission of the methylation of specific sequences, and the mechanics by which carcinogens may alter this process at the template level of by direct interaction with the enzyme. In this renewal, one goal will be to produce monoclonal antibodies against the major DMase specie(s). These will be used to simplify the purification scheme as well as detect active and inactive DMase during carcinogenesis, and to test for the possible presence of multiple enzyme species. These antibodies will also be used to identify DMase polypeptides in in vitro translation assays and colony lysates so that DMase cDNA can be generated, clone and detected in either expression (pUC) cDNA libraries or in nonexpression vectors (pBR). The purpose of obtaining these probes is to eventually isolate the DMase gene. Because our understanding of the role of methylation is hampered by the inability to preserve the methylation pattern on molecular cloning, one of our goals in cloning full-length DMase in an expression vector is to be able to genetically engineer the expression in a bacterial host for this purpose. In this way, it will be easier to test the function(s) of DNA methylation as controlled by DMase and to determine whether it is an obligate component in the development of cancer.
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{
"pile_set_name": "NIH ExPorter"
}
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The high affinity IgE receptor belongs to a class of receptor which lacks intrinsic enzymatic activity but activates non-receptor tyrosine and serine/threonine and phosphatases. This activations leads to phosphorylation and activation of phophatidylinositol-specific phospholipase C, an increase in intracellular calcium and ultimately the cell degranulation and the release of the mediators of allergic reactions. Last year we had identified a distinct role for the beta and gamma subunit: beta but not gamma associates with the tyrosine kinase lyn in resting conditions, and gamma but not beta can activate the tyrosine kinase syk. Based on our findings, we had proposed a model of Fc(epsilon)RI activation in which, upon receptor aggregation lyn phosphorylates both beta and gamma, and the phosphorylation of gamma is necessary to recruit syk and activate syk. We now have tested this model of activation by reconstitution the various elements in fibroblasts. This is accomplished by infection of the fibroblasts expressing the tetrameric Fc(epsilon)RI receptor with recombinant vaccinia viruses. In this system, Lyn is the only kinase required for receptor phosphorylation. Cotransfection of Syk does not alter the phosphorylation state of Fc(epsilon)RI due to Lyn alone. The presence of the tetrameric receptor and Lyn is also required for the formation of a receptor-Lyn-Syk complex, which is increased after receptor aggregation. Thus, the cooperation between the two kinases and the beta and gamma subunits is necessary for the full capacity of the Fc(epsilon)RI receptor.
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{
"pile_set_name": "NIH ExPorter"
}
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Experiments are proposed to explore the relationship between structure and function of the membrane protein responsible for anion transport across the human erythrocyte membrane. This protein carries out an important biological function, and is present in the membrane in quantities suitable for detailed biochemical analysis. The activity of the protein is inhibited by extracellular proteolysis with papain. The molecular origin of the inhibition will be investigated by characterizing the products of extracellular papain digestion of the anion transport protein, and comparing them with the products produced by extracellular chymotrypsin, which cleaves the protein but does not inhibit transport. The techniques used will include preparative and analytical polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and end group analysis. To facilitate these analyses, the intact protein or its major glycosylated peptide will be deglycosylated by enzymatic or chemical techniques. The kinetics of anion transport indicate that the transport step involves a conformational change between two distinct states of the protein, but the nature conformational change is completely unknown. The proportion of protein molecules in each of these two states may be varied by changing the Cl-gradient across the membrane. The chemical similarities and differences between these two conformations of the protein will be investigated by performing irreversible chemical modification of the protein, under conditions in which virtually all copies of the protein are in one state or the other. The chemical probes to be used include pyridoxal phosphate, 5-nitrosalicylaldehyde, FeO4, an lactoperoxidase iodination.
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{
"pile_set_name": "NIH ExPorter"
}
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The parent grant to this application evaluates the effects of a safer conception counseling (SCC) intervention to improve accurate use of safer conception methods (SCM) and prevention of horizontal transmission for HIV serodiscordant couples in a cluster randomized controlled trial in Uganda. This revised application leverages this clinical trial to inform integration of pre-exposure prophylaxis (PrEP) into SCC for HIV serodiscordant couples considering childbearing. Family planning services for people living HIV/AIDS (PLHA) in Uganda focus on preventing unplanned pregnancies and mother-to-child-transmission, and currently provide no services to support safer conception, despite high rates of fertility desires and pregnancy among HIV serodiscordant couples. Antiretroviral therapy (ART) greatly reduces transmission risks related to childbearing, but ~40% of PLHA in Uganda are not on ART and ~30% of ART clients have poor adherence. SCC to promote use of complimentary SCM, including timed unprotected intercourse (TUI) and manual self-insemination (MSI), and now PrEP as it begins to become more readily available, is much needed. While PrEP may be a desirable form of SCM for serodiscordant couples wishing to conceive, it presents unique challenges. Gaps in knowledge exist, as just 15% were aware that ARVs could be taken by the HIV-negative partner to reduce risk in our prior research; yet, when explained, 84% believed that uninfected partners would be willing to use PrEP while trying to conceive. The broader literature on PrEP points to barriers to uptake, including fears of stigma and side effects, doubts about its efficacy, and beliefs of negative effects on fertility, but also suggests that pregnancy desires may be a facilitator of PrEP use. Nevetheless, in-depth qualitative studies exploring how PrEP may influence decision making related to childbearing and selection of SCM, identifying facilitators and barriers to uptake and adherence to PrEP as a SCM, and informing strategies to integrate PrEP into SCC are absent in the literature. With nearly half of serodiscordant Ugandan couples wanting children, this research is key to optimizing the impact of PrEP. Since July 2018, TASO, our collaborating partner in the parent grant, has provided PrEP to over 100 serodiscordant couples at one of our study sites, presenting us with the opportunity to use mixed methods with our current study sample as well as new research participants to: (1) understand individual, interpersonal and structural factors that may influence use of PrEP among serodiscordant couples with childbearing desires, including how access to PrEP and childbearing desires may influence each other; (2) identify strategies to integrate and promote PrEP in SCC programs, overcome structural barriers and promote adherence; and (3) examine client factors that influence PrEP uptake, adherence and retention. To achieve these aims, we will use qualitative interviews with clients (and willing partners) (10 couples using PreP, 10 not using PreP, and 10 who have discontinued PrEP)(n~45-60); key informant interviews with TASO leadership, trainers and providers (n~16); and quantitative analysis of TASO?s routinely collected PrEP program data.
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{
"pile_set_name": "NIH ExPorter"
}
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Identified neurons of leeches and snails are being used as test objects for investigation into the membrane actions of convulsant and anticonvulsant drugs.
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{
"pile_set_name": "NIH ExPorter"
}
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Varicella-zoster virus (VZV) establishes latency in human sensory and cranial nerve ganglia during primary infection (varicella), and the virus can reactivate and cause zoster after primary infection. Unlike infections in humans where large quantities of cell-free virus are released, infection in vitro does not result in release of cell-free virus. Thus, better models are needed that recapitulate observations of VZV in humans. Autophagy is a cellular process in which cellular components are degraded and recycled. VZV has been shown to induce autophagy in transformed cells and keratinocytes. We observed autophagy in skin organ cultures between 14 and 28 days after infection with VZV; in contrast, autophagy in uninfected skin organ cultures was minimal. The results in VZV-infected skin organ cultures were similar to those observed in biopsies from persons with herpes zoster. Autophagy was induced at a similar level in cells infected with wild-type VZV or in cells infected with recombinant VZV expressing the herpes simplex virus (HSV) ICP34.5 gene which inhibits autophagy in cells infected with HSV. Thus, the autophagy blocking function of the HSV ICP34.5 gene was not active during VZV infection.
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{
"pile_set_name": "NIH ExPorter"
}
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Neuroelectric oscillations reflect rhythmic fluctuations of neuronal ensembles between high and low excitability states. These fluctuations clearly impact on sensory processing and cognitive operations, but their actual utility is unclear. Our broad goal is to explore the overarching hypothesis that low frequency neuronal oscillations can function as "instruments" for active selection of task-relevant inputs in visual cortex and for enhancement of their representation across processing stages. A key proposition of this hypothesis is that in dealing adaptively with ever-changing tasks, ranging from rhythmic (e.g., watching a person walking by) to random (e.g., waiting for a traffic light to change), the brain shifts dynamically between "rhythmic" and "continuous" modes of operation. This proposition makes specific predictions vis-`-vis neuronal oscillations, and these will be explored by "paired" linear array multielectrode recordings from V1 and extrastriate visual cortices in awake- behaving monkeys. We have 3 specific aims. Aim 1: investigate the mechanistic role of neuronal oscillations in inter-modal attention. Expt 1 will build on our earlier studies on intermodal selective attention (b.2.a;c.2.1). Specific new predictions will be tested using behavioral measures, along with laminar current source density and multiunit activity profiles sampled during task performance using linear array multielectrodes positioned in V1, V4 and inferotemporal (IT) cortex. Inter-areal interactions (prediction 4) will be evaluated using dual multielectrode recordings. Aim 2: evaluate the generality of oscillatory involvement in attentional function and dysfunction. Generalization of finding outside of intermodal selection is important to establish because most selective attention investigations focus on effects within one sensory modality. Thus, Expt 2 will use a visual feature- attention task. To maintain comparability, stimulus manipulations, recording methods and tests of predictions will otherwise generally parallel those of Expt 1. The paradigm in Expt 2 is adapted from studies examining the neurophysiology of attentional deficits in schizophrenia. Expt 2 thus has additional translational value, in that it can determine the physiological significance of ERP components and/or oscillatory characteristics that are shown to be abnormal under corresponding conditions in schizophrenia (b.3;b.5;b.6). Aim 3: investigate the relevance of rhythmic mode processing in natural vision. Expt 3 will use a visual search paradigm, along with a match to sample paradigm, to evaluate the hypotheses that: 1) the key feature of rhythmic mode processing, the rhythmic fluctuation of neuronal excitability, is also a dominant feature in natural visual behavior, and 2) that this is due, at least in part, to the rhythmic nature of visual (saccade/fixation) sampling behavior, and the powerful organizing effects of fixation onset on cortical oscillatory activity (c.2.3). Expt 3 will also examine effects of fixation onset on oscillatory synchrony across cortical layers and across cortical areas. PUBLIC HEALTH RELEVANCE: A fundamental theme of our research program is a close integration of "basic" visual-cognitive neuroscience in monkeys, with ERP and fMRI investigations comparing normal function with neuropsychiatric dysfunction in patients with schizophrenia. This "translational" emphasis will be reflected in this competing renewal proposal.
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{
"pile_set_name": "NIH ExPorter"
}
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The long term objectives of this proposal are: (1) Basic research in the area of solid state chemical sensors, namely CHEMFETs, leading to understanding and utilization of unique features of these devices; (2) Biomedical applications of CHEMFETs namely for in vivo monitoring of electrolytes in biological fluids and ex vivo monitoring of electrolytes and gases. This proposal will also provide basic support to other research programs in terms of devices and transfer of know-how. The specific aims for this grant period include: (a) design/development of multisensor chip with integrated supporting on-chip electronics. This design will be done concurrently with the separately funded microencapsulation effort which is essential for making these devices commercially available; (b) development of rapid microtechnique based on potentiometric stripping for trace analysis of heavy metals (Pb, Cu, Cd) in biological fluids. In this work we shall use our recently developed electrostatically protected ISFET; (c) Basic study of Langmuir-Blodgettt films deposited on the CHEMFET gates. This work should lead to better understanding of the electrochemistry of olfaction and ion flux modulation by adsorbing molecules on lipid bilayer membranes; (d) As in the previous period our devices will be made available to non-profit organizations at no cost.
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{
"pile_set_name": "NIH ExPorter"
}
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PROJECT SUMMARY In order to effectively treat disorders associated with the over 1.8 million female veteran patients and the growing proportion of women serving in the armed forces (14%), it is imperative to understand the mechanisms that regulate steroidogenesis. The long-term objectives of this research are to fully understand the cellular mechanisms of action of gonadotropins and the regulation of steroidogenesis. The short-term goals of this research are to discover new protein kinase A (PKA) signaling events initiated by LH, and to determine how these novel mechanisms contribute to progesterone synthesis. This research involves components of second messenger pathways and proto-oncogene products that are central to the regulation of sex steroid secretion. The proposed research is designed to investigate a novel sequence of intracellular signaling events that mediate steroidogenesis in ovarian cells. The proposed aims are designed to test the overall hypothesis that activation of protein kinase A inhibits the intracellular signaling intermediate glycogen synthase kinase 3 (GSK3), which contributes to the steroidogenic response to LH by increasing the availability of a 2-catenin, a transcriptional co-activator that promotes the expression of the steroidogenic acute regulatory protein (StAR). This hypothesis is supported by our exciting observations showing that intracellular signals from the cAMP/PKA pathway converge with the Wnt/GSK3/2-catenin pathway in luteal cells. Three specific objectives are proposed to test this hypothesis: One) To determine whether protein kinase A (PKA) reduces GSK3 activity and thereby increases levels of 2-catenin and steroidogenesis we will use approaches for the expression and siRNA knockdown of signaling intermediates to validate their contribution to progesterone synthesis. These studies will be complimented by examining the subcellular localization of PKA, GSK3 and 2-catenin. Two) To determine whether LH/PKA-dependent phosphorylation regulates the stability and activity of 2-catenin we will evaluate whether stimulation of PKA increases the phosphorylation of C-terminus PKA phosphorylation sites on 2-catenin and determine if this contributes to the stability and/or nuclear localization of 2-catenin. To determine whether 2-catenin cooperates with a critical transcription factor LRH-1 (liver receptor homolog-1) known to regulate StAR expression and progesterone synthesis we will perform co-immunoprecipitation and co-localization studies using mutant proteins. Our hypothesis is that 2-catenin serves as a transcriptional co-activator for the nuclear receptor LRH-1 to increase the expression of steroidogenic acute regulatory protein (StAR) and, as a result, increase progesterone synthesis. Three) To determine whether 2-catenin interaction with LRH-1 recruits additional transcriptional co-activators, such as CBP (CREB-binding protein), during the regulation of StAR expression and progesterone synthesis. Our hypothesis is that LH- and PKA-dependent interactions among 2-catenin, LRH-1, and CBP are required during the steroidogenic response. This research plan integrates a variety of approaches [expression or mutant molecules, siRNA knockdown, immuno-localization, enzyme assays, gene expression, protein degradation, protein-protein and protein-DNA interactions, ChIP analysis] to identify novel intracellular signaling components activated by LH, and their potential for enhancing progesterone synthesis. Understanding signaling these cellular processes is vital to understand the integration of signal transduction pathways that control steroidogenesis in endocrine cells. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Research relevant to women's health is an area of D.V.A. attention. In order to effectively treat disorders associated with over 1.8 million female veteran patients and growing proportion of women serving in the armed forces (14%), it's imperative to understand the mechanisms that regulate steroidogenesis. This research involves components of second messenger pathways and proto-oncogene products that are central to the regulation of steroid secretion. In both men and women steroids control many vital processes, including carbohydrate, lipid, and protein metabolism, and sexual function. Some metabolic disorders associated with aberrant ovarian steroid secretion are hypertension, diabetes, obesity, osteoporosis, polycystic ovary syndrome and neoplasms of the colon, breast, ovary, and uterus. Considering the importance sex steroids in health and disease, we must have a better understanding of the mechanisms controlling the synthesis of steroid hormones.
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{
"pile_set_name": "NIH ExPorter"
}
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Linkage disequilibrium (LD) analysis has been shown to be an ideal method for mapping complex disease genes in isolated founder populations. This application is designed to collect a sample of patients with schizophrenia who are descended from the founder population of Costa Rica, with the goal of mapping and identifying schizophrenia predisposition genes in this country. The Costa Rican population is ideal for this study, because it is a large population descended over 20 generations from a small group of founders, and predisposition genes for bipolar affective disorder have already been mapped in this country. Our sample consists of Costa Rican patients with multiple hospitalizations for acute schizophrenic episodes and with early age of onset. Diagnostic procedures include a blinded interview by a bilingual psychiatrist, using the DIGS (Diagnostic Interview for Genetic Studies), as well as a family history interview, semi-structured collection of medical records, and a best-estimate process. Genealogic workup is done for each proband to document birthplace of ancestors in the great-grandparents' generation. The goal of this next phase of the study is to recruit additional schizophrenic probands who meet the following criteria: 1) DSM-IV consensus diagnosis of schizophrenia; 2) two or more psychiatric hospitalizations; 3) ancestry from central valley of Costa Rica; and 4) age of onset prior to age 40. Our hope is to collect information on several subtypes of schizophrenia, such as paranoid, undifferentiated and schizoaffective types. DMA samples will be collected from probands and parents, to allow for dense genome wide association analysis. Additional genomic analyses will be performed across schizophrenic candidate regions identified in this or other populations. Relevance to public health: Schizophrenia is a severe, disabling psychiatric condition which affects approximately 2.5 million Americans. Schizophrenia is a brain disorder, and this illness is thought to be inherited. This grant aims to identify the genes which contribute to causing schizophrenia. By identifying the genes which contribute to the diagnosis of schizophrenia, scientists will be able to better diagnose and treat this disabling illness. [unreadable] [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
}
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Both estrogens and dioxins have potent immunomodulatory properties. They both induce immunosuppression and thymic atrophy. Estrogens have also been clearly associated with certain autoimmune diseases in humans and rodents, while dioxin exposure has been shown to induce certain markers of hyperimmunity. The kinetics of atrophy induction and reductions in lymphocyte stem cell targets associated with this atrophy are remarkably similar between these agents at pharmacologically or environmentally relevant doses. Unlike corticosteroid, single doses of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) or 17beta-estradiol valerate (E2) in mice cause an atrophy that takes several days to manifest itself but persists for several weeks, and appears to be related to nonthymic stem cell reductions. Both of these agents mediate their affects through their specific receptors, the aryl hydrocarbon receptor (AhR) or estrogen receptor (ER) which are transcription factors involved in modulation of gene expression in most cell types. Both E2, and TCDD not only cause thymic atrophy, but also, unlike corticosteroid or radiation, induced increased numbers of liver lymphocytes, many expressing T-cell receptors normally deleted in the periphery of selected mouse strains. The long range objectives are to determine how activation of these receptors can lead to thymic atrophy and the appearance of T-cells that could promote autoimmune disease. Will inhibitors of these receptors (partial or complete antagonists) delay or reverse normal age related thymic atrophy? Will such inhibitors prevent or delay autoimmune disease? Specifically, studies will be carried out on the mechanism of thymic atrophy induction by these agents by determining the cell types expressing these receptors in the thymus and thymic stem cell compartments. At time points determine to be critical to atrophy induction or recovery, the activation status of the receptors will be determined by their ability to bind to their specific DNA response elements, i the cell types believed to be candidates for proximal targets. The dosimetry at which specific anti-estrogens (such as ICI 164,384), and partial TCDD antagonists (e.g., a-napthoflavone) can prevent atrophy induction by these agents will be determined. The activity of the receptors during normal physiological aging, and whether the inhibitors can delay or reverse normal age related atrophy will also be determined. The increased populations of liver lymphocytes induced by these two agents will be phenotype, including determining, by use of the stem cell markers for the recombination activating genes (RAG-1 and 2) and terminal transferase (TdT) whether they arise de novo in the liver from stem cells, or whether they are migrants. Whether the inhibitors described above, as doses that can prevent thymic atrophy, can prevent the appearance of these liver lymphocytes will be examined. Studies will be extended on the potential for autoimmune disease production or acceleration by both E2 and TCDD in an adult autoimmune model (SWR x NZB, SLE model). A perinatal exposure model in B6 x AJ mice which develop organ specific autoimmune disease after 3 days old neonatal thymectomy will also be examined.
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{
"pile_set_name": "NIH ExPorter"
}
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Cryptococcus neoformans is a pathogenic yeast belonging to Basidiomycetes, a taxonomic Class phylogenetically remote from the rest of the pathogenic yeasts (Ascomycetes). C. neoformans causes fatal meningoencephalitis primarily in patients with T- lymphocyte dysfunction and is the only species in the genus Cryptococcus that has optimum growth at temperatures higher than 30C. Although extensive studies on the molecular bases of cell cycle and morphogenesis have been carried out in ascomycetous yeasts, such studies have not been conducted in C. neoformans. This project was initiated to study the molecular genetics of cell cycle associated genes in C. neoformans. In 1999, we cloned the CCN1 gene from a serotype A strain of C. neoformans (B-4551) that exhibited altered cell morphology at 35 degree celsius and failed to multiply at 37 C. This strain caused chronic granulomatouse lesions in the nasal cavity of a cat without spreading to other sites due to its inability to grow at 37 C. This year we tested this strain (B-4551) for its ability to cause systemic infection in a murine model and characterized the CCN1 gene and the mutations that could be of responsible for the temperature sensitive phenotype. The strain was unable to cause systemic infection in mice. The CCN1 gene was found to encode a protein containing 16 tetratricopeptide repeats (TPR) of 34 amino acids each. It showed high sequence similarity with two known essential genes, CRN and CLF1, belonging to the TPR family. The CRN of Drosophila is involved in neurogenesis and the CLF1(S. cerevisiae homolog of CRN) is required for the assembly of spliceosomes. Comparisons between the genomic sequence of the CCN1 gene from both a wild type and strain B-4551 revealed the presence of five mutations, three consecutive nucleotides deletions and two missence mutations in the coding region. Site directed mutagenesis indicated that deletion of three consecutive nucleotides, corresponding to the highly conserved 8th amino acid of TPR7, was responsible for the temperature sensitivity of B-4551.The strain B-4551 complemented with wild type CCN1 restored its ability to grow at 37 C and caused fatal systemic infection in mice. These results indicated that CCN1 is essential for C. neoformans for growth at the body temperature and causes systemic infection in warm-blooded hosts.
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{
"pile_set_name": "NIH ExPorter"
}
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The physical-chemical properties and heterogeneities of lipid membranes, which are important to many biological processes, are being investigated by atomic force microscopy (AFM) in this project with NIAID collaborators. Quantitative features of the main phase transition in 2-dimyristoyl-sn-glycero-phosphocholine (DMPC) have been resolved by tapping-mode AFM images in an environmentally controlled chamber at various temperatures. These AFM images reveal two membrane phases near the expected DMPC chain-melting temperature. We have quantified the marked thinning and mechanical softening of the DMPC membrane upon chain melting from precise AFM thickness measurements. We have also constructed a novel biophysical model, which permits an estimate of the thermodynamic transition enthalpy, entropy, and the membrane phase domain size from our AFM-acquired temperature-dependent phase distributions. The deduced intrinsic domain size is about 4.2 nm in diameter. The work on DMPC membrane has been extended to examine the more biological membrane microdomains, or rafts, in (1) tri-lipid mixtures of dipalmitoyl phosphatidylcholine (DPPC), dilauroyl phosphatidylcholine (DLPC), and cholesterol (Chol), and (2) red blood cell membranes. We found that the tri-lipid membrane formed both microscopic and nanoscopic domains over mica substrates; both the cholesterol level and the temperature affected the sizes and dynamic of these domains. We developed new video microscopy and image analyses to characterize the red blood cell flicker and edge dithering phenomena during the Plasmodium falciparum malaria infection process. We found that the parasitic infection markedly modifies cell membrane dynamics, in potential relevance to malaria disease mechanism in microcirculations. Overall, the AFM and related technology are being advanced further to elucidate membrane-associated biomolecular events critical to medically important processes.
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{
"pile_set_name": "NIH ExPorter"
}
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Fibroblast growth factor (FGF) signaling plays pleiotropic roles in mammalian development and metabolism, and disease. The paracrine FGF1, FGF4, FGF7, FGF8, and FGF9 subfamilies play essential roles in spermatogenesis, mesoderm induction, somitogenesis, organogenesis, and pattern formation, whereas the FGF19 subfamily acts in an endocrine fashion to regulate major metabolic processes including glucose, lipid, cholesterol, and bile acid metabolism, and phosphate/vitamin D homeostasis. The diverse activities of FGFs are transmitted by the FGF receptor (FGFR) subfamily of receptor tyrosine kinases (RTKs). Perturbed FGF signaling leads to numerous human diseases, including skeletal, reproductive syndromes, hearing loss, renal phosphate wasting, neurodegenerative disorders, and cancer. Several paracrine FGFs and all the endocrine FGFs are being pursued for drug development. The four specific aims of this competing renewal are: I. Characterize the structural basis by which epithelially-expressed FGF4 and FGF9 subfamilies attain their specificity towards mesenchymally-expressed FGFRc isoforms. II. Elucidate the structural basis by which a/bKlotho co-receptors promote signaling by the endocrine FGFs. III. Dissect the role of A-loop tyrosine phosphorylation in the hyperactivation of FGFR tyrosine kinase by pathogenic gain-of-function mutations. IV. Elucidate the structural basis by which FGFR recruits and phosphorylates FRS2a. Recombinant protein expression and engineering, x-ray crystallography, Surface Plasmon Resonance (SPR) spectroscopy, isothermal titration calorimetry (ITC), steady-state kinetics analysis, and time-resolved mass spectrometry will be used to accomplish the Specific Aims of this proposal. The structural and biophysical/biochemical results obtained will also be validated using cell- and animal-based assays. The data obtained under Aim I should provide molecular insights into the roles of paracrine FGFs in embryonic development and also facilitate the discovery of drugs for tissue repair and bioengineering, promotion of self- renewal and differentiation of human embryonic stem cells for cell-replacement therapy. The data generated under Aim II should enhance our understanding of the role of endocrine FGFs in human metabolism and provide blueprints for drug discovery for major human diseases including diabetes, obesity, hypercholesterolemia, colon cancer, and chronic kidney disease, most of which represent a huge burden on public health. The results of Aim III will enhance our understanding of the mechanism of action of pathogenic mutations in FGFRs and other RTKs as well as the regulation of tyrosine kinase activity of the entire RTK superfamily. Since substrate recruitment and phosphorylation by RTKs is a general event in RTK signaling, the mechanistic insights gained under Aim IV will also be directly applicable to the entire RTK superfamily. PUBLIC HEALTH RELEVANCE: Fibroblast growth factor (FGF) signaling plays essential roles in human development and metabolism, and when it goes awry it leads to a wide array of human diseases, including skeletal, olfactory reproductive syndromes, hearing loss, phosphate wasting disorders, neurodegenerative disorders, and cancer. The overall goal of this application is to elucidate the molecular basis for FGF signaling in development, metabolism, and disease. The proposed studies should not only enhance our understanding of the role of the FGF signaling in human physiology but also lay the foundations for the discovery of new drugs for the treatment of many major human diseases, which currently represent a huge burden on public health.
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{
"pile_set_name": "NIH ExPorter"
}
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Humans are born with a propensity to look at faces, yet face recognition undergoes a surprisingly prolonged development into the late teens. Likewise, neuroimaging studies discovered prolonged development of face- selective regions in ventral temporal cortex (VTC) during childhood and into the teens involving age-related increases in face selectivity. Understanding the neural substrates of the development of face recognition is important because of the significance of face procesing in children's visual, social and emotional development, and is a prerequisite for understanding clinical conditions involving atypical face processing. However, it remains unknown how face selectivity develops, or how neural developments in VTC lead to better face recognition. We propose to elucidate the functional and structural brain mechanisms that underlie the development of face recognition abilities, using cutting-edge neuroimaging and behavioral methods acquiring multimodal data in the same subjects, cross-sectionally and longitudinally. In Aim 1 we will determine the factors that drive the development of face selectivity in VTC and examine the relationship between structural and functional developments. Thus, we will acquire behavioral, anatomical and functional data in each subject comparing data across young children (5-7 year olds, yo), children (9-11yo), and adults (23-25 yo) cross- sectionally and follow these developments longitudinally to determine: (1) what is the role of recent and cumulative experience in shaping face selectivity, (2) if viewing faces with central vision drives the development of face selectiviy, (3) if development of white matter structures is linked to development of face selectivity in VTC and (4) what is the reliability, sequence and longitudinal trajectory of the development of face selectivity, eccentricity bias and white matter structures in VTC. Longitudinal measurements wil provide unprecedented spatial and temporal precision revealing the casade of developments asociated with the emergence of cortical selectivity. In Aim 2 we will determine how neural developments lead to better face recognition. Proficient face recognition requires fine discrimination among similar faces and identification of specific faces across their many possible appearances. However, the neural mechanisms that underlie the development of these two key processes are unknown. Thus, using cross-sectional measurements of performance and brain responses in children (7-11 yo), adolescents (12-16 yo) and adults (24-28 yo) we will determine (1) if developmental changes in neural tuning to face identity and view lead to beter face discrimination and (2) if developmental increases in specialization to faces in VTC lead to beter face identification. We expect these studies to significantly advance our knowledge of the neural mechanisms that underlie the development of cortical selectivity to faces and improvements in face recognition, as well as progress our understanding of the neural mechanisms of long-term cortical plasticity.
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{
"pile_set_name": "NIH ExPorter"
}
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Previous research indicates that patient, provider, and health system characteristics are important factors in the adoption of healthcare innovations, including new prescribing patterns and/or medication utilization. However, efforts to increase evidence-based prescribing have been only modestly successful. These efforts have been of two main types: administratively based formulary manipulations such as drug restriction or preauthorization, and educational efforts based on diffusion theory such as academic detailing of opinion leaders or high-volume prescribers. We propose that evidence-based prescribing can be enhanced by focusing such interventions for maximal effectiveness and efficiency, and that this depends on knowledge of the flow of information and influence among providers working within a specific organizational context with specific types of patients. Such knowledge requires, specifically: (a) early identification of change in prescribing behavior (surveillance), (b) identification of characteristics of prescribers with a propensity to early adoption while controlling for confounding factors (including patient characteristics), and (c) identification of specific points of intervention by determining the relative importance of social vs. administrative factors in a provider[unreadable]s decision to prescribe. Geographic methods provide an innovative, large-scale approach to understanding the process of diffusion of innovation because provider, practice environment (location), and patient characteristics can be integrated into one empirical model that can be used to design and target practice-change interventions. In particular, space-time cluster analysis (STC) allows us to identify groups of providers and/or locations that change their behavior first ([unreadable]early adopters[unreadable]). These geographic methods first construct STCs of prescription events. Specifically, STCs are defined as geographical areas characterized by relatively higher rates of prescribing in a given time interval. Hierarchical General Linear Modeling then allows us to explain the development of these clusters using patient, provider and facility characteristics. We therefore propose to identify STCs that describe the spread of prescribing of second generation antipsychotics (SGAs) for two serious mental illnesses of high cost and priority to the VHA: bipolar disorder and PTSD. Focusing on SGAs in bipolar disorder takes advantage of several discrete events in 2004[unreadable]new FDA indications for bipolar disorder[unreadable]that anchor the investigation of diffusion. PTSD provides an important complementary disorder by which to study innovation spread since SGAs have become widely used for PTSD[unreadable]though without FDA indications. We will utilize national VHA data from Decision Support System (DSS), the Personnel and Accounting Integrated Dataset (PAID) and related datasets to identify STCs of early adopter providers prescribing SGAs for bipolar disorder, and within these STCs to evaluate: (a) the demographic characteristics of prescribers;(b) the demographic characteristics of the patients who receive prescriptions, and (c) the structural and cultural organizational characteristics at the VISN, VAMC, and CBOC levels within which the prescribing occurs. We will then characterize the robustness of early adopter prescriber profiles by determining the consistency of early adopter characteristics across SGAs, and determining whether the same characteristics that identify early adopters for bipolar disorder also identify early adopters for PTSD. Finally, we will develop an integrated model that characterizes the relative strength of diffusion-based versus organizational factors in prescribers[unreadable] likelihood of adopting SGAs for bipolar disorder. We hypothesize that both geographic factors, consistent with classic diffusion theory, and organizational factors, as articulated in more recent applications of diffusion theory to dissemination within healthcare organizations, will shape SGA spread and, therefore, identify opportunities for intervention.
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{
"pile_set_name": "NIH ExPorter"
}
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Claustrophobia and disruptive patient motion are common impediments to MRI examination, but they may be prevented or ameliorated with a non-pharmacologic behavioral intervention administered by trained staff. The potential benefits of such an intervention are highly significant, considering that the alternatives are to cancel the study or administer sedation. Inability to complete their MRI scans adversely affects an estimated 700,000 patients every year in the US. These patients are either deprived of a diagnosis, subject to diagnostic delays, or are exposed to risks of pharmacologic sedation, including death. The imaging facilities in turn, typically cannot fill the suddenly vacated examination slots in time before the next scheduled patient and thus incur considerable lost revenue and efficiency. The long-term goal is to provide a validated, clinically feasible means for non-pharmacologic amelioration of claustrophobia and disruptive patient motion, achieved by training MRI staff to use advanced rapport skills and comforting language to help patients. Phase I will design and perform a formative evaluation of a Comfort TalkTM Training intervention so that definitive testing of the hypothesized benefits for the intervention can be accomplished in Phase II. In Specific Aim 1 the Comfort TalkTM intervention sequence will be designed to include training in advanced rapport skills, using basic comforting language and avoiding negative language for all facility staff (including receptionists, technologists, nurses, and physicians). A core of licensed healthcare professionals will be taught how to guide patients in self-hypnotic relaxation techniques, using scripts, found to be safe and effective in the radiology department. Techniques are designed to easily integrate into the normal workflow without adding time. Training will include 2x8 hrs live at three test sites. Acceptance, observation of staff behavior, qualitative feedback, and rates of noncompletion will be used to assess efficacy of training, guide prototype development, and develop train-the-trainer materials. Using in-market piloting techniques, niche applications with their metrics will be explored to develop situation-specific supplemental materials. In Aim 2 an interactive web-based electronic platform will be developed to supplement training and provide post- training support. It will provide scenarios and practice applications, guided dialogue options, functions for online live interactive classes, and interface with Smartphones. The prototype will be beta-tested with 20 experts in hypnosis and refined through user input from the test sites. Aim 3 is to conduct a formative evaluation of the entire Comfort TalkTM intervention. Effects will be evaluated in 3-months intervals and compared to baseline performance. Criteria will include acceptance, qualitative feedback, content mastery, staff behavior, rates of noncompletion, patient recall and satisfaction. Additional metrics identified through in- market exploration will be analyzed for their potential as economic drivers and evaluation instruments.
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{
"pile_set_name": "NIH ExPorter"
}
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Graphical user interfaces are widespread in the field of genome informatics, and are considered key to the success of many application programs. Currently, a developer must choose between creating one from scratch, or working within large-scale, highly integrated systems that may impose monolithic designs and involve high start-up costs. The investigators are developing a toolkit for interface design based on the notion of a domain-specific widget set, where widgets are high level objects that are concerned exclusively with graphical display, hiding the details of placement, drawing, motion, interaction, etc. from the application programmer to the extent possible. Using the Tcl/Tk graphical user interface design system, the investigators have prototyped chromosome, map, and sequence widgets, as well as a number of dialogs and utilities that should promote the development of communicating small applications with a consistent "look and feel." The investigators propose to: improve existing widgets and dialogs, making them significantly more robust and versatile; design new widgets, dialogs, utilities, and database interfaces to support a wider range of applications; reimplement for efficiency and compatibility, creating a compiled and highly tuned widget set for UNIX, Macintosh, and PC platforms, as well as additional software environments including the WWW; create applications in collaboration with biologists, as demonstrations and to drive widget design; and support and archive widgets, dialogs, utilities, and applications in a repository for software sharing.
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{
"pile_set_name": "NIH ExPorter"
}
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Using molecular genetic techniques, the chief objectives of this proposal are to home in on the disease gene in X-linked bipolar affective illness and to detect other major genes in the transmission of the disorder. These goals will be attained by 1. gene mapping and related DNA techniques; and 2. conducting linkage studies with DNA markers in suitable pedigrees segregating bipolar and related affective disorders. The study will also aim to characterize linked vs. unlinked cases on clinical measures in an attempt to identify and define homogeneous subsets of the disorder and to compare different populations and ethnic groups for possible genetic differences. Long-term goals will include cloning of the defective genes; determination of their structure; searching for their biological products; and assessment o( gene-environment interaction. Israel is the data collection site. There are six main reasons for this choice: 1. X-linked pedigrees have already been identified. 2. Availability of large multigenerational pedigrees with multiple affected cases that are readily accessible for study. 3. Low rates of alcoholism and drug use, conditions that confound the psychiatric diagnosis. 4. An opportunity to compare different populations and culture. 5. Previous successful collaboration and intriguing findings that require extension. 6. The relatively low cost of human research. Israel, probably uniquely, meets all these requirements. The availability of a unique population, coupled with recent methodological advances such as molecule genetic techniques, approaches to systematically obtaining family data, structured interviews, precise diagnostic criteria with improved reliability; and a range of genetic models, holds much promise for unraveling the genetic mechanisms that underlie affective illness. This, in turn, could have major implications for the etiology, nosology, pathophysiology and, possibly, prevention and treatment of these disorders.
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{
"pile_set_name": "NIH ExPorter"
}
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Cardiomyocytes experience a wide range of physiologic and pathologic stimuli, which can influence their cellular state. Although cardiomyocyte hypertrophy and hyperplasia are well known adaptive cardiac cellular responses, some cardiomyocytes also retain the capacity to reprogram (i.e. cardiac plasticity) in order to alter their differentiation state and identity to adapt to stress. For instance, cardiomyocyte de-differentiation is a component of the maladaptive response during heart failure; a portion of the right ventricle in Ebstein's anomaly, which is exposed to altered hemodynamic forces, becomes ?atrialized?; and both cardiomyocyte de- differentiation and trans-differentiation regulate cardiac regeneration under certain conditions. Thus, this cardiac ?adaptive cellular reprogramming? can act in not only pathologic but also beneficial circumstances. However, despite the importance of cardiac reprogramming in regulating adaptive responses to stimuli, our understanding of the intrinsic processes that control cardiomyocyte plasticity and the external cues that activate cardiac reprogramming to modify cardiomyocyte differentiation states and cell identities remains yet to be fully elucidated. Thus, the overall goals of these proposed studies are to illuminate the underlying mechanisms that 1) control cardiomyocyte plasticity, 2) activate cardiomyocyte reprogramming in plastic cardiomyocytes and 3) regulate the reprogramming of these cardiomyocytes. The results of these cardiac reprogramming studies will not only illuminate how cardiomyocytes may adaptively (or maladaptively) reprogram in response to cellular stress in vivo but also provide further insight into how to direct mammalian cells from various cell sources (i.e. fibroblasts, pluripotent stem cells, cardiac progenitor cells) into functional ventricular and atrial cardiomyocytes for human cardiac disease modeling and therapeutic screening in cell culture systems as well as for human cardiac regenerative therapies.
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{
"pile_set_name": "NIH ExPorter"
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The adduction of DNA by the environmentally and endogenously produced genotoxins vinyl chloride, acrolein, crotonaldehyde, and 4-hydroxynonenal (4-HNE), yields /nte/strand crosslinks, /nfrastrand crosslinks, DNA-protein conjugates, and regioisomeric mono-adducts. These chemicals likely contribute to background levels of inter- and /nfrastrand crosslinks and DNA-protein crosslinks in human cells. An inability to repair genomic damage correlates with human disease?e.g., cancer, premature aging, fatty liver disease, and atherosclerosis. Using materials prepared by Project 1 and the DNA Synthesis Core, this project will utilize NMR and crystallography to obtain high resolution structural data for DNA modified with these bis-electrophiles, to delineate the underlying structural basis for their mutagenicity and cytotoxicity. It will be determined how formation of /nterstrand crosslinks depends upon the identity and stereochemistry of substituents at C6 of proximal 1,N2-dG enal adducts. Using reduced crosslinks of the 6R- and 6S crotonaldehyde adducts it will be determined if the 6S crosslink creates a greater structural perturbation to DNA than does the 6R crosslink. The (6S,8R,11S) crosslink of the 4-HNE adduct possessing the same absolute stereochemistry as the 6R-crotonaldehyde crosslink will be characterized as to its chemistry. Work with reduced peptide-DNA crosslinks will focus on the conformation of the glycosyl torsion angle, which will orient the peptide in the minor vs. major groove. Our work will be correlated with that in Project 2 designed to understand crosslink repair. The chemistry of intrastrand crosslinks will be determined via incorporation of NMR-active isotopes. Using saturated analogs of these crosslinks, it will be determined if the intervening T in the 5'-GTX-3'sequence extrudes from the duplex. Residual dipolar coupling measurements in partially oriented samples, and gel electrophoretic mobility assays, will probe crosslink-induced DNA bending. The capacity of the Sulfolobus solfataricus Dpo4 polymerase to accommodate these crosslinks will be examined with crystallography of binary (Pol + DNA) and ternary (Pol + DNA + dNTP) complexes. The results will be correlated with site-specific mutagenesis experiments in Project 2. NMR studies will examine the hypothesis that all but one of the stereoisomeric 4-HNE adducts exist largely as 1 ,N2-dG adducts in the syn conformation about the glycosyl bond. Likewise, N1-dA and N3-dC adducts, and their N1-dl and N3-dU deamination products, may exist in the syn conformation about the glycosyl bond. Crystallography involving binary and ternary complexes of human Pol-iota and Pol-kappa with modified primer-template complexes will probe the sequential bypass of enal adducts by these polymerases, as observed by Project 2. It will be determined if hPol-iota exploits the syn conformations of these adducts for bypass. The role of hPol-kappa in positioning the primer 3'-OH to catalyze extension following insertion by hpol-iota will be determined. The potential for strand slippage during bypass of these lesions by Pol-eta, leading to frameshifts, will be determined.
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{
"pile_set_name": "NIH ExPorter"
}
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This project will test the hypotheses that 1) G protein activates VSM L-type Ca2+ channels via cAMP/PK-A pathway, and 2) cGMP may exerts its inhibitory effect on these Ca2+ channels via phosphorylation of Ca2+ channels or some regulatory proteins by PKG. These hypotheses will be evaluated by utilizing freshly isolated portal vein myocytes, transiently expressed VSM L-type Ca2+ subunits, purified G protein subunits, and variety of non- specific and specific protein kinase blockers. Results from this project will provide cellular events by which G proteins and protein kinases regulate Ca2+ entry in VSM cells following physiological stimulation to induce vasodilation and vasoconstriction. Ca2+ channels are targets for a number of 2drugs used in the treatment of hypertension such as dihydropyridines, and adrenergic agonists and antagonists. By further defining the signal transduction pathways regulating Ca2+ entry, new strategies could be developed to control high blood pressure.
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{
"pile_set_name": "NIH ExPorter"
}
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Physical Sciences Inc. (PSI) proposes to develop and commercialize a new type of ophthalmoscope based upon the principles of the confocal Scanning Laser Ophthalmoscope (SLO). Though the confocal SLO has many valuable diagnostic capabilities, commercially available devices are large, expensive and are impractical for hand-held applications. In the Phase I program, PSI designed and demonstrated a confocal line scanning laser imaging device, or LSLO, using a simpler, compact optical design, standard low-cost components, and fewer moving parts that current SLO instruments. Based on this work, the requirements for a compact prototype have been specific that is similar in size and weight to commercial hand-held digital video cameras. In phase II, we will develop and clinically test a compact engineering prototype device for several clinical applications and commercial development. This device will be suitable for fundus examination under a wide variety of conditions, including screening and field and emergency medical uses. The Phase II prototype will incorporate additional capabilities including dual wavelength and stereoscopic imaging, and will be useful for examination of the anterior segment of the eye.
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{
"pile_set_name": "NIH ExPorter"
}
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Fluorescence Spectroscopy is inherently a very sensitive technique; it already forms the basis of most non-radioactive real time assays like PCR. Our lab has collaborated with former fellows (now in biotech industry) to develop alternatives to PCR like CataCleave probes for SNPs, leading to previous publication. We continue to study the photophysics and proper coupling of DNA components to multilayer metal nanoparticles for much faster PCR analysis and the use of FCS (see MPM report) to quantify very tight protein-protein and protein-DNA binding in sub-microliter drops (analytes are present in sub-femtomole amounts). We previously examined the structural transitions of similar amounts of DNA between G-quadruplexed and linear forms, using both MPM-FCS and Time-Resolved Fluorescence tools, with the goal of developing very sensitive 'G-quad' and aptamer detection assays. We have numerically combined time-resolved fluorescence detection with translational mobility (FCS) to help identify free and bound signatures for assay. We have just recently installed a RICS /FCCS microscope for combining the techniques mentioned above.
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{
"pile_set_name": "NIH ExPorter"
}
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ABSTRACT Developing fetus in maternal womb can be exposed to environmental stress, and this may lead to the development of long-lasting neurological and behavioral changes. Uncontrolled Inflammation encountered in utero and its effects on behaviors of offspring have been modeled in rodents and subsequently coined as maternal immune activation (MIA). However, it is still unknown how the immune activation, which takes place in pregnant dams, is translated into neurological and behavioral changes in offspring. Using both genetic mutants lacking a particular subset of pro-inflammatory immune cells and blocking antibodies targeting their activities, we have recently found that pro-inflammatory T helper cells (Th17 cells) expressing intereukin-17a (IL-17a) in mothers induce MIA-dependent behavioral changes and abnormal cortical phenotypes in offspring. We also observed that the receptor for IL-17a (IL-17Ra) is expressed in the fetal brain and its expression is increased in the cortical plate upon MIA. These observations taken together suggest an exciting hypothesis that uncontrolled activation of IL-17Ra expressed in fetal brain induces abnormal cortical patches and these structural abnormalities eventually lead to the MIA- associated behavioral phenotypes. Thus, in this application, we propose 1) to determine if cortical abnormalities could predict behavioral phenotypes in MIA offspring, 2) to characterize cortical abnormalities in adult MIA offspring, and 3) functionally determine if the cortical phenotype is the underlying cause of the MIA behavioral abnormalities.
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{
"pile_set_name": "NIH ExPorter"
}
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Laser Induced Fluorescence (LIF) can be used as a diagnostic device in biology and medicine. In general, LIF has difficulties relating the fluorescence spectra to the concentration of the emitters within the tissue, in identifying the various species, and in the possibility of destroying the molecules being probed if the wrong excitation wavelength is used. Many of the problems associated with fluorescence can be eliminated by the use of Raman scattering. Raman techniques allow the measurement of multiple species even in aqueous solutions (not accessible by infra red techniques because of water absorption). However, spontaneous Raman scattering techniques generate very weak signals. Signals can be enhanced through the use of resonance Raman techniques. Both of these methods still suffer from the fact that spatial and to some extent temporal information is lost. Spatial information can be recovered through the use of confocal microscopy. The research in this proposal is intended to investigate a non-invasive, real-time, high resolution device for the measurement of the concentration levels of enzymes, metabolites or other critical molecules. This unique device is based on nonlinear optical methods that can simultaneously measure the concentrations and temporal evolution of several molecular species in individual biological cells. PROPOSED COMMERCIAL APPLICATION: The method proposed here can be used for the continuous measurement of several molecular concentrations in small samples. Therefore, it may have applications in the medical field, in drug monitoring and manufacturing, and blood gas analysis. Furthermore, it may find applications in environmental monitoring.
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{
"pile_set_name": "NIH ExPorter"
}
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Decision-Making For Infants with Complex Life-Threatening Conditions Abstract Infants with life-threatening conditions who earlier would have died in the first months of life are now receiving highly complex and sometimes experimental treatments designed to cure the condition or prolong life. However, these treatments have uncertain outcomes, the infants are at high risk for death, and if they live face a life-span of significant morbidity. Infants with these conditions experience an uncertain trajectory that typically involves multiple health crises requiring parents and health care providers to make critical decisions about the type and level of treatment. Decisions span the continuum from whether to initiate treatment, how to alter the treatment to respond to a medical crisis, whether to shift from aggressive curative care to symptom- focused palliative care, to whether to withdraw treatment. This 5-year study will use a longitudinal, case study design to examine the trajectory of decision making for infants undergoing life-sustaining treatment for complex life-threatening conditions (CLTC). Three groups of infants with CLTCs who are at particularly high risk for death and significant life-span morbidity are the focus of this study: extremely preterm infants (<26 weeks gestation), infants with complex cardiac anomalies, and infants with genetic diagnoses requiring a hematopoietic stem cell transplant (HSCT). Forty cases will be studied and each case will include the infant, parents, providers and the physical context of caregiving. Narrative interviews and self-report measures will be used with case members following birth or diagnosis and at least monthly thereafter until death or 1 year following enrollment. A within-case and cross-case analysis will be used to explore the trajectory of decision making for these infants and examine how and when decisions are across the illness trajectory. The study will identify transition points across the trajectory and identify factors influencing decision making, such as the level of involvement that parents' want; characteristics of parents who desire more or less involvement; and roles health care providers can play in decision making. The findings will be used to develop typologies of decision making and illness trajectory transition points for infants with CLTCs. These will provide a foundation for developing interventions to facilitate the decision making for these infants. PUBLIC HEALTH RELEVANCE: This study will use a longitudinal multiple case study design to examine the trajectory of decision making for infants with complex life-threatening conditions (CLTC) and influences on parents' decision making from birth or diagnosis until death or 12 months. This study will be the first study to prospectively examine the trajectory of all types of health-related decisions (treatment initiation and intensification, shifts in treatment from curative care to palliative care, withholding or withdrawing treatment) across a year-long illness trajectory, in multiple populations of infants with CLTCs, both those who die and those who survive. In addition, the sample of extremely premature infants (<26 weeks gestation), infants with complex congenital heart disease (CHD), and infants with genetic diagnoses requiring a hematopoietic stem cell transplant (HSCT) will allow for exploration of how decision making is both similar and different for infants undergoing experimental treatment for the three most common causes of pediatric death. We will identify transition points across the trajectory of decision making and identify the influences on decision making, such as the level of involvement that parents' want; characteristics of parents who desire more or less involvement; and roles health care providers play in decision making. The goal will be to identify transition points across the trajectory and provide a foundation for developing interventions to facilitate parents' decision-making for these infants.
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{
"pile_set_name": "NIH ExPorter"
}
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Our long-term goal is to better understand the etiologic roles of protein posttranslational modifications (PTMs). This project focuses on isoaspartic acid (isoAsp, isoD or beta-Asp), which is generated spontaneously from either asparagine deamidation or aspartic acid isomerization. IsoAsp has been found in myriad proteins (e.g., beta-amyloid in the brain and crystalline in the eyes). Notably, isoAsp imparts a D-configuration and beta-linkage into the peptide backbone; such alteration of peptide backbones is exceptionally rare and may drastically alter protein structure and function. While well-recognized in aging, its emerging roles in signaling, regulation and autoimmunity have yet been widely appreciated. A lack of comprehensive knowledge of isoAsp and the unique technical challenges in its analysis hamper progress in this field. For one thing, isoAsp formation is the smallest PTM by mass change (Da; isoAsp has the same mass as Asp). Recently, these technical barriers have been overcome by novel approaches that combine chemo-enzymatic labeling and enrichment with mass spectrometry. Four specific aims will be undertaken. First, proteomic analysis of isoAsp in samples with biochemical and medical relevance will be performed. Second, tight binders to isoAsp will be engineered and used as antibodies for applications that complement existing methodologies. Third, the substrate specificity of the repair enzyme protein isoaspartate methyltransferase (PIMT) will be probed, focusing most assiduously on poor substrates of PIMT. Isoaspartyl peptide library screening, enzyme kinetic characterization, proteomic analysis and protein structural modeling will be integrated to augur the fate of isoAsp in individual proteins and whole systems. Last, alternative metabolic pathways for isoAsp will be discovered. The proposed research will lead to the discovery of biomarkers of human diseases, and effective methods of prevention, intervention and treatment for age-related conditions, neurodegenerative disorders (e.g., Alzheimer's disease), autoimmune diseases and other isoAsp-related ailments. PUBLIC HEALTH RELEVANCE: Isoaspartic acid formation is one of the most common, yet under-appreciated, modifications of proteins, and may significantly alter protein structures and functions. A comprehensive knowledge of this ubiquitous protein modification will advance both our understanding of the etiological mechanism and the intervention of diseases, particularly neurodegenerative and age-related disorders.
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{
"pile_set_name": "NIH ExPorter"
}
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This study will test whether rhCNTF is superior to placebo in slowing or halting the progression of ALS. The study is double blinded. Patients will be given daily subcutaneous injections of drug or placebo. The primary measure of efficacy will be via Tufts Quantitative Neuromuscular Exam.
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{
"pile_set_name": "NIH ExPorter"
}
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To determine if ethnic differences exist in pharmacokinetic and pharmacodynamic responses to immunosuppressive drugs.
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{
"pile_set_name": "NIH ExPorter"
}
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In order to understand the mechanism(s) responsible for compartmentalization or acid hydrolases into lysosomes we are isolating and analyzing mutant CHO cells defective in this process. An initial step in compartmentalization involves interaction of the mannose 6-phosphate receptor with phosphorylated oligosaccharides on the hydrolases. We have examined the effects of several glycosylation defects on the ability of the oligosaccharide to serve as an acceptor of phosphate.
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{
"pile_set_name": "NIH ExPorter"
}
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Genetic mouse models of neurodegenerative disorders can provide valuable insight into the pathogenesis of the corresponding human condition, and can serve to address specific hypotheses about the biology of the relevant gene product in health and disease. Investigations into genes that cause inherited forms of Parkinson's disease (PD), the mechanisms involved in their cellular and molecular biology, as well as the downstream pathways that they trigger shed considerable light on our understanding the fundamental determinants of life and death in dopaminergic neurons. DJ-1 is one of five genes identified thus far to be definitely linked to inherited PD. The two original mutations identified in the DJ-1 locus in affected pedigrees are transmitted recessively suggesting loss of function. Although considerable knowledge about the anti- oxidant properties of DJ-1 has been generated, and its diverse functions in other systems have been studied, its role in the biology of dopaminergic neurons has been addressed in limited studies and remains poorly understood. Therefore, how DJ-1 protects cells and how its absence or point mutations result in dopamine neuronal death remain largely unanswered questions. To date, the cytoprotective activity of DJ-1 against oxidant damage has been attributed largely to its reactive oxygen species (ROS) quenching ability. However, our findings indicate that the ROS quenching potential of DJ-1 is weak compared to its robust cell preserving activity. Rather, our data point to an alternative mechanism for the cytoprotective function of DJ-1 which appears to have a critical role in inhibiting a specific kinase-mediated cell death-signaling pathway. The end result is protection against insults that activate this pathway, including oxidant damage. The pathogenic relevance of this proposed scheme in PD is strengthened by failure of a PD-causing DJ-1 mutant at each step of this pathway. This application proposes to investigate the in vivo validity of this hypothesis using a novel genetic mouse model. This proposal also addresses the relatively selective vulnerability of dopaminergic neurons to DJ-1 deletion despite its ubiquitous expression. For lay public: The goal of this project is to understand how mutations in DJ-1, a gene responsible for some forms of Parkinson's disease, cause degeneration of dopamine producing brain cells. Elucidating the steps in this process can help develop treatments that could block its progression. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
}
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The major aim of the National Heart, Lung and Blood Institute (NHLBI)/Suburban Hospital Cardiovascular MRI Research Project is to develop new approaches in assessing patients with cardiovascular disease with MRI technology. 1) Detection of acute coronary syndrome with MRI. Earlier this year, we published that a rest MRI scan had higher sensitivity and specificity for diagnosing non-ST elevation acute coronary syndrome than cardiac risk factors, ECG, and troponin. The sensitivity and specificity for detecting acute coronary syndrome was 84% and 85% by MRI, 80% and 61% by an abnormal ECG, 16% and 95% for ST depression or T-wave inversion, 40% and 97% for peak troponin-I, and 48% and 85% for a TIMI risk score >3. The MRI was more sensitive than strict ECG criteria for ischemia (p<0.001), peak troponin-I (p<0.001), and the TIMI Risk Score (p=0.004). The MRI was more specific than an abnormal ECG (p<0.001). Multivariate logistic regression analysis showed an abnormal MRI was the strongest predictor of acute coronary syndrome and added statistically significant diagnostic value over clinical parameters (p<0.001). We concluded that the resting MRI scan exhibited diagnostic operating characteristics suitable for triage of patients with chest pain in the emergency department. We have extended this work in a second protocol that used adenosine stress MRI to evaluate 141 consecutive patients with troponin-negative acute coronary syndrome. The overall sensitivity and specificity for detecting ischemic heart disease were both greater than 90%. An abnormal adenosine stress MRI had significant 1 year prognostic value. We have determined that gadolinium delayed enhancement cardiovascular magnetic resonance corrrelates with clinical measures of myocardial infarction. This study imaged patients with acute myocardial infarction an average of 2 days post-MI. The transmural extent of delayed enhancement predicted the recovery of regional myocardial function. We have started a project that aims to characterize recently ischemic myocardium and demonstrated that we can image the ischemic area at risk after myocardial perfusion has been restored. This can be described as a form of "ischemic memory imaging." 2) Characterizing myocardial viability with MRI. We also developed a phase sensitive reconstruction method which improves the quality of heart attack images and minimizes the influence of user selected parameters on the apparent size of the heart attack. Our histopathological validation of the phase sensitive reconstruction method and a validation study showing that a computer algorithm can accurately measure infarct size on in vivo and ex vivo images is now in press. We have also extended this work to characterize IL-2 myocarditis and community acquired myocarditis. We have extended our validations of the phase sensitive inverson recovery methods for imaging myocardial infarction with another study: Artifact suppression in imaging of myocardial infarction using B1-weighted phased-array combined phase-sensitive inversion recovery. 3) First pass myocardial perfusion imaging. We have extended our first pass perfusion methods to provide quantitative analysis methods. We have shown that the MRI can measure myocardial perfusion as accurately as microsphere injections (a gold standard method only usable in animal models). We are in the process of translating these methods to analyze human dipyridamole stress tests and have corroborated the basic findings from the Christian manuscript apply to humans. 4) MRI characterization of atherosclerotic plaque. We have extended our prior observations that gadolinium nearly doubles the ability to discriminate different portions of the atherosclerotic plaque. We have studied the kinetics with which contrast enters the fibrous cap and lipid core of carotid atheroma]. 5) We have reduced image artifacts on DENSE images, a method for imaging myocardial strain. This year, we demonstrated that parallel image processing can accelerate the rate of acquiring phase contrast methodologies.
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{
"pile_set_name": "NIH ExPorter"
}
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The aim of this project is to study the processes involved in the assembly of the extracellular matrix in connective tissues. Specifically, the project is designed to 1) examine interactions between fibronectin and procollagen on the cell surface during matrix formation, 2) identify cellular structures (i.e. microfilaments, filopodia) responsible for the formation of fibronectin and fibronectin-collagen filaments, and 3) determine the orientation of fibronectin in fibronectin and collagen containing filaments. Specimens will be plunge frozen, partially freeze-dried and coated at low temperature with chromium or tungsten. The specimens will then be viewed at low temperature in the FESEM.
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{
"pile_set_name": "NIH ExPorter"
}
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Despite that speech is often characterized by either spectral or temporal cues, its everyday perception involves the integration of temporal patterns present at different spectral loci. The long-term goals of the proposed research program are to further our understanding of the normal mechanisms employed during the processing of the spectro-temporal patterns in speech, and to characterize limitations that hearing impairment places on these mechanisms. Techniques combining psychoacoustic and speech perception research will be employed to extend a number of recent observations concerning spectro-temporal processing. These examinations will involve both listeners with normal hearing and those with sensorineural hearing impairment. The aims are: (1) To establish the spectral resolution employed to process speech (2) To examine the use of temporal information at different rates and spectral frequencies (3) To examine the spectro-temporal processing deficit in hearing impaired listeners The information gained has the ability to further our understanding of normal speech processing. It also has the ability to further our understanding of the impact of hearing impairment by providing a comprehensive examination of the ability to use the fluctuating temporal patterns in speech, and by examining the influence of broadened auditory tuning on the reception of particular speech materials in particular frequency regions.
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{
"pile_set_name": "NIH ExPorter"
}
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The small intestine actively secretes HCO-3 and actively reabsorbs HCO-3 (or secretes H+). These processes are important in the normal function of the small intestine and in pathophysiological circumstances such as diarrhea. Nevertheless, the underlying ion transport events are unresolved and the intercellular and extracellular control mechanisms are not known. Moreover, the transporting cells responsible have not been identified. A subpopulation of mucosal cells in the jejunum of the urodele (Amphiuma) has been found to contain acid vesicles when exposed to the fluorescent stain acridine orange (AO). pH and K+ sensitive microelectrodes will be employed to measure the ion content of the vesicles and cytoplasm of the secretory cells of in vitro intestinal segments. Hypotheses regarding the cellular mechanisms of acid secretion will be tested. Scanning and transmission electron microscopy will be used along with AO flurosescence to determine the morphology of the secretory cells and their associated vesicles and to distinguish whether the changes in acid secretion result from changes in acid storage of H+ transport. The identity of the cells responsible for the enhanced acid secretion caused by catecholamines such as epinephrine will be determine and the ion transport events induced by catecholamines including, apparently, Na-H exchange studied in detail. The effect of cholinergic agents to reduce acid secretion will be examined. The ability of cholinergic agonists to stimulate HCO-3 secretion in the duodenum and adrenergic agents to block the effect will be documented using the pH stat technique. The effect of these agents on Na and C1 transport will be determined. The possibility that methacholine acts through elevation of cyclic AMP will be examined. These studies will provide the first electrophysiological and morphological analysis of an identified acid secretory cell in the intestinal mucosa. Whether the catecholamine-driven acid secretion is from the same or a different cell will be determined. The ion transport mechanisms responsible for spontaneous and epinephrine-stimulated acid secretion will be determined.
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{
"pile_set_name": "NIH ExPorter"
}
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Glutamate receptors (GLRs) are ligand-gated membrane bound proteins which are prevalent in animal nervous systems where they reside on the post-synaptic membrane. This project will examine the role of glutamate receptor gene homologues in plants which have recently been discovered in Arabidopsis. To do this, I will use a reverse and forward genetic approach to study the role of these genes in Arabidopsis growth and development. With a reverse genetic approach,, transgenic Arabidopsis plants will be created with low or no expression of GLRs using both an antisense and gene knockout approach. By using forward genetics, I will screen for mutants insensitive to BMAA, a neuroactive plant derived agonist of GLRs that appears to inhibit photo morphogenesis in Arabidopsis. BMAA insensitive mutants (bim) may be disrupted in GLR genes or other genes involved in a GLR response cascade. Plants derived from both the forward and reverse genetic approach will be tested for alterations in light responses at the morphological and molecular level. Finally, the spatial and temporal expression patterns of Arabidopsis GLRs that are found to be important for normal plant function and development from these studies will be examined. This combined approach will help in deciphering the role of these genes in Arabidopsis and perhaps also in animals.
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{
"pile_set_name": "NIH ExPorter"
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PROJECT SUMMARY/ABSTRACT The goal of the Administrative Core (Core A) is to facilitate the administration and implementation of this Program Project Grant (PPG), by enabling the individual projects of this PPG to be performed in a more integrated and efficient fashion. The activities of this Core will include all scientific and administrative supervisory functions of the PPG, including the organization and supervision of the individual Projects and of the Core facilities. All fiscal records and accounts for the individual projects and for each core facility will be maintained and distributed to Project Leaders and Core Directors on a monthly basis. This Core also will provide administrative support for regular meetings of the Program Director with the Project Leaders, will organize and arrange monthly joint lab meetings of all PPG participants, will organize and arrange annual meetings of the External Advisory Board, and will arrange for seminar speakers to visit on a regular, bimonthly basis. A Project Administrator and a Staff Assistant will be supported to carry out the goals of this Core. The personnel of this Core will have offices in a single location, within the Digestive Diseases Section of the Department of Medicine.
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{
"pile_set_name": "NIH ExPorter"
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This study aims to document the work of a widely used and trusted network of health practitioners providing alternative health care to thousands of immigrants from Oaxaca, Mexico living in the Los Angeles area. The goal of the project will be to systematically identify and to document the application of traditional healing techniques employed by people of Mexican origin which are consistent with their own cultural heritage yet complement Western medicine for the treatment of diabetes, hypertension and cancer. The Zapotec and other indigenous groups from Mexico seek care for culturally defined illnesses they would rarely see a Western doctor for, such as susto (soul loss), evil eye and bad air. Qualitative methods will be used to study a sample of traditional healers and their patients living in Los Angeles. The healers wil include bonesetters, masseuses, spiritists and curers of soul loss and other supernatural illnesses. Ethnographic, in-depth interviews will be conducted with six healers. Interview topics will focus on how they view their work, their philosophy of healing, techniques employed to treat patients suffering from folk illnesses in conjunction with diabetes, hypertension or cancer. In addition, interviews will be conducted with eighteen patients who will also be observed during a treatment session with their healer. The explanatory models (EMs) as described by Kleinman (regarding etiology, symptom onset, pathophysiology, course, and treatment) that both healers and their patients have will be studied and compared. Content analysis of interview and observation data will be conducted using an inductive approach, by identifying themes and patterns that emerge. Taxonomies and cognitive maps will also be developed. Thus, the study will research the types of herbal and plant remedies, ritual, spiritual and body work that traditional healers employ to treat patients who also have been diagnosed by Western doctors with serious health problems. It will also compare for the first time, the Explanatory Models of indigenous Mexican immigrant patients with their traditional healers.
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{
"pile_set_name": "NIH ExPorter"
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[unreadable] Epigenetic mechanisms such as methylation may be very important in human disease, especially for diseases of older ages. Yet studies documenting epigenetic changes over the human lifespan, the vulnerability of epigenetic marks to environmental exposures, and the subsequent effects on human disease have been limited due to the absence of technical and biosample resources. A team has been assembled with the technology and resources to measure changes in methylation over time in the same individual for the purpose of examining a relationship between exposure and such changes, and assess the relationship between methylation and disease, by combining the epigenetic and epidemiologic expertise at Johns Hopkins with the resources of colleagues at the Icelandic Heart Association (IHA). The IHA has followed over 18K participants of the Reykjavik Heart Study since 1967 and has recently collected extensive cardiovascular and functional measures on 5758 of the surviving cohort as part of the AGES-Reykjavik study. Of these, 638 participants have DNA samples available for epigenetic measurement from two visits spanning 15 years, allowing for measurement of epigenetic changes over time in the same individual, which must occur if environment can influence epigenetic marks, but has never been directly observed in a human population. In addition, diet and smoking data and serum samples are available since 1967 as well as recent phenotype measures and disease outcomes for all 5758 AGES participants (2004-2005). The specific aims include assessing the: (1) Impact of environmental factors including dietary fish intake, serum folate levels, and smoking on inter-individual changes in whole-genome and candidate-gene specific methylation spanning 15 years; (2) Relationship between 15-year changes in methylation and subsequent disease-related quantitative measurements such as blood pressure, BMI, central adiposity, and coronary calcification; (3) Cross-sectional relationship between urinary cadmium (Cd) and candidate gene methylation; and (4) Cross-sectional relationship between candidate gene methylation and disease outcomes including CVD and cancer. This unique resource and team allows the first opportunity for longitudinal epigenetic measurement over 15 years in the same people, and connection of these epigenetic measurements to dietary and smoking exposures, as well as to cancer and cardiovascular disease consequences. [unreadable] [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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This research will determine the effects of acute infection with Plasmodium falciparum on the absorption, pharmacokinetics and metabolism of iron from iron supplements and other Iron preparations in women of childbearing age. Our project will combine measurements of iron absorption during and after successful treatment of acute uncomplicated falciparum malaria with characterization of the pharmacokinetics of the appearance of plasma non-transferrin-bound Iron (NTBI) and measurements of the iron reguliatory hormone, hepcidin, and other proteins of iron metabolism. We will examine iron supplements like those used in the Pemba supplementation trial as well as alternative iron interventions that could minimize or avoid the formation of plasma non-transferrin-bound iron. Our research will be based in southeast Asia, where nearly one billion people are now exposed to malaria and 25% of the world's clinical attacks of malaria occur. This project will take advantage of a unique convergence of resources and expertise at Mahidol University in Bangkok, Thailand: (i) the Institute of Nutrition, which has recently completed a series of stable isotope studies of iron absorption in collaboration with investigators from the Laboratory fpr Human Nutrition, Swiss Federal Institute of Technology (ETH), ZCirich, Switzerland, and (ii) the Bangkok Hospital for Tropical Diseases, Faculty of Tropical Medicine, a world-renowned malaria research facility with an established, decade-long alliance in studies of malaria with researchers at Columbia University, New York, N.Y., U.S.A. This research has three specific aims: (1) to characterize the pharmacokinetics of the appearance of non-transferrin bound iron in the systemic circulation after oral administration of an iron supplement or other iron intervention; (2) to determine the effect of acute uncomplicated falciparum malaria on absorption of iron from iron supplements and other iron interventions, using erythrocyte incorporation of stable:isotopes of iron; (3) to assess the effects of acute uncomplicated falciparum malaria on Iron metabolism by repeated measurements of serum hepcidin, transferrin receptor, ferritin, haptoglobin, and concentrations of pro- (Th-1) and anti- (Th-2) inflammatory cytokines, erythrocyte zinc protoporphyrin, and the complete blood count with absolute reticulocyte count and reticulocyte hemoglobin content (CHr). Because of the public health importance of assuring iron sufficiency in mothers, our studies are focused on women of childbearing age but the results should be broadly applicable to infants and children. RELEVANCE (See instructions): These studies of the effects of P. falciparum on iron absorption and metabolism will further our basic understanding of interactions of iron with malaria and other infections. Examining changes in plasma iron produced by conventional iron supplements could lead to development of new interventions tp maximize iron absorption and minimize risks associated with plasma non-transferrin-bound iron (NTBI). Our results could help improve the safety of prevention and treatment of iron deficiency in malaria-endemic regions.
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{
"pile_set_name": "NIH ExPorter"
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Aims: We are planning a clinical trial in which we randomly assign and then observe infants on soy formula with and without soy estrogens to see if their exposure to soy estrogen prolongs anatomical and biochemical evidence of estrogen exposure and response. The time course of these findings in infants is not well characterized, and detailed planning of the trial will require a substantial amount of pilot work.. Plausible effects include 1) anatomic expression of estrogen activity: breast buds, labial size, vulval anatomy, scrotal rugae, and, by ultrasound, small ovarian cysts and possibly prostate volume and 2) biochemical evidence of activity: decrease in gonadotropins, decrease in estrone/estradiol synthesis. We have begun planning the pilot studies. Term newborns have anatomic and sonographic evidence of in utero estrogen exposure. Over the first 6 months to a year of life, as the influence of maternal hormone wanes, these findings recede. Soy infant formula contains large amounts of isoflavones (daidzein and genestein) that can occupy estrogen receptor and act as estrogens in the laboratory. Circulating isoflavones in infants fed soy formula are at serum concentrations 3 or 4 orders of magnitude higher than estradiol, so that even if they are weak and readily excreted, there is so much that they should be active. Anatomic, sonographic, and other biochemical evidence of estrogen exposure have not been looked for in infants fed soy. Limited clinical trial data show increases in cholesterol synthesis consistent with an estrogen effect in infants fed soy formula vs. human milk or cow milk formula. An estrogen source in the diet might prolong the effect of maternal hormones, or interfere with hormonal homeostasis in the child. Accomplishments: This project is in the planning stage. We have reviewed the literature, contructed a preliminary protocol, completed external review, and has a planning meeting with outside experts.
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{
"pile_set_name": "NIH ExPorter"
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ABSTRACT Dr. Peyton Thompson is an Assistant Professor of Pediatric Infectious Diseases at the University of North Carolina (UNC) who is applying for a K08 Mentored Clinical Scientist Career Development Award. Dr. Thompson?s long-term career goal is to become an independent physician-scientist with expertise in vaccinology and the epidemiology of vaccine-preventable diseases in low-resource settings. The prevalence of hepatitis B virus (HBV) is unacceptably high in sub-Saharan Africa given that there is an effective vaccine against HBV. In order to design improved vaccination strategies to protect vulnerable individuals against HBV, we must first address two important HBV-related knowledge gaps: 1) Geospatial drivers of HBV infection in African children, and 2) HBV household transmission patterns in African children. This proposal will address these knowledge gaps using epidemiological data and dried-blood spot samples from a large national survey in the Democratic Republic of the Congo (DRC). This application will address the following specific aims: Aim 1) Locate hotspots of high HBV prevalence and low population-level immunity among Congolese children using existing DBS samples from a nationally representative survey; Aim 2) Identify patterns of HBV transmission at the household level using phylogenetic approaches; and Aim 3) Estimate the impact of targeted vaccination strategies in the DRC using modeling techniques. These aims align with Dr. Thompson?s career development goals: 1) Training in epidemiological and spatial analyses of large data repositories; 2) Training in vaccinology; and 3) Training in impact modeling. To achieve these goals, Dr. Thompson has assembled a highly qualified mentorship team comprised of: her primary mentor, Dr. Sylvia Becker-Dreps, a global health researcher with expertise in vaccine-preventable diseases; her co-mentor, Dr. Steven Meshnick, a skilled epidemiologist with vast experience in the DRC; an internal advisory committee consisting of experts in analysis of large data (Dr. Jonathan Parr), hepatology (Dr. Michael Fried), spatial analyses (Dr. Michael Emch) and vaccinology (Dr. David Weber). This multidisciplinary team of NIH-funded researchers has an impressive track record of training junior investigators toward independent research careers. Under their supervision, Dr. Thompson will achieve the research and career development aims in this proposal, and will be prepared to apply for an R01 to implement and evaluate spatially targeted vaccination strategies specific to the DRC.
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{
"pile_set_name": "NIH ExPorter"
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Nucleosomes are the fundamental DNA packaging units of eukaryotic chromatin. Dynamic interaction between DNA and histones in the nucleosome play important roles in controlling the structure of chromatin and subsequently the accessibility of genes, which is an essential part of gene regulation. Chromatin modification such as DNA methylation and histone acetylation is a crucial element in the mechanisms of gene regulation. Abnormal levels of DNA methylation or defective histone acetylation lead to various developmental/proliferative disorders including various types of cancer, leukemia and Rubinstein-Taybi syndrome. The long-term objective of this research is to elucidate how nucleosome dynamics contribute to the mechanisms of gene regulation through chromatin modification. The goal of this project is to reveal the link between nucleosome dynamics and chromatin modification. The aims are to test the main hypothesis that DNA methylation and histone acetylation may alter the dynamics of DNA wrapping/unwrapping around histones during nucleosome assembly/disassembly and consequently control the efficiency of nucleosome assembly/disassembly. Due to the difficulty associated with monitoring dynamic structural changes of a nucleosome based on ensemble-averaging measurements and static structural tools, the effects of chromatin modification on the nucleosome dynamics have never been clearly addressed. In order to accomplish these aims, we will employ single molecule multi-color fluorescence resonance energy transfer to monitor the real-time dynamics of DNA wrapping/unwrapping during nucleosome assembly/disassembly with and without the modifications in a single molecule level in a time resolved manner. Changes in the kinetic rates of DNA wrapping/unwrapping during nucleosome assembly/disassembly upon DNA methylation or histone acetylation will be examined in order to test the hypothesis. The proposed aims, when successfully accomplished, will greatly facilitate our understanding of the mechanisms of gene regulation by DNA methylation and histone acetylation from a dynamics perspective. Results from the project will provide a novel and unique basis for the development of diagnoses and treatments of the diseases and disorders originated from abnormal DNA methylation and defective histone acetylation. PUBLIC HEALTH RELEVANCE: Defects in DNA methylation or histone acetylation lead to lethal diseases and various developmental and proliferative abnormalities including various types of cancer, leukemia and Rubinstein-Taybi syndrome. The proposed project will make a novel and unique contribution to the development of diagnoses and treatments of the diseases and disorders associated with abnormal DNA methylation and defective histone acetylation.
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{
"pile_set_name": "NIH ExPorter"
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Are you hungry? Tired? Sad? These experiences all rely on interoceptive sensations sent from the body to the brain. Interoceptive sensations can be experienced as a homeostatic requirement of the body (e.g., need for food, drink, rest), or as an affective state characterized as feelings of pleasure-displeasure and with some degree of arousal. In turn, affect seamlessly integrates into normal perception and decision-making. Moreover, because sources of affect go unrecognized everyday life, it is easy to misattribute affect to a spurious source, which can lead to unhealthy behavioral patterns (e.g., discomfort from an argument at work can be mistaken as discomfort of hunger, and can lead to overeating). Furthermore, in addition to being central to healthy mental life, interoception and affective processing are severely altered in many mental diseases such as somatoform disorders, depression, anxiety, and addiction, and in physical diseases involving neuropathy such as diabetes. Although facets of interoception and affective processing have been correlated, there is a critical lack of knowledge regarding causality in these relationships. Here, this barrier is addressed experimentally by using two training approaches to enhance interoceptive sensitivity in healthy young adults, and by comparing pre- and post-training measurements of three facets of affective processing: (i) affective judgment of evocative images, (ii) physiological reactivity in response to evocative images, and (iii) the extent to whic affective feelings are misattributed during person perception. To enhance interoceptive sensitivity, Study 1 uses traditional heartbeat detection training, whereas Study 2 uses a novel approach, real-time functional magnetic resonance imaging neurofeedback, to enhance brain activity in the right anterior insula, which is involved in interoception. In both studies, interoceptive sensitivity will be assessed pre- and post-training using a heartbeat detection task. Furthermore, to tailor interoceptive sensitivity training to the needs of each individual, Study 1 explores two sets of training instructions that may modulate whether an individual experiences interoceptive sensations as either (a) emotional feelings or (b) physical sensations (e.g., an individual with somatoform disorder has an unhealthy bias in experiencing physical sensations and hence would benefit from emotional feeling training). Accomplishing these aims will inform future research designed to explore novel interoception-intervention strategies (e.g., based on the two approaches studied here) to prevent, detect, or treat mental illness, to address psychological effects of physical illness, and to promote healthy behaviors in everyday life.
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{
"pile_set_name": "NIH ExPorter"
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SUMMARY Squamous cell carcinoma (SCC) accounts for over 90% of head and neck (HN) cancer. HNSCCs in heavy smokers respond poorly to therapies and have the highest rate of relapse/recurrence among all HNSCC patients. Inhibitors of programmed death-1 (PD-1) are FDA approved to treat relapsed/recurrent HNSCCs, but are only effective in ~25% of HNSCCs, indicating additional immune suppressive/evasion mechanisms. We reported that transforming growth factor-?1 (TGF?1), an immune suppressor, is elevated in >60% of tobacco- associated HNSCCs. Unique to HNSCC, TGF?1 causes excessive inflammation with the majority of tumor infiltrating leukocytes being myeloid cells. Developing new therapeutic interventions that effectively target these tumor microenvironment (TME) characteristics is hindered by a dearth of HNSCC models with metastatic potential in an immune competent background. This application employs new therapeutic agents to target both PD-L1 and TGF? in HNSCC and metastasis, and analyze the underlying mechanisms. We have created a mouse model in which Smad4, a tumor suppressor frequently lost in tobacco-associated HNSCCs, is deleted (Smad4-/-) in head and neck epithelia. Smad4 loss causes SCC and metastasis, and compensatory TGF?1 overproduction. Preliminary data revealed that Smad4-/- SCCs also overexpress PD-L1 and short-term TGF? inhibition sensitized SCCs to anti-PD-L1. Further, in mice with SCC eradicated, re-transplanting the same SCC cell line failed to initiate new tumors, suggesting a memory T cell-dependent anti-tumor response. TGF? inhibition also reduced SCC lung metastases in immune compromised mice. Taken together, we hypothesize that attenuating a TGF?-induced immune suppressive and inflammatory TME in Smad4 mutant HNSCCs makes immunotherapy more effective, thus dual TGF??PD-L1 inhibition eradicates these HNSCCs via T-cell-dependent and -independent mechanisms. Aim 1 will perform experimental therapeutics using novel TGF?/PD-L1 inhibitor drugs on genetic mouse models and transplanted human HNSCCs to determine if Smad4 loss and TGF?1 overexpression predict therapeutic response to TGF?/PD-L1 dual inhibition in HNSCCs in immune competent and compromised conditions. Aim 2 will assess T cell- dependent mechanisms of TGF? inhibition on sensitizing or synergizing with anti-PD-L1-mediated SCC eradication, utilizing tumors generated in Aim 1 and patient HNSCC specimens to examine if Smad4 loss and TGF?/PD-L1 levels correlate with immune suppressive T cell profiles. Aim 3 will use tumors generated in Aim 1 to assess if myeloid cell-dependent targeting effects of TGF?/PD-L1 inhibition contribute to therapeutic efficacy in HNSCC and metastasis, and patient HNSCC specimens to examine if Smad4 loss and TGF?/PD-L1 levels correlate with increased myeloid cells and associated molecular markers. These studies will lead to a novel therapeutic strategy for HNSCC patients with high rates of recurrence and metastasis. Additionally, the mechanistic studies will offer novel insights into future biomarker-driven selection for future clinical trials of TGF?/PD-L1 dual inhibition in HNSCC patients.
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{
"pile_set_name": "NIH ExPorter"
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The long term goal of my research program is to understand the design of muscular systems. This involves performing experiments that facilitate the integration of information from molecular, cellular and whole animal studies. This approach is unusual and important because scientists generally work either on molecules, cells or whole animals but not on all three. This approach is crucial because there are large gaps in our knowledge of the principles by which animals use their muscles. An ultimate goal of this integrative approach is to understand enough about the molecular (e.g., crossbridges, Ca pumps) and macroscopic (e.g., joints, muscle arms) components of muscular systems, so that we can develop a comprehensive model that enables us to understand and predict how alterations in one parameter (e.g., crossbridge detachment rate) might affect motor performance. With the recent developments in biophysical, whole animal, and musculo-skeletal modeling techniques, we are for the first time in the position where we can relate molecular properties (e.g., crossbridge kinetics) to whole animal function in meaningful way. The frog, Rana pipiens, presents a superb model to proceed to this level. First, the jumping muscles are nearly pure in fiber type, and all the fibers are maximally recruited during jumping. Further, frog fibers are amenable to biophysical techniques, and jumping is amenable to biomechanical analysis. Our goal will be to 1) measure the sarcomere length changes and activation pattern of the major extensor muscles during jumping, 2) drive the isolated muscles through these in vivo length changes and stimulation pattern, and measure the resulting force and power output, 3) construct an anatomically and physically accurate model of the frog muscular system. This "virtual animal" will enable us to transduce our understanding of isolated muscle to whole animal locomotion and to test specific hypotheses about the design of the frog muscular system. 4) We will make a series of biophysical measurements on frog muscle fibers from which we will construct a simple molecular-based model of muscle which can be integrated into the overall model of the frog. We will then manipulate molecular properties in the model and observe the effect on jumping performance. Ultimately, elucidation of the principles of how healthy motor systems work may be useful in 1) understanding disease/injuries of the motor/cardiovascular systems, 2) designing computer systems for aiding movement in the handicapped, 3) designing pharmacological and genetic interventions for muscle disease states, 4) understanding the functional basis of training/ rehabilitation, 5) choosing appropriate skeletal muscle for heart pumping assist.
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{
"pile_set_name": "NIH ExPorter"
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The in vitro differentiation of clonal mouse embryonal carcinoma cells will be characterized biochemically using single cell assays for alkaline phosphatase, plasminogen activator, alpha fetoprotein and H2b antigen. The stability of the undifferentiated state will be determined by measuring the number of clones of different independently isolated embryonal carcinoma cell lines producing differentiated progeny from 1-7 days after plating. The early differential drug toxicity or cellular adhesion properties. The nuclear proteins of undifferentiated embryonal carcinoma cells will be compared by gel electrophoresis to those of primary cultures of early differentiated cell populations and to established endodermal cell lines. The possible induction of mesodermal derivates by primative endoderm will be investigated by co-cultivation of undifferentiated embryonal carcinoma and either primary cultures of endodermal cellular populations or established endodermal cell lines.
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{
"pile_set_name": "NIH ExPorter"
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Throughout history, placebo effects have been variously considered as tricks played upon the gullible by medical practitioners and powerful but mysterious healing forces. With the advent of direct measurements of human brain function, modern science has shown that placebo effects are neither of these. Rather, they reflect the principled impact of psychological and brain processes on diseases of the brain and body. Placebo effects represent an opportunity-because they provide a window into internal brain processes that influence health- and a challenge, because dozens of clinical trials have now failed to show the superiority of commonly prescribed drugs to placebo, at great cost to health care providers and consumers. This may not be due to the ineffectiveness of the medications, but rather seems to be related to high placebo response rates. Studies of the brain mechanisms underlying placebo effects can provide valuable insight into how the brain actively anticipates, responds to, and learns about active treatments, and the effects of these processes on pain and other aspects of health. In the previous period of this grant, functional magnetic resonance imaging (fMRI) of placebo and opiate drug effects was instrumental in establishing that placebo treatments impact the neurobiology of pain, including effects on the cortical and subcortical brain regions most closely associated with pain experience. The research also provided some of the first evidence on the interactions in the brain that give rise to placebo effects, and demonstrated the involvement of prefrontal cortical-subcortical-brainstem systems and the endogenous opioid system. The view that emerged is that placebo treatments engage brain systems related to both conceptual thought (explicit beliefs) and learning processes that change the value assigned to treatment cues and pain itself. In this Renewal application, we build on this foundation to address some of the many unanswered questions that need to be addressed before placebo effects are understood, harnessed in therapy, and separated cleanly from drug effects in clinical trials and practice. These include fundamental questions about a) the nature of the pain-related processes regulated by placebos (are they specific to pain, or general affective processes?) and b) the critical conditions required to create placebo analgesia. Our working hypothesis is that conceptual and learning processes interact, and that both are critical. Learning systems fundamentally shape the motivational value of pain and modulate nociception in subcortical-brainstem systems, and conceptual processes are needed to attribute perceived benefits to the placebo treatment and engage learning. To better understand these systems, we bring to bear new computational machine-learning tools that can identify and test placebo effects on patterns of brain activity specific to pain. We also test the involvement of several neurochemical systems-opioids, dopamine, and oxytocin-and their relationships with specific brain and psychological aspects of the placebo response-generation process.
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{
"pile_set_name": "NIH ExPorter"
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Immune deficiency following bone marrow transplantation or in AIDS leaves a patient highly susceptible to life threatening opportunistic infections. Recovery of the immune system, particularly T cell function, is often delayed for up to several years, especially in aged individuals. Thus far, while cytokines have been given clinically to accelerate myeloid recovery, very few agents have been demonstrated to promote multi-lineage immune cell recovery and fewer yet promote thymopoiesis and T cell recovery. Neuroendocrine hormones such as growth hormone (GH) and prolactin (PRL) have been demonstrated to exert pleiotropic effects on the immune system. We have previously demonstrated that GH and PRL promote T cell function and restore thymopoiesis in neuroendocrine hormone-deficient dwarf mice. This proposal will now evaluate the effects of GH and PRL on immune recovery in several BMT models, comparing the effects in both young (less than 10 weeks) and aged (greater than18 months) mice. Use of these mice will allow for the assessment on the role of age on the outcome following BMT. Toward this goal, we have developed 5 specific aims: Specific Aim 1 will assess the effects of GH and PRL on immune recovery after syngeneic BMT in young and aged mice. Particular emphasis will be given to effects on the thymus and T cell recovery. Specific Aim 2 will use a thymic organ culture model to determine the mechanism underlying the effects of hormones on the thymus. Specific Aim 3 will evaluate the effects of GH and PRL in allogeneic BMT models using T cell depleted BMC to ascertain effects on host rejection mechanisms and subsequent engraftment. Specific Aim 4 will examine the effects of GH and PRL on allogeneic BMT models in which graft-versus-host disease (GVHD) occurs to determine whether GVHD is worsened after hormone treatment. Finally, Specific Aim 5 will use nonmyeloablative BMT models to determine the effects of GH and PRL conditioning on thymic recovery and engraftment of donor-derived cells. All of the Specific Aims will use young and aged mice to ascertain if age significantly impacts hormone responsiveness. Thus far, even though immune reconstitution in the aged remains a significant and growing concern, very few preclinical studies have addressed the role of age and potential use of neuroendocrine hormones to circumvent the hampered immune recovery associated with age. This proposal will thus address both basic science (role of hormones in immune development) and clinically useful (role of age in BMT procedures and use of hormones to promote recovery) issues.
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{
"pile_set_name": "NIH ExPorter"
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Unraveling the genetic basis of common polygenic diseases, such as hypertension, diabetes and heart failure, will require fresh approaches to view how genes work together in groups rather than singly. In this proposal, we investigate gene network analysis as a promising new approach. Our goal is to identify specific expression patterns of gene modules, rather than single genes, which predict susceptibility to heart failure (HF). A network analysis of DNA microarray data typically groups 20,000 genes into 20-30 modules, each containing 10's to 100's of gene, drastically reducing number of possible candidates required to perform a gene network- based Gene Module Association Study (GMAS), which will be complementary to GWAS. To test the GMAS concept, we will use a systems genetics approach integrating DNA microarray analysis with physiological studies and computational modeling, to examine whether gene module expression patterns predict susceptibility to heart failure (HF) induced by cardiac stress. For this purpose, we will utilize a novel resource developed at UCLA, the Hybrid Mouse Diversity Panel (HMDP), consisting of 102 strains of inbred mice from which a common mouse cardiac modular gene network comprised of 20 gene modules has been constructed. Our preliminary findings reveal that different HMDP strains show considerable variability in both gene module expression patterns and phenotypic response to chronic cardiac stress (isoproterenol). Using biological and computational experiments, we will test the hypothesis that gene module expression patterns among HMDP strains represent different good enough solutions, all of which are adequate for normal excitation-contraction- metabolism coupling, but have different abilities to adapt to chronic cardiac stress. Three Specific Aims integrating experimental and computational biology and combining discovery-driven, hypothesis-driven, and translational elements are proposed, towards the goal of relating HMDP results directly to human heart failure.
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{
"pile_set_name": "NIH ExPorter"
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Stroke is the third leading cause of death in the United States and afflicts over 700,000 Americans each year. Treatment of acute ischemic attacks focuses on early intervention to minimize the degree of tissue loss in the brain. Magnetic resonance imaging (MRI) using proton based diffusion and perfusion techniques, has become an essential clinical service for evaluating the degree of stroke pathology. The capabilities of MRI in stroke evaluation would be enhanced by including sodium imaging. Tissue sodium concentration measured by sodium MRI would complement diffusion and perfusion MRI data by rapidly and directly evaluating regional tissue viability. Such data allows the identification of the volume of tissue that may be salvageable to be balanced objectively against the risk of hemorrhagic complications of thrombolysis by reperfusing already non-viable tissue. The present Phase I SBIR application proposes to develop a dual-tuned sodium/proton radiofrequency head coil at 3.0 Tesla for use in clinical service. This technology will be developed in three steps: 1) multi- conductor transmission line and finite element methods will be used to numerical simulate and optimize the electrical circuit design; 2) industry-standard mechanical CAD programming will be used to generate a 3-dimensional model of the coil and circuit layout, and 3) the coil will be evaluated for SNR, sodium quantification, spatial resolution, B1 homogeneity and acquisition times acceptable for sodium and proton imaging at 3.0 Tesla. The development of a robust, sodium/proton dual-tuned, head coil would help with the management, image acquisition, and diagnosis of critically ill stoke patients. The proposed technology will be used with MRI in a clinical service to diagnose, treat and manage critically ill stroke patients. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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With each heartbeat, a small amount of calcium entering the cell through membrane channels triggers the release of a larger amount of calcium from intracellular stores. The resulting increase in intracellular calcium has the dual effects of both: 1) enabling contraction and 2) influencing the ionic currents that shape of the action potential. Improper transport of calcium in cardiac myocytes can therefore contribute to both impaired ventricular function and lethal cardiac arrhythmias in disease states such as heart failure. Studies suggest that unstable calcium regulation and arrhythmias are associated with increased leak from intracellular stores to the cytosol. In particular, the inherited disorder catecholaminergic polymorphic ventricular tachycardia (CPVT) results in both increased leak and dangerous ventricular arrhythmias triggered by spontaneous release of calcium. This disease therefore represents an obvious example of the close links between ion transport and electrical signaling in heart cells. However, the mechanisms by which calcium link can increase arrhythmia risk remain unclear. We hypothesize that, because of the inherent complexity of calcium signaling and competing effects within cardiac myocytes, leak is only deleterious under certain conditions. A combination of innovative experiments and computational modeling will quantitatively determine the factors that control calcium leak and define the boundaries of when leak is dangerous and when it is protective. Together the studies proposed will yield significant insight into the factors that influence arrhythmia risk in CPVT and in heart failure. The project can be sub-divided into the following Specific Aims: Aim 1: Determine, in healthy cells, the factors that control calcium leak. Aim 2: Determine the mechanisms underlying increased leak in heart failure. Aim 3: Determine the mechanisms by which altered gating of ryanodine receptors can increase the risk of arrhythmia despite reduced sarcoplasmic reticulum calcium content. The work will provide fundamental new information concerning both the normal regulation of Ca2+ in healthy heart cells and the defects that occur in pathology. By developing a quantitative framework for understanding normal and defective calcium release, these studies can help identify thoughtful targets for cardiac therapies
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{
"pile_set_name": "NIH ExPorter"
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DESCRIPTION (provided by investigator): Although public policies continue to be implemented to minimize Environmental Tobacco Smoke (ETS exposure) in the work place and in public places, ETS exposure in the home, where children are most likely to be exposed, remains an issue of great concern. We are proposing to develop a comprehensive program to promote and assist healthcare providers in counseling and advising patients how to reduce ETS exposure in the home. Our product consists of a CME training component, a kit of performance support tools and procedures for providers and staff to use to initiate and follow up on ETS interventions, a self-help workbook for providers to give to patients and their families, and an online patient support component. We plan to design a "moderate intensity" intervention model of clinician-provided advice and brief counseling, adapted from more extensive services/products used in previous efficacy studies. For many years now, clinical interventions for smoking cessation have been developed. To our knowledge, there are no comparable intervention programs that target ETS exposure, although a substantial amount of clinical trial data indicates the efficacy of clinical interventions for ETS exposure reduction. We believe the results from these studies are sufficient to warrant transferring clinical interventions from an experimental setting to the field. The aim of this SBIR Phase I project is to develop a pilot version of the proposed residential ETS reduction program, and then to conduct a formative evaluation of the pilot product. We will focus our efforts on obtaining extensive feedback from providers and patients that will allow us to fully elaborate and refine our product. This feedback will guide the tailoring of the program components to make them suitable for integration into clinicians' daily office practice and potentially more effective and less expensive than what has been done in earlier efficacy studies. The information we obtain in this formative evaluation will inform the design and development of the final product during SBIR Phase II, and the Phase II research design. The end goal of the proposed program, to be evaluated in Phase II, is reduction of residential ETS exposure and smoking cessation. Related objectives include changing provider behavior to increase the rate and effectiveness of ETS exposure interventions, and changing patient behavior to reduce ETS exposure or to stop smoking entirely. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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Both the increased incidence of serious infections and increased death rate from pneumonia are known in chronic alcoholics. A wide variety of immune defects are well known in these individuals. Alcohol being a powerful immnosuppressive agent is known to influence both the humoral as well as cell-mediated immunity. Interferons are best known as immunomodulators. Nothing is known at the present regarding the status of the interferon system in chronic alcoholics. We will identify two groups of individuals: one group of chronic alcoholics admitted to the detoxification center at the Erie County Medical Center (ECMC); and the second group of moderate smokers as particiants of the Smoke Cessation Program at Roswell Park Memorial Institute (RPMI). Participants will be selected based upon guidelines established by NCI for smokers and by the Michigan Alcoholism Screening Test for chronic alcoholics. The following interferon parameters will be measured: 1) circulating serum interferon levels; 2) ciruculating serum interferon inactivator levels; and 3) inducibility of their WBC for interferon alpha and gamma. Identity (alpha, beta or tau) of serum interferon and presence or absence of acid-labile alpha interferon will also be determined. All methodologies for interferons studies are standard and are routinely carried out in our laboratories. These interferon parameters will be determined twice among chronic alcoholics - first when they are admitted to the detoxification center at ECMC and for the second time on a subgroup of these individuals who will be admitted to the Alcohol Treatment Program at ECMC. This will allow us to determine the imporvements in the interferon system in patients after a month of their succesful treatment for alcoholism. In order to clarify the role of alcohol consumption, a control group of smokers is include in our study primarily because nearly all alcoholics are moderate to heavy smokers. Measurements will be taken from three sources: 1) patient receords; 2) Michigan Alcoholism Screening Inventory; and 3) interferon parameters on blood samples. Data analysis will be conducted at the New York State Research Institute on Alcoholism in Buffalo. We have a VAX750 computer which is supported by standard data analytical statistical packages including SPSSX.
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{
"pile_set_name": "NIH ExPorter"
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CIDR is a valuable national resource for cutting-edge genetic research. CIDR was established at the Johns Hopkins University in 1996. CIDR provides high quality next generation sequencing and genotyping services to investigators working to discover genes that contribute to disease. On-site statistical geneticists provide valuable insight into analysis issues as they relate to study design, data production and quality control. Completed studies encompass over 200 phenotypes across 900 projects and over 1.2 million samples from over 300 different principal investigators located world-wide. The impact is evidenced by over 1,190 peer-review publications. The most recent contract for the NIH CIDR Program, High Throughput Genotyping and DNA Sequencing for Studying the Genetic Contributions to Human Health and Disease, was awarded
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{
"pile_set_name": "NIH ExPorter"
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The purpose of the proposed research is to test a theory of age differences in processing resources for semantic and episodic memory. Salthouse has hypothesized that processing speed is a processing resource. Allen has proposed that neural noise as measured by entropy provides an even more basic measure of age differences in processing resources. Specifically, increased neural noise reduces the available processing resources for cognitive processes. The goal of the present project is to determine why differential age differences occur for episodic and semantic memory tasks across peripheral and central processing stages by examining whether practice and/or task type affects these age differences. The present proposal contains eight experiments that are designed to determine how entropy (a measure of neural noise), processing speed, and processing accuracy are related to age differences in processing resources across different processing stages (peripheral vs. central) and tasks (episodic vs. semantic memory). Experiments 1-2 will examine the time-accuracy functions of healthy young and older adults on lexical decision and memory search tasks that vary exposure duration. Accuracy data will be used to derive entropy values. Experiments 3-4 will use Pashler's psychological refractory period paradigm to examine age differences in processing resources as the stimulus onset asynchrony is varied between two tasks (Experiment 3 will use dot location followed by a lexical decision task: Experiment 4 will use dot location followed by lexical decision or memory search tasks). Experiment 4 will also examine the effect of practice on age differences in processing resources by using four sessions per participant. Experiments 5-8 will consist of four within subject experiments in which the same stimuli (words or numbers) are used for both semantic and episodic memory tasks. Experiments 5-8 will rule out stimulus confounds that were present in earlier studies on this topic.
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{
"pile_set_name": "NIH ExPorter"
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Three dimensional (3D) fluorescence microscopy is a powerful tool for the study of living specimens. 3D images are normally obscured by out-of-focus blur and often artifactual due to the limited band of spatial frequencies that the conventional fluorescence microscope can image. These problems can be ameliorated by optical and/or computational methods. A recent renaissance in optical microscopy has led to a wide variety of approaches to overcome the out-of-focus blur and band-limitation (confocal and partially confocal scanning, 2- and 3-photon fluorescence excitation, standing wave, structured-fluorescence-excitation microscopes..). These approaches improve the imaging properties, but they are not blur-free and computational approaches further improve image quality. Computational methods have gained acceptance as an alternative or complement to optical methods, but their development has not enjoyed the fast pace of their optical counterparts. Few commercially-available deconvolution packages exist, and most are based on the first few algorithms developed for 3D deconvolution, including ad-hoc and unconstrained methods unable compensate for the band-limitation of microscopes. Significant improvements are possible with methods based on a thorough mathematical model of the process of image formation and recording. The long-term goals of the proposed research are: 1) To further develop computational algorithms that account for the most significant sources of degradation in the image. 2) To develop computational image estimation methods for the newly-developed structured illumination microscope. 3) To provide guidelines for the use of the algorithms and for their capabilities and limitations. To achieve this goal we will first derive algorithms based on an accurate model for the microscope and light detector then we will do a thorough evaluation of these and other algorithms to assess their relative merits and to provide guidelines for their use.
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{
"pile_set_name": "NIH ExPorter"
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The long range objective of this proposal is to further our understanding of the molecular basis for sickle cell anemia with particular emphasis on examining genetic factors that contribute to the variable clinical expression of the disease. The specific goals are: (1) to characterize sickle cell disease and the factors which impact on its severity in our recently developed mouse model of sickle cell anemia, whose red cells contain exclusively human HbS; and (2) to apply genome-wide mapping tools and quantitative trait analyses to identify genetic loci unlinked to the globin cluster that modify the severity of sickle cell disease. Our first goal relates to the transgenic/gene knockout mouse model of sickle cell anemia which we generated during the previous funding period. These animals, possessing exclusively human hemoglobin HbS and no adult murine hemoglobins, are born with extensive red cell sickling and associated morbidity. We will exploit this unique model to perform detailed investigations of the pathophysiology of the disease, unavailable for study in humans. We will examine the effectiveness of agents predicted to lessen disease severity. These studies will analyze factors which have an impact on gamma-globin gene expression (i.e., hydroxyurea) and vascular tone (i.e., nitrous oxide). The second goals will employ a genome-wide scan to identify loci that alter sickle cell disease severity and globin chain switching using inbred strains of mice. These studies are divided into two parts. They will identify modified loci, using different mouse genetic backgrounds, that: (1) affect the severity of murine sickle cell disease resulting from the expression of the same human sickle transgene; and (2) affect gamma-to beta-globin switching of the same human beta-globin YAC transgene. In both of these studies, we seek to identify genes unlinked to the globin cluster in mice which are likely to impact on the severity of sickle cell disease in humans. The studies are anticipated to contribute to our improved understanding of the molecular mechanisms responsible for variable clinical expression of sickle cell disease.
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{
"pile_set_name": "NIH ExPorter"
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PROJECT SUMMARY/ABSTRACT It is now well established that epithelial cancers arise in a multi-decade process from precancerous lesions. Esophageal adenocarcinoma, a cancer whose incidence has risen 5-fold since 1950, initiates with precancerous lesions known as ?Barrett's esophagus?, progresses to dysplasia, and finally emerges as malignant esophageal adenocarcinoma along a path that parallels the Correa Sequence for gastric adenocarcinoma. As the average life expectancy of patients diagnosed with esophageal adenocarcinoma is approximately one-year, considerable efforts are underway to define its Correa sequence, and especially its pre-malignant stages, as potential targets for interdiction ahead of the onset of malignant disease. Toward this end, we have applied novel stem cell cloning technologies originally developed in our laboratories for normal human gastrointestinal tract stem cells to reconstructing the Correa sequence in patients with early esophageal adenocarcinoma. Significantly, each of these patient-matched endoscopic biopsies of Barrett's, dysplasia, and esophageal adenocarcinoma yields discrete populations of stem cells that respectively yield intestinal metaplasia, dysplasia (but not tumors), and aggressive adenocarcinoma following transplantation to immunodeficient mice. From a detailed molecular genetics analysis of nearly 100 independent clones from across the Barrett's, dysplasia, and adenocarcinoma lesions of one patient, we have been able to reconstruct, at unprecedented resolution, both the direct phylogenetic sequence that led to this tumor as well as identify ?dead-ends? at both the Barrett's and dysplasia stages that did not contribute to the final tumor. Moreover, each of the cloned stem cells of Barrett's, dysplasia, and adenocarcinoma lesions represent permanent lines that have enabled powerful approaches to drug discovery that has culminated in leads that selectively target the entire Correa sequence while sparing normal esophageal stem cells. In three specific aims, we will 1.) clonally reconstruct the Correa sequence from 10 patients with early esophageal adenocarcinoma; 2.) establish high-throughput screens involving co-cultures of normal esophageal and Correa sequence stem cells for lead discovery; and 3.) develop in vivo xenograft models of patient-matched normal esophageal and Correa sequence stem cells for validating lead combinations targeting these lesions. Based on extensive preliminary studies, we anticipate the analysis of patient-matched stem cells of these progressive lesions will provide fundamental insights into the evolution of esophageal adenocarcinoma and as well as epithelial cancers in general. From the standpoint of filling important gaps in patient care, the drug discovery enabled by these sets of lesional stem cells offers promising and novel interventions to prevent to onset of esophageal adenocarcinoma as well as ones to address disease that has already taken hold.
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{
"pile_set_name": "NIH ExPorter"
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Antiretroviral therapy is finally reaching millions of persons with HIV infection in Africa. While countless lives have been saved, it is becoming increasingly clear that deaths in the first 6 months on ART are dramatically elevated as compared to industrialized settings, - perhaps claiming as many as five-fold more than in industrialized settings. Even these alarming estimates, however, are likely underestimates because high losses to follow up in resource limited settings mean that many deaths are not ascertained. The causes of these early deaths, furthermore, are not well explained. Therefore the proposed Aims seek to (1) evaluate a sampling-based technique that we have developed to understand the true magnitude of early mortality; (2) determine whether undiagnosed opportunistic infections are the cause of early mortality on ART by collecting biological specimens and investigating them intensively in San Francisco and (3) develop a clinical prediction rule to risk stratify individual patients for early mortality at the time or ART initiation. Overall, this study seeks to understand the magnitude and determinants of early mortality on ART in HIV infected patients in Africa in order to develop a targeted intervention in the future to reduce it. These Aims will also provide a platform for completing further career development toward independence as a scientist. RELEVANCE: Early mortality on ART may claim 1 in 10 patients of the millions of patients starting ART in Africa. These deaths are likely due to treatable infections. Therefore, a better understanding of early mortality on ART in Africa has the potential to improve ART delivery and save many lives.
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{
"pile_set_name": "NIH ExPorter"
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The objectives of this proposal are to study the factors controlling hematopoiesis in normal man and patients with malignant hematologic diseases. Specific objectives are (1) the purification and characterization of colony-stimulating activity to determine whether it is or is not a physiologic regulator of granulopoiesis, or merely a factor required for in vitro growth of myeloid stem cells; and (2) the critical examination of substances derived from mature granulocytes which are purported to have feedback regulatory activity (so-called "chalones").
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{
"pile_set_name": "NIH ExPorter"
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DESCRIPTION (PROVIDED BY APPLICANT: The goal of this project is to determine whether pharmacologic induction of Mrp3 can be used as a means of hepatoprotection in humans during cholestatic disease states. Mrp3, which is normally expressed at low levels, serves to export a wide range of organic anions from the liver, back to the blood, thereby decreasing their exposure and toxicity to the liver. Mrp3 expression is significantly increased both in response to certain microsomal enzyme inducers as well as during cholestasis in rodents. Human expression of Mrp3 is also increased in certain liver diseases. Mrp3 expression can be pharmacologically increased by certain microsomal enzyme inducers in both mouse and rat. However, the mechanism for the induction of Mrp3 appears to be a complex interaction where transcriptional studies performed in vitro are unable to accurately replicate the induction observed in vivo. Aims 1 and 2 are designed to determine whether human Mrp3 expression can be pharmacologically induced by this same mechanism. These are;1. Determine the common mechanism for the cholestatic and pharmacologic induction of Mrp3 expression. 2. Determine whether the human Mrp3 gene can be induced in vivo using a unique transcriptional activation assay and human liver slices. Cholestasis is a very complex disease with multiple etiologies that results in the accumulation of bile acids and other organic anions that cause profound hepatocellular damage. If Mrp3 can be pharmacologically induced in humans as a means of eliminating hepatic exposure to the toxic effects of accumulating bile acids and other organic anions, this would represent an important means of hepatoprotection during cholestasis. Aims 3 and 4 have been designed to determine whether Mrp3 induction can be used as a legitimate drug target in the treatment of human liver disease. These are;3. Determine whether microsomal enzyme inducers that increase expression of Mrp3 are capable of altering the disposition of biliary constituents and protecting against hepatotoxicity during cholestasis. 4. Determine whether the transgenic overexpression of human Mrp3 is hepatoprotective in models of cholestasis.
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{
"pile_set_name": "NIH ExPorter"
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The overall purpose of this project is to develop improved statistical techniques for using data from animal carcinogenicity tests conducted at high dose levels to estimate long-term human risk from chemical carcinogens at very low dose levels. Current research efforts have focused on such topics as the evaluation of competing risks and synergistic effects in the framework of multistage models for carcinogenesis as well as the question of species-to-species extrapolation.
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{
"pile_set_name": "NIH ExPorter"
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The proposed study attempts to explore cultural and social adjustment patterns of Korean immigrants in the Chicago area by using data collected among a sample of 500 Korean immigrants. In addition to simply describing the patterns, this study will test the following specific hypotheses for analytical purposes: (1) Social assimilation occurs concomitant with or subsequent to cultural assimilation; (2) Socio-economic status is positively related to the degree of cultural assimilation; (3) Socio-economic status is not related to the degree of social assimilation; (4) Status inconsistency affects the degree of both cultural and social assimilations; and (5) There is no direct linear relationship between the length of sojourn in the United States and the degree of cultural and social assimilation. Cultural and social assimilations will be measured by a series of attitudinal and behavioral items related to the immigrants' social interactional patterns and their adoption of American cultural traits. Analysis of variance will be used to test the hypothesis concerning the differential between cultural and social assimilations. The hypothesized additive effects of status variables on cultural and social assimilations may be ascertained by using multiple-correlation analyses. The hypothesis of status inconsistency will be tested by employing dummy-variables multiple regressions to isolate status additive effects on assimilation from inconsistency effects. To test the hypothesis concerning the effects of the length of sojourn in the United States on cultural and social assimilations, scattergram and correlation analysis may be used to find the nature and degree of the relationship between the two variables.
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{
"pile_set_name": "NIH ExPorter"
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Structure-function studies of the cellular retinaldehyde-binding protein (CRALBP) are proposed that focus upon functional domain analysis and development of protein preparations suitable for three dimensional structural analysis. The hypothesis of the proposal is that CRALBP plays a fundamental role in the metabolism of vitamin A in the visual cycle. CRALBP is a substrate carrier protein found only in visual tissue that carries 11-cis-retinol and 11-cis-retinaldehyde, retinoids that are only known to function in vision. The protein appears to play a regulatory role, influencing whether 11-cis-retinol is stored as retinyl ester in the retinal pigment epithelium (RPE) or oxidized to 11-cis-retinaldehyde and exported for visual pigment regeneration in photoreceptor cells. CRALBP stimulates oxidation and retards esterification in vitro, suggesting that the protein may help control retinoid flow at a key metabolic branch point in the visual cycle. A defect in the synthesis, regulation or structure of CRALBP could compromise vitamin A metabolism, resulting in impaired vision. Functional domain analysis will utilize human recombinant CRALBP and apply amide hydrogen/deuterium exchange and mass spectrometry for topological analysis to facilitate identification of buried domains associated with retinoid-binding and exposed regions involved with protein-protein interactions. Photoaffinity-labeling of rCRALBP will be performed to identify the retinoid-binding pocket and the crosslinked sites determined by Edman degradation and electrospray mass spectrometry. Targets for site-directed mutagenesis will be based upon results from these approaches and changes in specific amino acids used to define residues critical for retinoid-binding and interaction with 11-cis-retinol dehydrogenase. Limited alanine scanning mutagenesis will also focus around two residues in a random rCRALBP mutant that has lost the ability to bind retinoid. A procedure will be developed for crystallizing human rCRALBP for the long term goal of 3D structural determination by crystallography. Limited proteolysis will be used to identify the smallest fragment capable of binding retinoid. The retinoid-binding fragment will also be produced as a recombinant protein for solution structural analysis by NMR. The substrate carrier efficacy of proteolysis fragments and mutants will be assayed using bovine RPE microsomes as the source of 11-cis-retinol dehydrogenase. For comparative purposes, native bovine GRALBP will be used as a structure-function reference. The unifying hypothesis of the research is that by establishing a molecular basis for understanding the normal structure, function and regulation of CRALBP, questions concerning visual disorders with which the protein may be associated can be answered in specific terms.
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{
"pile_set_name": "NIH ExPorter"
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Whereas COS-1 cell-expressed human UDP-glucuronosyltransferase (UGT)-2B17 metabolizes primarily dihydrotestosterone (DHT) and its androstane-diol (ADT-DIOL) metabolite, we have evidence the prostate-distributed isozyme requires regulated phosphorylation by its reversible and irreversible inhibition following treatment with curcumin or general PKC inhibitors, respectively. The fact that treatment of COS 1-expressed 2B17 with PKCalpha-siRNA downregulates activity or treatment with PKCepsilon-siRNA, Src-siRNA or Src kinase-specific PP2 inhibitor upregulates its activity indicates PKCalpha activates 2B17 and that PKCepsilon and/or Src downregulate activity. PKC-site mutants--S172A-2B17 and S422A-2B17-- are null and unchanged, respectively; tyrosine kinase-site mutants --Y99F-2B17 and Y237F-2B17-- show preferential inhibition of DHT over ADT-diol glucuronidation at the 70/30 and 90/60 percent levels, respectively-- a phenomenon that converts to essentially 50 to 60/70 percent following expression in Src-free cells. Solubilized 2B17-His-transfected Src-free fibroblasts subjected to in-vitro 33PATP-dependent phosphorylation by PKCepsilon and/or Src followed by affinity purification, and SDS-gel analyzed revealed higher radiolabeling of 55- to 58-kDa 2B17-His compared to alpha and/or Src. Activity for all combined kinases reactions were additive. Collectively, evidence indicates 2B17 requires regulated phosphorylation by PKCalpha, PKCepsilon and Src, which is consistent with the complexity of synthesis and metabolism of its major substrate, DHT. Evidence indicates that the 95% identical prostate basal cell-distributed UGT2B17 and luminal cell-distributed 2B15 are overall inversely programmed by phosphorylation. Related manuscript: Combined positive PKCalpha and negative PKCepsilon and Src effects on regulated phosphorylation of human dihydrotestosterone-metabolizing UGT-2B17 depress its constitutive activity-- N.K. Basu, M. Basu, J. Park, R. Banerjee and I. S. Owens, J. Biol. Chem. 2B17 Under Revision- Human prostate basal cell distributed UDP-glucuronosyltransferase-2B17 (UGT-2B17) and its 97%-identical UGT-2B15 homolog metabolize both dihydrotestosterone (DHT) and its androstane- diol (Andro) metabolite. Mass spectrometry has confirmed UGT-2B17 has 4 of 5 predicted phosphorylation sites in luminal-cell-distributed UGT-2B15; dissimilarly, 2B17 contains the rare tri-phosphorylated-TYS sequence at position 98-100. Both isozymes undergo regulated phosphorylation for two different functions. Like UGT-2B15, anti-PKCalpha-immunocomplexed UGT-2B17-S172 confirmed phosphate-signaling enables a non-fixed active-site that catalyzes an unspecified number of substrates based on analysis of 6/19 human UGTs. Contrariwise, anti-PKCepsilon immnocomplexes of 2B17 generated dense smearing patterns, except for its Y99F mutant or following expression of constructs in Src-free cells. Hence, previously identified robust and critical signaling by the Src/PKCepsilon-partnership phosphorylation site, TYS, located at position 98-100 in 2B17 suggests its critical role. Following 2B17 expression in Src-/- versus +/- cells, glucuronidation of DHT versus its Andro metabolite indicates Src inhibits both by 50 %, which necessarily elevates anti-apoptotic DHT levels concomitantly. Exchanges of IYG in wt-UGT-2B15(IYG) and TYS in wt-UGT-2B17(TYS) at positions 98-100 followed by expression in COS-1 and PC3 cells enabled mt-UGT-2B17(IYG) to generated 4 to 10-fold greater in-cellulo caspases 8/3 activations over wt-UGT-2B15(IYG), while mt-UGT-2B15(TYS) suppressed activation of caspases 8/3 over 50% of wt-UGT-2B15 (IYG) levels. Treatment of LNCap cells that contain endogenous UGT-2B15/UGT-2B17 with Src inhibitor, curcumin, enabled a cycle of apoptosis and recovery within 16 h. Combined, evidence indicates the triple-phosphorylated TYS creates a signaling site involving Src and PKCepsilon that is anti-apoptotic, while the pro-apoptotic Src-specific binding/phosphorylation site, IYG, in UGT-2B15 at position 98-100 is blocked by Src. SIGNIFICANCE- This report provides evidence human prostate luminal-cell distributed DHT-metabolizing UGT-2B15 uses programmed phosphorylation-based signaling to carry out luminal-cell specific apoptosis. Contrariwise, basal-cell-distributed UGT-2B17 -- contained within a Basal-cell Compartment that houses intermediate stem cells with both basal- and luminal cell-surface cytokeratin markers-- utilizes programmed anti-apoptotic signaling to protect this population of cells. For the first time, we have identified the luminal-cell specific UGT-2B15 as the pro-apoptotic agent that removes challenged luminal cells before transformation ensues. Moreover we have identified basal-cell specific UGT-2B17 as the anti-apoptotic agent that protects intermediate stem cells containing both basal- and luminal- cell surface markers that replace the departed luminal cell and gives rise to a new basal cell. These findings indicate UGT-2B15 controls luminal transformation and that UGT-2B17 controls continuity of the prostate. Endogenously expressed UGT-2B15/-2B17 in LNCap cells show a cycle of apoptosis and recovery within 16 h after treatment with Src-inhibitor, curcumin. Evidence indicates the triple-phosphorylated TYS creates an anti-apoptotic Src / PKCepsilon signaling site, while pro-apoptotic Src-specific IYG signaling site in UGT-2B15 is blocked by Src phosphorylation. Hence, UGT-2B15 and -2B17 appear to be unique clinical markers. Related manuscript:Identification of a Pro-apoptotic and Anti-apoptotic Controlling Sequence in Human Prostate-distributed DHT-metabolizing UGT-2B15 and UGT-2B17 Respectively Submitted to: Proceedings of the National Academy of Science on 09-2014. In order to establish an in-vivo mammary-gland model that prevents depurination by 4-OH- catecholestrogens associated with initiation of carcinogenesis, we pursued studies to identify mouse homologs of highly effective human UDP-glucuronosyltransferase-2B7. By sequence analysis, we identified mouse Ugt-2b34 and -2b36 homologs that avidly metabolize test-agent 4-OH estrone with 2b35 expressing trivial activity. Unlike low 14M-Km 2B7, 2b34 and 2b36 metabolized 4-OH estrone with 90- and 430-M Km, respectively. Unexpectedly, the isozymes are distributed primarily in male hormone-responsive tissues, whereas 2B7 is found primarily in female hormone-responsive tissues. Also, we found 2b34 metabolizes the non-classical-estrogenic dihydrotestorerone-metabolite, androstane--diol, of DHT at a rate greater than for DHT, which is not known to be estrogenic. Notably, 2B7 essentially failed to metabolize xeno-estrogens; alternatively, 2b34 and 2b36 metabolized BPA and DES at superior rates. Finally, real-time PCR-based analysis of estrogen receptor alpha (ESR1) gene knockout in mouse prostate showed 50% and 63% decreases in Ugt2b34 and Ugt2b36 mRNA, respectively, compared to controls. Contrariwise, estrogen receptor beta (ESR2) k.o. revealed a 2.7/3.3-fold increase in 2b34 and 2b36 mRNA, respectively, in prostate. ESR1 k.o. completely suppressed 2b34 and 2b36 mammary-gland messages; ESR2 k.o. caused a 12-fold increase in 2b34 message without affecting 2b36. Hence it appears males benefit from both Ugts, while females benefit from only one Ugt, according to tissue-distribution studies. Findings for Ugt-2b34 and Ugt-2b36 suggest the two mouse isozymes are intrinsically programmed to protect against a more complex environment than that by human high-activity UGT2B7 and low-activity UGT2B4 and their known distribution patterns. Related manuscript:Prostate-distributed Ugt2b34 and Ugt2b36 Control Estrogenic Metabolites Derived from both Estradiol and DHT in Mice, A. Raychoudhuri, S. Jana, M. Basu, N. K. Basu, and I. S. Owens. Bioscience report (In Press)
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{
"pile_set_name": "NIH ExPorter"
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[unreadable] The Maryland Science Center (MSC), in collaboration with Johns Hopkins University (JHU), The University of Maryland, Baltimore (UMB), and Morgan State University (MSU), requests a Phasel/ll five year grant of $1,381,827 to develop CELLULAR UNIVERSE: The Promise of Stem Cells, a unique exhibition and update center with related programs that highlight the most current science in cell biology and stem cell research. Visitor surveys have shown that science museum visitors are very interested in learning about stem cell research, but know little about the science of stem cells or cell biology which forms the basis of stem cell research. The goal of this project is to help visitors learn about advances in cell biology and stem cells so that they will make informed health-related decisions, explore new career options and better understand the role of basic and clinical research in health advances that affect peoples' lives. Topics to be covered include the basic biology of cells, the role of stem cells in human development, current stem cell research and the clinical research process. This exhibition will also address the controversies in stem cell research. Our varied advisory panel, including cell biologists, physiologists, adult and embryonic stem cell researchers and bioethicists, will ensure the objectivity of all content. CELLULAR UNIVERSE: The Promise of Stem Cells will include interactive exhibits, a wet laboratory, updateable media center, and presentation space, and will be organized into four areas: [unreadable] 1. Cell World [unreadable] 2. The Cell Bio Lab [unreadable] 3. Stem Cells and Human Development [unreadable] 4. Stem Cells In the News/ Current Research and Issues [unreadable] Programs will be created to complement the exhibitions including: internship programs, live demonstrations, video conferencing with scientists, teacher programs, a website, and community outreach. [unreadable] The public health impact of the proposed project will be to create exhibits and programs that will help millions of science museum visitors make informed health-related decisions and serve as a model for creating engaging, unbiased science exhibitions on controversial health-related subjects like stem cell research. [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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The details of the mechanism for DNA melting are fundamental to many aspects of biology and biotechnology. Recently, it has been shown experimentally that there are at least three states involved even for relatively short structures. To help clarify the reaction coordinate and mechanism of the melting transition of DNA we propose to use theoretical and computational methods. We will simulate the melting process for both free oligomer duplexes and hairpins in explicit salt water at temperatures above the sequence specific melting temperature. Analysis of the trajectories will reveal the various biochemically important processes that occur on different time scales. Augmenting this with theoretical techniques we will use the coordinates to map out free energy surfaces. An outcome of this research will be to identify plausible reaction coordinates for the melting transition. How these differ for various geometries (free oligomers vs hairpins etc.) and sequences will be useful in identifying various relevant states and kinetic traps that occur during replication, transcription, recombination, and DNA repair as well as in biotechnological applications. PUBLIC HEALTH RELEVANCE DNA melting is the process by which double-stranded DNA separates into single strands by biological means or heat or the introduction of certain chemicals. Basic research into how DNA strands couple and uncouple has tremendous relevance to issues of public health as this mechanism is important for understanding DNA mismatches, disease expression, and associated gene therapies. Research into DNA melting has direct application to the development and treatment of catastrophic diseases such as cancer, diabetes, and other genetically inherited diseases, as well as the design of gene based diagnostic tools, like DNA microarrays.
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{
"pile_set_name": "NIH ExPorter"
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Secretory immunoglobulins. Using sheep colostrum as the model for exocrine secretions a study is being made of its immunoglobulin composition. Emphasis is given to a study of the presence of J-chains and secretory component in various classes of antibody. Antisera and physicochemical techniques are used for their detection and characterization. Human 13 S Immunoglobulin G and other M proteins. We are examining monoclonal IgG and IgM antibodies from plasma-cytomas. One such material is an IgM cryoglobulin with specific activity against secretory IgA. The nature of this interaction is under investigation. Another IgG myeloma is a 15 S aggregate which may require intact Fc regions for interaction.
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{
"pile_set_name": "NIH ExPorter"
}
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We have identified an estrogen-inducible protein which binds with high specificity to a segment of the 3'-untranslated region of the estrogen- stabilized mRNA encoding the egg yolk precursor protein, vitellogenin. Since this protein is widely distributed in different tissues of Xenopus laevis, is regulated by both estrogen and testosterone, and we have identified a human homologue, it likely plays an important role in steroid hormone control of mRNA degradation. Our approach is to clone, express and characterize the protein, to investigate its regulation by estrogen and other hormones, determine the precise sequence and structure of the mRNA binding site, identify additional mRNAs which bind the protein and investigate its role in the regulation of mRNA stability. The high specificity with which the protein binds to its recognition sequence, and our development of a nitrocellulose filter binding assay make direct screening of a cDNA expression library our preferred method for cloning and initial expression. If this proves impossible, we will use biochemical techniques including RNA affinity chromatography to purify the protein, obtain partial amino acid sequence, and use degenerate oligonucleotides to screen a cDNA library. The Xenopus cDNA clone will be used to isolate a human binding protein cDNA clone. Nucleic acid probes and antibodies to the expressed protein will be used to determine whether estrogen induces the mRNA and protein, or increases the affinity of pre-existing protein for its mRNA binding site. We will then carry out a more detailed analysis of the regulatory mechanism. To further characterize the mRNA binding site we will use an iterative in vitro selection method, RNase protection and mutagenesis. The in vitro selection provides an unusual approach to identifying a consensus mRNA binding site, and generating up-binding mutants of the binding site, and will facilitate modeling the secondary structure of the binding site. We will use the sequence and structure of the consensus binding site to identify other mRNAs whose stability is regulated which contain potential binding sites for the protein. We will determine the relationship between estrogen induction, binding of the protein to its recognition sequence and control of vitellogenin mRNA stability. We will also examine the role of the binding protein in controlling the stability of one human mRNA in which we identify a binding site. We will determine whether the initial RNase cleavage site is located near the protein binding site. These studies should provide important insights into steroid hormone control of mRNA stability.
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{
"pile_set_name": "NIH ExPorter"
}
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Support is requested for the 38th annual meeting of the International Narcotics Research Conference (INRC) to be held in Berlin, Germany from July 8-13, 2007. INRC has for many years been the most prestigious international meeting for the dissemination and exchange of information about biomedical research on opiate drugs and the endogenous opioid system. The topics range from molecular to behavioral and clinical studies. While the main topic is still opiate drugs and abuse, there is increasing discussion of other abused drugs, such as marijuana, cocaine, amphetamines, and alcohol, since extensively interactions between these drugs and the endogenous opioid system has been documented. Support is requested primarily for partial travel support to enable young scientists, i.e. graduate students, postdoctoral fellows, and junior faculty, to participate in the Conference. Preference will be given to young investigators who have not previously attended the meeting. A small portion of funds will support distinguished plenary speakers from other disciplines, relevant to research on addictive drugs, who will describe strategies, concepts or techniques likely to be useful to an INRC audience. Invited symposium speakers, not normally INRC participants, will receive partial support. This year INRC has five confirmed plenary speakers on various topics, including Drs. Jeffrey Mogil, Montreal, Canada, F. Benedetti, INAIL Direzione Centrale, Roma, Italy, and T. Jentsch, Universit[unreadable]t Hamburg, Germany. Drs. Volker H"llt, University of Magdeburg, Germany, and Horace Loh, University of Minnesota, were elected the Founders' Lecturer for the 2007 Meeting. Eight symposia will cover a wide range of topics described in the application, including neuroimmune interactions, genetic influences and opioid-nonopioid interactions in pain control. There will also be sessions dedicated to the biosynthesis and functional efficacy of new opioid analgesics as well as opioid receptor signaling and regulation by scaffolding/signaling complexes. A number of relevant contributed papers will be chosen for oral presentation at each symposium. There will be three poster sessions, at which there is always much exchange and discussion. For participants from many countries, this international meeting is their only chance to meet and exchange ideas. We feel that participation by many pre- and post- doctoral trainees is likely to continue to result in recruiting promising scientists to a career of drug abuse research. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
}
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The research goal is to identify factors associated with the maintenance of good health and well-being among women as they age. The study examines differences in the life histories and life patterns of women in different age groups and how these factors relate to health outcomes among the age groups or cohorts. The study uses social, sociopsychological, and demographic data from a household interview survey conducted in 1970-71, a 1982 follow-up survey, and computerized medical record data beginning three years prior to the 1970-71 survey and continuing to the present. The study has two interrelated sets of aims. The first set will be achieved using essentially a cross-sectional research design, while the second set uses a longitudinal or panel design. The first set of aims: 1) to examine cohort differences in family history, demographic characteristics, adult role sets, health beliefs, attitudes, and behaviors; 2) to assess the relationship of these variables to health outcomes within each cohort; and 3) to assess the possible independent, additive, or multiplicative nature of the relationship of these variables to health outcome. The second set of aims are: 1) to assess changes in role sets and attributes to roles including continuity of roles and relationships for each cohort in the decade between the surveys; 2) to assess the effect of these roles and relationship changes on health outcomes among the cohorts; 3) to incorporate findings from the analysis for the first set of aims regarding variables influential in health outcomes; and 4) to identify important intervening variables which may modify the health effects of particular independent variables. The study population is a 5 percent sample of female members of large HMO who in 1970 were age 25 or older. This sample of 1,189 women is divided into three age cohorts for the study. The basic approach to the analysis begins with an examination of bivariate relationships and moves towards multivariate assessments. The independent variables include categorical and ordinal level measures, while the dependent variables include ordinal and interval measures. For purposes of examining bivariate relationships which include categorical and ordinal variables, nonparametric statistics will be used. Where interal level dependent variables are examined analysis of variance will be used. When the objective is to determine the separate and combined effects of the independent variables on the dependent variables, multivariate techniques such as log linear models for multidimensional contingency tables and/or multiple regression and discriminant analysis will be used. The study will be the basis for more specialized studies, including later examination of these same subjects as they advance through the life cycle (since, as members of a panel, medical data will continue to be collected on them).
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{
"pile_set_name": "NIH ExPorter"
}
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The proposal will build upon an ongoing longitudinal study of 6,000 subjects who were tested up to three times during their junior high school years (1971-73) and again as young adults (between 1981 and 1986) in order to test theoretical models of the adoption and changes in level of involvement in more or less deviant adaptations to life stresses. By obtaining data from a second adult interview and from blood samples from a portion of these subjects (N=1800) we will test theoretically informed multivariate models of the mutually influential more and less proximate precursors of 1) infection with HTLV-III (indicated by ELISA, immunofluoresence, culture), 2) immune deficiency data (reflected in differentiation marker studies and functional tests), 3) changes in behaviors that are recognized as implicated in infectiion with AIDS virus, and 4) responses to threats to self-conceptions posed by the request to provide blood samples, the expectation of receiving information regarding the test outcome, knowledge of the test results, and symptoms associated with HTLV-III infection, using a range of multivariate analytic strategies. We aim to conduct four sets of analyses guided by previously formulated general theoretical paradigms and comprehensive reviews of the literature. Explanatory factors in the first set of models include parenteral drug use, homosexual patterns such as anal receptive intercourse, sexual promiscuity, as well as the psychosocial factors that increase the opportunity to learn and to become motivated to engage in these patterns. In the second set of models explanatory factors include various patterns of substance abuse, psychosocial stress, nutrition, other indicators of health status, and the biopsychosocial precursors of these factors that facilitate the learning, motivation to engage in, or experience of these patterns. These factors are not of the direct or indirect effects of HTLV-III infection or its precursors upon immunodepressive states. In the third set of models, explanatory factors further include beliefs about the consequences and the subjective evaluative significance of the behaviors that are in fact and/or believed to be implicated in the onset and course of AIDS and the risk status of the person for contracting HTLV-III infection. For the fourth set of models, explanatory factors include characteristic adaptive/coping/defensive patterns, personal and interpersonal resources (both instrumental and socioemotional), the evaluative significance of threatened social roles, and awareness of the potentially threatening AIDS-related stimuli, and the precursors of these factors.
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{
"pile_set_name": "NIH ExPorter"
}
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Treatment of chronic dermal ulcers is a significant problem that confounds many clinicians. This application examines the ability of a keratinocyte-populated microcarrier device (KPMB-device; patent no. 5,972,332) to modify the wound fluid environment into which it is placed and thereby enhance healing. The device consists of human keratinocytes grown on cell microcarrier beads placed into porous fabric bags. These bags can be placed into wounds and later retrieved. We hypothesize that modifying the wound environment to produce a milieu more compatible for host cells will promote healing of chronic wounds. The research will examine the ability of the device of modify wound fluid and relate these changes to the ability of the device to enhance wound healing. Determination of the expression and secretion of growth factors, protease inhibitors, and an extracellular matrix protein by the device keratinocytes will be performed to identify a marker molecule(s) of device activity. The ability of the KPMB-devices to modify wound fluid will be assessed focusing on changes in the level product release assay and to identify further manipulations of the KPMB-device to optimize the healing produced by these devices.
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{
"pile_set_name": "NIH ExPorter"
}
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Abnormal or deficient cysteine dioxygenase (CDO, EC 1.13.11.20) activity has been claimed to be seen in individuals with a variety of diseases, both non-neurological and neurological. Low CDO activity may result in problems, either because of insufficient supply of sulfate (or taurine) as products of cysteine catabolism that are needed for a variety of purposes including phase II conjugation reactions or because of accumulation of cysteine or toxic metabolites. We have identified CDO as the major hepatic enzyme involved in regulation of cysteine catabolism. The regulatory response is robust, with CDO activity being barely detectable in liver of animals fed low protein diets and increasing up to 170-fold in rats fed diets containing high levels of protein or sulfur amino acids. The long-term objective is to further elucidate the molecular mechanisms involved in the regulation of CDO and to understand the possible contribution of genetic or other impairments in CDO in the etiology of diseases such as rheumatoid arthritis. Proposed studies include (1) the characterization of the two CDO isoforms; (2) studies of CDO expression and regulation by cysteine in nonhepatic tissues (brain, kidney, and lung) as a step in understanding the relation of cysteine metabolism to the development and progression of certain neurological and nonneurological diseases associated with aging; (3) assessment of the mechanism by which CDO levels are increased -- an increased rate of mRNA translation or an increased stability of CDO protein in the presence of cysteine, and, if appropriate, examination of the possible formation of an alternatively processed mRNA transcript for CDO; and (4) assessment of the relationship of tissue cysteine concentration to CDO levels in intact rats. Experiments will involve dietary treatments of rats and assessment of the effects of diet on CDO expression in various tissues, studies of the molecular mechanisms involved in regulation of CDO abundance, and characterization of CDO protein and CDO mRNA isolated from these rats or from hepatocytes in primary culture.
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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. Cell development in higher organisms is controlled in part by communication between the nucleus and the surrounding extracellular environment, and accumulating evidence shows that changes in the structure of a cell''''s genetic material are central to the regulation of this communication. The inability of the extracellular environment to communicate properly with the nucleus is one of the hallmarks of tumor development. We have developed a model system in which normal mammary epithelial cells respond to a physiologically relevant three-dimensional, laminin-rich basement membrane (IrBM) by undergoing a functional differentiation. This assay will be used to determine how the extracellular environment influences the structure and organization of a cell's genetic material in a manner that regulates the expression of specific genes. To identify these changes, we will employ a combination of biochemical and microscopy techniques. Characterizing the role of the extracellular environment on mammary cell development will contribute towards a better understanding of normal breast cell development and gene expression and may also have implications for breast tumorigenesis and disease intervention.
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{
"pile_set_name": "NIH ExPorter"
}
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The goal of this project is to develop organoselenium compound with highest efficacy but with low toxicity, understand the mechanism of action of this agent in colon and mammary carcinogenesis and ultimately translate the results into human application. Our earlier studies have identified 1,4-phenylene-bis(methylene)selenocyanate (p-XSC) as superior to those organoselenium compounds evaluated previously in mammary and colon cancer models. Our studies also indicated that the chemopreventative efficacy and toxicity of selenium compounds depend on the chemical form in which they are administered, suggesting that their metabolism is importing in exerting biological effects. Preliminary studies suggest that glutathione conjugate of p-XSC (p-XSe-SG) in a putative intermediary metabolite and tetraselenocyclophane (TSC) is a metabolite of p-XSC. We hypothesize that arylselenol, active form of selenium formed from these metabolites is likely to be responsible for p-XSC's chemopreventative activity. In this proposal, the chemopreventative properties of p-XSe-SG and TSC in comparison to p-XSC will be evaluated in mammary and colon carcinogenesis. These agents will be synthesized, maximum tolerated doses (MTDs) of the compounds will be determined and 40 and 80% MTDs of p-XSe-SG and TSC and 80% MTD of p-XSC will be evaluated for their potential chemopreventative activities during initiation and post-initiation phases of mammary and colon carcinogenesis. Organoselenium compound showing highest chemopreventative index will be further evaluated for its efficacy when administered during promotion/progression in mammary and colon carcinogenesis. This agent will also be characterized with respect to its absorption, excretion and tissue distribution. Mechanisms of inhibition of colon and mammary tumors by this agent will be explored. Unified mechanistic hypothesis is that (a) organoselenium inhibits oxidative stress and lipid peroxidation which modulated transcription factors, thereby down regulating cyclooxygenase (COX-2) which in turn can enhance apoptosis and decrease tumorigenesis and (b) organoselenium induces apoptosis in the abnormal cells by increasing DNA cytosine methylation through inhibition of excess DNA methyl transferase.
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{
"pile_set_name": "NIH ExPorter"
}
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GRASP (GFP Reconstitution Across Synaptic Partners), originally developed in invertebrates, is a genetically controllable fluorescence-based system for identifying sites of contact between two cells (or cell populations). It relies on the expression of membrane-tethered split (i.e. non-functional) GFP modules that must come together to reconstitute GFP fluorescence (spGFP1-10 &spGFP11);since this functional complementation requires close apposition between membranes (<100 nm), GRASP is a powerful system to identify synapses between two cells. The objective of this application is to develop and validate GRASP methods for use in mammalian and Drosophila neural circuits. We will generate a battery of GRASP tools, including viral vectors for targeted expression of spGFPs, produce transgenic (and knock-in) lines expressing spGFPs under the control of the Cre or tetracycline inducible systems, and engineer Drosophila chromosomes harboring GRASP components under orthogonal expression control systems. Together, these reagents will afford temporal and spatial selectivity of GRASP expression, and provide a versatile platform for circuit mapping. We propose to test these components in two different models: a thermosensory circuit screen in flies and a Cre reporter screen in mice. We will also engineer a multicolor GRASP as a novel "synaptic fingerprinting" technique. We expect the results of these studies to significantly enhance the arsenal of tools available for circuit mapping, and be helpful in the characterization of normal and diseased nervous systems. PUBLIC HEALTH RELEVANCE: Mapping the connectivity of neural circuits is an important prerequisite to understand how neuronal ensembles process sensory stimuli and drive behavior. Towards this goal, we will develop and validate new strategies to more easily identify synapses between defined neuronal populations in the mouse. We anticipate that inducible and multi-color GRASP, as well as the novel Grainbow transgenic lines and viral vectors will provide a toolbox that will be of considerable value for the entire neuroscience community.
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{
"pile_set_name": "NIH ExPorter"
}
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We discovered a systemic problem with our whole-room indirect calorimetry suites (commissioned in August 2008). During the winter of this year, we detected significant artifacts in all three chambers that lead to unstable performances. After rigorous testing, we traced the issue to extreme low relative humidity of the building supply air as it introduced high differential partial pressure (of water vapor) between the chamber and reference air. We engaged with building engineers and officials from Office of Facility Management, Office of Research Facilities, and the Clinical Center. We confirmed our findings of low humidity and are currently working towards a three-prong approach to rectify the issue: (1) tighter humidity control of the Metabolic Unit supply air to provide a more stable environment by the OFM, (2) use the Clinical Center medical air as supply air for the calorimetry suites, and (3) revamp the calorimetry air sample and condition equipment, calibration gas mixing, and analyzer upgrades. With the strong support from NIDDK leadership and Clinical Center, we are making steady progress in these critical renovations. Research findings: In 25 young and normal-weight healthy volunteers, Dr. Celi and his colleagues have found that a 12-hr environmental temperature change (from 24 to 19 C) produced a 6% increase in resting energy expenditure as measured by our metabolic suites. This increase was likely a result of the activation of brown adipose tissue via the sympathetic nervous system, but less of an influence by the thyroid hormone. While expanded studies are underway to study obese and elderly individuals and to further quantify the brown adipose tissue activity by FDG-PET imaging, we have published the current findings. We are continuing our intra/extramural collaboration project with Dr. Naji Abumrad from Vanderbilt University to study the changes in energy metabolism, protein turnover, insulin action, and associated weight loss before and after Roux-en-Y gastric bypass surgery. The Vanderbilt study has stopped new recruitment but we following up the patients that we studied on whole-body metabolism during rapid weight loss. We have published a manuscript in Obesity recently. Services rendered (FY09 activities): 24-hr energy expenditure by whole-room respiratory chambers (299), resting energy expenditure by metabolic carts (364), exercise and physical function testing (144), body composition (581 Bod Pod measurements, 388 iDXA scans), objective physical activity monitoring (588), food-array experimental dinning (146), muscle biopsies (28), subcutaneous fat biopsies (79), and Health and Physical Exams (75). All activities are significantly up from FY08, despite of the humidity issue with the metabolic chambers.
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{
"pile_set_name": "NIH ExPorter"
}
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This proposal is based upon the desire of the University Medical Center to meet the requirements of the Animal Welfare Act of 1970 and N.I.H. guidelines as stated in the policy on the care and treatment of laboratory animals (N.I.H. Guide for Grants and Contracts No.7, June 14, 1971). The effort to upgrade animal resources at the University Medical Center was begun in 1967 by the establishment of an administrative unit, Laboratory Animal Facilities, and the hiring of a veterinarian experienced in laboratory animal medicine as the director. The institution provided the administrative structure and financial resources needed to meet all requirements of the Animal Welfare Act of 1966. In 1969 an additional effort to upgrade our facilities was made by application for an accreditation site visit by the American Association for Accreditation of Laboratory Animal Care based upon the N.I.H. Guide for Laboratory Animal Facilities and Care. In June, 1970, provisional accreditation was granted with a list of requirements necessary to gain full accreditation. Even though a number of these requirements have been met, lack of funds has prevented correction of the major problems. The combined factors of law, N.I.H. policy and our desire to get our facilities accredited, make it necessary to seek financial aid which is not locally available.
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{
"pile_set_name": "NIH ExPorter"
}
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Alterations in skeletal muscle sarcoplasmic reticulum (SR) calcium (Ca2+) release via the ryanodine receptor (RYR) Ca2+ release channel have been identified in a number of muscle diseases including malignant hyperthermia and central core disease. The regulation of SR Ca2+ release by endogenous ligands is also altered in these diseased muscles. This signifies the importance of understanding the regulation of RYR activity by endogenous modulators. Techniques utilizing laser scanning confocal microscopy and the Ca2+ indicator Fluo-3 have allowed the imaging of discrete localized Ca2+ release events (Ca2+ sparks) in functionally intact skeletal muscle. Examination of properties and frequency of occurrence of these events in response to either voltage or ligand stimulation is providing insight into the molecular control of SR Ca2+ release within the context of the functioning muscle fiber. The main objective of this proposal is to examine the modulation of the local control of SR Ca2+ release by calmodulin in functionally intact skeletal muscle fibers. Specifically, I intend to examine the effects of calmodulin on spontaneous and voltage activated skeletal muscle Ca2+ spark properties to gain insight into the role calmodulin plays in skeletal muscle excitation contraction coupling. The use of recombinant calmodulin, both wild type and a mutant that cannot bind Ca2+, will allow for the assessment of the role calmodulin plays in the activation, propagation and termination of these discrete localized Ca2+ release events.
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{
"pile_set_name": "NIH ExPorter"
}
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Enzyme I: GP, AP[unreadable] [unreadable] Deuterium labeling of proteins is a commonly used technique in structural studies. We would like to explore how deuterium labeling affects the stability and activity of enzymes. As a model protein in ths study we have selected the first enzyme of an important new nitrogen signal pathway of E. Coli, the enzyme I-Ntr (EI-Ntr). The His356 residue of EI-Ntr is phosphorylated in the autocatalytic reaction of EI-Ntr with PEP/Mg(II) and this high energy phosphate is transferred to His16 of NPr, a small carrier protein containing 90 amino acids. We have obtained thermodynamic parameters for dimerization of EI-Ntr and for interactions of EI-Ntr and its monomeric amino terminal domain EIN-Ntr with the acceptor protein NPr using isothermal titration calorimetry, analytical ultracentrifugation and light scattering methods. Comparing the stability of EI-Ntr with its deuterium labeled counterpart we observed that the stability of deuterated enzyme is compromised and the Tm of the deuterated EI-Ntr is lowered by 4 degrees K as shown by the differential scanning calorimetry. We continue to thermodynamically characterize interactions of the deuterated EI-Ntr with its ligands to understand what aspects of the activity of this enzyme are affected by deuterium labeling.[unreadable] [unreadable] [unreadable] Oligomeric state of the equine infectious anemia virus (EIAV) matrix protein. (GP, NT)[unreadable] [unreadable] Equine infectious anemia virus (EIAV) matrix protein domain (MA) is targeting the viral precursor polypeptide to the cytosolic side of the infected cell membrane during the virus maturation. Despite poor sequence homology, MA domains from various retroviruses are similar in size (14-15 kDa) and share a highly conserved tertiary structure composed of five helices. Interestingly, HIV-1 MA crystallized as a trimer while EIAV MA was crystallized in a nonsymmetric dimeric unit. Nevertheless, fluorescence studies suggest that EIAV MA exists as a multimer of two to three subunits. We carried out a series of analytical ultracentrifugation measurements to characterize the oligomeric form of EIAV MA in solution. Our results are consistent with a primary monomer-trimer equilibrium model for EIAV MA at micromolar concentrations.[unreadable] [unreadable] [unreadable] Conformational dynamics of the ClpA oligomers. (GP, MRM)[unreadable] [unreadable] ClpA, a Hsp100/Clp chaperone from E. Coli, forms a hexameric ring of subunits that unfolds native proteins in an ATP-dependent process and delivers the unfolded polypeptides to ClpP protease for degradation. The precise mechanism of ATP driven unfoldase activity of ClpA is important but not well elucidated. Previous calorimetric titrations of ClpA hexamer with SsrA target peptide have suggested that peptide binding may trigger conformational changes within the ClpA hexamer. This observations were confirmed by analytical ultracentrifugation studies indicating that ClpA hexamer conformational changes induced by SsrA peptide binding are reflected by sedimentation coefficient distributions.[unreadable] [unreadable] We continue to use this methodology to gain further insight into the role of individual ClpA domains. Each subunit of the ClpA hexamer has two ATP binding domains (D1 and D2) and the N domain located at the end of the central channel. Using the ClpA-deltaN and the ClpA-ND1 mutants, lacking the N-terminal domain and the ClpP docking D2 domain, respectively, we were able to establish that the ring-forming D2 domain, rather then the presumably flexible N domain, is responsible for most of the conformational changes associated with substrate binding by the ClpA hexamer. Models of the ClpA substrate processing mechanism based on those results were further supported by the data of the ATP-ase activity stimulation obtained with ClpA deletion mutants. In those experiments proteins showing large conformational changes by analytical ultracentrifugation were also stimulated in their ATP-ase activity and vice versa.[unreadable] [unreadable] [unreadable] Capping Protein - CARMIL Interaction Studies. (GP, TU, JAH)[unreadable] [unreadable] Capping Protein (CP) is a highly conserved actin-binding protein that is essential for normal actin dynamics which is an important part of many cellular processes, including immunological responses and cancer cell proliferation. CP binds to the barbed end of the actin filament blocking both association and dissociation of actin monomers. One potential regulator of CP is CARMIL, that might act as potent CP antagonist and inhibit CP interaction with actin filaments.[unreadable] [unreadable] Two regions of the CARMIL protein (CAH3-a and CAH3-b) were previously identified as crucial for interactions with CP. Cloned fragments of CARMIL with sequences encompassing both the CAH3-a and CAH3-b regions, with several single point mutations with either of them, were used to study CARMIL interaction with mouse Capping Protein (mCP) by Isothermal Titration Calorimetry (ITC). Results obtained by ITC were compared with those from an actin polymerization inhibition assay allowing for the identification of CARMIL regions contributing to binding affinity and those necessary for uncapping activity. We continue to use this effective strategy to locate mCP active sites for CARMIL binding by studying several single point mCP mutants in ITC titrations. Results of those experiments are compared with fluorescence titrations where the intrinsic Tryptophane fluorescence of mCP is used.
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{
"pile_set_name": "NIH ExPorter"
}
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