text
stringlengths 1
32.7k
| meta
dict |
|---|---|
Negative-stranded RNA virus (NSV) vectors have shown promise as vaccines against a variety of infectious diseases. While they have similar life-cycles, NSV vectors interact with and modulate innate immunity, such as type I interferon responses, by very different mechanisms. VSV is a highly cytopathic virus that shuts down cellular gene expression by virtue of its M protein. Rabies virus (RV) is a noncytopathic virus that down-modulates STAT function by virtue of its P protein. NDV is an avian paramyxoviruses that appears not to have specific mechanisms of down-modulating innate immunity in mammalian cells. All of these viruses replicate to different extents in vivo and elicit different host signaling programs in dendritic cells. Since it is unclear what constitutes an ideal vector for HIV vaccines, we will now compare these viruses and extract information on how their specific characteristics modulate immunogenicity. A comprehensive analysis of immunogenicity parameters induced by vectors with a similar backbone and replication cycle but with very diverse set of host-virus interactions has not been performed. The overall hypothesis for this application is that NSV vectors can be improved by altering their interactions with the innate immune system. We hypothesize that an effective HIV-1 vaccine must activate dendritic cells (DCs), which are an important link between innate and adaptive immunity. We will first study the impact of these vectors expressing HIV-1 antigens on DC-mediated immunity in-vitro, and identify parameters that would be expected to induce effective adaptive immunity in-vivo, such as efficient antigen presentation and activation. These initial studies will allow us improve the efficiency of our vectors, and provide us with a better basic understanding of what constitutes an optimal vector for the induction of HIV-1-specific immune responses. Once these parameters are determined, we will study the vectors for pathogenicity and immunogenicity in heterologous prime-boost approaches in mice. The most promising approaches identified in mice will be further analyzed in rhesus macaques. PROJECT 1: Examination of modified RV-based HIV-1 vaccines (Schnell, M) PROJECT 1 DESCRIPTION (provided by applicant): Rabies virus (RV) based vaccine vectors induce potent cellular and humoral anti-HIV responses in mice and in rhesus macaques and can provide protection against an HIV-1 like disease in monkeys. However, there is also evidence that such vectors can be improved. Here we perform a detailed study of the activation pattern of RV-infected DCs, which will guide us to construct improved RV vectors. Moreover, we will analyze the impact of type I interferons on DC activation and on CCR7 expression for DC migration. We will analyze the immunogenicity and pathogenicity of such vectors in a mouse model, compare them with Newcastle Disease virus and vesicular stomatitis-based vaccine vectors, and prioritize their use for studies in rhesus macaques. Two specific Aims are proposed: Specific Aim 1. Determine the activation state of DCs after infection with RV-based HIV-1 vaccine vectors and analyze the impact of type I interferon expression by a recombinant RV. Test the hypothesis that interferon type I increases DC activation. Determine the role of CCR7 expression on DC migration. Specific Aim 2 Immunizations of improved NSV HIV-1 vaccine vectors in mice and rhesus macaques. Determine the impact of the innate immune response on the adaptive anti-HIV-1 immune response after infection with optimized RV vectors. Test the hypothesis that activation of innate immune responses reduces vector pathogenicity. Test the hypothesis that two viral vectors induce more potent responses against the expressed HIV-1 antigens than a single vector. Study the efficiency of optimized viral vectors in the rhesus macaque / SIV challenge model system.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The baboon as a model for immunological studies of schistosomiasis mansoni will be further studied with emphasis on defining conditions of infection with Schistosoma mansoni leading to the development of immunity. The role of murine-like alpha 2-macroglobulin antigenic determinants on the parasite's surface will be examined in the contexts of evasion of host immune responses and molecular mimicry. Immunogenicity of these antigenic determinants in baboons and the extent of mimicry of host protein are of particular interest.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The objective of this research is to correlate the energy utilization of neuronal tissue with its functton. We are using the cockroach nerve cord because of the resistance of inverterbrates to low oxygen levels. We are correlating the levels of ATP in thoracic ganglia with spontaneous electrical activity and with behavior. We will also study regional glycolysis of the head and thoracic ganglia during hypoxia. BIBLIOGRAPHIC REFERENCES: Nelson, S.R., Walter, D.C., and Hathaway, P. Comparative studies of glycogen phosphorylase using isoelectric focusing. Sixth International Congress of Pharmacology, Helsinki, Finland, 1975. Walter, D.C., and Nelson, S.R. Energy metabolism and nerve function in cockroaches (Periplaneta americana). Brain Res. 94: 485-490, 1975.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Core A. ADMINISTRATIVE AND STATISTICAL CORE The purpose of the Administrative and Statistical Core is to provide the foundation upon which the proposed program project is built. This Core serves several functions vital to the implementation of the proposed studies. Core A is responsible for the following scientific and administrative functions: (1) Coordination of communications and scientific activity among the various projects and cores; (2) Coordination of the daily functioning of a complex interdisciplinary research activity; (3) Budgetary planning and control; (4) Data base management including all aspects of subject scheduling, tracking, and data security, as well as rules and procedures governing data sharing and joint publication policies; (5) Subject identification and initial screening; (6) Statistical consultation and collaboration including the development and/or utilization of analytic procedures tailored to the special problems that are encountered in the study of rare populations as well as the co-ordination of methodologies that result from interdisciplinary collaborations. Participants in the Program Project will be children ages 7 through 10 years 11 months in the following groups: Typically Developing Controls (TD), Specific Language Impairment (LI), Peri-natal Focal Lesion (FL), High Functioning Autism (HFA), and Williams Syndrome (WS). Subjects will be identified by Core A, screened and inducted into the study by Core B, and undergo MRI scans in Core C. Data from all subjects will be utilized by all Projects to test Project hypotheses. Based on our 20 years of experience with the administration of a large multidisciplinary research center, we have administrative procedures in place that are necessary to run this complex program project grant, and that will sustain an industrious and productive research center over the next 5 years.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The tight junctions at the boundaries of epithelial cells are of critical importance to the development and function of most tissues in multicellular organisms because they enable epithelia to work in the separation, protection, and shaping of internal organs and glands. These tight junctions act as physical and chemical barriers and also as fences, mediating differential transport of macromolecules, solutes, and ions that regulate body fluid composition. Tight junctions are composed of several groups of proteins, but three classes of tight junction integral membrane proteins (TJIMPs): occludin, claudins, and tricellulin; are thought to play a leading role in their architecture and function. Disruptions of TJIMPs are implicated in several human diseases, such as hepatitis, and cancer, as well as renal wasting disorders, ocular disease, and deafness. Tight junction barrier function may provide targets for manipulating drug transport. But the function of TJIMPs remains poorly understood. We hypothesize that select domains of TJIMPs dictate barrier and fence function, and that tight junction diversity in various epithelia is governed by the TJIMPs that constitute them. This proposal aims to understand TJIMP structure and function in molecular-level detail by: Aim 1: determining the crystal structures of one or more selected TJIMPs and, Aim 2: examining the physiological function(s) of TJIMPs. Bioinformatics will be coupled to high-throughput cloning, expression, and protein analysis technologies to select one or more targets with the best probability for successful X-ray crystallographic structure determination. Functional analysis will employ selective mutagenesis, cell adhesion assays, lipid-labeling strategies, protein localization, freeze-fracture electron microscopy, and electrophysiological means to assess barrier and fence function in situ. This research has widespread significance for understanding diseases related to the disruption of TJIMPs and provide targets for therapeutics, as well as promoting understanding of drug transport. PUBLIC HEALTH RELEVANCE: Epithelia separate, protect, and shape the tissues of the human body. Membrane proteins found at tight junctions of epithelial cells are essential to their ability to function as physical and chemical barriers while allowing differential transport of solutes and ions. The goal of this research is to determine the structures of these proteins and extend insight into their function, leading to a better understanding of diseases caused by tight junction disruptions and to the development of novel therapeutics.
|
{
"pile_set_name": "NIH ExPorter"
}
|
As realized by this RFA, there is a need to identify novel molecular targets for cancer drug discovery. Among these will be molecules that directly regulate the intrinsic apoptotic pathway. We recently discovered that the large GTPase dynamin-2 (dyn2) signals through p53 to trigger apoptosis, specifically in rapidly dividing cells. As little as five-fold overexpression of endogenous wild-type dyn2 will activate p53-dependent apoptosis and this is absolutely dependent on GTP binding. A mutation defective in dynamin's intrinsic GAP activity is even more potent. Thus, compounds that inhibit dynamin's GTPase activity or its intrinsic GAP may be effective proapoptotic, anti-cancer drugs. To identify these compounds we have developed a simple and robust high-throughput assay for dynamin GTPase activity and established a collaboration with Barry Sharpless, a leading chemist who has pioneered a new azido-based method of "click chemistry" that facilitates the generation of complex libraries of related compounds, allows for rapid modular SAR of identified leads and most importantly and uniquely allows the assembly of functional moieties to be selected for on the surface of the target molecule to generate customized, high-affinity, high-specificity inhibitors. We propose the following Specific Aims that will allow us to apply click chemistry to identify potential anti-cancer drugs targeted against the regulatory GTPase, dynamin. 1. To identify, characterize and optimize high affinity, high specificity compounds that inhibit dynamin's basal GTPase activity. 2. To identify, characterize and optimize high affinity, high specificity compounds that selectively inhibit dynamin's assembly-stimulated GTPase activity. 3. To apply click chemistry for in situ selection of very high affinity, very high specificity inhibitors on dynamin, itself.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Organisms rely on large molecular machines that convert nucleic acids between their deoxyribose and ribose'forms to transmit genetic information. This proposal aims to study these molecular machines from the standpoint of human disease. In the case of HIV, reverse transcriptase (:RT) is responsible for catalyzing the transfer of information from viral genomic RNA to DMA. This series of reactions are crucial steps, and are absolutely required for viral replication. RT is a proven drug target, as evidenced by the number of therapeutics that function by inhibiting RT. Mutations to current drug binding sites on RT which confer resistance is the greatest cause of failure for chemotherapeutic intervention, compelling the development of novel drugs which can function in the face of these mutations. The ribonuclease H (RNH) domain of RT represents an underutilized drug target despite the required activity of the RH domain for proper RT function. Furthermore, the RNH domain is located away from current drug binding sites, reducing the risk of cross-resistance with current therapies. This study will execute a multi-tiered strategy to discover and develop novel inhibitors that target RT. First, fragment based screening in conjunction with x-ray crystallography will be used as a platform for screening chemical space for small molecule scaffolds that bind in the RNH active site and disrupt activity. Information derived from fragment screening will be exploited to develop novel RNH inhibitors that will eventually be turned into therapeutics to treat AIDS. Second, the structures of an existing class of N-acyl hydrazone inhibitors of RNH function in complex with RT will be determined using x-ray crystallography. Information gained from these structures will be used to elucidate the design of the next generation of more potent inhibitors. Finally, the interactions of the existing class of alkenyldiarylmethane (ADAM) inhibitors of RT polymerase activity will be determined using x-ray crystallography. Although effective polymerase inhibitors already exist, this structural information will aid in the development of ADAM analogues that can retain activity in the face of resistance mutations. PUBLIC HEALTH RELEVANCE: Novel molecules that inhibit the function of an absolutely required HIV enzyme will be discovered and developed via structural methods. These new inhibitors will contribute to combating the emergence of drug resistant strains of HIV.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Our aim is to perform a double blind crossover controlled study in patients with diabetic neuropathy for a period of one year to determine the clinical effects of Mexiletine in diabetic neuropathy, monitoring for its side effects, and to demonstrate if Mexiletine is beneficial in relieving the symtoms of parethesias and pain for diabetic neuropathy.
|
{
"pile_set_name": "NIH ExPorter"
}
|
A specific genetic strain of Corriedale sheep is used as an animal model for the study of liver pathophysiology, specifically, hyperbilirubinemia (Dubin-Johnson Syndrome). This project is concerned with increasing the numbers of animals available for research by the controlled breeding of individuals which genetically transmit this character. This project is utilizing what is considered to be the only existing Corriedale sheep homozygotic for this trait. Efforts are being made to obtain both homozygous and heterozygous offspring from these highly inbred individuals. Semen collected artificially from the homozygotic rams was diluted in various cryoprotective extenders and then freeze preserved. This will ensure the long term availability of male gametes for artificial insemination. Overall this project allows prepetuation of this specific gene pool and ensures availability of research animal for future investigations of Dubin-Johnson Syndrome and related metabolic disorders.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The long-term goal of this proposal is to develop novel nanoparticles, "platelet-mimicking nanoparticles," as a drug carrier that can target and deliver a therapeutic agent to the injured vessel wall after cardiovascular interventions such as angioplasty. Our strategy is to mimic the binding of the glycoprotein Ib (GPIb) of platelets (a blood cell type) through either P-selectin expressed in damaged endothelial cells (ECs) or vWF deposited on injured subendothelium, which is critical for the initial interaction of circulating platelets onto the injured vessel wall under high shear conditions. The major advantage of using GPIb-nanoparticles for targeting drug delivery in our proposal, compared to current targeting strategies including anti-P-selectin antibodies, is that GPIb specially binds to both P- selectin expressed on damaged ECs and vWF deposited on injured subendothelium, thereby accumulating more nanoparticles as drug carriers to the injured wall site for effective drug delivery. To accomplish our goal, three specific aims are: (1) Develop drug (dexamethasone)-loaded biodegradable GPIb-nanoparticles using a standard double emulsion method. (2) Investigate the targeting activity and effectiveness of these nanoparticles in vitro using the parallel flow plate system, surfaces coated with P-selectin or vWF, and activated ECs. (3) Evaluate the efficacy of our novel platelet-mimicking nanoparticles in vivo using rat balloon injury models. The assessed parameters for these specific aims include the binding sites and stability of GPIb, changes in nanoparticle properties, the adhesion and uptake of GPIb-nanoparticles in activated endothelial cells under flow conditions as well as pharmacological activities of these nanoparticles in inflamed ECs and injured rat arteries. Cardiovascular interventions often injure the vessel wall, leading to the development of late pathological conditions such as inflammation and restenosis. The development of our novel platelet- mimicking nanoparticles is a unique strategy to rapidly target and deliver therapeutic agents to damaged ECs and subendothelium, despite the shear influence, for more effective therapies to treat these complications. [unreadable] [unreadable] [unreadable]
|
{
"pile_set_name": "NIH ExPorter"
}
|
Phenylketonuria (PKU) causes mental retardation in children. The underlying cause of PKU is not well understood. The critical biochemical parameters and reaction in the development of PKU is plasma phenylalanine elevation whereas tyrosine is maintained at a relatively low level. The serotonergic system has consistently been implicated in the pathogenesis of PKU. The goal of this research is to determine if changes in serotonin receptors (5-HT2C) contribute to the conditions associated with PKU by inducing the primary characteristics of PKU beginnings in 9-day-old rats. To achieve this goal, animal models of PKU will be used to examine a number of pharmacological parameters, including quantitation of serotonin levels by means of high performance liquid chromatography (HPLC), and radioligand binding assays that are coupled with determinations of blood phenylalanine levels. The findings of this work may contribute toward an understanding of the mechanisms involved in this disease.
|
{
"pile_set_name": "NIH ExPorter"
}
|
World-wide rates of obesity have reached epidemic proportions. In the United States alone 30% of the population is overweight and 30% is obese, contributing to an excess mortality of 300,000 deaths a year. Significant consequences of obesity include Type II diabetes and non-alcoholic fatty liver disease. Rates of Type II diabetes are rising in adults and worryingly, Type II is now also seen in adolescent patients. Despite the morbidity and mortality of obesity, few useful therapeutic agents have emerged and many current therapies used to treat diabetes induce additional weight gain. Fibroblast growth-fact 21 (FGF21) when administered pharmacologically to mice, has multiple beneficial effects including: improved glucose tolerance, activation of brown adipose tissue, browning of white adipose tissue, weight loss and anti-fibrotic and anti-inflammatory effects in the liver. Recent data suggest that beneficial effects are also seen in humans and thus FGF21 is considered a promising drug target. We found that in rodents FGF21 plays a role in browning of white adipose tissue in response to cold which is associated with beneficial metabolic consequences. Furthermore treatment with FGF21 mimics many of the effects of cold exposure. At least some of these effects are mediated by direct action through the central nervous system (CNS). We also found that FGF21 has anti- fibrotic and anti-inflammatory effects in the liver. However there are several significant uncertainties regarding it's mechanisms of action. In this proposal we will use an integrative physiologic approach to elucidate the effects of FGF21 on white adipose tissue, brown adipose tissue and liver. Many of the beneficial metabolic effects of FGF21 occur in an intact animal and cannot be reproduced in isolated tissue culture models. We believe that FGF21 integrates signals from the liver, brain and fat, and in the process of communicating between these 3 tissues, acts as a master regulator of whole body metabolism. We are particularly focused on distinguishing between effects mediated by the brain. We will focus on CNS mediated effects on the peripheral targets BAT, WAT & liver. We will also examine the differential effects of several potential downstream mediators. These include the fat derived hormone adiponectin and the mitochondrial-associated uncoupling protein 1 (UCP-1), an important mediator of increased energy expenditure, that can be activated through multiple pathways. We predict that FGF21 effects, such as weight loss and improved glucose homeostasis, depend on the ability of FGF21 to act in the CNS and activate browning through UCP-1. Other effects may reflect cell autonomous action on WAT, which may be mediated by adiponectin. Anti-inflammatory and anti- fibrotic effects on the liver will likely reflect direct ation on this tissue. We hope that with this physiologic approach we will be able to distinguish the integrative physiologic actions of FGF21 and uncover additional targetable systems for the therapy of metabolic disorders.
|
{
"pile_set_name": "NIH ExPorter"
}
|
A major instigator of radiation-induced pathology is cellular damage caused by reactive oxygen species formed in the first moments after exposure. Most therapeutic agents are free radical scavengers which must be present in high concentrations as exposure occurs, making their use as treatments problematic. In addition, the normal cell already has scavenger molecules present, such as glutathione. Glutathione accepts electrons from reactive electrophiles as part of the normal cellular defenses, but the resultant electrophilic glutathione conjugates still pose a threat to cellular integrity. Final elimination of risk then requires the transport and elimination of these reactive glutathione S-conjugates - a key function of RLIP76, a membrane-associated transport protein. Terapio has been testing RLIP76 as a systemic therapy for high-dose radiation poisoning. Preliminary studies in mice show that increasing cellular content of RLIP76 by systemic administration significantly increases survival even if the protein is administered AFTER radiation exposure, with a clear dose-response relationship between amount of RLIP76 administered and survival that extends to all but the highest exposure levels. Further, RLIP76 can be orally administered, increasing its ease of treatment. In order to move forward with clinical development, additional work regarding protein storage, stability, optimal dosing and schedule of administration is necessary. Further, although much is known about the protein's mechanism of action on a cellular level, organ-specific effects have yet to be determined. Therefore, this proposal will focus on three main areas. Specific Aim 1 will look at the stability of the protein under a variety of storage conditions to determine loss of activity and how that may affect recommended dosing in the field. Expanded studies of in vivo stability of the protein will be performed by following tissue levels of administered protein over time. Specific Aim 2 will enlarge understanding of the relationship between dose level, timing and schedule of administration and length of treatment in a mouse model. Specific Aim 3 will look at how RLIP76 affects specific organ toxicity from radiation and examine potential long term effects, again in a mouse model. The information obtained is necessary to plan an optimal treatment regimen for RLIP76 under different radiation release scenarios. PUBLIC HEALTH RELEVANCE: RLIP76 is a naturally-occurring protein that can be given orally and counteract the effects of radiation poisoning even if given AFTER exposure occurs. This project will investigate the most effective dosing of the protein and how to optimize packaging for long term storage.
|
{
"pile_set_name": "NIH ExPorter"
}
|
VRC-300 (NIH 03-I-0285) was approved by the NIAID IRB. Through this protocol, the VRC has initiated recruitment of healthy, HIV-negative subjects for a clinical trial of an investigational Ebola vaccine trial. The protocol may also be used in the future to recruit and screen for vaccine studies for infectious diseases other than HIV. The results of these screening studies will be used to determine if the subject meets eligibility requirements for participation in Phase I trials of preventative vaccines. Educational materials on vaccines are reviewed with and provided to subjects before enrollment into a study. Some of these vaccine studies will be part of the VRC Biodefense agenda.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Spatially defined sub-cellular heterogeneity determines neuron function. Thus, it is not surprising that disease origins can be traced back to the aberrant behavior(s) of dendritic filopodia that wire the brain. Interactions of the myriad filopodia extended by dendrites of individual neurons in their spatial contexts generate the remarkable range of functionalities of the human brain. Even adjacent filopodia encounter distinct local micro-environments and develop individual functionalities. Only by overcoming ensemble averaging of populations and measuring molecular signatures with single- filopodium resolution can we understand the interplay of the diverse intrinsic and extrinsic regulators, and explain the spectrum of neurological functions encompassing healthy and disease states. In particular, there is an unmet need for ways of probing of local regulators of filopodia during the emergence and sculpting of the dendritic arbor. This innovation proposal addresses this need by integrating our expertise in designing and fabricating nanoliter microfluidic environments for ultra-low density neuronal cultures with our expertise in the cell biology of neurons. We propose to use microfluidic device (FD) environments and high resolution image analysis to probe changes in localization, activity, and function of specific microRNAs (miRNAs) in developing hippocampal dendrites. miRNAs are short, non-coding RNAs that act as regulators of local protein synthesis, especially during dendrogenesis and local wiring of the nervous system. Our objective is to control the structure and function of individual dendrites within micro-channels of fabricated FDs to isolate individual dendrites. We will use this system to map and influence miR125b functioning in filopodia during their development and in response to glutamatergic stimulation. This novel set of studies will address the need for understanding with high resolution the localization, activation, and function of miR125b during wiring of the hippocampus. This approach will provide new insights on this putative regulator, new tools for studying properties of miRNA control of dendrogenesis in single neurons, and contribute to effective strategies for restoring defects in models of affective dysfunctions, chronic stress, Alzheimer's disease, and autism.
|
{
"pile_set_name": "NIH ExPorter"
}
|
In the past year we have continued our biologic and molecular characterization of different viral components isolated from Friend Murine erythroleukemia virus complex. Using a protocol developed in this laboratory for cloning in tissue culture Spleen focus forming virus (SFFV) free of helper virus, we have analyzed different isolates of SFFV obtained from several independent strains of Friend virus complex, including Lilly-Steeves, Mirand, and Axelrod strains of FV-P (polycythemia-inducing Friend Virus), and we have found that each of these isolated of SFFV contains xenotropic-MCF related env gene sequences. Furthermore, nonproducer cells containing each of these isolates of SFFV can be used to detect proteins coded for by SFFV. The relationship of a rapid undifferentiated leukemia induced by F-MuLV to erythroleukemia induced by SFFV has been explored.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Wild-type (WT) and IL-4-/- NOD.H-2h4 mice given 0.05% NaI water develop lymphocytic spontaneous autoimmune thyroiditis (L-SAT) with B and T cell infiltration of the thyroid, and production of anti-thyroglobulin antibody. IFN[unreadable]-/- and IFN[unreadable] receptor (R)-/- NOD.H-2h4 mice do not develop L-SAT, but all develop another autoimmune lesion characterized by thyroid epithelial cell hyperplasia and proliferation (TEC H/P). TEC H/P is very severe in 70% of IFN[unreadable]-/- mice given NaI water for 7mo. The thyroid is almost completely replaced by proliferating TEC, and there is extensive collagen deposition (fibrosis). Mice with severe TEC H/P produce anti-thyroglobulin antibodies, have low serum T4 and have some thyroid infiltrating T cells. Lymphocytes are required for development of TEC H/P. TEC H/P results from excessive proliferation of thyrocytes that occurs when IFN[unreadable] is absent. IL-4-/- mice are resistant to TEC H/P. The objective of this proposal is to define the mechanisms involved in development of severe TEC H/P and fibrosis, and determine how it can be inhibited. Thyrocytes of IFN[unreadable]-/- mice with severe TEC H/P strongly express TGF[unreadable], and all transgenic mice expressing TGF[unreadable] on TEC develop TEC H/P. These studies will test the following hypotheses: 1) TEC H/P is initiated by T cells able to migrate to the thyroid, 2) T cells produce cytokines in the thyroid (IL-4 or a cytokine that is low in IL-4-/- mice) that promote severe TEC H/P and fibrosis, and 3) overexpression of TGF[unreadable] in proliferating thyrocytes is important for development and maintenance of severe TEC H/P. These studies will use gene knockout, ie. IFN[unreadable]-/-, IFN[unreadable]-/- SCID, STAT-6-/-, and transgenic NOD.H-2h4 mice expressing TGF[unreadable] or dominant negative TGF[unreadable]RII on thyrocytes or T cells. Cell transfer experiments will be used to characterize the effector cells for TEC H/P, and to determine how cytokines such as IL-4, IL-13 and TGF[unreadable] interact with other T cells and TEC to promote development of severe TEC H/P and fibrosis. These studies will be applicable to understanding mechanisms involved in initiation and inhibition of abnormal cellular proliferation and fibrosis that can occur in the thyroid and also in other organs in some human autoimmune diseases such as systemic sclerosis and mixed connective tissue disease. This animal model provides a unique opportunity to clarify underlying mechanisms and may suggest novel therapeutic interventions for human diseases associated abnormal cellular proliferation and fibrosis.
|
{
"pile_set_name": "NIH ExPorter"
}
|
PROPOSAL: (Adapted from the applicant's abstract): Dr. Stein will develop a career in patient-oriented research by obtaining advanced training in clinical research design, biostatistics, lipoprotein biochemistry, and advanced ultrasound research techniques. He also will receive training in the ethical conduct of human subjects research. His long-term goal is to become a nationally and internationally recognized clinical investigator who develops therapeutic strategies and tests for the diagnosis and management of patients with or at risk for atherosclerotic vascular disease. As part of his research career development plan, Dr. Stein will complete the UWMS Clinical Investigator Preparatory Pathway (CIPP). As part of this program, he will obtain advanced training in research design, biostatistics, and epidemiology through courses and directed study in the UWMS Biostatistics and Medical Informatics Statistical Data Analysis Center. He will obtain training in lipoprotein biochemistry and lipid laboratory techniques through courses and directed study in the UWMS Hospital Lipid and Lipoprotein Laboratory. He will obtain training in advanced carotid ultrasound research techniques in the Center for Medical Ultrasound at Wake Forest University (WFU). Additional instruction regarding ethics and human subjects research regulations, leadership, teaching, and scientific writing are components of the career development plan. The research plan will determine whether the metabolic changes associated with use of human immunodeficiency virus (HIV) protease inhibitors are atherogenic, as determined by their effects on endothelial function (measured by flow-mediated vasodilation of the brachial artery) and early atherosclerosis (measured by carotid intimal-medial thickening). Abnormalities of these parameters, which are obtained by vascular ultrasound techniques, predict adverse cardiovascular events. Dr. Stein also will assess the effects of HIV protease inhibitors on atherogenic lipoproteins using the advanced lipid laboratory skills he obtains in the career development plan. Finally, Dr. Stein will determine if these medications impair postprandial clearance of triglyceride-containing lipoproteins by assessing lipid and lipoprotein responses to an oral fat load.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The African trypanosome, Trypanosoma brucei, is a protozoan parasite of medical and veterinary importance. The mitochondrion of T. brucei is able to undergo a life-cycle stage regulated biogenesis. In proliferative bloodstream forms, the mitochondrion is narrow, tubular and repressed since only glycolysis is required to provide energy for trypanosome survival. Over the course of two successive life-cycle stage differentiations, the trypanosome mitochondrion undergoes a biogenesis in which mitochondrial structure elaborates to support Kreb's cycle enzymes and fully functional cytochrome mediated oxidative phosphorylation. Mitochondrial homologs of dnaK (hsp70), dnaJ are the constituents of a universal chaperone machine central to the biogenesis of mitochondria. Disruption of the expression of these genes will have an effect on how the mitochondrion is constructed and may be able to prevent the mitochondrion being formed at all. In most eukaryotes dramatic disruption of mitochondrial biogenesis is lethal, however in the long slender form of the African trypanosome the repressed mitochondrion may not be an essential organelle. The African trypanosome is therefore likely to be a permissive system in which to dissect the functional relationships of these molecules. During the last year I have been successful in cloning and sequencing the T. brucei homolog of mitochondrial hsp70 and a novel mitochondrial dnaJ (tbmJ1). This grant will focus the basic characterization of tbmj1 expression, on the interactions of tbmj1 with tbmhsp70 and with other proteins, and on the effects of modulating the expression of the tbmj1 gene.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The flawless execution of cell division is essential to the generation and survival of all organisms. During every cell cycle, chromosomes must be accurately partitioned to daughter cells to prevent genomic instability and aneuploidy, a hallmark of many tumors and birth defects. Chromosomes segregate using their kinetochores, the specialized protein structures that assemble on centromeric DNA sequences and mediate attachment to microtubules. The foundation of all eukaryotic kinetochores is a conserved, inner centromere structure characterized by centromeric chromatin and its associated proteins. A hallmark of centromeric chromatin is Cenp-A, an essential histone H3 variant that epigenetically marks centromeres and is required for kinetochore assembly. The surrounding pericentromeric chromatin also makes various contributions to the fidelity of segregation. To fully understand the mechanisms that ensure accurate chromosome segregation, it is critical to elucidate outstanding questions about the functions and maintenance of centromeric and pericentromeric chromatin. This proposal will use purified kinetochores and a combination of biochemical, biophysical, proteomic and genomic approaches to address a number of outstanding questions about the contribution of centromeric chromatin to kinetochore function. 1) How do centromere-binding proteins contribute to the diverse functions of kinetochores? 2) How does pericentromeric chromatin regulate chromosome segregation? 3) What are the mechanisms that contribute to the exclusive localization of centromeres? The proposal will use budding yeast for these studies because they are amenable to biochemical, genetic and cytological studies, and the yeast kinetochore is the best characterized to date. Taken together, these studies of kinetochores and the underlying chromatin foundation in budding yeast will lead toward an understanding of the fundamental mechanisms of segregation in all eukaryotes. This work will not only elucidate important aspects about the process of segregation, but will aid in the design of better therapeutic interventions in the long-term. PUBLIC HEALTH RELEVANCE: All cells must inherit the right number of chromosomes every time they divide because the wrong number of chromosomes is a hallmark of cancer, birth defects, and other diseases related to problems in cell proliferation. We are therefore studying the process of chromosome partitioning to daughter cells when they divide to understand the basis for a number of human diseases.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Asthma, a pathological condition of reversible airways obstruction, comprises both inflammation of the lung as well as hyper-contractility of the bronchiolar smooth muscle. Such airway hyperresponsiveness (AHR) can exist in the absence of frank inflammatory infiltrates, however, suggesting that primary abnormalities in airway smooth muscle (ASM) contractility may exist in this disease. The major substances that induce bronchial smooth muscle contraction are agonists of G protein coupled receptors (GPCRs). Allergic reactions are initiated by allergen crosslinking of of high affinity IgE receptors on lung mast cells MCs sensitized by IgE, and this allergic mechanism is probably the most common inciter of the pathophysiological cascade in asthma. Many of the compounds contained in mast cell granules or synthesized by mast cells act on procontractile GPCRs to induce bronchoconstriction. Examples include histamine, cysteinyl leukotrienes (LTD4), endothelin 1, adenosine, and bradykinin. In general, these agonists induce activation of the heterotrimeric G protein G-alpha q, which increases the concentration of intracellular calcium in smooth muscle cells, promoting actin-myosin interactions. In contrast, ligands acting on G-alpha-s-coupled receptors, such as isoproterenol, increase intracellular levels of cyclic AMP (cAMP), facilitating ASM relaxation. A large family of Regulators of G protein signaling (RGS) proteins bind to the G protein alpha subunits Gi and Gq (but not Gs) through a conserved RGS domain and inactivate them by accentuating their intrinsic GTPase activity and by blocking downstream effector interactions. The physiological function of RGS proteins in the lung is unknown. The principal objective of this project is to determine which RGS proteins are expressed in specific cell types in the lung and to enumerate their functions in this organ. The first objective is accomplished primarily by immunohistochemistry and immunoblotting using specific antibodies. We have established RGS9 expression by PCR, immunofluorescence, immunohistochemistry, and immunoblotting in alveolar type II pneumocytes (TIIPs) identified in mouse lung sections and in a cultured human cell line with a TIIP phenotype. RGS5 was shown to be expressed by PCR and immunoblotting in human and mouse bronchial smooth muscle. In human cultured airway smooth muscle (ASM) cells, RGS5 expression appears to be affected by physiological regulators of ASM function such as endothelin-1, acetylcholine, bradykinin, and isoproterenol. The second objective will be addressed by studying RGS knockout mice and by using RNA interference to knockdown gene expression in cultured human cells. To elucidate how RGS5 regulates the ASM function in normal lung and in asthma, we are studying RGS5 knockout (KO) mice. The tracheas of these mice are excised and their contraction determined in a physiological salt solution bath connected to a pressure transducer at baseline and after agonist stimulation or electric field stimulation. Preliminary data indicate no significant differences in responses of wild type (WT) and KO tracheas to either acetylcholine (a bronchoconstrictor) or isoproterenol (a bronchodilator). Thus, future studies will examine tracheal contractility in these mice after allergen exposure or other treatments that upregulate Rgs5 levels as its expression appears to be low at baseline. In addition, airway responses of intact animals will be measured by whole body plethysmography before and after allergen exposure. We have also established siRNA duplexes that effectively reduce expression of endogenous RGS5 in cultured human ASM cells. We are currently examining the influx of intracellular calcium of siRNA-transfected human ASM cells and cultured mouse ASM from WT or KO mice. Contractility of cultured human airway smooth muscle cells will be studied in vitro by measuring distensibility of collagenn gel matrices connected to the pressure transducer. We also plan to study the proliferation and synthetic function (e.g. cytokine secretion) of cultured ASM cells directly. To determine the function of RGS9 in type II pneumocytes (TIIP), we employ RNA interference against both RGS9-1 and G-beta-5, its obligate binding partner. The most important physiological function of TIIPs is to secrete surfactant. Surfactant is composed of phospholipids important for the maintenance of alveolar surface tension and proteins involved in host defense. Abnormalities in number and function of alveolar TIIP have been suggested in asthma. Surfactant secretion can be mediated by GPCRs and is in general dependent on increases in intracellular calcium. We are examining the calcium responses of cultured TIIP transfected with RGS9-1 or G-beta-5 siRNA in response to physiological GPCR agonists. Future plans involve studying surfactant secretion in the human TIIP cell line transfected with siRNA or in primary cells isolated from RGS9 or G-beta-5 knockout mice. Finally, axin, a protein with a conserved RGS domain but with previously unknown G protein partners, was shown to interact with Gs and Gq. Axin association with Gs was shown to mediate prostaglandin (PGE2)-induced activation of the canonical Wnt-beta catenin pathway, which is important for many physiological processes such as colon cancer cell growth. In addition, axin was shown to be expressed in human airway smooth muscle cells and to be required for full beta-adrenergic receptor, Gs-mediated cyclic AMP (cAMP) formation. These findings indicate a potential role for axin in bronchial smooth muscle relaxation and/or contractility. We will determine the mechanism whereby axin modulates G-protein-dependent cAMP levels and establish whether axin affects Gq-mediated signaling pathways. Eventually, we will study how axin affects bronchial smooth muscle contraction or other functions of ASM cells by manipulating axin expression by RNA interference.
|
{
"pile_set_name": "NIH ExPorter"
}
|
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. Many papers are published that employ bottom-up proteomics to identify sites of potentail phosphorylation or other posttranslational modification. It is important to know the total ensemble of PTM for a protein at a particular time in the cell cycle. This information cannot be gleaned from the bottom-up strategy. Top down proteomics will be used to interrogate large parts of a protein (>10kDa) to discover the distributions of PTM during on proteins isolated from cells. The workflow will be developed by studying Chk2, an autophosphorylating kinase with more than 30 sites of phosphorylation. We will map out the distribution of phosphorylation from Chk2 captured at various times during the cell cycle.
|
{
"pile_set_name": "NIH ExPorter"
}
|
While changes in body composition-and often disastrous sequelae thereof-universally accompany old age, there is tremendous variability in their onset, pattern and severity. The long term objective of this project is to better understand the molecular basis for this heterogeneity, and thereby the pathways which underlie such changes. The immediate focus is whether some variability is due to genetic heterogeneity in the nuclear receptor RXRalpha (9-cis retinoic acid receptor). RXRalpha is essential for the action of many nuclear receptors (e.g. PPAR, THR, RAR) and lays significant roles in development and maintenance of bone, muscle and skin tissue (among other tissues). Ligand and artificial mutation suggest that alterations in RXRalpha activity or interactivity could have a variety of phenotypic consequences. We hypothesize that mutations in genes such as RXRalpha could contribute to (or protect against) severe or early onset declines in bone, muscle or skin. Specific aim #1 is to recruit small cohorts of well-characterized older individuals free of confounding conditions (e.g. glucocorticoid use) selected for severe and/or early onset: (i) osteoporosis, (ii) muscle weakness, (iii) decreased skin integrity, or (iv) absence of these at extreme old age. Specific Aim #2 is to perform SSCP analysis of RXRalpha on genomic DNA from 15 individuals of each cohort, and begin characterization of any variants identified. Beyond the scope of this pilot project is (i) screening these cohorts for variation in other genes that may influence musculoskeletal or dermatologic changes with age; (ii) performing functional analyses of large definitive association studies with any variants found from this screen; or (iii) assessing other conditions potentially influenced by variation in RXRalpha (e.g. thyroid function, vision). However, this project should form bases from which such questions could be explored. Within its scope, it may help to elucidate general pathways underlying age-related changes in body composition/function, which ultimately will lead to improved means of preventing and treating their sequelae (perhaps with the RXRalpha agonists/antagonists now being developed).
|
{
"pile_set_name": "NIH ExPorter"
}
|
The proposed study will continue a program of research aimed at empirically examing those variables theorized to affect the psychological health and well-being of women attempting to leave abusive partners. The study includes a longitudinal, experimental design, and will employ multi-method strategies to measure level of physical and psychological battering, depression, self-efficacy, locus of control, fear and anxiety, community resources, social support and psychological well-being over a two-year period following residence at a battered women's shelter. In order to examine hypothesized causal and correlative relationships, the effects of an intervention designed to help women mobilize community resources and social support will be examined. It is hypothesized that the increase in community resources and social support will lead to increased psychological health and well-being, as well as less abuse. The research utilizes a factorial design with two independent variables--time and the two levels of experimental condition. Interview outcome measures will be collected at six points--time zero, 10-weeks (post-intervention), 6-months, 12-months, 18-months, and 24-months. Archival data will also be collected from 1) the battered women's shelter, 2) the intervention agents' written weekly records, 3) police records, and 4) court records. This study will also examine the relationship between the following variables both within and across the six assessment periods: 1) depression, 2) self-efficacy, 3) locus of control, 4) fear and anxiety, 5) access to community resources and social support, 6) physical and psychological abuse, and 7) psychological well-being. Within the time period, the concurrent relationships among the variables will be examined. By also examining these relationships across time through path modeling, the potential causal ordering can begin to be examined. Funding is being requested to continue this research study for five years. The additional participants generated (projected N=160) will increase power in order to conduct meaningful within-and between- group analyses.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The major goal of this project is to determine the extent to which changes in plasma levels of specific lipoproteins, lipoprotein subspecies, and apolipoproteins are associated with quantitative changes in coronary atherosclerosis during the course of a controlled four-year risk factor intervention program. The study groups consists of 300 patients admitted to Stanford University Medical Center for management of coronary artery disease (transluminal percutaneous coronary angioplasty, coronary artery bypass surgery, or medical treatment). Coronary angiography and computerized quantitation of non-bypassed or non-dilated coronary artery segments have been carried out at baseline on all subjects and will be repeated at four years following medical management (all subjects) and intensive multiple risk factor is being carried out under separate funding at Stanford. The special intervention program at Stanford includes improved nutrition, weight reduction, increased aerobic exercise, stress management, and individualized treatment including pharmacologic reduction of low density lipoprotein cholesterol, elimination of cigarette use, and reduction of hypertension. Initial lipoprotein analyses on all 300 subjects were performed one to three weeks after medical stabilization, or six weeks after surgery, and are being repeated annually during four year of follow-up. In addition, pre-treatment samples will be obtained on al subjects beginning HMG CoA reductase-inhibitor treatment. Subfractions of plasma very low density (VLDL), intermediate density lipoprotein (IDL), low density (LDL), and computer-based quantitation procedure. LDL and HDL subspecies are also measured by AII and B in plasma, and apo B in LDL are measured by immunoassay. Analysis of lipid make it possible to identify those lipoprotein parameters most closely involved in the disease process. The results will also indicate whether detailed measurements of specific lipoprotein subclasses and apolipoproteins are more informative that conventional lipid measurements in predicting changes in coronary artery disease and in assessing the effects of strategies directed at its prevention and treatment.
|
{
"pile_set_name": "NIH ExPorter"
}
|
While many investigators have made great progress in developing methods for computer-aided detection and diagnosis (CAD) of lesions, current human interfaces for communicating the computer output to the user are inadequate. Intelligent workstations that aid radiologists in diagnosing cancer utilize an estimate of a lesion's probability of malignancy, usually obtained by training a classifier on an independent database. These estimates of the probability of malignancy are dependent on the prevalence of cancer in the training database, which most often does not correspond to the prevalence of cancer in the population from which the user has experience, e.g., the population seen in the user's medical practice. Thus, the user often has difficulty interpreting the computer-estimated probability of malignancy. This proposal aims to extend our intelligent workstation to include a transformation of the computer output that is either reader specific or radiology-practice specific. The specific aims of the study are (1) collect radiologists' rating data on a database of clinical mammograms and sonograms in terms of their assessment of the probability of malignancy, (2) develop models with which to transform computer output to those that would "match" the internal parameters of the reader, (3) compare the models using both the computer and human data across the two modalities, (4) use the results of the transformation to modify computer output in our design of clinically useful interfaces, and (5) perform the first test of CAD using enhanced intelligent interfaces customized to the particular radiologist or the particular radiology practice. The proposed study is highly relevant in that such enhanced human computer interfaces are expected to improve and expedite the use of computer aids in breast cancer imaging and interpretation. [unreadable] [unreadable]
|
{
"pile_set_name": "NIH ExPorter"
}
|
Genomic and transcriptional studies have now been completed that resolve human breast tumors into distinct subpopulations that progress and respond differently to aggressive chemotherapy. The breast tumor subtypes designated luminal/amplifier and basal respond least well to aggressive chemotherapy so our goal now is to develop more effective therapies against these two subtypes. This will be accomplished through work in three specific aims. Aim 1. An automated, high throughput approach will be used to assess responses to -100 FDA approved and experimental drugs (including those developed in other SPORE projects) in a collection of >50 breast cancer cell lines grown in two dimensional cultures in order to identify drugs that are particularly effective against the basal and luminal/amplifier subtypes. Drugs will be ranked for relative effectiveness in the basal and luminal/amplifier subtypes. Those that show high efficacy in either of these subpopulations will be further evaluated in additional breast cancer cell lines developed in this project and then in 3D cultures representative of the basal and luminal/amplifier subtypes. The most effective basalspecific drugs will be passed to the SPORE Project 3 for packaging into nanoparticle constructs that deliver them specifically to the basal tumor cells and/or tested as existing drugs in new trials via our I-SPY neoadjuvant network or in advanced clinical trials. Aim 2. CLIA compatible multi-gene molecular assays will be developed that define the luminal/amplifier and basal subtypes that can best be attacked using drugs and drug constructs identified in aim 1 in order to guide deployment of these drugs in clinical trials. Multi-gene assays developed in the last project period will be refined through analysis of formalin fixed paraffin embedded samples from the SPORE Tissue and Outcomes Core and then validated in 237 samples from the neoadjuvant I-SPY 1 Trial and further validated in 114 new samples resulting from the I-SPY 1 Amendment trial. Once basal and luminal/amplifier subtype specific drugs are identified, the multivariate assays will be refined to predict individual drug responses. Aim 3. Molecular mechanisms/pathways that influence response/resistance to the drugs selected in aim 1 will be assessed in order to facilitate selection of synergistic drugs and to guide elucidation of mechanisms of resistance.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Abstract Acute aortic dissection, particularly the type A dissection (AAD), is a life-threatening condition. Currently, there are no effective measures to prevent its onset and progression. A major barrier to satisfy these critical, unmet clinical needs is the poor understanding of the mechanisms that drive AAD development. AADs usually occur in aortas suffering progressive aneurysmal degeneration. However, compelling clinical evidence suggests that AADs and aortic aneurysms precede through distinct biological pathways. Yet, uncoupling these pathways has been a challenging task due to the silent onset of aortic dissections in patients coupled with a lack of animal models capable of mimicking the development of AAD reliably. To address this issue, we created two novel mouse AAD models, termed as ?aortic tear model? and ?aortic rupture model?, respectively. The ?aortic tear model? develops spontaneous aortic tears with few ruptures in mildly dilated ascending aortas, whereas the ?aortic rupture model? features acute aortic dissections with a high rate (40%) of aortic rupture in the first week. Using these models, we tested the long-standing, but unproved, hypothesis?disorders of immune response promote AAD formation. We found that 1) development of aortic tears is paralleled with an increased CD4+ T- cells and CD19+ B-cells in the AAD tissue as well as in the peripheral blood; 2) Th2 polarization via adoptive transfer of ex vivo expanded Th2 cells or neutralization of the Th1 signature cytokine interferon gamma (INF?) exaggerates AAD dilation; 3) complement components are upregulated and deposited in the medial layer of AADs; and 4) genetic shifting of T-cell-mediated immune response to a Th2 prominent immunity dramatically provokes aortic rupture (>90% in four weeks). These novel findings led to an overall hypothesis that skewing of the inflammatory response in the aneurysmal aortic wall to type 2 immunity promotes AAD development. In this project, we will use genetic, adoptive cell transfer, and pharmacological approaches to evaluate the role of T- cells, B-cells, and complement system in regulating AAD development, with profile of immune cell subsets and cytokine milieu characterized to understand the cellular and molecular events engaged in promoting AAD formation. Critical findings will be validated for their implication across different mouse models, and more importantly, their relevance to human AAD development. Completion of this project will lay a solid foundation for future studies to develop immunotherapies to prevent AAD formation.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The overall goal of this project is to develop a safe, non-invasive non-ablative focused ultrasound therapy (NAFU) which can be used for the treatment of primary carcinoma and prevention of metastases by increasing tumor immunogenicity. NAFU treatment be used as an adjuvant therapy with current therapies, such as chemotherapy, immunotherapy, surgery, radiotherapy, and HIFU. This proposal will establish the role of NAFU in generating systemic anti-tumor immunity and define specific protocols for inducing ER stress and augmenting tumor immunomodulatory markers, such as heat shock protein 70, calreticulin, ATP and HMGB1. NAFU will be combined with two different novel immunotherapies: a dendritic cell-based vaccine and a Listeria-based prostate cancer vaccine. Preliminary data indicates that NAFU-treatment increases cell surface immunomodulation, which when cultured with dendritic cells ex vivo will result in an enhanced autologous vaccine. Additionally, preliminary studies demonstrate that NAFU-treatment of a live, attenuated Listeria-based vaccine for prostate cancer enhances phagocytosis of the bacteria, resulting in a more robust immune response and control of tumor burden. Together these projects will solidify non-ablative focused ultrasound as an effective cancer treatment option which will induce systemic anti-tumor immunity, treat primary tumors, and reduce metastases.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Congenital cardiovascular malformations are the leading cause of neonatal and infant death and a major cause of adult cardiac insufficiency. Valvular stenosis and hypoplastic left heart syndrome are the most common malformations occurring in eight out of 1000 live births and constituting 25-30% of all cases of human cardiovascular malformations. There is a critical need to understand the mechanisms that cause these diseases. Our long-term goal is to identify the mechanisms and causes of these cardiac diseases and to develop tools to prevent their incidence. The objective of these studies is to determine whether an association between genetic and environmental factors is the ultimate cause responsible for disease incidence. Our central hypothesis is that prenatal exposure of mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD;dioxin), an organochlorinated environmental pollutant, mediates the aryl hydrocarbon receptor (AHR)-dependent repression of the homeobox transcription factor NKX2.5, a key determinant of cardiac morphogenesis. Our rationale is that mutations in the NKX2.5 gene and maternal exposure to halogenated hydrocarbons, dioxins and polychlorinated biphenyls during pregnancy are the main known risk factors for these human developmental cardiac malformations. Key in this context is our finding that AHR represses NKX2.5 expression. Completion of our specific aims will achieve the following short-term goals: (1), to characterize the AHR role in cardiovascular development and NKX2.5 regulation;(2), to identify regulatory gene expression changes during cardiovascular development that result from TCDD exposure;(3) to determine if TCDD exposure during embryonic development causes long-lasting cardiovascular malformations. These aims test the working hypothesis that organochlorinated environmental agents disrupt AHR-regulated functions during cardiac embryonic development and cause long-lasting cardiovascular injury. We will use promoter-mediated selection methods to model the gene-environment interactions established between NKX2.5, AHR and dioxin exposure and use global gene expression analyses and chromatin immunoprecipitation to identify genetic and epigenetic signatures characteristic of TCDD effects during differentiation. We will probe tissues of newborn and adult mice exposed to TCDD in utero for the expression of target gene clusters, epigenetic changes and incidence of cardiac malformations. The significance of this work lies on the identification of the molecular mechanisms by which AHR and TCDD exert their cardiac effects, providing a test of the causal connections between AHR, dioxin and cardiovascular disease. The novel approach that we propose will define a path applicable to many similar studies of other environmental agents. Our work is innovative because it proposes to use a combination of molecular tools and ES cell research never before employed in the study of the mechanisms of action of environmental agents. We expect to establish a strong mechanistic link between TCDD exposure, the genes that the AHR activates and cardiac malformations. PUBLIC HEALTH RELEVANCE: The exposure of human populations to aryl hydrocarbon receptor organochlorinated ligands has been associated with a number of disease outcomes, including cancer, chloracne, cardiovascular disease, diabetes, endometriosis, neurocognitive deficits, immunotoxicity and especially, cardiac developmental abnormalities. Two central questions that need to be addressed in this context are, (i), what are the mechanisms responsible for the causation of these diseases, and (ii) what are the long-term health consequences of developmental exposure on the susceptibility to environmental disease in the adult. By focusing this renewal application on the potential consequences of fetal exposure on congenital cardiac malformations and incidence of adult disease, we will address these two most critical issues of environmental health research today.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The factors involved in the regulation of myelin sheath formation, which is essential to the proper functioning of the nervous system, are still poorly understood. In this proposal, we will test the hypothesis that the timing and localization of myelination are controlled by regulating the development of myelinating oligodendrocytes. We will specifically focus on the role of the Notch signaling pathway, which has recently been shown to strongly inhibit oligodendrocyte differentiation in vitro. Oligodendrocyte precursor cells constitutively express the Notch1 receptor. Nearby retinal ganglion cells express the Notch ligand Jagged1 early in development, and downregulate its expression in a manner which temporally parallels the onset of optic nerve myelination. Two hypotheses were suggested by this data, which we propose to test here. These two hypotheses are: (1) the Notch pathway may control the timing of optic nerve myelination, and (2) the Notch pathway may control the localization of myelination (i.e determine which axons are myelinated and which remain unmyelinated). We will take two main approaches to address these questions. First, we will attempt to determine whether we can promote myelination in co-cultures of oligodendrocytes and neurons by disrupting Notch-mediated signaling. Second, we will attempt to perturb Notch signaling in vivo by a variety of methods to observe what effects, if any, this has on the timing and localization of myelination in the developing nervous system. A greater understanding of the mechanisms that normally regulate myelin formation may allow for the development of new treatments for demyelinating diseases such as optic neuritis and Multiple Sclerosis.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The regulation of beta-lymphoma growth by antibodies directed at membrane IgM (anti-IgM) has been studies using a panel of murine B-cell lymphomas. Previous evidence in our laboratory indicates that anti-IGM antibodies are able to inhibit growth of these lymphomas at a point in early G1 such that these cells arrest near the G1:S interface, after which they undergo apoptosis and die. In these cells, the retinoblastoma growth suppressor gene product, pRB, is underphosphorylated in the presence of anti-IgM, in a manner that mimics the effects of TGF-beta. However, recent data suggest that this effect may be TGF-beta independent. We have recently demonstrated that this process is consequent to the modulation of c-myc and dependent on the activity of the blk tyrosine kinase gene product. During this grant , we propose to inhibit/modulate tyrosine kinase activity by anti-sense oligos and by transfection of different tyrosine kinase genes, either in a constitutively active or an inactive form. In addition, analysis of c-myc protein levels and localization (nuclear vs. cytoplasmic) will be performed in conjunction with establishing the kinetics of PRB phosphorylation events. These studies will provide information on neoplastic B-cell regulation and the role that these kinases and oncogenes/anti-oncogenes play in this process.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Four cytogenetic techniques were compared to detect the clastogenicity of Mitomycin C in somatic or germinal cells of Rhesus monkeys (Macaca mulatta) in vivo: peripheral lymphocyte and bone-marrow cytogenetics, the micronucleus test in bone-marrow, cytogenetic evaluation of primary spermatocytes. Only the lymphocyte test failed to detect clastogenicity probably due to absence of DNA replication in these cells during treatment. The micronucleus test also failed in samples collected by biopsy. The bone marrow test proved to be as sensitive as the micronucleus test and could be performed with biopsy material. The lymphocyte test may also be potentially useful with testing of chemicals that are not dependent on DNA replication for their action. The cytogenetic test of primary spermatocytes was positive only when treatment was applied during the pre-meiotic S-phase. The pre-meiotic S-phase is 17 days before diakinesis-metaphase I.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The long term goal of this study is to identify essential components in the IRBP promoter required for full protein expression. These components include cis-elements, a basal promoter, small effector molecules and trans factors. Previous work has identified a short (less than 70 bp) essential IRBP promoter region, demonstrated that chick retina primary cell cultures provide a suitable in vitro assay system for IRBP promoter activity, identified a specific trans factor and devised a model of the IRBP promoter. The primary strategy is to exploit the promoter activity measurements in the retina cell culture of mutated IRBP promoters. The specific aims are: (1) to test whether the two cis-activators and basal promoter exist as proposed; (2) to test whether the putative retinoic acid receptor/estrogen receptor element shows responsiveness to these effector molecules in IRBP promoter activation; and (3) to test whether the first two proposed trans factors exist and to identify, clone or purify the above mentioned trans factor for the -55 to -50 cis-element. The experiments will either prove the proposed model correct or lead to appropriate revisions. The research will be significant in understanding of the basics of IRBP gene expression, clarifying of transcriptional issues and obstacles for gene therapy in the retina, creating practical tools for future photoreceptor-specific gene therapy, and understanding the coordination of photoreceptor-specific gene transcription.
|
{
"pile_set_name": "NIH ExPorter"
}
|
We are now screening, staging and intervening first degree relatives of patients who have Type I diabetes for anti-islet cell antibodies to detect those subjects who have a moderate risk of developing diabetes in the future; further determination of the risk for Type I diabetes by examining the first phase of insulin secretion during an IV glucose tolerance test. An algorithm based on historical data will then be used to select those who have a greater that 50% risk of developing Type I diabetes; risk subjects will be randomized into an intervention study. Those with a 5 year risk between 25 and 50% are currently being randomized into a blinded trial of po insulin.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The function of osteocalcin, a most abundant non-collagenous protein in bone containing the vitamin K dependent calcium binding amino acid, gamma-carboxyglutamic acid (Gla), is being investigated. Osteocalcin has been isolated and characterized as a 6000 MW protein from many species, however its biosynthetic pathway remains to be elucidated. Studies in bone organ culture and in bone microsome preparations indicate a precursor form of approximately 70,000 MW occurs. This suggests that the vitamin K dependent carboxylation reactions in bone may be analogous to the liver microsomal carboxylation of precursor prothrombin and may regulate conversion of proenzyme to active enzyme forms. The synthesis of osteocalcin in specific bone cell types is being monitored and the effect of bone hormones on osteocalcin synthesis and secretion is being studied. The relation of osteocalcin to the formation of the mineral phase of bone is being examined by measuring its distribution as a function of the density of bone and by comparing the synthesis of the Gla precursor and osteocalcin in normal, vitamin D and vitamin K deficient, 6 week chick bone. Cellular alterations in bone tissue and changes in mineral content are being examined during vitamin K antagonism in chick embryos treated with warfarin. Since calcified cartilage tissues of elasmobranchs are also rich in Gla, vitamin K carboxylated proteins may have a generalized function in calcification and purification of the protein my reveal certain similarities with bone osteocalcin. All pathologically calcified tissues contain Gla residues and the origin of the Gla protein in subcutaneous calcified plaque associated with scleroderma and dermatomysitis is being investigated. To gain further insight into the role of osteocalcin in bone metabolism, the turnover of osteocalcin in patients with bone related disorders is being examined by measuring urine Gla excretion and serum osteocalcin levels. Patients with ectopic calcification disorders excrete 2-3 fold more Gla than controls; rachitic (osteomalacia) children excrete less than their controls.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Work from several laboratories over the past 10-15 years has established that the monomeric end-products of lysosomal digestion, e.g. amino acids, monosaccharides, nucleosides and (probably) lipid components traverse the lysosomal membrane to the cytoplasm through the agency of specific carriers rather than by simple diffusion. The importance of these carriers to animal metabolism is indicated by the existence of two human disorders, viz., cystinosis and sialic acid storage disease, which owe their origin to inherited defects in the lysosomal transport systems for cystine and sialic acid,, respectively. To date, none of the aforementioned lysosomal transport systems has yet been purified or characterized by direct chemical approaches, nor has any of these carriers been cloned by genetic linkage studies or other recombinant DNA techniques. Although direct purification of membrane transportors is hampered by the lack of a suitable assay system, such difficulty has been overcome in the past by the use of reconstituted lipid membrane systems or proteoliposomes, i.e., lipid vesicles incorporating the membrane proteins and which retain the original transport function. As a preliminary to employing this methodology to the purification of the lysosomal cystine carrier, we have prepared lysosomal membrane vesicles from rat liver and have investigated their characteristics with respect to cystine transport. Our studies have indicated that these vesicles possess cystine transport activity and may therefore serve as useful starting material in the preparation of functional proteoliposomes for purification of the cystine carrier.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Projects included are: 1) A follow-up of young black men. In the current year we studied the development of childhood deviance in these men, identifying causal sequences of behavior. 2) A study of school and police records over two generations. We found parents' arrests to be powerful predictors of delinquency in their children. Conformity in the extended family tended to ameliorate the parents' effects to a small degree. 3) A study of depression, drinking, and suicidal ideation in 219 older black and white patients and controls. Equally as many blacks as whites had depression, but fewer attempted suicide or belived that suicide was ever justified. 4) A record net for identifying an unbiased sample of alcoholics. One hundred seventy-seven black men without alcoholism in 1965 have been traced through police and hospital records to see whether those whose earlier records resembled records of alcoholics did develop alcoholism. 5) Screening interviews have been developed for five psychiatric diagnoses (hysteria, alcoholism, depression, anxiety neurosis, and antisocial personality) and validated by retrospective and prospective studies for the first two diagnoses. 6) In the course of a follow-up study of Vietnam veterans, information about drugs, alcohol, depression, and deviance had been collected. Pre-service non-drug deviance has been found to be a powerful predictor of liability to readdiction in former narcotic addicts who resumed use after return.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The objectives of this study are to utilize recently developed methods for the successful in vitro culture of rat trophoblast to establish predictable relationships between certain structural and functional events that occur in the cultured trophoblast cell. These events will be correlated with the development and differentiation of the trophoblast in vivo and in vitro. We will also determine if these processes are regulated or occur autonomously. Rat trophoblast, in utero, hatches and the trophoectoderm then attaches to the endometrium and insinuates itself between epithelial cells. By the process of invasion the cells then penetrate the decidualized stroma to establish a relationship with maternal vascular sinuses. Blastocysts cultured in vivo follow a similar pattern. This provides a very advantageous model for analyzing trophoblast development by studying these processes in defined conditions which can be experimentally altered. After hatching trophoectoderm of cultured blastocysts attach to co-cultures of uterine stroma cells. Initially they insinuate themselves before invading the stromal monolayer. Cells cultured without a substration will grow out on the plastic dish. We propose to characterize these cellular functions of cultured trophoblast by analyzing available developmental landmarks. Microtubule assembly and distribution (morphogenesis and outgrowth) will be correlated with evidence of invasiveness (plasminogen activator) and with indices of biochemical differentiation (steroidogenesis of progesterone, testosterone and estradiol; production of rat placental lactogen). Realization of the objectives of these studies of structural and functional correlates of trophoblast development are strengthened by the availability of a productive model (in vitro culture of rat trophoblast). Additional reinforcement derives from preliminary observations in cultured trophoblast of microtubule distribution, synthesis of testosterone and estradiol and production of rat placental lactogen. These are described here for the first time.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Triple negative breast cancer (TNBC) accounts for 15% of all breast cancers, and patients with this disease have an increased likelihood of distant recurrence and shorter overall survival compared to non-TNBC patients. The standard of care for TNBC is neoadjuvant therapy (NAT), consisting of a panel of cytotoxic therapies, followed by surgery. However, the field currently lacks a consensus on the appropriate combination of therapies and an ability to predict how any patient will response to a given therapeutic regimen. This proposal addresses these issues through construction of a mathematical model utilizing tumor-specific imaging data. Several attempts have been made to capture tumor growth and treatment response within a mathematical framework, but many of those attempts have relied on parameters and data that are difficult or impossible to measure with the requisite temporal and spatial resolution. This effort is distinguished by proposing a model parameterized exclusively with experimentally available data. Specifically, the proposal builds on recent advances in time-resolved automated fluorescent microscopy and diffusion-weighted magnetic resonance imaging (DW-MRI) to populate the proposed model. Fluorescent microscopy can track cell populations in two dimensions over time, and DW-MRI can provide quantitative information on cell density in three dimensions. We have developed in vitro assays to leverage each of these imaging modalities to track tumor status noninvasively throughout the course of therapy. We hypothesize that this data can be used to initialize a computational model to predict: 1) the temporospatial response of TNBC to therapy and 2) optimal NAT regimens for a given tumor. To test this hypothesis, we propose three specific aims: 1) to measure and model TNBC cell line response to NAT in 2D using fluorescent microscopy, 2) to measure and model TNBC cell line response to NAT in 3D using MRI, and 3) to evaluate model predictions in an in vivo model of TNBC. The proposal will subject a representative sample of TNBC cell lines to a panel of clinically relevant therapies evaluating tumor response in both 2D and 3D. Preliminary data indicates that data collected via fluorescent microscopy can describe tumor-scale data collected via MRI with appropriate temporospatial scaling factors. The last decade has produced significant advances in the genetic and molecular characterization of tumors and their response to therapy. Our proposal will provide insight into how these cell-scale observations translate to clinically relevant measures of tumor status. Further, this proposal will quantitatively characterize the response of TNBC to NAT. The field requires this quantitative understanding to properly evaluate next- generation therapies. Finally, this proposal will demonstrate the utility of a computational approach to therapy design through in vivo experiments. Ultimately, these Aims will move the field towards the goal of precision medicine: delivering the optimal drug in its optimal dose on an optimal schedule to each patient.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Heart disease is the leading cause of death in the United States. One million Americans die from heart disease or about one person every 33 seconds. Modification of cardiac muscle performance through gene transfer holds promise as one mechanism to prevent or correct acquired and inherited cardiac diseases. The goal of this proposal is to optimize gene transfer through adenoviral gene delivery in vivo in working myocardium. Adenovirus vectors for expression of important myocardial proteins represent a potentially efficient strategy for correcting deficiencies in the failing heart. In Phase l, we have optimized the conditions for maximal efficiency of gene transfer in vivo in the rat model. In this proposal, we will optimize the conditions for gene transfer and examine closely the expression and inflammatory response induced by adenoviral gene transfer. In addition, we will examine the effect of overexpressing key proteins in murine models of heart failure. A primary concern centers on the stability of gene expression in the myocardium. To date adenovirus reporter gene transfer experiments show that transgene expression can be transient. PROPOSED COMMERCIAL APPLICATIONS: There is a rapidly growing market for the ability to test gene therapy vectors. The optimization of a delivery system as well as technology for quantification of clearly defined end-points in well characterized animal models of disease are of high market value to the biotechnology and pharmaceutical industries as well as individual investigators.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Heavy metals such as arsenic, nickel, and chromium as well as polycyclic aromatic hydrocarbons (PAHs) as benzo(a)pyrene are common environmental contaminants. Human exposure to such toxicants greatly increases cancer risk. The carcinogenicity of PAHs has been attributed to their covalent binding to DNA, which can result in mutations that ultimately lead to carcinogenesis. The mechanisms of metal-induced carcinogenesis are less clear. It has been found that nickel and arsenic exposure can induce DNA hypermethylation. Such non-mutational epigenetic changes could also result in suppression of tumor suppressor genes, such as the p53 gene, triggering tumorigenesis. Recently, we have found that the guanines at C5 cytosine methylated CpG sites are the major DNA targets of many PAHs, including benzo(a)pyrene diol epoxide (BPDE). In fact, we have found that the BPDE binding spectrum in the tumor suppressor p53 gene coincides with the mutation spectrum of this gene in lung cancer. It has been found that a mutated p53 gene is sufficient to trigger carcinogenesis in animal models, and that more importantly, 50% of human cancers have a mutation in the p53 gene. These findings have lead us to hypothesize that targeted DNA damage, rather than selection, is the major determinant of the p53 mutation spectrum in cancer, and that the CpG selection, is the major determinant of the p53 mutation spectrum in human cancer, and that the CpG methylation status of an individual p53 gene may determine the susceptibility of this gene to DNA damage and mutation. These findings have led us to hypothesize that targeted DNA damage, rather than selection, is the major determinant of the p53 mutation spectrum in human cancer, and that the CpG methylation status of an individual p53 gene may determine the susceptibility of this gene to DNA damage and mutation. In light of these findings, we propose that the carcinogenicity of nickel, arsenic and chromium may be partly due to their ability to induce DNA hypermethylation, which in turn enhances the susceptibility of methylated tumor suppressor genes and protooncogenes to bulky carcinogen-induced DNA damage and mutations. The objective of this research is to test the hypothesis using two state-of-the-art technologies; UvrABC-ligation-mediated-PCR to map DNA adducts at the single nucleotide level, and the p53 GeneChip to detect mutations and cytosine methylation. We will determine: 1) the p53 gene methylation status in lymphocytes of different individuals and assess its relationship with the susceptibility to BDE damage, 2) whether nickel, arsenic, and chromium induce changes in the methylation status of the p53 gene and whether nickel, arsenic, and chromium induce changes in the methylation status of the p53 gene and, consequently, in the susceptibility of this gene to BPDE induced-DNA damage, and 3) the effect of nickel, arsenic and chromium treatment on the repair of BPDE-DNA adducts in the p53, beta-actin and HPRT gene.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The multiple genetic markers (allotypes and idiotypes) of rabbit immunoglobulins and homogeneous antibodies will be used in studies to define the number and nature of genes encoding antibody variable regions. Segregation of VH allotypes with idiotypes of homogeneous antibodies have indicated linkage between these markers. Observations of unexpected combinations of these markers have suggested, however, that the genes encoding the binding site (idiotypes) are distinct from those encoding constant portions (allotypes) of the variable region. Further studies on idiotypes and group a allotypes will attempt to more completely define the genetic relationships between these putative V region genes. Recently described marker for the VL region will be used in experiments to obtain similar data for the antibody light chains. Studies on the subspecificities of the group a allotypes will be continued to determine whether these serologic variations within one allotypic determinant are caused by structural differences in the constant parts of the variable region or by modulation of identical determinants by differences in the hypervariable regions of idiotypically distinct molecules. The use of sensitive radioimmunoassays has shown the presence of group a allotypes not detected by quantitative typing nor anticipated from breeding data in the sera of about 50 percent of the rabbits tested. These latent allotypes were shown to occur in a transient and sporadic fashion. The molecules carrying latent allotypes will be fully characterized and studies will be carried out to determine whether the group b allotypes on the light chain behave in a similar nonallelic fashion. Sera containing latent allotypes will be examined for the presence of antibodies directed against these allotypes.
|
{
"pile_set_name": "NIH ExPorter"
}
|
This study investigates the safety and tolerability of losartan administered concurrently with the usual dose (10mg BID) of enalapril in patients with with NYHA Functional Class III to IV heart failure treated previously with an ACE inhibitor. Additionally, to investigate the effects of the three treatment regimens of losartan administered in addition to usual dose enalapril and high dose enalapril on submaximal exercise tolerance, neurohormone activation and quality of life.
|
{
"pile_set_name": "NIH ExPorter"
}
|
This grant will partially support travel costs and conference fees for speakers and session Chairs who have been invited to participate in the Molecular Membrane Biology (MMB) Gordon Conference to be held July 8-13, 2007 at Proctor Academy in Andover, NH. This meeting brings together scientists working on all aspects of membrane structure and function, including vesicle trafficking, organelle biogenesis and structure, lipid biology, membrane protein structure and function, and protein translocation. Hence, MMB has great value in that it facilitates detailed discussion and scientific exchange between investigators focused on understanding the complexity of membrane function. Proposed session topics include: Entering the ER; Traversing the Golgi; Lipid Transport and Localization; Vesicle Formation; Pathogens and Membrane Traffic; Organelle Structure and Dynamics; Membrane Domains; Membrane Fusion; and Reaching the Plasma Membrane. We have invited a diverse group of speakers, many of whom are early in their careers and 22% of whom are female. We have left open at least 19 slots for additional short presentations chosen from submitted abstracts, increasing our opportunities for hearing the most recent and exciting results and diversifying our speakers. We have invited several established investigators (33% female) to serve as session Chairs, and have set aside 20 minutes at the end of each session for the Chairs to present an overview and to lead a general discussion of current controversies, unresolved issues, and future directions, in a manner that will engage a large percentage of the audience. This meeting is generally oversubscribed and is considered a 'must-attend' for many in the field. Research conducted by attendees is funded by several NIH institutes, including NIGMS, NCI, NEI, NIDDK, NICHD, NIMH and NIAID. Thus, the work discussed is of general significance to human health and disease. [unreadable] [unreadable] [unreadable]
|
{
"pile_set_name": "NIH ExPorter"
}
|
PROJECT SUMMARY The overall mission of the National Center of Neuromodulation for Rehabilitation (NC NM4R) will be ?fostering the success of the NM4R researcher?. The principal objective of the NC NM4R Pilot Studies Program is to prioritize, provide and manage short-term funding for scientifically meritorious, new and innovative projects through a competitive program with open solicitation and peer review. The goal is to provide adequate resources, support and training for pilot investigators to develop sufficient preliminary data to prepare a competitive extramural grant application as Principal Investigator that will directly utilize one or more of the NC NM4R resources. The program will focus on enabling NC NM4R affiliates (e.g., investigators who have participated in NC NM4R workshops and/or collaborative opportunities) to conduct proof-of-principle or feasibility studies and/or explore an area that is distinct from their currently funded research using neuromodulatory methodologies supported by the NC NM4R. Not only will this component provide a pilot studies program for qualified researchers (Aim 1), we will also have extensive interactions with these investigators before submission and after they receive their award (Aim 2). This program will solicit pilot study applicatons annually by broadly advertising a request for applications (RFA) five months in advance of the anticipated award date. Methods of distributing the RFA will include announcements on the NC NM4R web site, e-newsletter, listserv announcements to Center affiliates and other targeted avenues with national reach. Submission will be web-based with a two-phase application process including a Letter of Intent (LOI) followed by a full application after interactions with the NC NM4R. Applications will be peer-reviewed and scored using the NIH scale, then prioritized according to four criteria: 1) quality of the research (scientific merit, innovation and impact); 2) relevance to the mission of the Center and direct involvement of one or more of the NC NM4R resources in the research plan; 3) qualifications of the research team to lead the research effort toward securing extramural funding, and 4) stimulation of collaborative efforts with NC NM4R investigators. We plan to fund an average of four new or competitive continuation pilot studies per year with 12-month project periods. Applicants must be eligible Principal Investigators as defined by their home organization and have the skills, knowledge and resources necessary to carry out the proposed work plan; they also must be eligible to submit an NIH R01 or equivalent as PI from their institution. Investigators will receive one year of research support along with access to NC NM4R cores, equipment and other resources as needed to perform an investigator-initiated project related to the mission of the NC NM4R. We envision that the Pilot Studies Program will enable investigators to form interdisciplinary, interactive, sustainable research projects that will impact NM4R research.
|
{
"pile_set_name": "NIH ExPorter"
}
|
In order to determine the prevalence, severity, and extent of endocrine dysfunction in patients with acute and chronic illness, a study will be undertaken to compare the responsiveness of various anterior pituitary disease and normal controls.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The research proposed here will identify several components that are deemed essential for the development of alcohol-related culturally appropriate preventive interventions that target Mexican Americans. The proposed project has the following specific objective: To identify the culture- specific attitudes, expectancies, and norms held by Mexican-Americans regarding: (a) alcoholic beverage consumption in general; (b) heavy drinking; and (c) driving under the influence of alcohol (DUI). In order to meet this objective, random samples of Mexican Americans and nonHispanic Whites in Los Angeles, California and in San Antonio, Texas will be interviewed over the telephone in order to identify their culture-specific attitudes, norms, and expectancies related to alcoholic beverages consumption, heavy drinking, and DUI.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Present approaches are unable to cure rheumatoid arthritis (RA) and other chronic autoimmune diseases. The current proposal plans to develop a novel approach that tests the central hypothesis: CD4?? regulatory T cells (iTregs) generated with IL-2 and TGF-2 are stable in an inflammatory milieu and are able to treat collagen-induced arthritis (CIA). While thymus- derived, naturally-occurring CD4???? (nTregs) suppress Th1- or Th2-cell-mediated autoimmune diseases, these cells are less successful in controlling (IL-17-producing) Th17 cell- mediated diseases such as CIA. Additionally, unlike iTregs, nTregs have increased cell plasticity, and can be converted into Th1, Th2 or Th17 cells while losing their suppressive activities in the presence of pro-inflammatory cytokines. We and others have established TGF-2 is able to convert naove CD4? cells to iTregs that share similar phenotypic and functional characteristics with nTregs. Interestingly, our recent studies revealed that unlike nTregs, iTregs did not make conversion to Th17 and Th1 cells in the presence of pro-inflammatory cytokines. In addition, iTregs but not nTregs maintained the suppressive activity against T cell response in the presence of IL-6 in vitro. Moreover, iTregs but not nTregs even prevented other T cells from becoming Th17 cells in the presence of IL-6 and TGF-2. However, pretreated nTregs with IL- 2/TGF-2 or atRA alters the plasticity and restore functionality of nTregs. Based on these preliminary data, we anticipate that iTregs and pretreated nTregs are stable following adoptive transfer into the established autoimmune arthritis. We expect iTregs and pretreated nTregs can significantly ameliorate the clinical signs of the established arthritis following treatment. We also expect that combination of all-trans retinoic acid (atRA) and TGF-2, or antigen-specific iTregs can enhance the therapeutic effects of iTregs on the established arthritis. We believe that iTregs not only directly suppress T cell response, but also induce the formation of tolerogenic DCs and these DCs produce IL-27, atRA and/or IDO that eventually restrain Th17 cell differentiation and function. Accordingly, the project has three specific aims: 1) Determine the relative stability of nTreg and iTreg cells when adoptively transferred into established collagen-induced arthritis (CIA). nTreg or iTregs will be sorted or induced from DBA/1 Foxp3-GFP knock-in mice or Foxp3-GFP/IL-17-RFP double knock-in mice. nTreg or iTregs cells will be adoptively transferred into DBA/1 or C57BL/6 mice at day 14 or day 28 after immunization with collagen II (CII) and Complete Freund's Adjuvant (CFA). The migration, distribution, survival, phenotype (Foxp3) and conversion into T help cells (Th1, Th2 or Th17 cells) of Treg subsets will be monitored with GFP and RFP expression. 2) Compare the therapeutic effect of both nTreg and iTreg cells on development of collagen-induced arthritis. nTreg, iTreg or control cells will be administrated to DBA/1J mice on day 14 or day 28 after immunization with CII/CFA. The protective effect of these cells will be judged by arthritis incidence and severity, levels of anti-CII IgG2a antibodies in sera and histological examination of arthritic limbs. 3) Define the cellular and molecular mechanism(s) by which iTregs are resistant to Th17 cell conversion and regulate Th17 cell differentiation and function in the inflammatory milieu. We will examine whether the transcription factor T-bet and Th1 cytokine expression in iTregs are responsible for their resistance. We will also examine whether these factors or tolerogenic DCs induced by iTregs contribute to suppressing Th17 differentiation and function. The results from this study will promote the understanding of therapeutic effects of Treg cells in the prevention and cure of rheumatoid arthritis. If successful, this project will also have a direct clinical relevance and possibly provide a novel approach to treat RA and other autoimmune diseases which may not have the severe side effect(s) characteristic of current therapies.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The proposed studies would allow extensive evaluation and development of technetium-99m stannous pyrophosphate (99mTc-PYP) techniques for acute infarct sizing in patients, the further development of noninvasive means of measuring total and segmental ventricular ejection fraction and ventricular wall motion alterations in patients with ischemic heart disease and provide for the additional development of noninvasive techniques for both "hot spot" and "cold spot" myocardial imaging estimates of infarct size. In addition, the proposed studies would allow for the development of additional new means of radionuclide sizing of infarcts, the evaluation of additional possible means of identifying injured myocardial areas during myocardial ischemia and infarction, the development of animal models of unstable angina pectoris and an assessment of the feasibility of using antibodies developed in our laboratory against the myocardial specific CK-MB isoenzyme and human heart myoglobin for myocardial imaging. In addition, these studies would allow us to attempt to develop means of identifying the presence or absence of collagen using radionuclide techniques as a potential tool for quantitating the amount of scar tissue in hearts of experimental animals and man with ischemic heart disease. Altogether these studies performed in both clinical and experimental animal settings of myocardial ischemia and infarction should provide for considerable additional developments of noninvasive radionuclide techniques for sizing myocardial infarctions, assessing the functional impact of infarct size on ventricular performance and attempting to develop new means of identifying injured and/or scarred myocardium.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The human immunodeficiency viruses (HIV-1 and HIV-2), along with their simian counterpart, simian immunodeficiency virus (SIV), belong to the lentivirus family and are the etiological agents of acquired immune deficiency syndrome (AIDS). The regulation of HIV-1 and HIV-2 mRNA expression is determined by cis-regulatory sequences located in the long terminal repeat (LTR) region of the provirus. The HIV-1 LTR contains a core enhancer located at -79 to -109 bp, which has overlapping NFkappaB and ETS binding sites (EBS) (GGGACTTTCC) in a direct repeat configuration. An examination of promoters and enhancers that contain dual or multimeric EBS reveals that two distinct orientations exist for EBS: i) direct, in which the purine rich DNA strand has a linear, "head to tail" orientation, and ii) palindromic, in which the EBS are found to exist in a "head to head" orientation. The spacing between the two EBS cores in the HIV-1 enhancer is ten nucleotides, however, the distance between two EBS cores varies in different promoters.
|
{
"pile_set_name": "NIH ExPorter"
}
|
This competitive renewal of our T35 program is targeted towards undergraduate, graduate, and medical students. Our purpose is to provide and environment that will stimulate these trainees to pursue careers in biomedical research in fields related to heart, lung, or hematologic diseases. The success of our program over the past 10 years has allowed us to expand our program to include clinical and basic science investigators who are highly interactive and studying areas that include lung infections, regulation of immune responses, vascular biology, gene regulation and gene therapy, epithelial and endothelial cell biology, exercise, and airway physiology. These investigators span numerous departments including Medicine, Pediatrics, Microbiology and Immunology, Cellular &Integrative Physiology, Biochemistry and Molecular Biology, Pharmacology and Toxicology, and Pathology. The wide range of laboratories and faculty in our program will provide trainees with outstanding exposure to fields related to heart, lung, or hematologic disease. Progress Central to our success has been that the T35 served as a "link" to combine training experiences for minority high school students up through postdoctoral fellows. Our "multi-tiered" training program has emphasized the importance of having trainees from several educational levels work with students who have exceeded at the next level. Seeing that the next level is attainable empowers the junior trainee and provides a higher level of confidence to pursue careers in science. Further, research methods are reinforced for the senior trainee who teaches a junior trainee. This approach facilitates the development of a student network that persists beyond the end of the training period. An outcome study clearly showed that the program serves as a "pipeline" for recruitment of minorities into biomedical sciences. To recruit and retain minorities in biomedical science we propose several specific aims: 1) The student will have a high quality scientific experience examining an area related to heart, lung, or hematologic pathophysiology. 2) The student will actively participate in the multi-tiered training program. 3) The student will become part of a "network" crossing multiple education levels. Our "multi-tiered" approach to training is novel and provides a model for successful training of minority students in biomedical research.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The long-term goal of this research is to elucidate how bacterial pathogens suppress host innate immunity to cause disease using the model Arabidopsis-Pseudomonas syringae pathosystem. Through evolution, plants and humans have developed powerful immune systems to effectively prevent colonization of most microbes; however, they can be highly susceptible to a subset of microbes that have evolved specific mechanisms to overcome host immunity. One such mechanism, used by many human and plant pathogenic bacteria, is the type III secretion system, which injects potent effector proteins into the host cell to promote infection. Recent studies have begun to show exciting results regarding how these bacterial effectors attack the host immune system. In particular, the Pseudomonas syringae effector HopM1 degrades a regulator (MIN7) of immunityassociated host vesicle traffic in a host 26S proteasome-dependent manner. Although HopM1 is the first bacterial effector reported to trick the host ubiquitination/26S proteasome system into degrading a host protein, the exact mechanism of HopM1 action remains to be elucidated. HopM1 is functionally redundant to another P. syringae effector, AvrE, which contains two WxxxE motifs found in many effectors of human pathogenic bacteria. The specific goals of this project are: (i) to characterize the mechanism by which HopM1 manipulates the host ubiquitination/proteasome system to degrade MIN7;(2) to conduct live cell imaging to determine dynamic subcellular focal accumulation of MIN7 during the immune response;(3) to identify immunityassociated GTPases regulated by MIN7;and (4) to characterize the host targets of AvrE. An integrative approach using molecular genetic, cell biological, transgenic, microscopic, and pathogenesis methods will be taken to achieve these goals. Elucidating the mechanisms by which HopM1 and AvrE modulate host vesicle traffic promises to illuminate common and novel protein biochemistry used by different pathogens to engage eukaryotic innate immunity components, and to provide fundamental knowledge for the development of novel disease intervention strategies.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Type 2 diabetes is one of the most prevalent diseases in the United States, inflicting more than 20.8 million people and expanding at epidemic rates in some areas of the country. The key diagnostic of Type 2 diabetes is the presence of amyloid fibers in the pancreas. These fibers are composed of the human islet amyloid polypeptide (hIAPP) and many in vitro and in vivo studies have linked them to the disease. Even so, the mechanism by which hIAPP inhibits pancreatic 2-cell function and insulin production is not understood. A growing body of evidence points to hIAPP interacting with the cell membrane as the cause of cell dysfunction rather than the fibers themselves. One piece of evidence for this hypothesis is that lipid vesicles catalyze fiber formation and in doing so become permeable and leak. Thus, it appears that understanding the disease mechanism requires structural characterization of hIAPP during membrane association, folding, and fiber formation. However, since the mechanism is both kinetic and involves membranes, conventional structural approaches such as NMR are difficult to apply. As a result, almost all experimental structural information comes from circular dichroism measurements, which provide only a rudimentary characterization of the peptide structure. It is not even definitively known which part of the peptide associates with the membrane. Considering the importance of understanding the structural changes of hIAPP with lipid membranes, we propose to use FTIR and 2D IR spectroscopy, in conjunction with 1-13C=18O isotope labeling, to yield site-specific structural information on hIAPP during the kinetics of folding in the presence of lipid vesicles. We will gain residue-level information on peptide association with the membrane, insertion and orientation, secondary structure formation, and test whether pores in the membrane form. The kinetics of structure formation will help reveal the catalytic mechanism for amyloid fiber formation. We seek to obtain a detailed structural characterization of hIAPP membrane catalyzed kinetics that is not currently possible with other techniques. Membrane catalyzed amyloid formation in diabetes studied with 2D IR spectroscopy
|
{
"pile_set_name": "NIH ExPorter"
}
|
The University of Rochester was fortunate to be one of the successful applicants following the original RFA in 2005, and became one of the eight Centers for Medical Countermeasures against Radiation that make up the current CMCR network: the Center for Biophysical Assessment and Risk Management following Irradiation (CBARMFI). As has been demonstrated to date, the Administrative Core will maintain the UofRCMCR's responsibilities regarding National Institute of Allergy and Infectious Diseases (NIAID) programmatic issues and goals, and will facilitate actions within the Center when deemed appropriate. The U of R's Administrative Core has provided leadership within the CMCR network, including the planning and organizing of national, network, and open meetings and workshops and will continue to contribute significantly to the leadership and functioning of the CMCR National Network of Centers, as well as to the national reputation and development of the CMCR program. The Administrative Core facilitates oversight and evaluation of progress through development of documentation for Project Leaders and External Scientific Advisors regarding stated goals, milestones and timelines, comparability of experimental results, etc. Our PI, Dr. Williams, will work closely with her financial team (Brian Martin and Amber Bessette) through monthly meetings so as to maintain strict budgetary oversight. Annual meetings/retreats of the UofR-CMCR group with its External Scientific Advisory Group (ESAG), which will be arranged prior to submission of the required Progress Reports, will overview progress and provide direction for each subsequent year. Finally, since all of the projects will require services from members of the office of Radiation Safety (e.g., assistance with security, dosimetric measurements, storage of radioactive materials, training of personnel, and removal of contaminated animal bedding), the University of Rochester's Radiation Safety Officer, Dr. Tom Morgan, together with a member of his team, will join the Administrative Core in order to provide coordinated services and centralize the administrative and financial requirements.
|
{
"pile_set_name": "NIH ExPorter"
}
|
We postulate that in women, as well as other mammalian species, uterine quiescence is maintained by increased progesterone receptor (PR) transcriptional activity, and that spontaneous labor is initiated or facilitated by a concerted series of biochemical events that activate inflammatory response pathways, reduce coactivator levels and negatively impact PR function. In recent studies, we observed a marked decline in the PR coactivators, CREB-binding protein (CBP) and members of the steroid receptor coactivator (SRC) family, and in histone acetylation in myometrium of women in labor and in uterine tissues of pregnant mice at term. CBP and several SRC family members have histone acetylase activity, which maintains an open chromatin structure. Pregnant mice injected with a histone deacetylase inhibitor near term manifested increased histone acetytation in the uterus and delayed parturition, suggesting the functional importance of the decline in coactivators in the initiation of parturition. We also obtained intriguing data to suggest that the major lung surfactant protein, SP-A, a C-type lectin involved in innate immune response, that is developmentally regulated in fetal lung and secreted into amniotic fluid near term, signals the initiation of labor. SP-A activates amniotic fluid macrophages to express nuclear factor KB (NF-KB) and intedeukin-ll3 (IL-113). These macrophages, which are of fetal origin, migrate to the pregnant uterus leading to an inflammatory response and increased uterine NF-_:B activity. We suggest that the increase in NF-KB within the maternal uterus both directly increases expression of genes that promote uterine contractility and negatively impacts the capacity of the PR to maintain uterine quiescence, contributing to the onset of labor. Based on these findings, the following research objectives are proposed: (1) to further define the role of SP-A and of the related surfactant protein, SP-D, in the initiation of labor;, (2) to characterize the receptors and signaling mechanisms whereby SP-A at term activates macrophages in amniotic fluid, resulting in activation of NF-_B in the maternal uterus; (3) to determine the cellular and molecular mechanism(s) for the decline in expression of coactivators within the myometrium at term, and;(4) to decipher the molecular mechanisms whereby progesterone and NF-KB regulate target genes that control quiescence/contractility of the myometdum. We believe that this research will provide important insight into the molecular mechanisms that mediate the decline in coactivators and the role played by maturation of the fetal lung and secretion of pulmonary surfactant in activation of inflammatory response pathways within the pregnant uterus that culminate in parturition.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Many reports have described a high incidence of a positive red cell antiglobin reaction (direct Coombs test) in patients taking the antihypertensive drug, alph-methyldopa. Based on our previous work showing that cytochrome P-450 generated superoxide anion oxidized alpha-methyldopa to a reactive arylating agent, we were interested to see if human erythrocytes could activate the drug in a similar manner. Incubation of intact or lysed human red cells with H3-alpha-methylled to covalent binding of the drug to red cell constituents. Further studies showed that oxyhemoglobin was required for the binding reaction. Because oxyhemoglobin can be visualized as a ferrihemoglobin-superoxide anion complex similar to the monoreduced form postulated for oxygenated cytochrome P-450 in the liver, we believe that superoxide anion from oxyhemoglobin oxdizes the catechol nucleus of alpha-methyldopa to semiquinone radicals or quinones which covalently bind to the erythrcytes. The denatured macromolecules may then be antigenic and lead to the production of a direct Coombs test and immune hemolysis. The mechanism for this drug allergy is now being studied by attempting to determine the natuue of the antigenic component.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Lentivirus-based gene transfer systems represent a promising gene delivery technology, as they integrate into the genome of the target cell and mediate sustained expression of the transferred gene. Advanced Vision Therapies, Inc. (AVT) has developed a proprietary lentiviral vector system based on the bovine immunodeficiency virus (BIV), an animal lentivirus not associated with human disease. The BIV vectors combine the transduction efficiency of the HIV-based vectors with the safety advantages of animal-based lentiviral vector systems. Importantly, BlV-vector mediated delivery of an anti-angiogenic transgene efficiently blocked retinal neovascularization in a relevant rodent model, suggesting that the AVT vector is suitable for clinical applications. Lentiviruses are routinely pseudotyped with heterologous viral envelopes to broaden vector tropism. The most widely used envelope is derived from the vesciular stomatitis virus glycoprotein (VSV-G). However, VSV-G has several limitations including cytotoxicity and inactivation by human complement. Therefore, a variety of alternative envelopes have been explored for use in pseudotyping lentiviral vectors including the baculovirus gp64. AVT was recently successful in the generation of a gp64 envelope protein-pseudotyped BIV vector. High vectors titers were obtained, the vectors were stable, and importantly, gp64 can be constitutively expressed in cells without toxicity (an important consideration in the development of a BIV producer cell line). However, gp64 is inactivated by human complement. In this Phase I application, AVT will explore the use of a novel envelope derived from the Thogoto virus. Thogoto virus is transferred to human blood through ticks, and therefore, may display resistance to human complement. Interestingly, the Thogoto gp75 envelope glycoprotein displays significant homology to the baculovirus gp64 envelope glycoprotein, potentially due to the tropism of Thogoto for arthropods and human hosts. AVT has successfully generated a high titer BIV vector pseudotyped with the Thogoto virus gp74 envelope glycoprotein. This Phase I project will focus on the further evaluation of this novel vector. There are 3 specific aims for this Phase I project. Specific Aim 1: Evaluation of cellular tropism of Thogoto virus gp75- pseudotyped BIV vectors. A panel of cell lines and primary cells will be evaluated for transduction efficiency using Thogoto gp75-pseudotyped GFP. Specific Aim 2: Evaluation of Thogoto-pseudotyped BIV vectors in rodents. Both VSV-G and baculovirus gp64 envelope glycoprotein- pseduotyped vectors specifically transduce RPE cells following a subretinal injection in rodents. The tropism of the Thogoto-pseudotyped vector will be evaluated in rodents. Specific Aim 3: Determination of the human complement resistance of the Thogoto-pseudotyped BIV vector. Phase II studies will further evaluate BIV vector physical properties, including stability, Thogoto gp75 cytotoxicity, and vector purification strategies. [unreadable] [unreadable] [unreadable] [unreadable]
|
{
"pile_set_name": "NIH ExPorter"
}
|
5.7 Abstract - CHEMICAL LIBRARY SCREENING (Core Group B) The Chemical Library Screening Shared Resource (CLS) (Core Group B) offers Cancer Center scientists the ability to develop and conduct small- and large-scale chemical library screens and perform hit optimization and validation for the generation of selective probes of biochemical and cellular processes of tumor biology. This resource, located within the large Conrad Prebys Center for Chemical Genomics at SBMRI (a national Comprehensive Center for both the NIH MLPCN and NCI CBC programs), provides Cancer Center faculty with access to technology, expertise and infrastructure resources to develop novel means for characterizing cellular targets involved in tumor pathogenesis and tumor onset and to advance the development of new lead molecules for anti-tumor therapies. CLS consists of four specialized but highly integrated facilities: 1) the High Throughput (HT) Assay Development Facility provides technical and scientific support in the development, miniaturization, and implementation of cell-based and biochemical HT assays, assists with characterization of compounds identified in primary screens through development and execution of secondary and orthogonal assays, as well as performs structure-activity-relationship, selectivity-panel and mechanism-of-action studies; 2) the Compound Management and HT Screening Facility manages the Institute's natural product and small molecule chemical compound libraries (over 700,000 compounds), maintains automation and detection equipment, and executes large-scale screening campaigns; 3) the High Content Screening Facility supports all aspects of development, execution, and image-data analysis of image-based high content screens, as well as aids with high-throughput microscopy assays for non-screening applications; and 4) the Medicinal Chemistry Facility provides technical support and expertise in the areas of medicinal chemistry, combinatorial chemistry, scale-up for advanced studies, and determination of physical properties. Over the past 5 years, a total of 36 Cancer Center members used the services of the CLS Shared Resource, carrying out 26 successful Cancer Center chemical biology projects, 12 advancing to hit-to-lead optimization, and 7 successfully generated leads with sufficient potency and drug-like properties to be explored in vivo animal models of disease. This direct access to experts and technology in chemical biology and early-stage drug development for CLS has provided consultation and technical sections for 57 grant proposals submitted by Sanford-Burnham Cancer Center PIs, of which 26 were awarded. CLS staff has been included in 44 Cancer Center member publications as co- authors, reflecting their important contribution to these scientific discoveries. In addition, data produced by CLS, access to chemical libraries and advanced instrumentation, consulting, and other services, contributed to at least 37 additional publications. The CCSG funding (10% of the total CLS budget), leverages substantial institutional investment in this extensive drug discovery infrastructure, and provides Cancer Center researchers with priority access to this technology strongly supporting both discovery and early translational research.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Bioactivation of xenobiotics to toxic intermediates through cytochrome P450 oxygenation mechanisms is a well recognized process. However, the production of electrophilic intermediates by several P450 enzymes (e.g. 1A2, 2B6, 2E1, 2F1, 3A4, and 4B1), through dehydrogenation pathways has only recently been investigated, and the mechanisms that govern selective dehydrogenation rather than oxygenation are not established. Several of the dehydrogenated intermediates are so reactive that they inactivate the P450 enzymes, generally through alkylation of active site nucleophilic residues. Research concerning the catalytic behavior of these specific P450 enzymes and their propensity to dehydrogenate rather than oxygenate substrates is vitally needed. The hypothesis of this research is: the unique catalytic mechanism(s) offacilitated electron transport that determines dehydrogenation by certain P450 enzymes results in xenobiotic-mediated injury and altered drug metabolism in humans. The specific goals of this application are to determine the characteristics of the enzyme active-site environment that direct dehydrogenation mechanisms of specific cytochrome P450 enzymes, and to define the substrate structural features that regulate selective dehydrogenation rather than oxygenation. These goals will be realized through the following aims: 1) To determine the structures of the reactive intermediates that are produced by dehydrogenation of prototypical substrates, and characterize enzyme preferences for dehydrogenation vs. oxygenation of the substrates;2) To characterize the mechanisms of inactivation of each P450 enzyme by its specific inactivator;3) To define the active-site parameters that control the mechanisms of dehydrogenation and bioactivation of toxicants by P450 enzymes;and 4) To use the dehydrogenation substrates that covalently modify the P450 apoproteins to elucidate critical active-site residues that direct the dehydrogenation mechanism, or that control inhibitor/substrate access channels, binding, or product release. The enzyme/substrate pairs are CYP2F3/3-methylindole, CYP3A4/zafirlukast, CYP2El/capsaicin, and CYP2B6/tamoxifen. The long-term goals of this research are to elucidate the mechanisms of cytochrome P450-mediated dehydrogenation of xenobiotics in processes that generate toxic electrophilic intermediates, to assess the potential harm engendered by these toxic intermediates to human health, and to utilize mechanistic information to predict dehydrogenation, and concomitant toxicities and/or enzyme inactivation (altered drug metabolism), of new drugs and xenobiotics.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The major goals of this project are to characterize the host's immune response to helminth infections and to relate these findings to the pathogenesis of clinical disease. Field studies of bancroftian filariasis suggest that initial exposure to these parasites elicits vigorous cellular immune responses but with continued reexposure, infected individuals cannot manifest significant cellular reactivity to filaria products. This defect in responsiveness is filaria-specific and limited to cellular immunity. Similar findings come from studies of human schistosomiasis mansoni. Patients evaluated within three months of initial infection demonstrated marked cellular reactivity to schistosome antigens, whereas lymphocytes from those with chronic infection were unable to respond to schistosome products. Studies of the cellular responses to helminth infections by lymphocytes and by eosinophil leukocytes as well as the mechanisms underlying the diminished reactivity to parasite antigens found in schistosomiasis and filariasis will continue both in experimental animals and in man.
|
{
"pile_set_name": "NIH ExPorter"
}
|
We will exploit the multicenter Long Life Family Study (LLFS), a unique resource for research on human longevity and healthy aging, to find genetic variants associated with these traits. In the current period, we successfully enrolled and extensively phenotyped 4,953 individuals in 539 two-generational families that demonstrate clustering for exceptional survival in the upper generation. Fewer than 1% of the Framingham Heart Study (FHS) families (a roughly random sample of families) would meet the minimal entrance criteria for exceptional survival required in the LLFS. Thus our least exceptional families show more clustering for exceptional longevity than 99% of the Framingham families. Further, the children's generation have significantly lower rates of major diseases of aging including diabetes, chronic pulmonary disease, peripheral artery disease and show significantly more favorable profiles of quantitative mariners of healthy aging such as blood pressure, lipids, functional performance, and cognitive indices compared to FHS. These endophenotypes show greater clustering (with high heritability) in the LLFS familiesthan in FHS. Thus, LLFS has likely greatly enriched the prevalence of any gene variants for longevity and healthy aging endophenotypes, thereby increasing detection power. Most importantly, the family design of LLFS provides additional power and analytic opportunities to discover genetic influences than would be possible in a study of unrelated individuals, especially with regard to rare alleles. Our specific aims are to: 1) continue phenotyping by assaying biomarkers of healthy aging on stored samples, annually tracking subjects for new significant medical and health events, and comparing Medicare (and Danish equivalent) disease and utilization data with reference samples; 2) identiy common genetic variants for healthy aging and excepional survival using GWAS; 3) identify rare variants for exceptional survival and healthy aging by targeted sequencing; and 4) more clearly dissect the genetic architecture of exceptional survival an healthy aging through a systems approach involving genet networks and pathways, to better understand the complex interplay between genetic variants, exposures, and covariates in the development of endophenotypes. Taking a multidisciplinary approach involving clinicians, demographers, geneticists, epidemiologists, and computational scientists, we propose to capitalize on the investments already made in creating this unique cohort to further our understanding of the nature of exceptional survival and healthy aging.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The Neddylation pathway was recently validated as a cancer target. The SENP8 protease processes the precursor of Nedd8 and is essential for its activation. Based on the high resolution crystal structure of Nedd8 bound to SENP8 the substrate has two fundamental interaction sites: at the C-terminus of Nedd8 in the catalytic center, and interactions with the bulk of the Nedd8 protein covering a substantial surface of the protease - the exosite. Analysis of the SENP8 structure reveals cavities in the exosite, indicating the presence of a putative allosteric binding site. Our preliminary results for this SENP on uHTS of the ~ 330,500 MLSMR small molecule libraries using a penta-peptide based assay identified inhibitors binding to the catalytic center and not selective against other SENPs. In order to find selective SENP8 ligands and to explore this additional allosteric binding site, we developed a novel Fluorescent Intensity uHTS assay that utilizes a physiological protein substrate Nedd8 substrate. To study the function of this therapeutically important enzyme, we propose to screen for selective mechanistically novel SENP8 inhibitors using this newly developed assay. Based on available literature, this would be the first time that a representative protease targeting ubiquitin-like proteins is interrogated using a full-length substrate following an uHTS screen that targets only the catalytic site. Our application addresses an important unmet need for identification of specific modulators of SENPs. We will utilize the compounds identified in the project for characterization of physiological involvement of this class of enzymes in maintaining homeostasis of normal cells and their role in cancer. These compounds will be made available to other research labs, permitting acceleration of research in the SENP field. PUBLIC HEALTH RELEVANCE: Nedd8 is an ubiquitin-like protein, and its attachment to target proteins, by a process known as neddylation, is essential for specific cellular pathways. Previous work has shown that targeting the neddylation cycle with a small molecule can decrease tumor burdens, and this small molecule is now in clinical trials. To study the function of this therapeutically important enzyme, we propose to deploy a novel Fluorescent Intensity uHTS assay that utilizes a full length Nedd8 protein substrate to discover potent and SENP8 selective inhibitors that may prove to be allosteric using this newly developed assay.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The major objective of the proposed research is to understand the interrelationship between the structure and function of a newly identified nuclease: human flap endonuclease-1 (FEN-1) in the context of DNA repair. Current biochemical and genetic data have demonstrated that FEN-1 is a structure specific endonuclease. It may play critical roles in DNA replication, repair, and recombination. However, sufficient evidence is not available for this class of nucleases to establish a structural basis for their unique biochemical and physiological functions and their structural and functional relationships. Our preliminary data on the analysis of biochemical domains has suggested that there are three functional domains in human FEN-1 protein responsible for the functions of DNA substrate binding, catalysis, protein-protein interaction, and nuclear localization. The proposed research is designed to establish the detailed structural understanding of the catalytic center for DNA substrate recognition, binding and cleavage. and to analyze the structural elements responsible for protein-protein interaction and nuclear localization. We will examine functional alterations through site-directed mutagenisis of each functional domain. The mutant proteins will be characterized by our newly developed kinetic flow cytometry, biochemical assays and crystallographic structural analysis, and green fluorescence driven microscopy.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The overall goal of the Principal Research Core (PRC) of the Advanced Center to Improve Pediatric Mental Health Care (ACIPMHC) is to conduct effectiveness and implementation research that informs improvement of pediatric mental health care in the public service sectors of child welfare and mental health, by achieving greater integration of evidence-based practices (EBPs) and usual care. The research program is built upon a 17 year history of clinical epidemiology studies, and a more recent set of effectiveness and implementation studies conducted in a collaboration between services researchers in San Diego and intervention researchers at the Center for Research to Practice (CR2P: linked to the Oregon Social Learning Center) in Oregon, as well as with other intervention researchers across the country who are involved with parent-mediated interventions for youth with externalizing behavior problems/disruptive behavior disorders. Interventions central to existing and proposed studies include: The Incredible Years (Webster-Stratton), Parent-Child Interaction Therapy (Eyberg), Multidimensional Treatment Foster Care/Parent Management Training (Chamberlain, Fisher, Reid), and Multisystemic Therapy (Henggeler, Schoenwald). The overall PRC goal will be addressed through two specific aims: 1) To use an interdisciplinary research network linking intervention and services researchers to develop effectiveness and implementation research on strategies to integrate EBP and usual care to improve children's MH care. The core uses a multiple-stakeholder framework emphasizing community and organizational contexts, interchange of multiple perspectives, diverse methods of multi-level research spanning qualitative and quantitative traditions, and emerging theoretical frameworks. 2) To conduct developmental and pilot studies that inform full-scale effectiveness and implementation studies in the CW and MH sectors. The PRC will link investigators from different methodological and conceptual approaches and will link closely to proposed workgroups in the RMC as well as the CMC Board to conduct studies in collaboration with community stakeholders in natural laboratories at the local, state and national levels. Two developmental studies and two small pilot studies are proposed to demonstrate the type and scope of work intended in this core.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Multiple lines of evidence support a key role for abnormal synaptic connectivity in schizophrenia, but the molecular mechanisms underlying its pathogenesis are not known. Understanding these mechanisms may allow us to identify new targets for therapeutic intervention, especially early in the course of illness. The application wil focus on dendritic spines as cellular substrates of brain connectivity, because the majority of excitatory synapses are located on spines, and reduced spine density has been extensively documented in schizophrenia. Mounting evidence indicating that known schizophrenia susceptibility genes regulate spines and that regulators of spine plasticity are implicated in schizophrenia, strongly support the model that perturbations in the molecular network underlying spine plasticity are critically involved in the pathogenesis of schizophrenia. However, the mechanisms through which genetic alterations in this network underlie specific neurobiological phenotypes related to schizophrenia are not known. Recent data indicates that rare variants (including amino acid mutations) cumulatively account for a significant fraction of the missing heritability in schizophrenia, and cluster in gene networks that control synapses. Because a large fraction of such mutations are estimated to impair protein function, many are expected to cause brain circuit alterations. Thus, we propose that by identifying, testing for association, and characterizing rare variants enriched in schizophrenia, we will provide critical new insights into disease pathogenesis, because such mutations provide detailed knowledge about the affected molecular and cellular functions. Based on our preliminary data, we hypothesize that rare coding variants in genes that control dendritic spine plasticity, cumulativel enriched in subjects with schizophrenia, disrupt cortical connectivity and impact neuromorphological and cognitive measures in carriers. Using a multidisciplinary translational approach that combines human genetics, molecular and electrophysiological studies in cellular models, functional validation in mice, and cognitive assessment and structural brain imaging in patients, we will pursue these specific aims: 1) To assess the cellular impact of mutations in spine plasticity genes identified in schizophrenia subjects. 2) To determine the impact of mutations in spine plasticity genes on glutamatergic synaptic transmission. 3) To determine the impact of mutations in spine plasticity genes on cortical ultrastructure and functional connectivit in mice. 4) To assess the relationships between mutations in spine plasticity genes and phenotypic measures in patients. PUBLIC HEALTH RELEVANCE: Using a multidisciplinary and integrated translational approach we will test the hypothesis that rare protein coding mutations in genes that control dendritic spine plasticity in the cerebral cortex, which occur in subjects with schizophrenia, disrupt synapse structure and function within frontal cortical microcircuits, and affect specific neuromorphometric and cognitive measures in carriers. Data generated will provide new mechanistic insights into pathways that underlie abnormal brain connectivity in schizophrenia that will allow us to identify therapeutic targets.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The sex steroid hormones (estrogen and progesterone) stimulate growth, maturation and the development of new biochemical capacities in their reproductive target organs. These steroids play a major role in maternal physiology and fetal differentiation and their relationships to reproductive tissue malignancy are well established. Although steroid hormones exert major influences on the synthesis of nucleic acid and protein, the exact biochemical processes by which sex hormones regulate growth and function in target tissues are not yet clear. The general objectives of these studies are to define the mechanism of action of reproductive steroid hormones in regulating cell replication, morphologic differentiation and biochemical specialization in their target tissues. This will be accomplished by coordinating a network of separate in vivo and in vitro investigations in a single experimental animal model system in which specific biochemical events can be studied easily -- the chick oviduct. These studies will involve aspects of cell biology, nucleic acid and protein chemistry, immunology, and related intermediary metabolism; emphasis will rest heavily on regulation of nucleic acid and protein synthesis. The ultimate goal of this project is the generation of a cell-free system composed of purified reconstituted components, in which sex steroid effects on "coupled" gene transcription and translation can be completely defined. It is expected that the understanding derived from this project will be relevant to the biology of the natural reproductive hormones relative to maternal physiology, fetal development and the design of new contraceptive agents. The following proposed studies should also be pertinent to development of more precise theories for the biochemical mechanism of action of hormones in general, and more specifically steroid sex hormones. BIBLIOGRAPHIC REFERENCES: Buller, R.E., D.O. Toft, W.T. Schrader and B.W. O'Malley. Progesterone-binding components of chick oviduct: VIII. Receptor activation and hormone dependent binding to purified nuclei. J. Biol. Chem. 250: 801, 1975. Buller, R.E., W.T. Schrader and B.W. O'Malley. Progesterone-binding components of chick oviduct: IX. The kinetics of nuclear binding. J. Biol. Chem. 250:809, 1975.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Objectives: (1) To study the relationship between the level of superoxide dismutase (SOD) present in lung tissue and the ability of the animal to become "tolerant", i.e.: survive in pure oxygen or lethal doses of other pulmonary oxidants and (2) to determine the histological changes occurring in lungs exposed to oxidants and to identify the specific sites and characteristics of biochemical and physiological adaptation in "tolerant" animals.
|
{
"pile_set_name": "NIH ExPorter"
}
|
We have moved all aspects of this umbrella project forward during 2013. We discovered that C-terminal domains within ATPase proteins that dock against the proteasome's core particle undergo dynamic exchange between an expected 4-helix bundle and a partially unfolded state. We provided evidence that this exchange is important for interactions with chaperones involved in proteasome assembly. In unpublished work, we have moved forward the resolution of the 3D structures for multiple protein complexes, including a new ubiquitin binding motif. During 2013, we also characterized a small molecule that restricts proliferation of multiple cancer cell lines. This work was collaborative with the Roden lab at Johns Hopkins University and led to the characterization of ubiquitin receptor Rpn13 as a potential target for certain cancer types.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Aging of multicellular organisms typically involves progressive decline in the body's ability to maintain homeostatic cell replacement and to regenerate tissues and organs after injury. Skeletal muscle, in particular, regenerates robustly through most of adult life but fails to do so in old age. Age-acquired defects in muscle function profoundly impact the health of older individuals, resulting in a high incidence of age-associated muscle deterioration (sarcopenia) and inefficient or incomplete recovery from injury in the elderly. Precisely how aging causes deterioration of muscle function is poorly understood, but several lines of evidence, including preliminary data from my lab, suggest that loss or functional impairment of skeletal muscle stem cells directly contributes to age-dependent failures in tissue repair. In light of these data, the primary focus of this application is to identify age-regulated genes and pathways that can be manipulated in aging muscle to reverse the detrimental effects of age on muscle stem cell number and improve muscle stem cell function. To this end, we have generated extensive preliminary data that strongly suggest that the age-related impairment of muscle stem cell function may be mediated by increased exposure to a pro-inflammatory environment. In particular, we have found that aging of muscle stem cells is accompanied by induced expression of multiple inflammation-associated genes. In addition, we have found that restoration of myogenic function, which can be induced by heterochronic parabiosis, is accompanied by normalization of expression of at least some of these age-regulated, pro-inflammatory targets. Thus, the experiments described in this application are designed to (1) better understand the systemically regulated induction of inflammatory genes that occurs in aged skeletal muscle stem cells, (2) examine whether inhibition of inflammation can prevent or reverse age-associated suppression of muscle stem cell proliferation and muscle regenerative function, and (3) identify the physiological mechanism(s) that ultimately result in enhanced, chronic inflammation in aged muscle. These studies will use well-established mouse models already available to us and cell isolation strategies pioneered by my lab, and will provide a solid basis for clinical extension into novel treatments for human age-associated muscle disease. PUBLIC HEALTH RELEVANCE: Our data in mice suggest that progressive loss of muscle stem cells and dysregulation of their function is an important underlying cause of muscle deterioration in old age. Therefore, in these studies, we will use genetic and biochemical approaches to identify the mediators of age-associated dysfunction of muscle stem cells, as well as factors that can restore their "youthful" function. This work holds tremendous promise for halting and potentially reversing age-related defects in muscle regenerative function.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Cytochrome P-450 appears to catalyze the in vitro formation of phosgene (COCl2) and carbon dioxide (CO2) from chloroform (CHCl3) in rat liver microsomes, since these reactions are NADPH dependent and inhibited by carbon monoxide and SKF 525-A. Moreover, the cleavage of the C-H bond appears to be the rate determining step in this process since deuterium labeled chloroform (CDCl3) is biotransformed into COCl2 slower that CHCl3. This oxidative dehalogenation pathway is believed to have general metabolic and toxicological importance for other halocarbon drugs and environmental chemicals.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The proposed 3-day NYAS-sponsored conference will bring together, for the first, time, chemical, electrical and biomedical engineers, pharmacologists, pharmaceutical scientists and chronobiologists to discuss the reasons for the development of drug delivery systems that can automatically administer temporally-optimized patterns of a single drug, multiple drugs or genetically-engineered protein molecules. The purpose of this meeting is ultimately to stimulate the development of safer and more effective therapies for a wide range of disorders by considering administration timing or patterning in time. The meeting will include 8 major lectures covering essential topics in depth and about 30 other talks between 10 and 20 minutes in length. A poster session (50 posters) for the first of 3 evenings will serve to get participants communicating freely early in the conference. Posters will be displayed throughout the meeting to serve as a focus for interaction and communication. The six scientific sessions will, respectively: relate the physiologic need for pulsatile delivery of hormones and the need for retrieval of medical information present within high frequency cardiovascular rhythms; the afternoon will detail the importance of circadian physiology and will focus upon the development of circadian-based drug delivery systems; on day 2 the necessity for circadian-based delivery of cancer treatments will be followed by an afternoon devoted to drug carriers, chemical-physical aspects of drug delivery and how these systems can be temporarily controlled; the morning of the third day will discuss the temporal coordination of mechanical and computerized electronic delivery systems; the meeting is to be concluded by a panel discussion which focuses upon clinical study designs required for elucidation of optimal temporal delivery patterns, successful drug-specific chronotherapies, device-based time-qualified treatments; finally, regulatory implications of the development of time-specified therapeutic preparations will be considered.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Vascular smooth muscle plays a pivotal role in the physiology and pathology of the blood vessel. Alterations in the contractile properties and phenotype of the smooth muscle cell have been implicated in such common vascular disease processes as atherosclerosis and hypertension. Despite its central role in vascular pathophysiology, very little is known about the contractile apparatus and in particular about Myosin, the principal force-producing protein. Our recent studies have focused on identifying two distinct smooth myosin heavy chain isoforms that are the products of alternate RNA splicing of a single gene. These two isoforms are co-expressed in a number of smooth muscle tissues. In addition, we have new data to indicate that a novel myosin isoform is expressed in certain smooth muscle tissues. This proposal will continue to elucidate the molecular structure, heterogeneity and the factors regulating myosin expression. A major focus of this proposal will be to understand the role of growth factor(s) (PDGF) in regulating myosin gene expression. The primary objectives of this proposal are to: I. Determine whether alternate RNA splicing events occur at the 5' end of the SMHC gene encoding SM1 and SM2 MHC isoforms. II. Characterize the new SMHC isoform at the genetic and biochemical level. III.Complete the characterization of the SMHC gene promoter by DNA sequencing and gene transfer analysis. IV. Study the effect of PDGF and serum on MHC gene expression in aortic smooth muscle cultures. V. Identify upstream promoter elements which interact with trans-acting regulatory factors to direct tissue specific transcription of the smooth muscle MHC gene. VI. Isolate master regulatory genes involved in determining smooth muscle phenotype. We anticipate that these studies should provide direct insight into the mechanisms governing vascular function and modulation at the subcellular level.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Alcoholic fatty liver is considered as the earliest pathological alteration of progression in alcoholic liver disease (ALD) from hepatic steatosis to hepatitis, fibrosis, cirrhosis and even hepatocellular carcinoma. Hepatic reverse transport of free fatty acids (FFAs) derived from adipose hyperlipolysis resulting from alcohol ingestion plays a critical role in fatty liver formation. However, the mechanisms by which alcohol regulates adipose tissue lipolysis are unclear. Recently, fibroblast growth factor 21 (FGF21) has emerged as a hepatic regulatory factor that acts on multiple targets, including the liver itself in an autocrine fashion, and importantly on white adipose tissue to regulate lipid homeostasis. We have found that FGF21 plays a critical role in alcohol-induced adipose lipolysis and in hepatic fat accumulation in animal models of ALD, and circulating FGF21 levels are significantly increased in patients with ALD and in experimental animal models. However, how FGF21 regulates adipose tissue lipolysis during alcohol exposure is not known. The aims of this project are to investigate the role of FGF21 in adipose tissue lipolysis in response to alcohol exposure to improve our understanding of the molecular mechanisms in the regulation and action of this hormone in ALD, and to assess the therapeutic potential in alcohol-induced fatty liver formation. To achieve this goal, we will carry out the following specific aims. In aim 1 we will determine whether FGF21 plays a stimulatory role in adipose tissue lipolysis and the underlying mechanisms associated with hormone sensitive lipase and perilipin pathway during alcohol exposure using FGF21 knockout and transgenic overexpression mouse models. In the second aim, we will test our hypothesis that adipose tissue specific disruption of FGF21 decreases FFA release and hepatic uptake and attenuates alcohol-induced fatty liver formation using adipose selective ?-klotho knockout mice.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Defects in neocortical neurogenesis and migration cause severe brain developmental disease. LIS1, mutations in which cause lissencephaly (smooth brain), was the first neuronal migration gene to be identified. LIS1 functions in the cytoplasmic dynein pathway, indicating that microtubule motor proteins play a role in brain development. In earlier work supported by this grant we identified multiple discrete LIS1- and dynein- requiring stages in neurogenesis and migration, leading to a comprehensive model for the cellular basis of classical (type I) lissencephaly. We also found LIS1 to be required for the long-mysterious cell-cycle- dependent interkinetic nuclear migration (INM), a general feature of neuroepithelial and radial glial progenitor cell (RGPC) behavior. We have determined further that INM requires the activity of opposite-directed microtubule motor proteins, the plus end-directed unconventional kinesin Kif1a and cytoplasmic dynein. This model appears to explain the underlying mechanism for INM, and should allow us to address further basic and long-standing questions regarding its function and purpose. The Specific Aims are to determine the mechanism of nuclear transport by Kif1a; to determine how specific inhibition of basal and apical INM affect cell cycle progression and cell fate; and to determine the mechanisms for cell cycle control of INM using small molecule protein kinase inhibitors and other reagents. These issues have important implications for understanding how brain size, composition, and organization are controlled, and how stem cell proliferation is regulated under normal or neoplastic conditions. The analysis of genes responsible for INM and the use of small molecule cell cycle inhibitors will also identify potential targets for modulating neurogenesis and migration during early brain development.
|
{
"pile_set_name": "NIH ExPorter"
}
|
This interdisciplinary project has as its objective the elucidation of the biochemical mechanism by which aminoglycosidic antibiotics (neomycin, streptomycin and related compounds) cause permanent hearing loss. It begins with the hypothesis that these antibiotics bind to polyphosphoinositide lipids and displace calcium disturbing calcium homeostasis and cell membrane function. The hypothesis will be investigated by a combined in vivo/in vitro approach. In vivo, aminoglycosidic antibiotics, fragments and derivatives will be tested by cochlear perfusion in the guinea pig for their effect on cochlear microphonic potentials and phospholipid labeling by 32P-orthophosphate. In vitro experiments will be conducted with synaptosomes from brain to study the influence of these drugs on enzymes and substrates of phosphoinositide metabolism. This in vitro system will also be investigated for its potential usefulness as an in vitro correlate of in vivo toxicity. Studies with monomolecular films of phospholipids will focus on drug/calcium interactions, on the stoichiometry of the drug/lipid complex and on the determination of those sites on the antibiotic which are involved in its ototoxic action.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Alzheimer's disease (AD) is a neurodegenerative disorder that leads to the progressive loss of memory and other cognitive functions. At this time, there are no approved treatments that are capable of delaying its onset or slowing its progression. We have developed a novel category of drug-like, small molecule compounds that specifically target a cell surface receptor that is expressed by neurons affected in AD, known as the p75 receptor. These p75 receptor ligands activate survival-promoting signaling and inhibit degenerative-promoting signaling of the p75 receptor. In tissue culture studies, these ligands are capable of blocking the ability of amyloid beta (A[unreadable]) to activate degenerative signaling within neurons affected in AD. The protective effects of these compounds occur at low nanomolar concentrations and have been verified to occur through their action at p75. Moreover, our ligands block the toxicity of A[unreadable] oligomers, the A[unreadable] species thought to be most toxic to neurons and most relevant to AD. In studies of normal middle aged mice, our lead compound has been demonstrated to reach the brain following daily oral administration and has been found to have no toxic effects including studies of hepatic, cardiac and DNA toxicity. In these mice, our lead compound demonstrates a significant neurotrophic effect of reversing or preventing basal forebrain cholinergic atrophy of the type that occurs during aging and AD in both human and rodent systems. In pilot trials in a well characterized AD mouse model, our lead compound appears to be improving memory function and to be reducing pathological features typical of AD. In this application we will complete the following three milestone-driven projects: i) verification of efficacy in Alzheimer's mice and assessment of potential mechanism-based side effects; ii) cGMP scaled up synthesis and purity necessary for an Investigational New Drug (IND) application to the FDA; and iii)toxicology and pharmacology studies designed to complete an IND application. Completion of these three projects will allow an IND application with the overall goal of obtaining IND approval for conducting the first Phase I trials humans. Completion of the proposed project will also establish a new chemical entity (NCE) and a novel, first in class, drug compound for development in AD therapeutics. [unreadable] [unreadable] [unreadable]
|
{
"pile_set_name": "NIH ExPorter"
}
|
Research in Disorders of Sexual Development (DSD) has played a major role in understanding the genetic control of sex determination and the opposing pathways controlling gonadal development. In the testis pathway, SOX9 expression is critical to testis induction. Conversely, extinguishing SOX9 expression appears essential for normal ovarian development. For example, in XX DSD patients with SOX9 duplications, testes develop in XX individuals lacking the Y-linked testis-determining gene, SRY. Recent studies of the ovary pathway have not yet identified the long sought mechanism by which SOX9 transcription is normally extinguished in XX gonads. This could be a key step in the ovary pathway that opposes the testis pathway. Our goal is to identify a causative mutation in the subtype of XX DSD in which XX individuals develop testes while their XX siblings develop ovotestes. Studies in these patients have been impeded because the disorder is uncommon, family sizes are small, and there are no rodent models. The canine model is the only model of this XX DSD subtype. In the canine research pedigree, XX DSD is an autosomal recessive trait with expression limited to XX siblings, which develop testes or ovotestes. Early studies suggested that the testis pathway is incompletely suppressed or inappropriately activated in these gonads. Using GWAS, we have identified and resequenced a region significantly associated with canine XX DSD in the model pedigree. This region overlaps the regulatory region of SOX9. Our Specific Aims are: Specific Aim 1 Hypothesis: The causative mutation for canine XX DSD lies within a genetic switch that normally extinguishes SOX9 transcription in XX gonads. To test this hypothesis, we will produce transcriptomes [RNA-seq] to complete a pilot study comparing gonadal gene expression in XX DSD embryos to that of normal XX and XY controls. If our hypothesis is correct, SOX9 expression will be greater in XX DSD gonads than those of XX controls, perhaps approaching levels observed in XY gonads. If our hypothesis is incorrect, the gonadal gene expression data from this project will be valuable for constructing alternative hypotheses. Specific Aim 2 Hypothesis: The causative mutation for canine XX DSD lies within a genetic switch that is conserved in mammals. To test this hypothesis, we will use a combined comparative genomics and bioinformatics approach to compare two existing datasets, a microarray dataset from XX DSD patients and resequencing data from the canine XX DSD model. If our hypothesis is correct, we will identify orthologs in XX DSD individuals (humans and dogs) that are evolutionarily conserved, yet contain nucleotides that are different from the reference genomes. Those nucleotide differences will be our candidate mutations. Results from this project will remove two major obstacles to the study of XX DSD by identifying candidate mutations in human patients and the canine model, and confirming that the canine XX DSD model is an appropriate model in which to further characterize key elements in the testis and ovary pathways.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The objectives of this proposal are to investigate molecular and functional properties of synaptic junctions (SJs) and alterations in these properties during synapse formation. Functional interactions will be examined between membrane surface glycoproteins and underlying components of the postsynaptic density (PSD). We plan to elucidate functional interactions between postsynaptic transmitter receptors and calmodulin-dependent protein kinase II (designated CaM-kinase II), both of which are concentrated in SJs. Studies will focus on SJ-associated CaM-kinase II (of which the major PSD protein [mPSDp] is the major subunit) and its endogenous substrate proteins; how this dynamic system functions and is assembled at developing synapses. We will determine the in situ state of phosphorylation of SJ proteins and determine if the phosphorylation of these proteins or CaM-kinase II is altered by membrane depolarization or neurotransmitters. Highly specific antibodies will be used to examine the expression and localization of mPSDp and postsynaptic membrane proteins of hippocampal pyramidal neurons in culture. Neurons will be grown under conditions that favor (high density) or greatly limit (low density) the formation of SJs. Antibodies will be used as specific probes to study the molecular composition and functional properties of mature and developing synapses. Monoclonal antibodies have been obtained against antigens that are; (a) neuron-specific, (b) highly enriched in SJs, (c) cell-surface, and (d) developmentally regulated, i.e. appear during synapse formation. We will obtain a cDNA probe(s) for the mPSDp/CaM-kinase II. This will be used to; (a) identify mPSDp-specific mRNAs and measure their levels during brain development, and (b) obtain primary sequence information for mPSDp and determine its relationship to the 60 kDa subunit of CaM-kinase II. These studies will provide important information on the regulation of gene transcription and mRNA processing for this important protein kinase. The ultimate significance of these studies lies in their potential to provide explanations about; (1) molecular and cellular mechanisms that underlie synapse formation and the establishment of synaptic functions, (2) molecules that distinguish synapses from the remaining neuronal surface, and (3) relationships among molecular, functional and genetic properties of synaptic proteins in the CNS.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIAID), Division of AIDS (DAIDS) is funding the Regional Prospective Observational Research in Tuberculosis (RePORT) International Coordinating Center (RICC), which will serve as the point of contact and the coordinating entity for RePORT International related activities as needed. RePORT International began as a cooperative strategy between the U.S. Division of AIDS (DAIDS)/NIAID/NIH and interested governments to address the threat of TB, which affects the lives and well-being of people across the globe and poses an increased risk for people living with HIV and AIDS. Each RePORT consortium is designed to support in-country data collection, specimen biorepositories, and associated research with the goal of adding additional regional consortia to encourage worldwide TB (drug-susceptible and drug-resistant) prevention and treatment research.
|
{
"pile_set_name": "NIH ExPorter"
}
|
This R25 research education application entitled: 'Frontiers in Aging and Regenerative Medicine (FrARR),' responsive to PAR-12-016, trains and mentors promising junior and senior undergraduates from predominately underrepresented communities in sophisticated aging research, and encourages and supports them as they embark on graduate studies. FrARR offers: first, dynamic training courses, then nurtured Mentor-Trainee partnerships enhanced by guidance from Senior Mentors and peer-peer mentoring, enriched by annual symposia and scientific retreats, with quantitative tracking of career trajectories. The courses are offered at HBCU institutions, i.e. Xavier University (years 1 & 4), Morehouse (years 2 & 5) and Meharry (year 3), under the overall directorship of Gerald Schatten (Pittsburgh) and S. Michael Jazwinski (Tulane), with Shubha Kale Ireland (Xavier), Winston Thompson (Morehouse), and Ayman Al-Hendy (Meharry). Our Advisory Board of Scientific Counselors, composed of acclaimed experts in the fields of aging, regeneration and health disparities, oversee this initiative. The five specific aims are: I. Engendering the enthusiasm, passions and commitments to scientific careers embraced by the faculty provides intellectual and emotional underpinnings for supporting undergraduate trainees as they complete their MSTEM degrees. This is accomplished via conceptual education and laboratory training, focused on diseases and disorders relevant to aging and African- American communities. Didactic training includes: A. Biology of Aging and Stem Cell Models; B. Neurobiology of Aging and Alzheimer's disease; C. Muscle and Bone Aging; D. Reproductive Aging; and E. Epigenetics and Environmental Modulators of Aging, along with complementary labs. II. Fostering candid regarding current barriers to the recruitment and retention of our most talented researchers and training in responsible conduct of research, ethical, legal and societal implications, and especially problems of minority health disparities in research. III. Motivate, Enable and Sustain Trainees as they transition to professional careers, with mentored research projects and on-going career advice. Enhance their growing interests by their presentations at the Annual Reunion Symposia at Woods Hole contemporaneously with the MBL's Aging Course. Course alumni provide peer-peer mentorship. IV. Encouraging and empowering trainees and alumni by guiding them through the graduate application and matriculation processes so that they are rapidly and smoothly able to initiate doctoral studies in aging research and regenerative medicine. V. Evaluate, Improve and Monitor FrARR's strengths and avoid weaknesses with quantitative independent mechanisms, track trainees' achievements comprehensively and longitudinally, to ensure that these precious funds can be quantitatively demonstrated as wise, cost-effective and fruitful investments. In summary, our overall goals are to provide comprehensive sophisticated training in research strategies and career launching for underrepresented trainees to advance the scientific workforce pioneering the Frontiers in Aging and Regenerative Medicine.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Mitochondrial aconitase is highly sensitive to oxidative damage during aging. It reversibly converts citrate to isocitrate in the tricarboxylic acid (TCA) cycle. The cubane [4Fe-4S]2+ cluster of aconitase is essential for its catalytic activity, but it also renders the enzyme highly vulnerable to oxidative stress. We have discovered that isolated wild-type Saccharomyces cerevisiae mitochondria contain a nucleotide (GTP, NAD(P)H and ATP)- dependent machinery for iron-sulfur cluster (ISC) biogenesis of aconitase (Aco1p). The cluster biogenesis occurs in the mitochondrial matrix by a multi-step process requiring multiple components. In Aim I, we will biochemically dissect these steps and determine the nucleotide requirements of each. In Aims II-IV, we will study proteins that we found are directly relevant to nucleotide-dependent processes in ISC biogenesis of Aco1p. These include a GTPase (Mtg1p), a NADH kinase (Pos5p), a NADPH-requiring reductase (Arh1p), and an ATPase (Ssq1p). Mitochondria lacking any of these proteins are deficient in ISC biogenesis of Aco1p and display greatly reduced aconitase activity. However, the causal defects in these mutant mitochondria must be different since these proteins have different functions. Mtg1p is a GTPase in the mitochondrial matrix, and we will test our hypothesis that Mtg1p mediates the effects of matrix GTP on ISC biogenesis of Aco1p (Aim II). Pos5p is a NADH kinase and is required for NADPH production in the matrix. We will determine if the role of Pos5p in ISC biogenesis of Aco1p is mediated by its effects on NADPH levels in the matrix (Aim III). NADPH is likely utilized by reductase(s) such as ferredoxin reductase (Arh1p), which may provide reducing equivalents for one or more steps in cluster biogenesis, and this will be tested as part of Aim III. Ssq1p is an Hsp70 chaperone with ATPase activity in the matrix. Therefore some of the effects of ATP on ISC biogenesis of Aco1p are likely mediated by Ssq1p, and this will be explored (Aim IV). In addition to Ssq1p, yeast mitochondria contain an abundant Hsp70 (Ssc1p) that is involved in protein import. In contrast, human mitochondria contain a single Hsp70 (hSSC1), and thus it may participate in both protein import and ISC biogenesis, and we will test this possibility as part of Aim IV. Aco1p is essential for mitochondrial DNA (mtDNA) stability, and this activity is independent of its enzymatic activity in the TCA cycle. Aim V is to investigate interaction of yeast and human mitochondrial aconitases with mtDNA. We will determine if human mitochondrial aconitase, like yeast Aco1p, is also bifunctional and participates in the maintenance of mtDNA. Nucleotides and/or redox status of the matrix may play a critical role in distributing aconitase between the TCA cycle and mt-nucleoids, and we will examine these possibilities. The mechanism underlying the biogenesis of ISCs of mitochondrial aconitase in yeast is likely to be very similar to that in human. Homologs of most, if not all, yeast proteins that participate in the process also exist in human. Thus, conclusions from yeast studies proposed here will be informative to human physiology. Public Health Relevance: Mitochondrial aconitase requires a 4Fe-4S cluster for its enzymatic activity in the tricarboxylic acid (TCA) cycle and is highly sensitive to oxidative damage during aging. In yeast, aconitase is also essential for the maintenance of mitochondrial DNA, and this activity is independent of its catalytic activity in the TCA cycle. This proposal seeks to investigate the molecular mechanism of Fe-S cluster biogenesis of aconitase and its interaction with mitochondrial DNA. [unreadable] [unreadable] [unreadable]
|
{
"pile_set_name": "NIH ExPorter"
}
|
Aberrant DNA methylation in the promoter region of genes is found in a variety of human cancers and is thought to be associated with gene silencing. Restriction Landmark Genomic Scanning (RLGS) is currently the only technique that allows the scanning of thousands of promoter sequences for aberrant DNA methylation in human cancer. Aberrant DNA methylation was recently shown to be a major contributor and an early event in tumorigenesis, especially in the development of acute myeloid leukemia (AML). In this application we propose to investigate the role of DNA methylation in AML with special emphasis on clinical correlates. The samples that will be used for this study will come from the CALGB Leukemia Tissue Bank. Our hypothesis is that epigenetic changes (DNA methylation) are equally important as genetic alterations in leukemogenesis but have been underestimated in its extend. Since methylation changes could affect the transcription of genes it is likely that these epigenetic differences contribute to the molecular defects that underlie normal karyotype AML. Subsequently, aberrantly methylated targets can be used to identify novel diagnostic or prognostic biomarkers in AML. To test this hypothesis our specific aims are (1) to study methylation profiles in a subset of normal karyotype AML with blast counts >50 percent. (2) Rigorous statistical and bioinformatical analysis will identify diagnosis and relapse specific methylation events, candidate subclass predicting methylation events and finally methylation targets that correlate with clinical data such as duration of complete remission. (3) A small subset of highly informative methylation targets will be studied in detail by bisulfite sequencing. MS-PCR tests will be developed that allow (4) screening of larger patient samples. Statistical analysis will be performed to determine the value of a methylation event as a diagnostic biomarker or as a marker with predictive value.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Abstract: Our archival data indicates that 27% of older adults hospitalized with major depression and suicidality continue to report suicidal ideation (SI) three months after discharge. Having persistent post- discharge SI places individuals at very high risk for suicidal behaviors and death. Cognitive and affective mechanisms likely determine the course of post-discharge SI, but interrupting these processes remains challenging. This is, in part, because the upstream modifiable risk factors that exacerbate problems with cognition, affect, and SI in the post-discharge period are poorly understood. We propose that sleep-wake disturbances are potentially important contributors to the post-discharge prognosis. Sleep-wake disturbances plausibly influence the cognitive and affective mechanisms that underlie SI. But many potentially relevant sleep-wake factors have been identified, and there is not yet evidence regarding which mark or drive the mechanisms that perpetuate SI after discharge. To begin filling these gaps, we propose a prospective observational pilot study examining sleep-wake disturbances, alongside other putative suicide risk factors, in the critical post-discharge period. This Exploratory/Developmental Research (R21) proposal brings together experts in sleep-wake rhythms and late-life depression (Smagula, PhD, PI), sleep medicine (Buysse, MD), late- life suicide (Szanto, MD, Co-I), cognitive aging (Butters, PhD), and time series analytics (Krafty, PhD, Co-I). We will monitor the course of suicidality over 12-weeks in 70 adults (age 55-75, with non-psychotic major depressive disorder, current active SI, and a recent psychiatric hospital discharge). Over 6 weeks, we will perform high-resolution data collection including: assessing 24-hour sleep-wake patterns (using actigraphy and diary); measuring daily suicidality levels (passive ideation, active ideation, and planning) and affect (depression and anxiety); and administering weekly home-based tests of cognitive functions previously linked with sleep and suicide (i.e., attention, response inhibition, and reversal learning performance). Our testable hypotheses based on existing evidence are that: (1) weekly measures of short sleep duration and sleep-wake rhythm disruption will temporally precede and independently predict a worse course of post-discharge SI; and (2) these sleep-wake factors will relate to the course of SI, in part, via their adverse effects on cognition and affect. Given their important roles, we will also evaluate the effects of psychosocial factors and assess their relationships with sleep. Collecting these novel data will enable analyses: (1) ranking the effect sizes of putative risk factors including sleep-wake, psychosocial, affective, and cognitive measures; (2) examining the temporal relationships between risk factors; and (3) preliminarily testing mediational models. Results from this study will inform the development of confirmatory studies ultimately leading to novel, evidence-based, interventions. If sleep-wake factors have a role in determining post-discharge suicide risk, the potential for clinical translation is high, given that sleep-wake risks could be modified and/or monitored after discharge.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Addiction places a tremendous burden on individuals and society, and an improved understanding of the underlying genetic and environmental factors that contribute to this disease will aid prevention and treatment efforts. This project will use three large, genetically informative datasets that include comprehensive assessments of substance dependence, comorbid disorders and environmental factors: the Collaborative Study on the Genetics of Alcoholism, the Family Study of Cocaine Dependence, and the Collaborative Genetic Study of Nicotine Dependence. This project has four aims: Aim 1: To study genetic findings in three databases in the search for common and specific factors involved in the development of addiction. Genetic findings will be examined within and across datasets so that a better understanding of common and specific genetic factors in the development of addiction can be developed. Aim 2: To examine refined phenotypes, such as quantitative phenotypes of smoking, alcoholism, and polysubstance dependence in genetic analyses. The refinement of quantitative phenotypes such as a polysubstance symptom count will be undertaken in order to further the study of genetic polymorphisms and addiction. Because these three studies share a common assessment, these refined phenotypes can be applied to all databases. Aim 3: To examine the influence of ethnicity, gender and psychiatric comorbidity on the "at risk" genotypes. Phenotype and genotype correlations will be examined to determine whether distinct dependence subtypes are associated with specific "at risk" genotypes. This will include an examination by ethnicity, gender, additional substance dependence, and psychiatric comorbidity. Aim 4: To examine complex genetics of substance dependence and quantitative phenotypes using additional genetic analyses of pleiotropy, gene-gene, and gene-environment interactions. The candidate is an independent investigator who has already made important contributions to the study of the genetic epidemiology of addiction. This Independent Scientist Award will allow her to continue to pursue her research on the human genetics of addiction. She will continue to collaborate with investigators at Washington University and nationally in order to apply state of the art approaches to genetic analysis of this complex disorder. By comparing and contrasting findings in three datasets, a more thorough understanding of common and specific factors underlying human addiction will be developed. [unreadable] [unreadable] [unreadable]
|
{
"pile_set_name": "NIH ExPorter"
}
|
Skin cancer is the most common human cancer and the number one cancer in terms of incidence in the USA. Ultraviolet (UV) irradiation in solar light elicits various biological responses in the skin, including inflammation, pigmentation, erythema, and cell death, and therefore the role of UV light as a major etiologic factor is of particular concern. The use of sunscreens or sun blocks has not been effective in preventing skin cancer, and therefore new mechanism-based approaches are critically needed to treat UV-induced skin cancer. This project is aimed toward investigating the role of leukotriene A4 hydrolase (LTA4H) in solar ultraviolet (UV) - induced skin carcinogenesis. LTA4H was shown to exhibit high levels of protein expression in certain types of cancers and its inhibition leads to a reduced cancer incidence in various animal models. Importantly, our preliminary data indicate that suppressing LTA4H expression and activity decreases skin carcinogenesis in the 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) two-stage mouse skin carcinogenesis model. We also reported that [6]-gingerol or resveratrol binds with LTA4H to suppress colon or pancreatic cancer cell growth, respectively, in vivo. We hypothesize that inhibition of LTA4H by [6]-gingerol or resveratrol will result in suppression of solar UV-induced skin carcinogenesis.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The purpose of this project is to establish a physicochemical basis of taste reception. Using the techniques of surface physical chemistry we have demonstrated that charged monolayer of phospholipids change surface pressure in a manner which correlates with electrophysiological and psychophysical responses to various tastants. Our studies indicate that surface pressure changes may play a role in normal chemoreception in taste. This has lead to the prediction, born out psychophysically, that surface active agents may be powerful taste suppressants. This work has also suggested that known taste suppressants such as gemnemic acid may be surface active. This has also been verified. Future work involves physicochemical, electrophysiological, and psychophysical studies to illucidate further the role of surface activity in taste.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Squamous cell Carcinoma of the head and neck region (HNSCC) is the sixth most frequent cancer worldwide with an estimated 30,000 new cases and 8,000 deaths reported in the United States each year. Surgery and radiation are used to treat the primary tumor with chemotherapy reserved for disseminated disease. Unfortunately, there is a lack of transgenic HNSCC models with which to test new potential therapies. Traditional rodent models of HNSCC have relied on application of carcinogens to the hamster cheek pouch or xenografting of human cancer cells into immunodeficient mice. While these models have been useful, carcinogen exposure produces multiple DNA lesions which complicate the determination of which genetic events are required for HNSCC tumorigenesis. Xenograft studies do not replicate the early stages of carcinogenesis and are limited to human cancer cell lines that can proliferate in the murine environment. A key genetic event in most human cancer (including HNSCC) is mutation and inactivation of the tumor suppressor p53. This protein responds to DNA damage by inducing cell cycle arrest or apoptosis. The absence of functional p53 has been associated with tumor formation in mice. This proposal will develop two existing transgenic mouse strains in which p53 was inactivated by different mechanisms as animal models of HNSCC. The first model is the p53 null mutant mouse in which both alleles are inactivated by homologous recombination. The second model is the K14-HPV mouse in which the keratin 14 promoter directs expression of human papillomavirus early genes to the basal layer of oral mucosa. The HPV E6 gene product binds to and inactivates p53 protein. These models provide the advantage of monitoring turnorigenesis starting with a defined genetic lesion (i.e., p53 inactivation) and will prove very useful in future clinical and molecular studies of HNSCC.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The onset of asthma is often during childhood; and when the child is atopic, the child is more likely to continue to have asthma as an adult. Diseases such as asthma have a higher prevalence in childhood; and management that alters the morbidity of allergic diseases in children may impact disease outcomes in future years.[unreadable] [unreadable] This study examines techniques for the detection of inflammation in children with respiratory diseases due to allergy or immunologic dysfunction. Emphasis is on non-invasive methods. We will use information gathered to determine if there are inflammatory markers that may be diagnostic or representative of disease severity, and thus useful in management.[unreadable] [unreadable] Preliminary data supports the concept of ongoing airway inflammation in asymptomatic children with asthma. It also supports the existing use of nitric oxide as a marker of airway inflammation that may or may not correlate with bronchoconstriction as demonstrated by spiromertry. Furthermore, an elevated expired nitric oxide seen in association with airway inflammation may be associated with a decrease in airway pH, previously documented during an asthma exacerbation. A similar pattern was not demonstrated in healthy matched controls. [unreadable] [unreadable] Of interest, a small representative sample of exhaled breath condensate was analyzed for several inflammatory cytokines and the results are pending. [unreadable] [unreadable] We are completing the data collection in this longitudinal study of non-invasive measurements of airway inflammation in children with asthma and healthy-matched controls.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Aneuploidy is a hallmark of the vast majority of human solid tumors. Mutations in mitotic checkpoint genes such as BUB1, BUBR1 or MAD2 have been shown to be involved in generation of aneuploidy. These checkpoint gene products forms an intricate signaling network called spindle assembly checkpoint (SAC) that delays the segregation of sister chromatids until all chromosomes are properly attached to the mitotic spindle apparatus and aligned at the metaphase plate. Work from lower organisms such as yeast has clearly demonstrated that loss of the checkpoint function causes chromosomal instability manifested as gains or losses of chromosomes. A more detailed molecular picture of SAC is emerging from analyses in both yeast and higher eukaryotes. However, the function of SAC and its various components at an organismal level remains to be elucidated. Likewise, mechanistic dissection of SAC in mammals is lacking. In an effort to start to molecularly dissect SAC in mammals and to determine its function in preventing chromosomal instability and oncogenic transformation, we generated mouse strains that are defective in SAC to different extents. These strains are securin deletion, non-phosphorylable separase knockin, and Mad2-noninhibitable Cdc20 knockin (AAA-Cdc20). We have found that our AAA-Cdc20 mice were tumor-prone. We propose to address the mechanism by which AAA-Cdc20 promotes tumorigenesis and the role of p53 in limiting tumor development in SAC mutants. PUBLIC HEALTH RELEVANCE: This project focuses on spindle assembly checkpoint's role in the prevention of genome instability and tumorigenesis. Results obtained from the proposed experiments may help the treatment and diagnosis of cancer.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The mechanism of gonadotropins and eicosanoids action in luteal cells is complex and not fully understood. The action of these agents may not completely be explained by cell surface receptor binding and activation of adenyl cyclase. These agents internalize and associate with receptors present in one or more of the intracellular organelles. In our efforts to determine the biological significance of nuclear gonadotropin and PGs binding sites, we have succeeded in demonstrating the presence of gonadotropin, PGE and PGF2a responsive nucleoside triphosphatase (NTPase), an enzyme involved in nucleocytoplasmic transfer of mRNA, in bovine luteal nuclear membranes. These findings raise a number of additional questions, which this grant proposal aims to answer. Briefly, the effect of gonadotropins and PGs on mRNA transport from isolated luteal nuclei, whether gonadotropin and PGs responsive NTPase plays a role in this transport and identification of transported mRNA using cDNA probes will be investigated. PGs also bind to nuclear membranes and stimulate NTPase activity. But this response has not been characterized in detail and it will be done in the present studies. Whether adenosine, which amplifies gonadotropins action with respect to cyclic AMP and progesterone production, can also amplify NTPase response to hCG and PGs will be examined. Basal NTPase activity and its response to gonadotropins and PGs during periods of rapid luteal growth, structural and functional maturity and regression and in corpora lutea rescued by establishment of pregnancy will be studied. Slices of corpora lutea will be incubated with and without gonadotropins and PGs and then nuclear membranes will be isolated for measurement of NTPase activity. NTPase in bovine luteal nuclear membranes will be solubilized and purified to answer whether this enzyme can bind gonadotropins and PGs and whether these ligands can stimulate the purified enzyme. The proposed experiments are a logical extension of our most recent findings. This project offers several new ideas and can potentially contribute novel concepts in the area of the molecular mechanism of gonadotropin and PGs action in corpus luteum. Such concepts do not necessarily conflict with the importance of cell surface binding in the action of gonadotropins and PGs and eventual degradation of ligands and/or their receptors in lysosomes of luteal cells. The new concepts can be complimentary to or additional mechanisms of gonadotropin and PGs action.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The goal of this research project is to understand the mechanism by which antibody catalysts accelerate the reaction and induce stereoselectivity. Subsidiary goals are to determine experimentally the absolute stereochemistries of Diels-Alder adducts obtained in these reactions, to determine relationships between amino acid sequences and binding site structures of different catalytic antibodies, and to develop methods for the calculation of antibody binding and catalysis. The stereoselectivity of Diels-Alder catalysis by antibodies is very high, but the directions of enantioselectivity and sometimes even diastereoselectivity are unknown. We will establish experimentally the stereochemistries of these reactions, develop understanding of the origins of stereoselectivity and catalysis through modeling, compare the immune response to different types of haptens and the binding sites produced by the immune system to achieve these catalytic functions, and predict how more effective functionalized antibody catalysts can be developed. The research will involve both experimental and computational studies designed to reveal the basic organic chemistry of antibody catalysis, and the development of computational methods to achieve better methods for the exploration of antibody structures and binding by the immune system.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Many tumor-bearing animals develop antibodies to unique antigens associated with the oncogenic virus causing the tumor. These antigens, called "neoantigens," have been found in tumors caused by papovaviruses, adenoviruses, and herpes viruses. Hepatitis B virus, a hepadnavirus with suspected oncogenic potential, cannot be transmitted to non-primates but patients with HBV-associated hepatoma might be expected to have antibody to a HBV-associated neoantigen if one exists. Using a hepatoma cell line that contains integrated HBV DNA, we sought immunofluorescent antibody in sera of hepatoma patients. Approximately seven percent of sera from HBsAg-positive hepatoma patients contained an antibody that reacted with a nuclear antigen in the hepatoma cell line. This antigen was found in another hepatoma cell line that also contained integrated HBV genome but not in two other hepatoma cell lines lacking HBV genome. The antigen ("hepatitis B virus-associated nuclear antigen": HBNA) is being further characterized to determine if it is the product of a transforming gene. A different nuclear antigen was found in a human hepatoma cell line that did not contain HBV DNA. It was identified with serum from a patient with HBsAg negative hepatocellular carcinoma. The new antigen has characteristics similar to those of HBNA.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Amino acid are the main components of proteins and are important elementary nutrients for living organisms. They are also valuable synthetic intermediates for the preparation of a large array of biologically active compounds important to the pharmaceutical industry. Examples of drugs based on amino acids are L-DOPA, L-tryptophan, and beta-lactam antibiotics. Unfortunately, typical production methods for amino acids require expensive and time-consuming optical resolutions to obtain optically pure products. A more desirable way to prepare amino acids would be to enantioselectively hydrogenate carbon-nitrogen double bond (C=N) groups of amino acid precursors. This direct synthesis route could lower costs because only the desired product is formed avoiding the expense of a chiral separation. Unfortunately, the current catalysts generally are not sufficiently active or selective enough to be industrially useful. The objective of this proposal is to prepare optically pure amino acids by enantioselective hydrogenation of compounds with C=N bonds and to show that a variety of amino acids can be economically produced on an industrial scale. PROPOSED COMMMERCIAL APPLICATION This research will lead to the development general method for the production of optically pure amino acids by the enantioselective hydrogenation of compounds with C=N bonds. This will allow the economical production of valuable amino acids for use as pharmaceuticals and synthetic intermediates.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The project has two Aims in Phase I. The first Aim is to modify an existing adaptive optics fundus imager to create the first Super Resolution Fluorescein Angiography Retinal Imager (SFARI) device. This modified instrument will allow fluorescein retinal imaging with up to 3 microns spatial resolution at rates of 10 images per second over a 20 deg field of view. The second Aim is to conduct laboratory testing using artificial eyes and standard references to quantitatively measure the spatial and temporal resolution gains that this new SFARI device provides and certify the instrument for use on human subjects, and to demonstrate the performance of the SFARI using two normal human subjects. The proposed SFARI device is a revolutionary step forward in the field of clinical in-vivo imaging of the retina and retinal substructures at micron-scale spatial resolutions and with video (10 Hz) frame rates. The project introduces innovative adaptive optics technology to an important new application, fluorescein angiography. Successful completion will represent a significant contribution to clinical and research ophthalmology. Fluorescein angiography and indocyanine green imaging of the retina are a common procedure in most ophthalmology clinics making the commercial potential high with the device having a market that includes every major eye clinic in the U.S. and abroad.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Investigations will be continued into the conditions of production and the chemical nature of the reticulo-stimulatory substance in vaccines from anaerobic coryneforms (C. parvum vaccines). A continued search will be made for other bacteria which have similar activity.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Control of tumor growth has been reported for several human tumor xenograft models and for a few clinical trials using radioactivity delivered to tumors by linking a radioactive element to an antibody that accumulates in tumor and is retained in tumor tissue for many days, thereby delivering a continuous low dose of radiation in a cancer treatment called Radioimmunotherapy (RAIT). There is a need to understand the effects of RAIT on tumor physiology in order to improve results and to determine why some tumors fail to be controlled by the therapy. The radio-antibodies localize in the tumor near the blood vessels and possibly cause changes in vascular function that may influence intratumoral pH and pO2 and intratumoral interstitial pressure. The net effect of these changes may not only impact on the accumulation of additional doses of radioantibody in a multiple cycle scheme, but may also influence the uptake of other anti-tumor agents (e.g. drugs, or biological response modifiers). In this study we are investigating the effects of low-dose rate radiation on tumor pO2. Low pO2 could mean a tumor is less sensitive to radiation and also potentially less accessible to drug therapy due to a damaged blood supply. Methods to intervene could then result in a more appropriate therapy for individual tumors. Electron paramagnetic resonance (EPR) oximetry with solid paramagnetic materials implanted in the tumor will be used to monitor pO2 over the long term delivery of radiation to determine whether the tumor oxygenation changes during this new form of radiation therapy. Animals to be studied are Nude mice (T-cell deficient) bearing one of six different human tumor xenografts grown s.c on the flank including GW-39, LS174T, HT-29 and GS-7 human colonic carcinomas or ME-180 human cervical carcinoma or the CALU- 3 non-small cell lung adenocarcinoma. Tumors will be implanted with paramagnetic material, 400 um piece of gloxy at one site, or 25 microliters of a slurry of sterile paramagnetic material. After implantation of this material, pO2 will be monitored in the tumor using EPR. Twenty four hours after the first pO2 measurement, mice will be injected with radioactive I-131-intact IgG (~100-200microliters) i.p. I-131 is a radionuclide that deposits its energy within a 1-mm range. Then pO2 will be monitored daily for the first 72 hrs and at about 7 day intervals after that for the duration of the experiment.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Inborn errors in cholesterol (Chol) biosynthesis comprise a group of severe, often lethal, metabolic disorders, of which the Smith-Lemli-Opitz Syndrome (SLOS) -the fourth most prevalent recessive human disease - is the most well-known. Prior work has established a SLOS rat model, which exhibits a progressive retinal degeneration in which photoreceptors seem more susceptible than RPE cells or Muller glia. While the exact disease mechanism is not yet known, the initial biochemical defect involves inefficient conversion of 7- dehydrocholesterol (7DHC) to Chol, which is catalyzed by DHCR7 (3b-hydroxysterol-D7-reductase, the DHCR7 gene product). We have proposed that this leads to multiple sequelae, including altered gene expression, lipid and protein oxidation, caspase activation, and perturbed membrane structure, which then result in progressive cellular dysfunction and demise. 7DHC is the most oxygen-labile lipid known, and readily forms oxysterols (some of which are extraordinarily toxic to cells), and 7DHC-derived oxysterols tend to be more cytotoxic than are Chol-derived oxysterols. The native environment of the retina (high oxygen tension, iron, and incident light) presents ideal conditions for oxysterol formation. We hypothesize that the demonstrable rise in retina/RPE 7DHC levels with blockade of DHCR7 leads to in situ oxysterol formation, resulting in progressive dysfunction and death of photoreceptors, while RPE and Muller glia are relatively spared. Preliminary data strongly support this hypothesis, which we will test further via three Specific Aims: 1) Using transformed, retina-derived cell lines, we will examine whether 7DHC-derived oxysterols differentially alter gene expression and viability of photoreceptors, vs. RPE and Muller glia, in culture, and also will examine the protective effects of antioxidants; 2) We will examine whether photoreceptors are more sensitive to intravitreally injected 7DHC- vs. Chol-derived oxysterols compared to other retinal cell types in vivo; and 3) We will examine the impact of cell-type specific disruption of cholesterol biosynthesis on retinal structure and function in vivo, selectively knocking out Dhcr7 in rods, RPE, or Muller glia. This will markedly advance our understanding of the SLOS-associated retinal degeneration mechanism, as well as provide new insights into the development of more effective therapeutic interventions for such diseases. PUBLIC HEALTH RELEVANCE: The proposed studies will provide a mechanistic basis for understanding the pathobiology of retinal degeneration associated with the Smith-Lemli-Opitz syndrome (SLOS) and, by inference, allied cholesterol deficiency disorders, for which there currently are no cures and only partially effective treatments. If the hypothesis proves correct, blocking the formation of cytotoxic oxysterols (e.g., with a suitable antioxidant regimen) in conjunction with cholesterol supplementation would likely provide a markedly improved therapeutic intervention for such diseases.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The Dahl salt sensitive (S) rat will be studied as an animal model of post-menopausal hypertension to investigate the mechanisms responsible for the hypertension. The pressor systems that will be evaluated are the renin-angiotensin system (RAS) and the sympathetic nervous system (SNS). The role of estrogen, aging, and salt intake as modulators of these pressor systems via their effects on the nitric oxide (NO) system will be investigated. The first specific hypothesis is that the aging process with the accompanying loss of estrogen activity is associated with a down regulation of the nitric oxide system resulting in hypertension. To test this hypothesis, Specific Aim 1 of this study is to monitor blood pressure and the level of activation of the NO system in intact, ovariectomized (OVX), and OVX+estrogen-treated Dahl salt sensitive and Dahl salt-resistant (R) female rats as they age from 3 month to 20-22 months of age. The second specific hypothesis is that the factors maintaining the hypertension associated with the loss of estrogen activity in Dahl S rats is determined by the level of salt intake. OVX performed at young, middle and old age will cause an increase in blood pressure, but the rise will be attenuated with increasing age. However, the level of blood pressure and the activity of the pressor systems contributing to hypertension will be higher pre-OVX because of the effects of aging on the RAS and SNS. Two specific aims will address this hypothesis. Specific Aim 2 will be to establish that the Dahl S elderly and OVX females maintained on low salt intake will become hypertensive as a result of an activation of the RAS. Estrogen administration will maintain activation of the NO system to suppress the RAS. Blockade of NO formation in low salt animals will increase RAS function, especially in the OVX+estrogen animals. Specific Aim 3 is to determine that high salt fed Dahl S elderly and OVX animals will develop a hypertension that is dependent on the activation of the SNS. As with the low salt animals, estrogen supplement will suppress the SNS stimulation through a NO-mediated mechanism. Together these studies will provide evidence that the arterial pressure of the Dahl S rat is sensitive to the removal of estrogen through OVX or aging suggesting a useful model of post-menopausal hypertension. By investigating the relationship of estrogen, NO and salt, important new information will be gained in the mechanisms whereby estrogen provides protection against hypertension. Importantly, a greater understanding will be obtained addressing why the protection disappears with the aging process.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Stroke is the leading cause of serious long-term adult disability in the U.S. and third leading cause of death, and has a 2-fold greater incidence in Blacks compared to the majority Americans. Thrombolytic revascularization treatment administered within a maximum of 3 hours from symptom onset reduces morbidity, mortality and cost; however, only 3% of patients arrive at the hospital within 3 hours, mostly due to the public's lack of knowledge concerning stroke symptoms, and the appropriate response when they are recognized, which is to call 911. We propose to reduce these delays using a novel behavioral intervention to improve symptom recognition and response in a high-risk, minority, economically disadvantaged population. Expensive mass media stroke education campaigns are not sustainable for this purpose, particularly in economically disadvantaged populations. Instead, we propose to intervene in school classrooms with children aged 9 to 11 years, to teach the five cardinal stroke symptoms, the correct course of action when they occur, and to highlight the potential therapeutic benefit of early hospital arrival, with the intent that the children will then educate their parents. To help accomplish this, we have developed a program called Hip Hop Stroke (HHS), which is comprised of stroke rap songs and two animated musical cartoons that incorporate stroke knowledge. We have found that children aged 9-11 years can rapidly learn the information, and retain the information well for at least 2 years. One recent pilot study showed that 74% of children in the pilot (N=182) communicated the material to a parent, and that this communication significantly improved the parent's stroke literacy. Having demonstrated the efficacy of the HHS intervention in a small sample, we now propose a randomized controlled trial to test the likelihood that children can and will educate parents or adult caregivers about stroke in a high-risk, economically- disadvantaged, minority population, and parents' ability to recall knowledge transferred. Utilizing children as a transmission vector for carrying out interventions aimed at their parents has the potential to serve as the basis for intervention in any number of other areas such as healthy eating and weight loss. Thus, the significance of the proposed trial addresses the public health problem under study - stroke symptom identification and response - as well as development and refinement of a more general model of intervention.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.