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The development of an HIV-1 vaccine is a critical global health priority. Yet, despite over 30 years of research, only three HIV-1 vaccine concepts have completed clinical efficacy testing in humans. The RV144 study showed that an ALVAC prime, gpl20 protein boost regimen afforded 31% protection against acquisition of HIV-1 infection in a community-based study in Thailand, suggesting that the development of an HIV-1 vaccine is possible. However, additional novel vaccine concepts need to be tested in clinical trials, including clinical efficacy trials. In this IPCAVD program, we propose to evaluate our Ad26/MVA and Ad26/Protein vaccine candidates in SIV challenge studies in rhesus monkeys and in phase 1 clinical trials in humans. Specifically, we hypothesize that a heterologous prime-boost regimen combining novel serotype adenovirus vectors with poxvirus vectors and/or Env gp140 trimers will induce potent and protective antitbody and T lymphocyte responses. The goal of these studies is to define the optimal vaccine candidate for potential phase 2 clinical development. To accomplish this goal, we propose the following Projects and Cores: Project 1: Preclinical Evaluation of Novel Ad/MVA and Ad/Protein l-IIV-1 Vaccines Project 2: Clinical Evaluation of Novel Ad/MVA and Ad/Protein HIV-1 Vaccines Project 3: Manufacturing and Regulatory Support of Ad/MVA and Ad/Protein HIV-1 Vaccines Core A: Administrative Core; Core B: Immune Monitoring Core. Project 1: Preclinical Evaluation of Novel Ad/MVA and Ad/Protein HIV-1 Vaccines Project 1 Leader (PL): Dan H. Barouch DESCRIPTION (provided by applicant): Adenovirus vectors have proven highly potent for inducing both humoral and cellular immune responses. The failure of an Ad5-Gag/Pol/Nef vaccine in the Step study has led to the development of novel serotype Ad vectors, which are biologically very different and arguably substantially superior to Ad5 in terms of seroepidemiology, cellular receptor usage, in vivo tropism, innate immune responses, transcriptional profiles, adaptive immune phenotypes, and protective efficacy against SIV in rhesus monkeys. Moreover, the inclusion of Env may be critical for an HIV-1 vaccine. Based on our preclinical and clinical preliminary data, we have prioritized Ad26/MVA and Ad26/Protein regimens for further development. In Project 1, we will conduct the key preclinical studies to inform the clinical development program described in Project 2. These studies will help define the lead vaccine candidate and will help determine the mechanism of protection afforded by our optimal vaccine regimen. Specifically, we will test the hypothesis that adding a gp140 trimer protein boost will augment the partial protection afforded by our optimal vaccine vectors against acquisition of highly stringent, heterologous SIV challenges. We will also test the hypothesis that vaccine-elicited Env-specific antibodies are critical for this protection. To evaluate these hypotheses, we propose the following two Specific Aims: 1. To compare the immunogenicity and protective efficacy of Ad26/Ad26 and Ad26/MVA vector regimens with and without a gp140 trimer protein boost against repetitive, heterologous, neutralization-resistant, intrarectal SIV challenges in rhesus monkeys; and 2. To define the mechanism of blocking acquisition of stringent SIV challenges by conducting antigen formulation and adoptive transfer studies in rhesus monkeys.
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{
"pile_set_name": "NIH ExPorter"
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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long-term goal of this study is to investigate the anticarcinogenic properties of lycopene at the cellular level. Our first hypothesis is that one of lycopene's anticarcinogenic properties is due to its ability to suppress the proliferation of cancer cells. Our second hypothesis is that one of lycopene's anticarcinogenic properties is due to its ability to increase or renew gap junctional connexin (Cx) protein expression. Therefore, lycopene maintains or creates gap junctional communication from cell to cell, which can control proliferation of neoplastic cells from surrounding normal cells. Our third hypothesis is that there are additional receptors and ligands in cells that can bind lycopene, which may be determined with computational chemistry. Specific Aim 1 for the first hypothesis: we will determine if the anticarcinogenic properties of lycopene include the ability to reduce cell proliferation in the cancer cell lines. Specific Aim 2 for the second hypothesis: we will determine if lycopene, in a dose-dependent fashion, affects the mRNA and protein expression of gap junctional connexins and gap junctional communication (GJC) in cultured cancer cells and nonmalignant cells. We will determine if lycopene treatment is restoring the gap junction function and expression in cancer cells compare the function in nonmalignant cells. Specific Aim 3 for the third hypothesis: we will investigate the potential binding of lycopene to cancer-related targets by using computational chemistry. This will help to determine what receptors may be binding to the lycopene. Epidemiological studies have shown that lycopene is associated with a reduced risk of cancer. Lycopene has been promoted and sold as a dietary supplement with claims about its anticancer and other health benefits. The efficacy and safety of lycopene has not been specifically evaluated nor has the mechanism of lycopene action been determined.
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{
"pile_set_name": "NIH ExPorter"
}
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The mechanisms by which highly reactive epoxides hydrolyze in aqueous media are often complex, and can vary as a function of the pH of the solution. The pH-rate profile for the hydrolysis of a given epoxide provides essential information for determining the mechanisms by which the hydrolyses occur. Many of the kinetic studies, however, have been carried out by determining the rates of epoxide hydrolysis in aqueous solutions held at constant ionic strength by addition of electrolytes such as potassium chloride. We have recently shown that potassium chloride induces specific effects in the hydrolysis of indene oxide and a K-region arene oxide, phenanthrene 9,10-oxide. The objective of this report is to study the hydrolysis reactions of several highly reactive epoxides (1,3-cyclopentadiene oxide and 1,3-cyclohexadiene oxide), and also initiate studies on the hydrolysis of several epoxydiol metabolites of the carcinogen benzo(a)pyrene. pH-Rate profiles for the hydrolyses of these compounds will be generated in solutions held at constant ionic strength by potassium chloride and by other less-nucleophilic salts in order to test for the presence of specific salt effects. Such effects could lead to erroneous assignments of hydrolysis mechanisms if not taken into consideration.
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{
"pile_set_name": "NIH ExPorter"
}
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PROJECT SUMMARY Older adults exhibit a substantial reduction in propulsive power generation. This change in walking mechanics is associated with slower walking speeds and greater metabolic energy expenditure ? features that negatively affect independence and quality of life. Age-related reductions in propulsive power are often attributed to reduced ankle mechanical power during the ?push-off? phase of walking. Yet, strengthening the calf muscles has been an ineffective strategy for improving push-off intensity, walking speed, and/or metabolic cost, motivating the need for new and modifiable targets for preserving mobility in our aging population. Recent studies have determined that the foot, independent of the ankle, plays a critical role in governing push-off intensity. The interaction between active structures (e.g. muscles within the foot) and passive structures (e.g. plantar aponeurosis ? an elastic structure spanning the bottom of the foot) of the foot are vital to economic locomotion, but it is unclear how this interaction facilitates forward propulsion, or how this interaction is changed with age. We propose that age-related changes in passive and active contributions to foot stiffness may contribute to reductions in push-off intensity in older adults, both directly via deficits in foot power and indirectly via the misappropriation of ankle power. Aim 1: Our proposal will leverage a novel dual-probe ultrasound imaging technique to determine the role of foot stiffness and plantar intrinsic muscle contractile dynamics in governing foot-ankle interaction dynamics in young adults. Aim 2: We will then characterize the local energetic and mechanical response to changes in plantar aponeurosis stiffness by closely integrating our experimental data from Aim 1 into a computational model of the lower extremity. Aim 3: Finally, we will determine the effects of age on foot stiffness and its role in walking economy and functional mobility. This study has the potential to influence a paradigm shift in our biomechanical understanding and clinical management of age-related mobility impairment. Moreover, our findings will have immediate impact on targeted mobility intervention opportunities and innovation in the design of wearable integrated foot and ankle devices for enhanced mobility toward improving the health and welfare of our aging population. Finally, our technological advancements in musculoskeletal imaging will revolutionize the use of in vivo ultrasound during functional locomotor behavior, with broad implications in humans and other animals.
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{
"pile_set_name": "NIH ExPorter"
}
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This application is in response to the President's Brain Research through Advancing Innovative Neurotechnologies (BRAIN) initiative. A central goal of the BRAIN Initiative is to understand how electrical and chemical signals code information in neural circuits and give rise to sensations, thoughts, emotions and actions. Existing technologies are not sufficient to accomplish this goal and have to be significantly improved or novel tools should be introduced to analyze circuit-specific processes in the brain, leading to transformative advances in our understanding of the brain function and behavior. RFA-EY-15-001 seeks technology at the very earliest stage of development, which can assist recording and/or manipulating neural circuit activity in human and animal experiments. The specific goal of the proposed work is to introduce and validate a new voltage-sensitive upconverting photoacoustic imaging (VSUPAI) technique. It is based on voltage-sensitive dye (VSD) imaging, which exploits change of optical properties of dye associated with a cell membrane with variation of a membrane potential, allowing for real-time probing of the neuronal activity via non-invasive optical methods. VSDs have limited use in deep brain imaging, because they require excitation in the visible range. This proposal addresses the current limitation of VSD imaging through the convergence of photoacoustic tomography (PAT), biocompatible upconversion (UC) nanoparticles, and VSDs. In the proposed method, we exploit the voltage-sensitive change in dye absorbance to produce a change in the photoacoustic signal, as opposed to fluorescence-based probing with conventional VSDs. In our proposal, the PAT technique will involve NIR excitation and ultrasound detection, while UCNPs will serve as nanotransformers that convert skull penetrating NIR light to VIS light, which will be absorbed by the locally administered VSDs, allowing us to monitor changes in their absorption, induced by changes in action potentials, and, correspondingly, map the deeper brain neuronal activity.
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{
"pile_set_name": "NIH ExPorter"
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Frontotemporal lobar degeneration (FTLD) is the second leading cause of dementia in individuals younger than 65. Loss-of-function mutations in the progranulin (PGRN) gene have been shown to be one of the leading causes of FTLD. Mutations in PGRN lead to a neuropathology which is characterized of by the presence of ubiquitinated TAR-DNA binding protein (TDP-43) inclusions. A major unresolved question in the field is the exact relationship between PGRN and TDP-43. Clues to this relationship lie with the TDP-43 inclusions. The presence of ubiquitinated TDP-43 suggests a problem with TDP-43 degradation. We have shown that a major mode of TDP-43 degradation is through autophagy. We have obtained exciting preliminary data showing that basal autophagy induction is reduced in 6-month-old PGRN knockout mice. Additionally these changes correlate with a significant decrease of the ubiquitin binding protein p62, which serves as shuttling factor for select ubiquitinated proteins, such as TDP-43, towards autophagy degradation. Together these findings lead us to the following working hypothesis: Lack of progranulin leads to TDP-43 accumulation by (i) impairing autophagy induction and by (ii) lowering TDP-43 targeting to autophagic degradation via a p62- mediated mechanism. To test this hypothesis, we will use multidisciplinary approaches that will allow us to identify the mechanistic links between the lack of PGRN and TDP-43 accumulation. To elucidate the pathways linking PGRN and autophagy, we will use a lentiviral vector to increase ERK signaling to rescue autophagy in PGRN knockout mice. Further, we will measure autophagy induction in primary neurons and aged mice to investigate the role of PGRN in autophagy. We will investigate the role of p62 in TPD-43 accumulation. Primary neuronal cultures will be used to determine the signaling pathways leading to reduced p62 levels in PGRNko/ko mice and the molecular mechanisms by which reduction of p62 levels leads to TDP-43. We also will test the hypothesis that autophagy dysfunction facilitates TDP-43 accumulation. We will use a pharmacological approach to increase autophagy in transgenic mice expressing mutant TDP-43 that exhibit cognitive and motor deficits as well as TDP-43 accumulation. These experiments will test whether increasing autophagy may represent a possible therapeutic approach for the treatment of FTLD-TDP and will further elucidate the role of autophagy in TDP-43 pathogenesis. Successful completion of these proposed studies will represent a significant contribution to the field, and the data obtained has the potential to identify novel therapeutic targets in the fight against FTLD and TDP-43 proteinopathies.
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{
"pile_set_name": "NIH ExPorter"
}
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Our goal is to define how a bacterial cell integrates the timing of DNA replication and the polar positioning of the chromosomal origin region to control the temporal and spatial expression of cell cycle events. A lynch-pin in the temporal coordination of replication initiation and the transcription of genes encoding global cell cycle regulators is DnaA. This protein functions both to initiate replication and to activate the transcription of over 40 cell cycle-controlled genes. DnaA is controlled at multiple levels, including transcriptional regulation by differential methylation of its promoter that is linked to the progression of the replication fork, and the control of its activity by the replisome-associated HdaA protein. We will determine if changes in DNA methylation state that functions to 'clock' the sequential transcription of both dnaA and ctrA controls the temporal transcription of multiple cell cycle regulated genes, including those controlled by GcrA master regulator. We will also determine if a temporally-controlled dnaA anti- sense transcript contributes to the control of dnaA expression. The dynamic spatial deployment of proteins and the origin region of the chromosome are critical factors in cell cycle control. We have shown that upon replication of the chromosomal origin, the actual DNA sequence that moves toward, and is captured by, the cell pole is parS bound to the ParB partition protein. If the ParB/parS complex is not anchored to the pole, segregation of the rest of the chromosome is impaired and the FtsZ division ring is misplaced. We have recently identified PopZ as a protein that forms a polar polymeric network and functions to anchor ParB/parS to the new cell pole. Important questions are how the polar ribosome free zone formed by the PopZ network is established and how it functions. To define how ParB/parS moves across the cell to be captured by PopZ, we have initiated an analysis of the essential ParA segregation protein. We have generated multiple mutants of ParA and visualized ParA behavior both in vivo and in vitro. We will now identify and characterize the factors that mediate its role in DNA movement and determine the mechanism that drives chromosome segregation. PUBLIC HEALTH RELEVANCE: Based on our elucidation of the genetic circuitry that runs a bacterial cell cycle, we designed a new class of boron-based antibiotics that are in phase two trials. In addition, we identified a small molecule inhibitor of the MreB bacterial actin, with the goal of using this as the basis for a new family of antibiotics.
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{
"pile_set_name": "NIH ExPorter"
}
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More than 80% of hospice patients experience distressing symptoms that significantly impact comfort in their final days of life. Previous research demonstrated that complimentary therapies such as massage can alleviate symptoms for minutes to hours, but none have examined cumulative effects of massage therapy or cost-effective methods of providing massage to hospice patients. We propose a randomized clinical trial (RCT) comparing usual care versus usual care plus 5 daily massage therapy sessions given by licensed massage therapists (LMT) on symptom outcomes including: a) symptom distress, b) pain, c) mood state and d) sleep quality in a diverse group of 200 hospice patients with cancer. In a pre-test/post-test design with repeated measures preceded by a 5-day run-in period, patients will complete computerized assessments. All patients will receive usual home care level of hospice care, and patients randomized to the experimental group will receive daily massage therapy for 1 week. Outcome scores will be aggregated for each variable from day 2 to day 6 and analyzed using MANOVA, with post-hoc comparisons if significant group results are obtained. Study findings will be used to guide hospice care providers in selecting evidence-based complimentary interventions to relieve distressing symptoms in people at the end of life. A second aim is to determine the costs of the massage intervention and compare the effects of usual hospice care and usual care plus massage on hospice reimbursement and utilization costs. We will use a 2-step activity-based costing methodology for determining the costs of the intervention and the effects on cost outcomes. We hypothesize that the hospice reimbursements and utilization costs will not differ, meaning that the intervention will be cost neutral. A third exploratory aim is to describe the feasibility and the caregiver strain of LMT training 60 family caregivers to give daily massages at home to 60 of the hospice patients who completed Aim 1 procedures. Feasibility will be evaluated by: a) caregiver massage proficiency; b) massage frequency and length given by caregivers; c) caregiver and patient self-report about the massage experience. Strain is defined as the 1-week change in caregiver strain. We expect that family caregivers will be trained to give massages at a proficient level after 2 sessions with training, evaluation and feedback from a LMT. We also expect that caregivers will give 3 daily massages, each for 20 to 30 minutes and report no significant increase in their caregiver strain. Descriptive statistics and content analysis with Atlas.ti will be used to evaluate the feasibility of family caregivers providing massages and to design a clinical effectiveness study using family members as a cost-effective alternative to LMTs.
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{
"pile_set_name": "NIH ExPorter"
}
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JMJD2C demethylates the transcriptionally repressive H3K9Me3 mark on the histone H3 within nucleosomes, which are the basic building blocks of chromatin. JMJD2C promotes cellular proliferation in several different types of cancer due to genomic amplification and overexpression. JMJD2C overexpression causes increased transcription of several oncogenes, including Mdm2, c-MYC, and NOTCH1. It is therefore possible that JMJD2C promotes oncgenesis by direct catalytic activity at the loci of target oncogenes. The objective of this work is to develop a potent and selective small molecule inhibitor of JMJD2C, and use this inhibitor to investigate the mechanism by which this demethylase impacts cancer cell proliferation and survival. We hypothesize that elaboration of a small-molecule fragment identified by molecular docking will enable the development of an effective JMJD2C inhibitor, and that inhibition of JMJD2C will exhibit selective toxicity to cancer cells by preventing the transcription of oncogenes regulated by this demethylase. In Aim 1 we will synthesize derivatives of our hit compound. In Aim 2 we will test the potency and selectivity of these inhibitors against a panel of demethylases and related proteins using in vitro inhibition assays in order to establish structure-activity relationship (SAR). Finally, in Aim 3 we will test he ability of selected compounds to inhibit the proliferation of cancer cells. We will also explore th mechanism of toxicity to cancer cells by determining changes in expression levels of cancer associated genes directly transcriptionally controlled by JMJD2C. The overall goal of this study is to develop a potent and selective small molecule inhibitor of JMJD2C that will be used in cells to validate JMJD2C as a therapeutic target for cancer, and to reveal mechanism(s) by which JMJD2C promotes oncogenesis.
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{
"pile_set_name": "NIH ExPorter"
}
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No Abstract Available
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{
"pile_set_name": "NIH ExPorter"
}
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PROJECT SUMMARY/ABSTRACT We recently used the Cre/loxP system to selectively delete dopamine D1 receptors from GABAergic neurons from the Nkx2.1 lineage within the medial ganglionic eminence. These cells largely produce a subset of crucial cerebral cortical interneurons within the medial frontal cortex. Although these interneuron-localized receptors represent just a small fraction of cortical dopamine D1 receptors (most are on excitatory glutamatergic projection neurons), these conditional knockout mice exhibit a remarkable phenotype in one functional domain ? mood. Specifically, GABA-D1-cKO mice exhibit decreased immobility in the forced swim test, and decreased latency to consume a palatable liquid in a novel stressful environment; these are both indicative of an antidepressant-like effect. These data suggest a new mechanism by which cerebral cortical interneurons may contribute to mood regulation, and importantly, this mechanism may be ?druggable?. The current proposal seeks to validate this model using additional behavioral measures and stressors (Aim 1), define the developmental timing components of this neuroadaptation (Aim 2), and begin to assess the cellular and molecular underpinnings of this response (Aim 3). Success of this current project will position us to subsequently develop unique therapeutic strategies to selectively manipulate these receptors through genetic and pharmacological approaches that are limited to specific neuronal subpopulations.
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{
"pile_set_name": "NIH ExPorter"
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The purpose of the mutagenesis core is to provide assistance to each of the research projects in the administration of mutagens and the subsequent follow-up and observation of the animals until they are assayed. The mutagens to be used in commin are ENU and 4-aminobiphenyl. DMBA will be used by Drs. Tishfield and Doetschman, and ionizing radiation by Drs. Stringer and Tishfield. The Cobalt-60 source, however, is not part of the mutagenesis core, but is available through Dr. Denman, a radiation biologist, in Radiology (see attached letter). The core also will provide adduct analysis, particularly for Dr. Doetschman's project.
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{
"pile_set_name": "NIH ExPorter"
}
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The role of the transcription factor Brachyury (Bra) in notochord formation is well documented and evolutionarily conserved throughout the chordate phylum. However, still little is known about the transcription factors controlled directly or indirectly by Bra and about the function of factors acting in concert with Bra in notochord development and evolution. In particular, the knowledge of structure, common features and modalities of regulation of cis-regulatory modules (enhancers) directing expression in the notochord is fragmentary and poor. We have exploited the experimental advantages offered by the ascidian Ciona intestinalis as a model system to isolate and characterize a collection of notochord enhancers from genes that have already been described as Bra targets and from genes whose relationship with Bra is unknown. Our preliminary data, gathered from the comparative analysis of the notochord enhancers, lead us to hypothesize that notochord formation in Ciona relies upon additional, mostly uncharacterized notochord transcription factors that fall into two categories: Bra-downstream factors, controlled directly or indirectly by Bra and acting as its transcriptional intermediaries and factors acting in concert with Bra. We will test these hypotheses through the following specific aims: (1) Isolate minimal regulatory sequences necessary for notochord expression;(2) Identify the activators controlling the minimal enhancer sequences and study their role in notochord development;(3) Identify the enhancers controlling, in turn, selected notochord activators. The broad scope of the proposed research is to rapidly untangle the gene networks underlying the different steps of notochord development and differentiation and to gain valuable insights into the molecular mechanisms controlling gene expression in the notochord during its formation and evolution.
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{
"pile_set_name": "NIH ExPorter"
}
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Elevated intracellular free Zn2+ concentrations ([Zn2+]i) have been implicated in the neuronal death caused by stroke. Therefore, a therapy that restores low [Zn2+]i has the potential to improve the outcome of stroke. The goal of proposed research is to design such a therapy. Preliminary studies on hippocampal neurons indicate that an intracellular acidification similar to that taking place during stroke causes [Zn2]i elevations due to Zn2+ release from intracellular stores. We will determine the role of low affinit Zn2+ ligands, such as adenine nucleotides, in these [Zn2+]i elevations and establish whether neuronal viability can be improved by a specific chelation of acid-induced [Zn2+]i elevations. In particular, we will design a method of Zn2+ chelation that specifically targets these acid-induced [Zn2+]i elevations. The research will be conducted on primary hippocampal neurons from mice. Aim 1 will clarify which low affinity intracellular Zn2+ ligands release this ion when the pH drops into a stroke-characteristic range. In Aim 2, a method will be developed to specifically chelate acid-induced [Zn2+]i elevations. Aim 3 will determine whether this method of Zn2+ chelation improves the viability of neurons exposed to oxygen and glucose-deprivation. If it does, our method will provide a framework for the development of therapy that counteracts excessive [Zn2+]i elevations during stroke.
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{
"pile_set_name": "NIH ExPorter"
}
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Our goals are to enhance quality of life and to integrate effective EOL care throughout the course of treatment for adolescents living with HIV/AIDS, as well as to test an intervention to support HIV positive adolescents and their family members in order to prevent depression and anxiety. These goals are consistent with the NIH 2006 Plan for HIV Related Research (NIH, 2004, pp. 260-261). Our specific aims are 1) to adapt and standardize interventions already established as efficacious for use with a new group: HIV positive adolescents and their families;2) to pilot test the feasibility and acceptability of the Family Centered ACP intervention;3) to estimate the effect of the intervention on adaptive coping, psychological adjustment, quality of life, plans. Stage of illness will also be studied. More than 30,000 adolescents in the U. S. die annually from the effects of all chronic illnesses (Mineno, et al., 2002). EOL care for these patients is a public health issue (Freyer, 2004; Rao, et al., 2002), because of its high burden and potential for the prevention of suffering. We will recruit from two sites and enroll 40 (N=80) adolescent/legal guardian or surrogate dyads. Temporarily not ready dyads will be placed in an observational group. We will randomly assign 35 dyads to either the Family Centered ACP intervention (n=20) or the Adolescent Health Control (n=15). The format is three 60 to 90 minute sessions: (1) structured EOL survey; (2) Family Centered ACP interview; (3) optional Family Problem Solving. Standardized, self-report measures will be administered at baseline, immediate post intervention and 3 month follow-up. To assess the feasibility and acceptance of the intervention in Aim 2, frequency distributions in Stata 8 will be used to estimate each of the indicators of performance from enrollment to data completeness. Analysis of covariance models will be used to estimate the effect of the intervention on hypothesized outcomes in Aim 3. The NIH Strategic Plan (IOM, 2003) to reduce health disparities is relevant to this study, which aims to improve the quality of life for a condition that disproportionately affects minorities, as is the NIH Statement on Improving EOL Care (Dec.6-8, 2004) to increase the study of minorities and children who are underrepresented in EOL research. Careful research in psychologically sensitive areas such as EOL care is consistent with IOM recommendations (IOM, 2002) and the NIH Roadmap intention to discourage "risk-adverse" research and to encourage innovation (IOM, 2003).
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{
"pile_set_name": "NIH ExPorter"
}
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The influence of genetic predisposition to diabetes mellitus on susceptibility to coxsackievirus B4 infection, and on virus induced pancreopathy will be studied. Dietary restriction prevents the expression of diabetes mellitus in inbred mice with recessive and dominant inheritance for diabetes, i.e., C57BL/Ks db/db -- the diabetic mutant mouse; C57BL/6J ob/ob -- the obese mouse; and the C57BL/6J Ay/a -- the yellow obese mouse, respectively. Susceptibility to coxsackievirus B4 infection in these animals under dietary restriction will be established. Similarly, coxsackievirus B4-induced pancreatic pathology during acute, convalescent, and post-convalescent stages of infection will be determined by classical histopathological techniques, special stains, and electron microscopy, with special emphasis being given to beta cell pathology. Virus localization in the pancreas of animals with diabetic heredity will be done by fluorescent antibody and peroxidase-anti-peroxidase procedures. The effects of a single and multiple episodes of viral pancreatic injury in animals with genetic predisposition to diabetes on precipitating onset of diabetes mellitus will also be ascertained. The experimental results will determine the influence of diabetogenic factors, i.e., specific mutations -- db, ob, Ay, and background modifiers -- C57BL/Ks, C57BL/6J on coxsackievirus B, (1) susceptibility, (2) pancreatic pathology, and (3) pancreopathy.
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{
"pile_set_name": "NIH ExPorter"
}
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SBIRT for Substance Abuse in Mental Health Treatment Settings Project Description An estimated 40% of patients in mental health treatment settings engage in hazardous alcohol and/or drug use. Yet mental health clinics often do not provide intervention or referral to specialty substance use disorder (SUD) treatment for these patients. One model of intervention that has been shown effective in medical settings to reduce alcohol use and/or promote engagement in addiction treatment is screening, brief intervention, and referral to treatment (SBIRT). Despite the effectiveness of SBIRT for risky alcohol use in medical settings, there has been limited research on the effectiveness of SBIRT for drug use and no research on the effectiveness of SBIRT in mental health treatment settings. Given the proportionately large number of mental health patients who also engage in hazardous substance use and the limited attention given to substance use issues in mental health clinics, research is needed to find an appropriate and effective substance use intervention for patients in these settings. The proposed study uses a randomized controlled trial to examine the extent to which the World Health Organization's SBIRT model, the ASSIST (Alcohol, Smoking, and Substance Involvement Screening Test) and its associated brief behavioral intervention, leads to reductions in substances prevalent in mental health settings: alcohol, cannabis and stimulants (i.e., cocaine and methamphetamine). The study will also examine the effect of SBIRT on improvement in psychiatric symptoms, improved quality of life and for those whose level of substance misuse indicates a need for treatment, initiation and engagement into SUD treatment services. Eligible participants will be mental health patients who report any past year use of cannabis or stimulants or at least one heavy drinking day in the past year. An exploratory aim of the study is to identify effect sizes of SBIRT for patients whose highest risk is for alcoho, cannabis or stimulants. Mental health patients (n = 1,080) who meet eligibility criteria will be enrolled and randomly assigned to either the SBIRT intervention condition or to a health education attention control condition. Participants will be assessed at baseline on substance use, psychiatric symptoms and quality of life. Each participant will be assessed at 3-, 6- and 12- month follow up points for alcohol and drug use, involvement in SUD treatment services, severity of psychiatric symptoms and quality of life. Random effects growth models will be used to test the study hypotheses. If successful this study will yield valuable new knowledge about the effectiveness of SBIRT in mental health treatment settings and will promote improved well being of mental health patients. Further, the study will provide evidence on the effectiveness of SBIRT for reducing illicit drug use. Results from this research will be used as the basis for broader dissemination and of SBIRT in mental health settings.
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{
"pile_set_name": "NIH ExPorter"
}
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Previous studies have provided compelling evidence that the Growth Hormone (GH)-IGF-1 axis is a conserved pathway important for determination of both healthspan and lifespan in diverse organisms. Data from several rodent models indicate that lifelong deficiency of GH and/or IGF-1 or perturbations that suppress their signaling pathways result in increased lifespan, reduced age-related pathologies and beneficial effects on cardiac and cognitive function. Nevertheless, the concept that reduced levels or signaling of these hormones are beneficial for the aging organism remains controversial since there are numerous studies that have reported beneficial effects of GH and/or IGF-1 replacement on the aging cardiovascular system and an overwhelming database indicating beneficial effects of these hormones on the aging brain. The question of whether reduced GH and IGF-1 levels are directly associated with increased longevity and whether GH and/or IGF-1 replacement have beneficial effects on individual organ systems (e.g. healthspan) remains a seminal question in the field of biogerontology. This controversy will continue until appropriate and translationally relevant animal models that can be used to regulate GH and IGF-1 levels throughout the lifespan are available. The necessity of new models for advancement of the field has been recognized by us and our colleagues in recent consensus reports. The proposed experiments are in response to PA-10-014 (Development and Characterization of Animal Models for Aging Research) and our goal is to conduct translationally relevant studies as well as basic lifespan, metabolic, behavioral and pathological analyses to address the hypothesis that deficiencies of hepatic IGF-1 initiated at different stages of the lifespan have unique effects on survival, end-of life pathology and metabolic parameters. The following specific aims are proposed: 1) Analyze the effects of hepatic IGF-1 deficiency initiated peri-natally by crossing alb-cretg/+ and igff/f mice or post-natally at 30 days, 6 or 16 months by injecting the igff/f mouse with MUP-iCre-AAV8 or control vector and assessing effects on lifespan and end-of-life pathology. 2) Determine the effects of hepatic IGF-1 deficiency initiated peri-natally or at 30 days, 6 or 16 months of age on a) accumulation of age-related pathological changes using a cross-sectional design, b) paracrine IGF-1 gene and protein expression, c) circulating hormone levels (thyroxine, leptin, insulin and glucose) as well as whole body insulin action, glucose production and rates of glucose utilization in individual tissues, d) kidney and liver function, as well as e) basic measures of food/water consumption, activity and lean/fat mass. Effects of IGF-1 deficiency on these endpoints will be assessed at 6, 16 and 26 months of age. 3) Assess whether hepatic IGF-1 deficiency initiated peri-natally or at 30 days, 6 or 16 months influences cellular redox homeostasis and inflammatory processes in vasculature, heart, liver, brain, muscle and kidney during aging. The studies presented are designed to produce one of the most comprehensive and rigorous analyses of the effects of IGF-1 deficiency on pathology and lifespan using a pre-clinical, translationally relevant approach.
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{
"pile_set_name": "NIH ExPorter"
}
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The primary goal of this research project is to delineate genetic controls of development of the embryonic axis and patterning of the neural tube in mammalian embryos. Genes postulated to play key roles in these processes, including Wnt-1 and 3a; Engrailed-1; nodal: hedgehog; and hepatic nuclear factors alpha and beta, will be disrupted in mouse whole embryo culture during gastrulation and neurulation stages of development using antisense oligonucleotides. Specific Aims include: 1. To determine the function of Wnt-1 and 3a, engrailed-1, Sonic hedgehog, nodal, and hepatic nuclear factors alpha and beta in axis formation and neural tube patterning. a. Antisense oligonucleotides targeted to each of the genes will be microinjected into the amnion of 3-5 somite stage mouse embryos undergoing the processes of neurulation and gastrulation. Embryos will be grown in whole embryo culture and the effects of the targeted gene disruptions on development will be monitored. b. Functional redundancies between genes and the interaction of genes in signalling pathways responsible for axis formation and neural tube patterning will be determined by targeting 2 or more genes simultaneously. c. Effects of targeted antisense exposures on gene expression will be monitored using quantitative PCR, quantitative in situ hybridization, and immunohistochemical procedures. 2. To determine the morphological alterations at the cell and tissue level that are altered by a targeted gene's disruption and that contribute to the abnormal phenotype. These studies will be completed using detailed histological and scanning electron microscopic techniques. 3. To determine the underlying biological effects produced by targeted gene disruption that may play a role in production of altered phenotypes. These studies will include analysis of cell death and cell proliferation patterns. Together these studies will provide information on the functional roles of these genes during embryogenesis, assist in defining genetic signals responsible for key morphogenic events, and identify cellular processes involved in development at stages when many birth defects are induced. In turn, identification of roles for these genes will provide data that will be important in defining the origins of birth defects, such as spina bifida and caudal dysgenesis.
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{
"pile_set_name": "NIH ExPorter"
}
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We propose to conduct a phase I study of high-dise radioimmunotherapy using the anti-breast cancer monoclonal antibody humanized-bre-3labeled with 90-yttrium followed by aahpcs in patients with metastatic breast cancer that are refractory to conventional or high dose chemotherapy. Bre-3 labeled with III-indium will be used to imaging these patients. The clinical endpoints of the study will be to define the maximum tolerated dose and dose limiting toxicities of the radioimmunoconjugate. In addition we will study the dosimetry and pharmacokinetics of the radiolable and we will also obtain preliminary estimates of its efficacy in preparation for a phase II study in the future.
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{
"pile_set_name": "NIH ExPorter"
}
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Lipoproteins allow for transport of insoluble lipids in aqueous environments and transport the majority of cholesterol and triglyceride in the bloodstream. Alteration of normal lipoprotein levels can lead to human diseases, including atherosclerosis and coronary heart disease, one of the leading causes of mortality in developed countries. Dietary lipids are digested and taken to the endoplasmic reticulum (ER) in intestinal cells where they are processed before secretion to the bloodstream. An essential step of lipoprotein secretion is COPII-dependent transit from the ER to the Golgi in the form of chylomicrons in the intestine and very low density lipoproteins (VLDL) in the liver. These lipoprotein cargos are actually larger than canonical COPII vesicles, and thus pose a special problem for the secretory machinery. Despite the paramount importance of this process to human health and basic cell biology, the molecular mechanism of how COPII enables large lipoprotein cargo export from the ER is largely unknown. Chylomicron Retention Disease/Anderson's Disease (CMRD), a disease where newly formed lipoproteins are unable to exit from intestinal cells, offers an entry point to address this question. CMRD patients carry mutations in SAR1B, a component of the COPII coat; however no patients were identified with mutations in its highly conserved paralog, SAR1A. The functional difference between the two SAR1 paralogs may provide important insights into the mechanism of COPII secretion of large lipoprotein cargos. Because other COPII components have paralog-specific functions, these components, and their effectors, may also have essential roles in lipoprotein secretion. In order to elucidate the mechanisms by which large lipoprotein cargos are secreted, we will: 1) Define the amino acid residues responsible for functional differences between SAR1 paralogs and their effect on COPII coat formation and VLDL secretion. 2) Use in vitro transport vesicle budding assays to determine the requirements for specific COPII coat components for VLDL secretion. 3) Determine whether KLHL12, which ubiquitinates COPII component SEC31A and alters its ability to secrete the large cargo collagen, is required for transport of large lipoproteins. The experiments proposed here will provide novel information about the mechanisms of intracellular transport large lipoprotein cargos and the unique roles of COPII component paralogs. This information has implications for the etiology of CMRD and may provide insights into how to regulate VLDL secretion as a potential treatment for atherosclerosis and other human diseases.
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{
"pile_set_name": "NIH ExPorter"
}
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The antibody response to HIV-1 that is mediated by B-1 B cells is a largely unexplored area in HIV vaccine development. However, recent progress has raised important questions about whether the conventional B-2 B cell-oriented immunization strategies are solely responsible for induction of HIV-1 broadly neutralizing antibodies (bnAbs), [including those that are highly specific for carbohydrate epitopes and those that are characteristically polyreactive and autoreactive, as these antibodies are often seen in the B-1 repertoire.] Importantly, such antibodies represent dominant classes of HIV-1 bnAbs identified from a portion of HIV-infected subjects using the advanced technologies of single-cell cloning and deep sequencing. Our team has been studying carbohydrate antigens and anti-carbohydrate B cell responses for years. Recently, we obtained evidence that oligomannose-based HIV antigens are immunogenic in vivo under certain conditions. In essence, we observed that autoantibodies targeting Man9 clusters were significantly increased in the blood circulation of men with aggressive prostate cancer, in the cerebrospinal fluid of patients with multiple sclerosis, and in the serum of mice with experimental autoimmune encephalomyelitis (EAE). Interestingly, co- immunization of SJL/J mice with a Man9-KLH conjugate at the time of EAE induction elicited highly significant levels of anti-Man9-cluster autoantibodies. These anti-sera are strongly cross-reactive with HIV-1 gp120 glycoproteins (see Preliminary Results). Moreover, we discovered that immunization of non-human primates (NHP) using human RV144 HIV vaccines elicited significant levels of antibody responses to broadly HIV- neutralizing glyco-epitopes, including oligomannoses and other N-glycan cryptic antigens (Preliminary Results). Most importantly, we recently uncovered a significant population of oligomannose (Man9) cluster-binding B cells (BMan9+) in the naive B cell repertoire that are readily reactive with oligomannose clusters. These B cells represent approximately 12% of CD19+CD45+ B cells in the blood circulation of healthy human subjects. Interestingly, the BMan9+ B cell population is also present in mice, constituting approximately 10% of B-1a (CD19+CD5+) and 2% of B-1b (CD19+CD5-) B cells in the peritoneal cavity. Given that B-1 and B-2 B cells differ significantly in responding to an antigenic stimulation and that most HIV-1 vaccine strategies were developed based on B-2 responses without consideration of the B-1 potential, we propose to conduct a focused investigation of the B-1 B cell-mediated antibody responses to HIV-1. [In essence, we will apply a key HIV vaccine, RV144, to probe the B-1/ BMan9+ B cell responses in multiple species, including human, NHP, IgH-allotype chimeric mice, and humanized mice. Specifically, we will examine whether human RV144 vaccine elicited the characteristic profile of anti-carbohydrate antibody responses as observed in NHP models (Aim 1); examine contributions of murine B-1 and B-2 B cells to production of anti-HIV-1 antibodies in responding to RV144 vaccines (Aim 2); determine whether the human BMan9+ B cells in humanized mice contribute to anti-oligomannose responses and other anti-HIV antibody responses (Aim 3); and explore potential molecular signatures of B-1/BMan9+ B cell-mediated anti-carbohydrate antibody responses by a comparative IgV region sequence analysis (Aim 4).] [In summary, this revised R01 application focuses on identification of molecular and cellular signatures of the B-1/BMan9+ B cell-mediated immune responses to HIV-1 and exploration of the potential of this newly recognized B cell subset in human HIV vaccine development. If successful, this project could lead to the strategic development of novel preventive and/or therapeutic vaccine strategies against HIV infection and AIDS.]
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{
"pile_set_name": "NIH ExPorter"
}
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An ever-expanding family of ets-related genes has been found in the human genome, as well as in that of other species. Features of this ets family include a DNA-binding domain, as well as a helix-loop-helix motif, which has been postulated to be a site for protein-protein interaction. The goal of this project is to identify proteins which interact with individual ETS proteins, and thus give rise to functional differences among the members of the ets family. To this end, defined coding regions of the human ETS1 and ETS2 genes have been placed into prokaryotic pET vectors, which can be induced to overexpress the fusion protein, which then can be rapidly purified. These engineered proteins are radiolabelled and used to probe expression libraries to identify proteins with which ETS proteins interact. Preliminary results have revealed several clones which appear to interact with ETS1. This interaction will be further investigated to define the domains required for interaction. These methods will also be employed to identify proteins which interact with other members of the ets family.
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{
"pile_set_name": "NIH ExPorter"
}
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Over the past three decades, research supported by this grant has had a major impact on our understanding of how cells respond to damage to their DNA. Many of our findings have been relevant to cancer. It has made particularly important contributions to our knowledge of the crucial role that translesion DNA polymerases play in DNA damage tolerance and mutagenesis and of SOS responses to DNA damage. The proposed research addresses critical problems concerning the roles of translesion DNA polymerases, mechanisms of DNA damage removal, and cell responses to DNA problems. We have discovered that Y Family DinB-related DNA polymerases - E. coli DinB (DNA pol IV), its mammalian ortholog Pol kappa, and its archaeal orthologs - have a striking ability to preferentially carry out accurate translesion synthesis (TLS) over certain adducts typified by N2-furfuryl-dG. We have termed these stealth lesions since the high ratio of DinB to replicative polymerase makes them largely invisible to DNA replication, but they remain in the DNA to cause problems with transcription. The proposed experiments will yield new detailed insights into the properties of DinB that endow it with this special characteristic and test whether the associated costs are DinB's propensities to -1 nt deletion mutations and to incorporate oxidized nucleotides in a mutagenic fashion. The experiments will also offer fresh insights into the complex regulatory processes that regulate access of potentially mutagenic TLS DNA polymerases to termini. We have discovered unanticipated roles in DNA repair and damage tolerance for NusA, a component of elongating RNAP polymerases. We have proposed a previously unrecognized pathway of NusA-dependent transcription-coupled repair (TCR) that is of particular importance for the removal of stealth lesions from the transcribed strand of expressed genes. We have also proposed a model for NusA- dependent transcription-coupled TLS (TC-TLS), the first for any organism, that can help cells deal with transcriptional problems created by gaps in the transcribed strand that result from lesions in the non transcribed strand. Our experiments will define the relationship between a lesion's ability to be preferentially bypassed by DinB, to block transcription, and to be recognized by nucleotide excision repair (NER). They will also provide in vivo and in vitro tests of our models of NusA-dependent TCR and TC-TLS. Hydroxyurea (HU), an inhibitor of class I ribonucleotide reductases, is widely used to block DNA replication. We made the unexpected discoveries that cells die after HU treatment because a sequence of cellular events results in the production of toxic hydroxyl radicals and that alteration of a TLS DNA polymerase can prevent the lethality associated with HU treatment. The proposed experiments will offer key insights into the molecular details of the processes that underlie these phenomena and could lead to the identification of new drug targets.
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{
"pile_set_name": "NIH ExPorter"
}
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Type 2 diabetes imposes a heavy burden on public health in the United States. This burden falls disproportionately upon African Americans. The prevalence of type 2 diabetes is over 50% greater in African Americans than in whites, and African Americans are at even higher risk for severe diabetic complications including end-stage renal disease and lower extremity amputation. Possible causes of this excess risk in African Americans include inadequate access to optimal primary health care and suboptimal health behaviors. We are therefore conducting a randomized, controlled trial to test two primary-care-based interventions to reduce risk in diabetic African Americans: 1) a community outreach intervention and 2) a nurse case-manager intervention. Now in the fifth year of a 5-year grant from NIH-NIDDK, we have completed recruitment of 186 African Americans with type 2 diabetes who reside in East Baltimore and receive primary health care from sites affiliated with Johns Hopkins and its internal medicine training program. Each participant has been assigned to one of four parallel arms: 1) usual care only, 2) usual care + community outreach, 3) usual care + nurse case-manager, and 4) usual care + community outreach + nurse case-manager. As interventions continue, we are in the process of gathering yearly follow-up data on participants. Over the next 12 months, we will continue these interventions and ultimately compare their effectiveness in improving blood glucose, blood pressure, and serum lipid levels. The support of the Johns Hopkins OPD-GCRC continues to be absolutely crucial to the success of this project. It provides space and a staff for all screening and data collection visits. It also supports laboratory analyses, data entry, and data processing.
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{
"pile_set_name": "NIH ExPorter"
}
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This project identifies a number of strategies that will be used to increase the power of direct phasing methods for applications to larger molecular structures than are currently determined in a routine fashion. In addition to the time efficient improvements to a number of computational algorithms described in this proposal, insights will be provided with regard to how the availability of more accurate and higher resolution data may or may not be advantageous to strengthening various phasing strategies. Native X-ray data will be recorded and examined for an appropriate base of both solved and unknown macromolecular structures to test these new methods. Computer graphics techniques will play an important role in strengthening the phasing procedures used for these large macromolecular structures and graphics software will be developed to help recognize and extend the structural patterns that exist in marginally phased electron density maps. On the experimental side, preliminary studies indicate that low temperature investigations of single native crystals can provide direct phase information that was previously thought to be unavailable. An analysis using a room temperature and a liquid nitrogen temperature data set, recorded from two different crystals of the orthorhombic gramicidin structure, measured on two different X-ray diffractometers, demonstrates that the precision required of the data to successfully apply this method is not insurmountable. Analysis of several difficult structure determinations, including that of the gramicidin data, moreover, shows that large blocks of phases can be secured with a minimum of effort provided that as few as a dozen well-chosen and identifiable pivotal invariants upon which the structure solution depends can be unambiguously determined. These results are an encouraging indication that complex crystal structures containing as many as 300-500 atoms ban be determined with far less difficulty than is currently experienced for structures containing 100 atoms or less.
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{
"pile_set_name": "NIH ExPorter"
}
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The overall objective of the proposed project is to investigate the process of resolution of grief following the loss of the spouse in an elderly population. There are four general objectives: To describe the patterns of bereavement in an elderly population over a two-year period; to explore the function of supportive networks and coping strategies used by the elderly on physical and emotional adjustment to bereavement; to identify critical time periods in the bereavement process. The design of the study requires a randomly selected sample (N equals 100) of the bereaved persons (59 males, 50 females) for the study group. In addition, two control groups will be selected: a random bereaved control group, and a matched married non-bereaved control group. Personal interviews (unstructured and structured will be done at six intervals with the bereaved sample: 3 weeks, 2, 6, 12, 24 mo. post-bereavement. The major variables to be measured will be: circumstances of the death, strategies for managing grief, perceived helpfulness of persons in the supportive network, physical symptoms, depression, unresolved grief, self-esteem, life-satisfaction. Both control groups will receive mailed questionnaires at various points over two years to assess physical and emotional well-being. Content analysis and case study method will be used for the qualitative data analysis. Correlation and analyses of variance for repeated measures will be the principal statistical techniques employed.
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{
"pile_set_name": "NIH ExPorter"
}
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SUMMARY Our goal is to determine the molecular mechanism of soluble guanylate cyclase (sGC) activation by NO via discovery of Long-range Interaction NetworKs of amino acids (LINKs) using a novel activating-mutation reporter screening assay. Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The only effective therapies aim to lower intraocular pressure (IOP) to limit disease progression. However novel molecular targets and therapies are necessary to halt or reverse disease progression and improve patient quality of life. The sGC enzyme, already an established drug target in peripheral vascular disorders, plays a prominent role in the development of POAG. sGC controls the NO-sGC-cGMP pathway by producing cGMP, which plays a key role in IOP regulation. The enzyme is regulated by nitric oxide (NO) binding to its regulator domain transmitting a yet to be characterized activating signal to the catalytic domains that increases cGMP output several hundred fold. Despite the recent determination of a low-resolution structure of full-length sGC, the mechanism by which NO allosterically enhances sGC activity remains unknown, limiting the utility of sGC as a therapeutic target for POAG. Key to elucidating the structural requirements for NO activation of sGC is the mechanism by which the catalytic domains achieve their high-activity conformation. We propose to use a retro-mechanistic approach to first define the high-activity structural transitions in the catalytic domains while subsequently tracing them back to the NO-binding event in the regulatory domain. We hypothesize that LINKS play a critical role in NO- dependent formation of an active site conformation that is optimized for full enzymatic activity. We have already defined parts of these LINKs in our new preliminary data confirming a novel role for residues located in an interfacial ?-flap as key modulators of the orientation-dependent activation of the catalytic domains. Fully defining sGC-activating LINKs will be crucial to (i) understand the structural transitions required for sGC activation and how they are modulated by NO and (ii) design small molecule modulators of sGC that modulate these transitions. Results from our studies will identify new sites (referred to as ?LINKs? in our proposal) that promote transitions between the inactive and active sGC catalytic states. Defining these transitions will be the first step to understand how NO allosterically activates full-length sGC. In the long-term, results from this research will allow us to specifically modulate the NO-sGC-cGMP pathway that plays a key role in regulating IOP, crucial to the etiology of POAG.
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{
"pile_set_name": "NIH ExPorter"
}
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Although 7T systems have been available since the late 1990s, progress for brain imaging at 7T has been slowed by the transmit performance of conventional head coils (i.e. a large transmit only volume coil with a receive only phased array). At 7T large single drive transmit head volume coils suffer from poor homogeneity (40-50%) and low efficiency and limited SNR. The use of receive only phased arrays within these coils significantly enhances SNR and enables parallel reception but does not improve the transmit performance. These limitations can be addressed by the use of transceiver arrays which provide both independent transmission and reception. Transceiver arrays using parallel transmission and/or RF shimming offer improved homogeneity, spatially tailored excitation, gradient independent outer volume suppression and reduced SAR. To date, the number of coils in these transceiver arrays has been limited to the number of independent transmit channels (typically 8) and the small size of these coils required to maintain optimal SNR. To address these limitations we will: i) eliminate the need for equal numbers of transmit and receive channels for the multiple row transceiver arrays by developing new pulse sequence methods which utilize RF multiplexing to drive multi-row transceiver arrays and reduce power deposition; ii) enhance the efficiency of multi-plane MRSI data collection and reduce SAR by developing multi-band acquisition MRSI acquisitions. To evaluate the methods developed we will study veterans with mild traumatic brain injury (mTBI) arising from blast exposure. It has become clear that veterans exposed to mTBI from blast injury display delayed neurological deficits, often without clear imaging correlates. In the absence of objective confirmatory imaging evidence, poor performance on cognitive evaluations can be attributed to poor subject effort, complicating diagnosis, management, rehabilitation and raising questions as to validity of the reported disability. Our recent work has demonstrated that in veterans with blast related mTBI, significant metabolic alterations are seen in the hippocampi which correlate with assessments of effort and cognitive performance. Using the enhanced spatial coverage afforded by the methods developed we will evaluate the hypothesis that in veterans exposed to blast related mTBI, the presence and severity of cognitive and neurologic deficits are correlated with metabolic abnormalities/impairments in the functionally linked brain regions.
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{
"pile_set_name": "NIH ExPorter"
}
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Musculoskeletal Diseases comprise the most frequent ailment for primary care physician visits in the United States, and the increases in incidence of musculoskeletal diseases with aging (particularly osteoporosis and osteoarthritis) is higher in women than in men, and leads to a significant amount of disability and reduced quality of life. Epidemiologic data clearly demonstrate the proportion of women affected my musculoskeletal diseases is higher than in men with aging, yet the biologic explanation for this sex difference remains unclear. The objective of this interdisciplinary, multi-institutional proposal, entitled Sex Differences in Musculoskeletal Conditions Across the Lifespan, is to integrate cutting-edge basic science regarding sex differences in the physiology relate to acquiring peak bone mass, epidemiologic study on the relation of sex differences in bone shape to occurrence, severity and prognosis of osteoarthritis, clinical study of sex differences in high resolution ultrasound in diagnosis and prognosis of carpal tunnel syndrome with conservative and surgical treatment, and a randomized trial of sex differences in response to a physical activity intervention for kyphosis. The overarching goal of this Specialized Center of Research is to inform and transform preventive efforts and clinical practice in diagnosis and treatment of these musculoskeletal conditions in both sexes and lead to improvements in women's health. The four projects that compose the Center will conduct critical, innovative research to characterize sex differences in musculoskeletal conditions via: 1) a mechanistic study of sex differences in progesterone receptors that are related to regulation or influence peak bone mass, 2) a prospective clinical cohort study using novel diagnostic technology to examine sex differences in the results of this technology to diagnose carpal tunnel syndrome and sex differences in standard treatments for this condition, 3) an epidemiologic imaging study to assess sex differences in bone shape and the influence of bone shape on the development, severity and prognosis of osteoarthritis of the knee, and 4) a randomized clinical trial of sex differences in response to an exercise intervention for the treatment of kyphosis. The Center's research results will be translated to the local and national medical community's through presentations by Center researchers at a number of different forums, including UC Davis and UCSF continuing medical education programs, as well as local grand rounds and national meetings. PUBLIC HEALTH REFERENCE: This translational SCOR grant, Sex Differences in Musculoskeletal Diseases Across the Lifespan, focuses on four musculoskeletal diseases or syndromes that differ by sex and include peak bone mass (a laboratory based project), carpal tunnel syndrome (epidemiologic and observational), osteoarthritis of the knee (observational), and kyphosis (exercise intervention). Each project will carefully determine the sex differences in relation to the musculoskeletal diseases and inform preventive and clinical practices.
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{
"pile_set_name": "NIH ExPorter"
}
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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Heme plays an important role in many cellular processes and dysregulation of its synthesis contributes to multiple diseases. Cellular heme homeostasis is regulated by multiple mechanisms that have typically been attributed to a combination of synthesis and degradation. Our recent findings extend this paradigm to reveal that a mitochondrial ABC transporter, Abcb6, regulates heme/porphyrin biosynthesis. A large proportion of heme usage in the liver is accounted for by cytochrome P450 isoforms, many of which are associated with drug metabolism. Each P450 is composed of a heme and an apoprotein moiety, whose cellular synthesis are tightly coordinated. Similar tight coordination of synthesis is also observed with other hemoproteins such as cytochrome c oxidase subunits of the mitochondrial respiratory chain, catalase and liver tryptophan 2,3-dioxygenase (TDO). In most of these cases, heme has been established as a transcriptional and translational activator of these hemoproteins. The modification of the enzymatic activity of the cytochrome P450 (CYP) enzymes by inhibition, induction, or activation is of great interest due to the potential of these alterations to greatly change the metabolism of the drug substrate for that enzyme and thereby alter the biological activity of the drug leading to the potential for harmful drug-drug interactions. Indeed P450 activity is impaired in several disorders of heme synthesis, such as in acute porphyrias and in acquired heme synthesis, such as in lead poisoning. Based on these observations we hypothesize that overexpression or interference with Abcb6 function might regulate hepatic CYP450 expression and/or function. Knowledge regarding the impact of Abcb6 on CYP450 activity has both immediate implications on the metabolism and disposition of pharmacological agents and environmental toxins and long term implications on drug clearance and drug toxicity.
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{
"pile_set_name": "NIH ExPorter"
}
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The overarching goal of the Biostatistics Shared Resource is to establish and maintain the highest possible standards of statistical and scientific quality in the cancer research conducted by the NCCC Programs. The resource provides expert statistical consultation and analysis for all NCCC members; study design, statistical review and interim monitoring for clinical trial protocols; statistical design and expert collaboration for large scale cancer control and epidemiology studies; experimental design and specialized statistical methods for integrative biology and laboratory-based research; statistical data management expertise; and specialized statistical software support and hardware access. The resource is staffed by seven faculty statisticians and three statistical/data management/programmer analysts. Areas of faculty statistical expertise and research include longitudinal data analysis and study design, measurement error methods in clinical research and epidemiology, statistical methods for clinical trials and epidemiology studies, statistical methods for high dimensional genomic and imaging data, clinical decision modeling, cost-effectiveness analysis, and diagnostic test assessment. The BSR faculty provides statistical expertise to the NCCC Clinical Cancer Research and Safety and Data Monitoring Committees for approving and monitoring clinical protocols. The Director of the BSR serves as a member of the Cancer Research Committee and meets weekly with the other NCCC core leaders. The BSR faculty members are active collaborators on numerous NCI-funded projects throughout NCCC and participate in all regularly scheduled Program meetings. Information technology and data management activities at NCCC are managed in coordination with the BSR, NCCC Administration, the Office of Clinical Research, and the Bioinformatics Shared Resource. The BSR data management specialist serves as the NCCC Director of Clinical Information Systems, and provides technical leadership for the ongoing development of clinical research data systems. New initiatives planned for this grant period include the further development of statistical educational seminars and workshops, for researchers at all levels within NCCC, and development of interactive statistical reporting based on the new clinical information system to provide real-time clinical trial safety and data monitoring and access to research data.
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{
"pile_set_name": "NIH ExPorter"
}
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The Department of Maternal and Child Health of the School of Public Health, the University of North Carolina at Chapel Hill (UNC) proposes to develop a training program in perinatal health research in collaboration with the Department of Obstetrics and Gynecology at Wake Forest University School of Medicine (WFUSM) and the Latin American Center of Perinatology (CLAP) as the Major Foreign Collaborator. The program will also involve work with the Departments of Obstetrics and Gynecology and Pediatrics at UNC. The title of the proposed training is the Latin American Perinatal Health (LAPH) Training Program. The main objective is to enhance perinatal health research training opportunities for scholars and clinicians from Argentina and Uruguay. Our secondary objective is to encourage greater interagency collaboration and boost infrastructure development in centers already involved in maternal and perinatal research. The training program will seek the participation of individuals involved in the perinatal research activities of the CLAP network. These individuals will be recruited from CLAP headquarters in Montevideo, Uruguay; the main research coordinating center in Argentina, the Rosarian Center for Perinatal Studies (CREP); and from collaborating hospitals throughout Argentina and Uruguay. The LAPH Training Program will provide training opportunities over a 5-year period for: 2 predoctoral students for up to 4 1/2 years (including one year of in-country research at CLAP headquarters), 2 postdoctoral trainees for 2 1/2 years each (including 1/2 year re-entry grants), 12 clinicians in short-term training activities, and clinicians and researchers from all over Latin America who have access to the Internet. The training plan for predoctoral students will involve the Maternal and Child Health Department and the Departments of Obstetrics and Gynecology and Pediatrics at UNC and will include UNC-based courses, preparation of the dissertation proposal, and one year of mentored research at CLAP headquarters in Montevideo, Uruguay. Postdoctoral trainees will receive individualized research training and participate in the ongoing research activities of core faculty members at the Department of Obstetrics and Gynecology, WFUSM. Clinicians involved in the short-term training program will attend courses provided by CLAP in Montevideo, Uruguay. By the fourth year of the program, the short-term training course will be available on the LAPH web site as a self-guided tutorial.
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{
"pile_set_name": "NIH ExPorter"
}
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Neurogenesis occurs in the dentate gyrus (DG) of the hippocampal formation (HF) of adult mammals and is altered in both health (aging) and disease (stress, neurological damage) states. Decreased neurogenesis is an emerging model for the etiology of major depressive disorder (MDD). Antidepressant treatment (ADT) increases neurogenesis in the DG of rodents. Therefore, restoration of neurogenesis may be an antidepressant therapeutic effect. However, this has yet to be examined in the human brain. Studies will be carried out in three groups: 1. Patients with Mood Disorder (MD, n=20) who were on antidepressants (ten treated with selective serotonin reuptake inhibitors, SSRI, ten treated with tricyclic antidepressants, TCA) or lithium (n=6) at the time of death; 2. MD patients (n=20) who were not on antidepressants for e 3 months and 3. normal, non-psychiatric controls (n=18). The three groups will be matched for sex, age (range 24 to 62 years in the whole sample), postmortem interval (PMI), suicide and race. We will also match the MD groups on proportions with comorbid diagnoses and the percentage that have MDD or bipolar disorder. All cases, including controls, will have psychological autopsies determining DSM Axis I and Axis II diagnosis, as well as neuropathologic examination and brain toxicological screen. Immunocytochemistry for Ki67, a cell cycle protein expressed during mitosis, will be used to identify dividing cells. Double labeling with antibodies for multipotent progenitor cells (Nestin, Pax6, GFAP), neuronal progenitors cells and neuroblasts (TUC-4, TUJ-1, PSA-NCAM), or immature glia (Vimentin) will be used to establish the phenotype of the dividing cells. The HC from MDD patients with and without SSRI, TCA or lithium and controls will be assayed. Stereology will be used to estimate the labeled cell number in the DG of the HF. The number of dividing cells (Ki67-immunoreactive [IR]), multipotent progenitors (TUC-4), and neural progenitor/neuroblasts (TUC-4-IR) will be examined along the antero-posterior axis of the hippocampus, and compared across the lifespan, between MD and normal controls and between MD patients without treatment and MD subjects who were on SSRI, TCA or lithium. The rostrocaudal extent of the right dentate gyrus will be used for the study and series of sections at 2mm intervals will be assayed for each antibody. We will test the hypotheses that neurogenesis: 1. is more pronounced in the anterior versus posterior HC; 2. is decreased in MD versus controls, and 3. MD patients receiving SSRI or TCA may have more Ki67- IR and Nestin-IR cells compared to MDD patients without treatment; on the other way we hypothesize fewer TUC-4-IR cells in antidepressants treated versus untreated cases. In an exploratory analysis we test the same effects for or lithium. These results have implications for understanding the pathophysiology of depression and the mode of action of antidepressants and lithium treatment. PUBLIC HEALTH RELEVANCE: Major Depression (MDD) is a serious public health problem and antidepressant treatment (ADT) is not effective in all cases. It has been shown that neurogenesis in adult rodents is necessary for memory and emotional responses, but it can be reduced by stress and improves with ADT, so that impaired neurogenesis may lead to MDD. We propose to examine neurogenesis in MDD patients, with and without ADT, compared to normal healthy controls, evaluating whether neurogenesis is reduced in MDD and restored by ADT.
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{
"pile_set_name": "NIH ExPorter"
}
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The prevalence of benign prostatic hyperplasia (BPH) increases as men age. Although BPH is not life threatening, the symptoms increasingly interfere with the quality of life, with lower urinary tract symptoms such as frequent urination, the sensation of incomplete bladder emptying, interrupted orweak urinary stream, and urinary leakage. One major component of the urinary bladder that is responsible for many aspects of voiding behavior is bladder smooth muscle. Unfortunately our understanding of the regulation of smooth muscle in normal bladder is incomplete which has hampered investigations into the changes that occur during diseased states. Therefore the focus of this proposal is to fill these gaps by developing a detailed mechanisitic understanding of excitation-contraction coupling of bladder smooth muscle. The global hypothesis that this proposal will test is that stimulation of bladder smooth muscle produces activation of signaling pathways leading to contraction as well as modulatory pathways that alter the final level of force produced for a given stimulus. These pathways include primarily the myosin light chain (MLC) kinase, the MLC phosphatase, activated and constitutively active Rho kinases, and protein kinase C (PKC). The specific hypothesis that this proposal will test is that bladder smooth muscle contains a constitutively active stretch dependent Rho kinase that sets the tone on the muscle cells in the unstimulated bladder wall. Stimulation of the bladder smooth muscle then initiates a series of events involving calcium dependent MLC phosphorylation, activated Rho kinase, and PKC which by linear and cross-talk pathways fine tune the levels of force produced via actions on the MLC kinase and phosphatase. The fact that smooth muscles subjected to cell culture phenotypically modulate into a non-muscle phenotype has hampered smooth muscle research. In order to circumvent this problem, we have developed a novel organ-cultured bladder smooth muscle tissue that maintains viability and the contractile phenotype and allows introduction of siRNA. The first aim of this proposal will be to continue the development of this organ-cultured preparation and demonstrate the utility of siRNA knock-down. The second aim will test the hypothesis that protein kinase C is an integral part of the signaling in the regulation of bladder smooth muscle contraction and that specific isoforms of protein kinase C are involved in specific steps in this signaling. We will also test the hypothesis that protein kinase C affects the activity of Rho kinase. The third aim will examine the role of Rho kinase in the regulation of bladder smooth muscle contraction. We will test the hypothesis that a constitutively active, stretch-dependent Rho kinase sets the basal tone in bladder smooth muscle and that a second Rho kinase is involved in the regulation of MLC phosphatase activity. These studies will allow us to develop a detailed, mechanistic model of bladder smooth muscle regulation which we will use to construct studies aimed at understanding the changes in regulation that occur in disease states such as BPH.
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{
"pile_set_name": "NIH ExPorter"
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People with neuropsychiatric disorders, particularly those with schizophrenia and depression, tend to be heavy smokers. It has been suggested that this behavior is directly related to the reinforcing properties of nicotine in the brain. For example, nicotine induces the release of the neurotransmitter glutamate, the brain's major excitatory neurotransmitter, from sites implicated in arousal and attentive behavior. It also facilitates dopamine release from areas that activate the brain reward system. Nicotine has also been shown to reduce the sedative and sometimes aversive effects of antipsychotic medications by increasing their metabolism and clearance. Smoking also reduces, in some patients, the parkinson's-like rigidity caused by neuroleptics. The conclusion drawn from these studies is that patients self-medicate with nicotine to improve their concentration, reduce boredom and anxiety, and ameliorate the negative symptoms of schizophrenia. Recently, a number of neurpathological changes in nAChR levels and expression have been described in brains from schizophrenics, adding to the interest in studying the interaction between nicotinic receptors and psychosis. It is the objective of this proposal to determine the identity and location of the nicotinic iacetylcholine receptor(s) involved in nicotine-induced dopamine release and to characterize the molecular mechanisms that regulate this nicotinic effect. Our hypothesis is that nicotine plays a direct role in some of these effects by regulating dopamine release in selected brain areas and that this release is accomplished by a specific nicotinic acetylcholine receptor (nAChR) subtype(s) located presynaptically on doparninergic neurons. Therefore, Our specific aims are 1) to determine the distribution and levels of expression of nAChRs and dopamine transporters in mesolimbic, nigrostriatal, and mesocortical target areas, 2) to determine the co-expression of nAChR subunits upon dopaminergic nerve terminals at the ultrastructural level, and 3) to determine the mechanism of nicotine-induced responses of the dopaminergic system to short- and long-term presence of nicotine. All of these studies will be performed in control (sham) animals and in an animal model of schizophrenia, the neonatal ventral hippocampal lesion (NVHL) model of schizophrenia.
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{
"pile_set_name": "NIH ExPorter"
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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Spontaneous diabetes mellitus has been reported in several nonhuman primate species, but has not been documented for the mangabey. The Yerkes National Primate Research Center has a colony of mangabeys with a high prevalence of diabetes. A review of pancreatic tissues, collected at necropsy from 1969-2008, revealed a greater than 50% incidence of insular amyloidosis, a hallmark of diabetes in nonhuman primates, in animals over ten years of age. We believe the screening of our colony of over 200 mangabeys for clinical diabetes, as well as, an investigation of the clinical, genetic, and diagnostic manifestation of the disease, would provide important information about diabetes mellitus and its pathogenesis in the sooty mangabey. To date, we have collected samples from approximately 185 mangabeys to screen for diabetes as well as 100 animals for fructosamine levels, 28 animals for glycosylated hemoglobin and 60 samples to evaluate insulin levels. Initial anaylsis of these samples confirms an approximately 15% incidence of diabetes and / or a potential pre-diabetic state within the colony. In addition, characterization of normal serum chemistry and complete blood count reference ranges for mangabeys will aid in clinical evaluation of diabetes and other diseases affecting the population. We have collected over 100 CBC and serum chemistry samples from mangabeys to build a database of normal values. These samples demonstrate some values for mangabeys that are different from the normal chemistry values of other species of non-human primates. Finally, identifying which is the best clinical test for diabetes melltus in the mangabey would allow for early diagnosis and clinical management of the disease in our colony. Initial analysis of this information reveals that fructosamine levels appear to be a more reliable indicator than glycosylated hemoglobin as a diagnostic tool for diabetes in the sooty mangabey.
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{
"pile_set_name": "NIH ExPorter"
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Approximately 135,000 new cases of non-small cell lung cancer (NSCLC) are diagnosed each year in the USA, yet only 20%-25% of those patients will be diagnosed at a sufficiently early stage to be cured by surgical or other procedures. When used in concert with radiographic imaging, a sensitive and specific blood test for NSCLC could be a clinically valuable and cost-effective tool for early diagnosis. The immune system responds to tumor-associated protein by secreting autoantibodies long before metastasis occurs. Autoantibody profiling for this important group of circulating tumor markers in peripheral blood has lead us to the identification of appropriate target peptides. We have developed a panel of 21 short peptides which were formatted as a protein microarray for use in a simple capture immunoassay. When probed with a blood sample the array can identify the presence of tumor-associated antibodies in NSCLC but not in normals with both sensitivity and a specificity exceeding 90% at early and later stages of the disease. As used to measure tumor-associated antibodies in serum from a cohort of cancer patients and risk-matched controls, this test affords predictive accuracy that exceeds that of currently available circulating NSCLC- associated protein markers. The overall purpose of this application is to prepare microarrays of a panel of peptide targets and to demonstrate that they enable measurement of autoantibody profiles that can distinguish cancer from normal blood samples with unprecedented sensitivity and specificity. Microarrays of the purified peptides will be used for rigorous optimization and standardization of all technical and clinical performance measures for the multi-target immunoassay. Our initial clinical application will be early detection of lung cancer, although multiple applications in lung cancer management are rational. Our data shows feasibility and proof of concept that supports the rationale for further development and testing of this approach. A subsequent Phase II application will lead to use of an appropriate platform for definitive marker validation trials for application to clinical screening of NSCLC. Thus the primary goal of this application is to develop a novel blood test for NSCLC that can be rapidly translated into clinical practice. A blood test for early detection of lung cancer could provide a cost-effective screening approach, and in concert with CT scanning would enable a viable strategy for reducing the severe mortality rate of this disease. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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Project Summary Ablation of germ cells (GCs) extends lifespan in various species, providing paradigms for tradeoffs between reproduction and longevity, modulation of aging by signaling between tissues, and responses to metabolic perturbation. We recently identified a critical mechanism in this pathway in C. elegans: a response to fat overload that occurs when lipids that were produced for reproduction are not consumed. In the digestive system counterpart (intestine), this lipid accumulates and induces nuclear localization of the transcription factor SKN-1, the Nrf protein ortholog. SKN-1 activates lipid metabolism genes, reduces fat levels, and increases lifespan. SKN-1 and Nrf proteins are well known to respond to stress from reactive oxygen species (ROS), but GC ablation activates SKN-1 through a different pathway that is largely uncharacterized. This novel pathway involves fatty acid (FA)-dependent signals, specific lipid transport and metabolism activities, the gasotransmitter H2S, and 150 genes we have identified by RNA intereference (RNAi) screening. It is possible that this pathway may be triggered by particular FAs, or by bioactive lipid mediators (LMs) derived from them. In C. elegans we have collaboratively detected low-abundance LMs that promote or resolve inflammation in humans. GC ablation appears to alter their profiles, suggesting an ?inflammatory?-like response. Our findings provide a new genetically tractable platform for (1) uncovering how organisms respond to lipid overload, and (2) elucidating how specific lipid-based signals regulate gene expression and possibly aging. They also may represent a paradigm for understanding how mammalian Nrf proteins protect against fatty liver disease. This project will address a number of exciting questions. Aim 1 will elucidate lipid-dependent effects of GC ablation. Analysis of tissue-specific gene expression in the intestine and studies of particular SKN-1 isoforms will reveal FA-dependent effects that are mediated by SKN-1, and other regulators. Studies of FA profiling and ?-oxidation will reveal whether SKN-1 mediates effects that correlate with these gene expression changes. Our LM detection effort will identify candidate LMs that are modulated by GC ablation, and will enable study of these important regulators in this genetically tractable organism. Aim 2 will reveal how GC ablation and lipid signals activate SKN-1 and possibly other regulators. A combination of FA biosynthesis enzyme knockdown with administration of specific FAs and LMs should identify specific lipids that mediate these effects. Completion of our RNAi screening will suggest additional mechanisms through which GC ablation activates SKN-1. Finally, in the centerpiece of this project, epistasis and model-driven experiments will place H2S and lipid signals within this new pathway, and will elucidate the involvement of candidate regulatory mechanisms that our results have identified. Completion of these aims will provide new insights into lipid-based gene regulation, control of SKN-1/Nrf, and longevity assurance, thereby having major impacts in the fields of aging, stress responses, lipid metabolism, and lipid-based signaling.
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{
"pile_set_name": "NIH ExPorter"
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The mission of the Clinical and Molecular Immunology Group within the Clinical Immunology Section is to perform cellular, molecular and clinical studies aimed at understanding the basis of immune tolerance. A long-term objective is to develop new therapies for allograft transplantation and the treatment of autoimmune and inflammatory diseases. I. CD28 Signaling Signaling through the CD28 receptor during T cell activation exerts a profound influence on the outcome of T cell receptor (TCR) engagement. Failure to receive a costimulatory signal through CD28 results in an unresponsive state termed anergy or in T cell death; both of which contribute to the induction and maintenance of immune tolerance. Thus CD28 signaling is a critical determinant of T cell activation and is an important factor in the development of both auto- and allo- immunity. While signal transduction through the T cell receptor has been extensively characterized, the CD28 signaling pathway is poorly understood. To decipher this pathway, we've utilized a mouse model system in which CD28 signaling is responsible for greater than 99% of T cell IL-2 production. Earlier work had shown that this CD28 dependent regulation of IL-2 expression is not at the transcriptional or translational levels but rather a consequence of increased IL-2 mRNA stability. Our earlier work using sequence tagged genomic IL-2 reporter constructs demonstrated that sequences within the 3' untranslated region of the mouse IL-2 mRNA are responsible for mRNA instability but cannot confer CD28 responsiveness upon a heterologous reporter mRNA. In addition, we discovered the presence of an additional mRNA instability element located within exon 3 and that sequences within exon 2 and the coding region of exon 4 are required for CD28 mediated IL-2 mRNA stabilization. Within these same regions of the IL-2 mRNA we have identified a sequence motif that may be critical for CD28 responsiveness. To test the role of this putative CD28 response element (RE) nearly a dozen mutants have been generated and stable cell lines carrying these mutations have been established. Unexpectedly, analyses of these constructs indicate that CD28 mediated stabilization of the IL-2 mRNA in the cytoplasm appears to be coupled to splicing of the pre-mRNA in the nucleus. While this finding has complicated our studies on the role of the exonic CD28REs, we are actively pursuing this novel finding to determine how and why these two processes are coupled. The results of these studies will assist us in the identification of RNA binding proteins that interact with cytokine mRNAs in a CD28 specific fashion, thus providing us with a protein probe to help elucidate the CD28 signal transduction pathway. Biochemical studies carried out to identify proteins that bind the IL-2 mRNA initially focused on the 3'UTR of the mRNA. We identified one such protein to be HuR, the mammalian homolog of the Drosophila ELAV (embryonic lethal abnormal vision) gene. It is widely held that HuR binding stabilizes labile mRNAs such as c-myc and IL-3, however, we found no association between HuR binding to the IL-2 mRNA and its CD28-mediated stabilization. The controversial nature of our observation has prompted us to determine the biological significance of this finding. One hypothesis that is being pursued is that such binding is related to transport of the mRNA from the nucleus to the cytoplasm. This proposal is supported by our observation that HuR appears to translocate from the nucleus to the cytoplasm upon T cell activation. Along these lines we have generated several IL-2 deletions that would be postulated to abolish HuR binding and the phenotype of these mutants is being characterized. In addition to furthering our elucidation of the CD28 costimulatory pathway, these observations may have important consequences for our understanding of how IL-2 gene expression is regulated at both the transcriptional and post-transcriptional levels. The initial results of these studies have been submitted for publication. II. Molecular Consequences of IL-2 Receptor Blockade Laboratory investigations to understand the mechanism(s) by which blockade of CD25, the high affinity IL-2 receptor (a therapeutic modality in transplantation, allergic, and autoimmune disease) inhibits immune activation have completed their initial phase of study. We found that receptor blockade inhibits the expression of multiple cytokines (both Th1 and Th2) implicated in the pathogenesis of autoimmune disease and graft rejection. We have gone on to show that inhibition of IFN-gamma production occurs through both IL-12 dependent and IL-12 independent pathways. Furthermore, inhibition via the IL-12 dependent pathway is a consequence of blocking CD40L expression, which itself is critical for the induction of IL-12 from monocytes. Furthermore, our results reveal for the first time that both IFN-gamma production and CD40L expression are biphasic and that the latter, but not the initial phase of expression, is highly dependent of IL-2R signaling. These findings have important implications for the choice of immunosuppressive regimen (e.g. anti- IL-2R vs. anti- IL-12) employed in the setting of transplantation or autoimmune disease. These observations are being extended to understand at the molecular level the activation pathways involved in CD40L expression. We expected early expression to occur on memory CD4 cells and late expression to be on naive cells. Though CD40L expression on resting cells is restricted to the CD4 memory population, early expression on activated cells occurred proportionately on naive and memory T cells while late expression was predominately on naive cells. Early expression, like late expression, is dependent on cell-cell contact with monocytes, but through different cell surface receptors. In contrast, early expression is independent of cytokines while late expression is wholly dependent on IL-2 and partially dependent on INF-gamma. Furthermore, the late phase can be completely restored in cells that have not been CD28 costimulated by exogenous IL-2. Since others have shown that blocking CD40L alone can induce long-term tolerance in a primate transplant model, it will be of critical importance to understand the pathways controlling the expression of this important determinant of immune tolerance. Recently, a great deal of scientific interest has focused on a subpopulation of CD4 T cells that are characterized in part by expressing CD25 on their surface. These resting cells, in contradistinction to activated CD25+ CD4 T cells, appear in the mouse to play an important role in the homeostasis of the immune system and in establishing tolerance. An analogous population has been defined in humans but their in vivo significance is unknown. We have a patient population that has been treated with a monoclonal antibody against CD25 for over 4 years, but contrary to what might have been predicted from the mouse experiments, these patients are healthy; showing no evidence of immune dysregulation. This patient population presents a unique opportunity to study the functional significance of these CD4/CD25+ regulatory cells in humans. We have begun to do so by further characterizing these cells in normal blood donors in an attempt to better define this population phenotypically. By using 4-color multiparameter flow cytometry, we?ve been able to enrich this population 20-fold. We are currently assessing whether this phenotypic population is also enriched for regulatory cell function.
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Translation termination is the final stage of protein synthesis. It includes at least two essential functions, stop codon recognition and polypeptide chain release. In eukaryotic organisms, the class I release factor eRF1 recognizes each of the three termination codons (UAA, UAG, and UGA) and mediates release of the nascent polypeptide chain. The class II release factor eRF3 assists the termination process in a GTP-dependent manner. The long-term goal of this project is to better understand the process of translation termination so therapeutic strategies aimed at the suppression of disease-causing premature stop mutations can be developed. The eRF1 protein contains three discrete domains. A consideration of structural and genetic data led to the proposal that domain 1 mediates stop codon recognition; domain 2 interacts with the peptidyl transferase center of the ribosome to facilitate polypeptide chain release; and domain 3 mediates the interaction between eRF1 with eRF3. Competing models argue that either the TASNIKS motif or the YCF motif in domain 1 is critical for stop codon recognition. The first aim of this proposal will identify key residues of eRF1 domain 1 involved in stop codon recognition to test the relative merits of these competing models. The yeast SUP45 gene encodes eRF1. We recently discovered that the half-life of the SUP45 mRNA is regulated by the efficiency of the termination process. This mechanism leads to an increase in the eRF1 protein level when termination is compromised. The second aim of this proposal will test this model and explore how this novel regulatory mechanism controls SUP45 mRNA and eRF1 protein levels. We recently found that the previously uncharacterized protein Tpa1p influences the efficiency of translation termination, mRNA poly(A) tail length, and mRNA half-life in yeast cells. This led us to propose a model in which Tpa1p couples translation termination to the deadenylation of cellular mRNAs. The third aim of this proposal will test various aspects of this model so we can better understand the important interplay between translation termination and mRNA stability. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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T cells in the intestinal mucosa face continual antigenic challenge from the contents of the intestine. Therefore, specific mechanisms of immune regulation must have evolved to prevent uncontrolled immune responsiveness in the gut. In fact, there is evidence that loss of proper immune regulation in the intestine leads to inflammatory bowel disease (IBD). Many T cells in the intestinal mucosa of mice express a homodimer form of CD8a chains instead of an W13 heterodimer. Evidence presented in the Preliminary Results section shows that the TL antigen, a class I molecule which is expressed almost exclusively by intestinal epithelial cells in mice, strongly prefers to bind to CD8cw. By contrast, other class I molecules show preference for binding CD8ab. Our data further suggests that the interaction between CD8aa and TL can inhibit immune reactivity. Our overall hypothesis is that the TL-mediated engagement of CD8aa, separately from the TCR-MHC complex, down regulates TCR-mediated signal transduction and thus prevents chronic stimulation. The experiments outlined are designed to explore the TL-CD8acz interaction. In aim I, the functional consequences, in terms of T cell proliferation, cytotoxicity and cytokine release, will be analyzed in vitro using TL positive presenting cells. In aim 2, the consequences of this interaction will be explored in vivo. Several transgenic and gene knock out strains, that differ with regard to their TL-CD8aa interactions, will be analyzed for mucosal T cell number and function. In aim 3, the biochemical basis for preferential binding of the TL antigen to CD8aa will be characterized. In aim 4, the membrane proximal signal transduction events altered by CD8 aa engagement will be analyzed. The experiments in this application therefore will characterize a novel regulatory mechanism for mucosal T cells. As regulation of and by intestinal T cells is crucial in controlling intestinal inflammation and preventing IBD, in the induction of oral tolerance, and in the surveillance for cancers of epithelial cells, the results from these studies could have important ramifications for understanding normal homeostasis and treatment of pathological conditions in the intestinal mucosa. Furthermore, in mice and humans, CD8aa can be expressed by activated T cells and NK cells, and in vitro studies show that engaging CD8aa on systemic T cells can be inhibitory. Therefore, the mechanisms outlined here could have implications for cancer and other diseases that extend beyond the mucosa.
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{
"pile_set_name": "NIH ExPorter"
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[unreadable] Dr. Jane Liebschutz is an academic physician in general internal medicine/primary care with experience studying health effects of interpersonal violence using both qualitative and quantitative methods. She seeks a K23 award to become a leader in the development of treatment approaches for persons with co-occurring substance use disorders (SUD) and post-traumatic stress disorder (PTSD) in the primary care setting. To advance her goals, she will also focus on chronic non-cancer pain, a condition both associated with SUD and PTSD and a common clinical presentation of patients with these conditions in the primary care setting. As part of her K23 award, she will conduct a cross-sectional study of primary care patients with chronic noncancer pain to describe the prevalence of co-occurring SUD and PTSD and treatment needs. (AIM 1) Using qualitative research methods, she will conduct a focus group study of primary care physicians to explore preferences for primary care based interventions for patients with co-occurring SUD, PTSD and chronic pain. (AIM 2) She will utilize information gathered in these two projects in conjunction with established behavioral therapies for patients with SUD and PTSD to develop a multi-disciplinary behavioral intervention for co-occurring SUD, PTSD and chronic pain for the primary care setting. (AIM 3) [unreadable] [unreadable] Career development activities for this award include mentoring by individuals with extensive research experience in addiction, dual diagnosis and PTSD, directed readings, graduate level coursework, immersion in specialty settings (PTSD training center, substance abuse treatment program, pain rehabilitation program), attendance at specialty conferences, and the research itself. Completion of the proposed work will prepare Dr. Liebschutz to independently investigate the treatment of co-occurring substance use disorder and PTSD in the primary care setting. [unreadable] [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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This proposal requests funding for a Global Reseach Training in Population Health (GRTPH) program that links the University of Colorado Population Program and two outstanding research and training groups in sub-Saharan Africa - at the University of the Witwatersrand in Johannesburg, South Africa (Wits) and the African Population and Health Research Center in Nairobi, Kenya (APHRC). These two institutions each run longitudinal health and demographic studies, one rural, the other urban. The program is intended to address needs for trained demographers and population scientists in low- and middle-income countries who can be involved in the measurement, monitoring, and evaluation of central population health issues and who can conduct research that meets international standards on population health, migration, environment, and their interrelationships. It builds on the demonstrated strengths of each unit in carrying out multidisciplinary and interdisciplinary research in these areas and the commitment each has to training. It is based on continuing relationships among institutions, rather than single investigators. Specifically, it builds on the African Population Studies Research and Training Center at CU, a collaborative effort among these three institutions. The program currently consists of (1) a postdoctoral fellow partnership program; (2) intensive visits to CU by African trainees; (3) African student mentoring and involvement in collaborative research; (4) conference on training/curricula for programs based in African institutions; (5) an annual Colloquium in which all three groups participate; (6) small grants for pilot collaborative research; (7) grant preparation and administration assistance; (8) strong continuing institutional ties and senior faculty/ administrator involvement: and (9) ongoing collaborations across institutions emphasizing health, migration, and environment and their interconnections. To this existing program, the GRTPH program would add: (1) Postdoctoral fellowships open to African scholars with industrialized-country Ph.D.'s to return to one of the two partner institutions; (2) Fellowships for graduate students at Wits; (3) Internships for Wits graduate students at APHRC; and (4) Short courses at Wits and APHRC offered by APHRC senior staff and CU staff. This program is especially intended to increase research and training capacity at Wits and APHRC while it increases African research and collaborations at CU. Through continued interactions, training, and collaborative research, this network is well-positioned to contribute in powerful ways to expanding training opportunities of African junior investigators and cultivating current and forging new collaborative research and training relationships, aimed at contributing to fuller and better understanding and, ultimately, to improvement of African population health. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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The Henry Ford Health System is applying for designation as a Developmental MEDTEP Research Center on Minority Populations. The focus of the center will be on the clinical outcomes of Black Americans. The HFHS is a vertically integrated health system supplying care to one half million people in Southeast Michigan. Our system includes 5 hospitals, our own health maintenance organization, a 900 person medical group practice, and an extensive network of 30 ambulatory care centers. Our patient population is representative of the general population of Southeast Michigan, with 28% of all patients being Black and a much larger percentage 80% within the City of Detroit service area. For its research activities, the HFHS has the organizational resources, the computer data systems and the scientific personnel and experience to contribute to the understanding of differences in health outcomes of Black Americans. Our training activities will expand on our substantial commitments to Black physicians within HFHS as well as provide a range of opportunities for researchers outside of HFHS to learn and perform research on clinical outcomes. We will provide technical support in the areas of research design and analysis as well as support for outcomes methodology, data base analysis and grant writing. Our dissemination efforts will build on our record of national conferences on urban health issues and of outcomes management. Finally, through collaboration with the Agency for Health Carte Policy and Research and our own efforts we will become a full center by the end of the three years of our developmental phase.
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{
"pile_set_name": "NIH ExPorter"
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There is now compelling evidence that regulatory T cells (Tregs) suppress immune activation and are perturbed during HIV infection. Exploiting the mechanisms of Treg function is relevant for controlling chronic immune activation during HIV infection or conversely in boosting HIV-specific immune responses. However, several key questions and gaps remain in our understanding of Treg cell biology, especially in the context of HIV pathogenesis. The analysis of Tregs in HIV patients is complicated due to lack of specific markers. It is also still not clear whether Treg cell loss contributes to inflammation during HIV infection or whether they are detrimental by suppressing HIV-specific immune responses. During the previous grant period we made major advances in understanding Treg cell differentiation and established methods to expand them in vitro. We discovered a cell surface molecule, called GARP, highly specifically expressed on bona fide activated Tregs. We also found that receptors for proinflammatory cytokines IL-1 and TNF and their respective decoys are preferentially expressed on human Treg cells. Additionally, we developed a novel technology to expand and genetically engineer Tregs with HIV-specific T cell receptors (TCRs) to determine the role of TCR signals in their suppressive function. In this proposal we aim to use these innovative tools and technical advances to: (1) Determine association of Tregs with immune activation status of HIV-infected individuals either with or without treatment in a prospective study; (2) Determine the function of Tregs in sensing and suppressing inflammation and whether this function is compromised during HIV infection; (3) Determine the specificity and suppressive role of Tregs in manipulating HIV-specific immune responses; and (4) Develop an experimental framework to decode the role of peptide affinity and concentrations in regulating Treg responses through TCR signaling. Together, these new approaches will have wide range of implications for using bona fide Tregs as biomarkers during HIV infection, defining their role in immune activation and in suppressing HIV-specific immune responses during HIV infection. This knowledge can also be exploited to design more potent HIV vaccines.
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{
"pile_set_name": "NIH ExPorter"
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Abstract: Huntington's disease (HD) is a fatal neurodegenerative disease for which no therapy has been proven to delay onset or to slow progression of the disease. The development of therapies is challenged by lack of clear understanding of disease mechanisms and limited availability of biomarkers which could be used to monitor the effectiveness of therapies. MicroRNAs (miRNAs) are short non-coding RNA molecules which are appealing for biomarker discovery and development because of their relative abundance and remarkable stability in biofluids. We hypothesize that, miRNA markers associated with neurodegeneration in HD brain can be detected in the CSF and plasma of HD patients and have preliminary data demonstrating disease-related miRNA signatures in both biofluids. Here we propose examining the miRNA signatures present in concurrent CSF and plasma samples from HD patients and comparing them with each other and with miRNA changes in postmortem HD brain. Our goal is to understand the inter-relationships of the plasma and CSF miRNome in HD patients and the overlap of these with brain miRNA. We hope to identify candidate plasma and CSF markers that could correlate with neurodegeneration in brain.
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{
"pile_set_name": "NIH ExPorter"
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Cathinone and methcathinone are the -ketone analogs of amphetamine and methamphetamine, respectively, and like their amphetamine analogs, cathinone and methcathinone function as monoamine releasers that selectively promote the release of dopamine (DA) and norepinephrine (NE) over serotonin (5-HT). Cathinone and methcathinone produce amphetamine-like, psychostimulant effects and are classified as Schedule I drugs of abuse by the United States Drug Enforcement Agency (DEA). Recently, synthetic cathinone analogs have emerged as designer drugs of abuse in Europe and the United States and have been marketed under deceptively benign names like bath salts in an attempt to evade legal restriction. These dangerous, recently emergent and novel drugs of abuse display varying selectivity to promote release of DA/NE vs. 5-HT, and selectivity for DA neurotransmission is believed to correlate with abuse liability. This F30 application proposes preclinical research to examine the structural determinants of abuse-related behavioral and neurochemical effects produced by synthetic cathinone bath salts. To evaluate structural determinants of the abuse-related stimulant effects of cathinone derivatives, this application will focus on one feature of the methcathinone scaffold: the para substituent of the benzene ring. A series of six novel methcathinone derivatives will be examined in order to evaluate how physicochemical parameters (steric volume, lipophilicity and electron-donating/withdrawing capacity) of the para substituent of methcathinone derivatives influence in vitro monoamine transporter selectivity as well as in vivo neurochemical and behavioral effects. We hypothesize that electronic features of the para substituent will correlate with neurochemical selectivity to promote DA vs. 5-HT release as determined by in vivo microdialysis and behavioral selectivity to produce abuse-related effects as determined by intracranial self-stimulation (ICSS). Completion of this project will provide an improved understanding of the mechanism of synthetic cathinone analogs' abuse-related effects, allowing for development of novel strategies for treating bath salts intoxication and improvement of clinical outcomes associated with bath salts abuse.
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{
"pile_set_name": "NIH ExPorter"
}
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In contrast to progress for autism, bipolar disorder, and schizophrenia, discerning the biological basis of major depressive disorder (MDD) has been difficult. Genetic studies of MDD using genome-wide linkage, candidate gene, and GWAS (with Ns > 20,000 subjects) have not been successful in identifying risk loci that meet contemporary standards for replication. Major lessons are that genetic approaches for higher prevalence/lower heritability diseases like MDD may be suboptimal given the likely role of etiological heterogeneity. We propose here an alternative strategy to identify networks and pathways involved in the etiology of MDD. To minimize heterogeneity, we will study postpartum depression (PPD), a more homogenous type of MDD. Although most women have mild mood symptoms in the first few days to weeks postpartum (the baby blues), these symptoms usually resolve spontaneously. In contrast, PPD is a severe and persistent form of MDD that is one of the most frequent complications of childbirth (prevalence 15-20%) and is the leading cause of maternal death. Practically, pregnant women are straight-forward to identify, as they have frequent contact with the healthcare system and are willing to participate in research on perinatal problems. The study of PPD should decrease heterogeneity in multiple ways (females only, age-banded, all subjects exposed to the same biopsychosocial event). Moreover, we have moved to the rigorous study of the biomarker space to identify the distinguishing features of PPD of potential clinical relevance. DNA methylation markers are appealing because methylation is directly related to gene expression. RNA expression signatures add complementary data on the state of a tissue and even of an organism. Finally, the comprehensive study of neuroendocrine hormones is of clear salience for PPD. We propose: (1) to ascertain and sample 1,000 PPD cases and 1,000 euthymic controls, all self-reported African-American; (2) conduct discovery analyses using an unbiased/screening biomarker assessment for 500 PPD cases and 500 controls (methylomics and transcriptomics via next-generation technologies on peripheral blood), and state-of-the-art statistical analyses will identify multivariate biomarker signatures for PPD; and (3) conduct validation assays of biomarkers meeting liberal significance criteria in independent samples (500 PPD cases and 500 controls). Our focus on African American women is optimal as this US minority group is manifestly under-studied, the prevalence of PPD is higher in African-American women, and in contrast to genetic studies where ancestry heterogeneity is a major source of false positive signals, it can be a strength in biomarker studies. MDD is a first-rank public health problem due to the associated morbidity, mortality, and personal/societal costs. Studying the MDD sub-form PPD is particularly appealing due to the inherent control for multiple sources of heterogeneity - we propose to study women of child-bearing years who are all exposed to the major risk factor of pregnancy. Moreover, PPD is itself an important and under-studied human health concern, particularly in African-American women. Successful completion of the proposed research will yield strong and replicable biomarker signatures for PPD. This new knowledge would provide insight into state-related alterations characteristic of PPD and potentially to trait-related vulnerabilities to MDD (particularly in combination with GWAS findings). Future research could evaluate whether a biomarker signature is of predictive utility and, if so, rational primary prevention may become feasible. PUBLIC HEALTH RELEVANCE: Postpartum depression causes enormous human suffering and cost to society. Our goal is rapidly to learn more about the biological basis of postpartum depression by studying the blood of African-American women with and without postpartum depression.
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{
"pile_set_name": "NIH ExPorter"
}
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Altered Genes Expression In Rat Mammary And Mouse Lung Models: Comparison With Altered Expression In Humans And Modulation of Gene Expression by Known Chemopreventive Agents. Substantial genetic instability, as reflected in LOH or aneuploidy, appear to be a hallmark of most human cancers and even many preinvasive lesions, e.g. DCIS in breast, dysplastic lesions in lung, colon adenomas, CIN 2 and CIN 3 in cervix etc. In contrast a more limited number of oncogenes or tumor suppressor genes [P53, Ki Ras, APC, Rb etc] have been fully characterized for possible mutations and most cancers are characterized for a limited number of mutations in these same genes. The objective of this study is to use methodologies which allow the simultaneous examination of altered RNA levels in hundreds of genes simultaneously and which additionally allows one to clone and perform partial sequencing of the cDNAs coding for these genes in a relatively easy manner. The rat mammary tumor model is being used to readily determine whether effective chemopreventive agents alter levels of the presumptive RNAs identified in the screening mode. 1) Extensive screening of MNU rat mammary tumors for altered expression of various RNAs 2) Compare using a more limited subset of RNAs (40-50) altered expression of these genes in rat mammary tumors induced by MNU, DMBA and tumors induced by radiation plus estradiol 3) Determine again employing a limited number of RNAs whether one sees a substantially different pattern in MNU induced tumors bearing a mutation in the HaRas oncogeny versus those tumors not bearing a mutation in the HaRas oncogene 4) Determine employing a subset of RNAs whether the expression of these genes is modulated by known chemopreventive agents.
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{
"pile_set_name": "NIH ExPorter"
}
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Human T cell leukemia virus type I (HTLV-I) is an exogenous retrovirus that is the etiologic agent of adult T cell leukemia in man. The overall goal of this study is to map antigenic sites on gp46 and gp21 envelope glycoproteins of HTLV-I that give rise to virus neutralizing antibodies and to evaluate in animals a synthetic vaccine containing these neutralizing sites. This study will also attempt to identify antigenic sites on HTLV-I proteins that are identified by T helper cells with peripheral blood leukocytes of HTLV-I+ patients and to incorporate these T cell sites into a synthetic vaccine. This study will also evaluate the prevalence of HTLV-I infection in individuals with AIDS, in hemophiliacs, and in individuals from sexually-transmitted disease (STD) clinics in North Carolina. Synthetic peptides containing hydrophilic amino acid sequences from HTLV-I gp46 and gp21 envelope proteins will be used to raise antisera in rabbits to viral envelope antigens. Anti-peptide antisera will be evaluated for reactivity to viral envelope and for the ability to inhibit both HTLV-I-induced synctium formation and the infectivity of HTLV-I (VSV) pseudotype particles. Antibodies in HTLV-I+ patient sera will be tested for reactivity to env-encoded synthetic peptides in radioimmunoassay (RIA) to map immunogenic sites in man. Techniques and reagents developed during the first three years of this grant will be used to purify gp46 from lysates of HTLV-I infected cells. Proteolytic fragments of gp46 characterized by amino acid sequence analysis will be used to immunize mice for the production of murine monoclonal antibodies to defined regions of gp46. Purified gp46 and polymers of gp46 will be tested for binding to surface of T cells and for the ability to induce resting peripheral blood T cells to undergo blast transformation. Purified gp46 will also be used with cross-linking agents to identify cell surface receptors for HTLV-I. Synthetic peptides containing sequences of HTLV-I proteins that are predicted to have amphipathic helical structures will be tested for the ability to induce blast tranformation of peripheral blood leukocytes from HTLV=I+ patients. RIA and immunoblot assays will be used to evaluate the prevalence of HTLV- I infection in AIDS patients, in hemophiliacs and in individuals from STD clinics in North Carolina.
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{
"pile_set_name": "NIH ExPorter"
}
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During the first three years of this award, we have characterized mouse models of lung adenocarcinoma initiated by doxycycline-inducible transgenes encoding the two common forms of mutant EGFR found in the corresponding type of human cancer. These mice develop tumors indistinguishable from the human cancers;the tumors regress when the oncogene is de-induced or when the mutant kinase is inhibited with drugs used in patients, and they develop drug resistance under certain conditions, in some cases as a result of a secondary mutation in EGFR also found in about half of drug-resistant tumors in patients. We now propose to extend our work with these mouse models in several ways with the goals of (i) evaluating genes and proteins (including tumor suppressor genes, members of the EGFR family, and phosphotyrosine-containing proteins) that might influence EGFR-initiated lung tumorigenesis;(ii) discovering additional genes that contribute to tumor formation, progression or drug resistance;(iii) assessing the potential of tumors with various genotypes to metastasize and become established cell lines in culture;and (iv) studying the origins and functions of inflammatory cells observed in these mouse tumors. To pursue these goals, we will take advantage of several conditional mouse mutants, results from our own proteomics surveys, the Sleeping Beauty transposition system, and methods for characterization of immune cells. We expect our findings to offer new insights into EGFR-mediated lung carcinogenesis, tumor progression, secondary drug resistance, and the role of immune cells in solid tumors. In addition, we may generate new mouse tumor cell lines for experimental use and identify genes that are useful for diagnosis, classification, and treatment of lung cancers. PUBLIC HEALTH RELEVANCE: Lung cancer is the major cause of death from cancer in the United States and the world, and adenocarcinoma is the most common form of lung cancer. Our laboratory is among those who found that about ten percent of patients in the US with lung adenocarcinomas respond to a new set of drugs--- drugs that inhibit a type of enzyme called a protein-tyrosine kinase---because their cancers are caused by one of two characteristic mutations affecting a growth factor receptor called EGFR. Unfortunately, the beneficial response to these drugs is short-lived;in about half the cases, the acquired drug resistance is due to a second mutation in the EGFR gene that we discovered a few years ago. To study this type of lung cancer more thoroughly than is possible in human patients, we have manipulated genes in mice so that the animals develop lung cancers that are very similar to human lung cancers with EGFR mutations;as we have shown during the first phase of this grant, the cancers respond to the same drugs, develop similar drug resistance, and disappear if the mutant gene is turned off. We now propose to use these powerful models to study a number of related aspects of lung cancer. First, we will look at the roles that might be played by other genes when mutant EGFR causes lung cancer. These other genes include well-known tumor suppressor genes and genes identified through studies we have done to characterize proteins that are modified by EGFR or related enzymes in lung cancer cells. In the course of this work, we will also ask what is required to make lung cancer cells metastasize to other sites and attempt to develop mouse lung cancer cell lines from the metastases. To seek novel genes that might contribute to accelerated growth of a lung cancer or to the development of drug resistance, we will make use of a moveable DNA unit that can identify such genes. Finally, we will explore the striking appearance of immune cells in mouse lung tumors, in an effort to understand how such cells arrived in the lung tissue and what functions they serve to promote or retard tumor development.
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{
"pile_set_name": "NIH ExPorter"
}
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Monocyte-derived macrophages are critically involved in the initiation and progression of the atherosclerotic plaque in man as well as in experimental models. Plaque rupture is thought to be the trigger event for acute coronary syndromes in man, and several mouse, models of plaque rupture have been described. Macrophages have been implicated in plaque rupture by releasing de-stabilizing proteases. Macrophage apoptosis is prominent in advanced atherosclerotic lesions, but it is controversial whether macrophage apoptosis is beneficial or detrimental. In this proposal we will examine the mechanisms and consequences of macrophage apopotosis in experimental atherogenesis. We propose that macrophage accumulation and activation in the intima contributes to smooth muscle cell death and thereby promotes plaque rupture. Therefore, we will test the primary hypothesis that macrophage apoptosis reduces intimal lesion progression and ultimately contributes to plaque stabilization. As secondary hypotheses, we also propose that Fasmediated signaling plays an important role in macrophage apoptosis and activation in atherogenesis. In Aim 1, we will identify components of the Fas signaling complex regulating activation versus apoptosis in macrophages in vitro, using isotope-coded affinity tag and tandem affinity purification with mass spectrometry. In Aim 2, we will determine the role of apoptosis proteins in macrophage activation and apoptosis in vitro. We will investigate the effect of the anti-apoptotic proteins, c-FLIP, Bcl-2, dominant-negative FADD, and p35 caspase inhibitor, on macrophage activation or apoptosis in response to Fas ligation or atherogenic stimuli. In Aim 3, we will determine the effect of blockade of macrophage apoptosis on lesion progression and plaque rupture in LDLR-/-and ApoE-/-mice. We will transduce hematopoietic stem cells (HSCs) with a novel retrbviral vector incorporating the macrophage-restricted human CD68 promoter and encoding 1 of the antiapoptotic proteins. We will then determine the effect of transplantation of HSCs transduced with a virus encoding 1 of these anti-apoptotic proteins on early and advanced lesions and plaque rupture in LDLR-/- mice. Positive results with transplantion of transduced HSC in the LDLR-/-model will be extended to lesion initiation/progression and plaque rupture in the ApoE-/-model by generating transgenic ApoE-/- mice overexpressing Bcl-2 selectively in macrophages.
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{
"pile_set_name": "NIH ExPorter"
}
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The overall aim is to study the role of endogenous growth-promoting peptides, particularly EGF and transforming growth factors (TGF), in mouse embryonal development. TGF-like peptides have been proposed as normal gene products during embryogenesis that are inappropriately expressed in tumor cells. EGF receptors appear as early as 6 1/2 days after fertilization on trophoblast cells; later in gestation nearly all fetal tissues gain the ability to bind EGF. In vitro responses of cells growing on plastic appear to be different from in vivo responses. Cell geometry, cell interactions and synergisms between stimulating factors may account for the difference. The role of EGF and similar factors will be examined in mouse fetal liver in several different culture systems employing various cell geometries. Responses to EGF and other growth factors including TGF are compared by analyses of growth and production of tissue-specific proteins. The placenta is a rich source of identified factors that support the fetus, as well as of TGFs. Several oncogenes are expressed in the murine and human placenta and other extraembryonal tissues in a stage-specific manner. The role of the oncogenes, c-fos and c-fms will be studied by making antibodies to synthetic peptides, by in situ hybridization and by in vitro translation of mRNA extracted from placenta. The hypothesis is that oncogene protein products will have a role in growth control or in differentiation/development of fetal or extraembryonal tissues.
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{
"pile_set_name": "NIH ExPorter"
}
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Despite the availability of highly active antiretroviral therapy (HAART or ART), opportunistic infections (OIs) remain the leading cause of considerable morbidity and mortality in HIV infected persons as stated by the CDC in MMWR, April 10, 2009. This is particularly important with the leading OI, pneumonia (PCP), caused by Pneumocystis jirovecii. Trimethoprim/sulfamethoxazole (TMP/SMX), the combination of a dihydrofolate reductase (DHFR) inhibitor (TMP) and a dihydropteroate synthase (DHPS) inhibitor (SMX) for PCP is the first- line agent. The failure of this option due to adverse reactios and resistance to the sulfa drug as well as TMP along with the failure rate and adverse reactions of second-line agents necessitates the urgent need for alternate agents. Our group has isolated and characterized the elusive DHFR from the human pathogen Pneumocystis jirovecii (pjDHFR) and shown it to be distinct and different from its surrogate Pneumocystis carinii (pcDHFR) (occurs in rodents), with respect to inhibitory activities of therapeutic agents. n addition, we have identified two series of compounds (1, 3, 5 and 6) with selectivity for pjDHFR over hDHFR of 19-99-fold with picomolar and nanomolar Ki and IC50 values for pjDHFR. To our knowledge, we are the only group with access to pjDHFR and compounds that have both high selectivity (19-99-fold) and potency (picomolar and nanomolar) against pjDHFR (compared to hDHFR). Recently we have also cloned and expressed clinically relevant, TMP-resistant double mutants of pjDHFR and found that our compounds retain nanomolar inhibition against the double mutant pjDHFR in the face of TMP's 500-fold resistance, and will also use these in our evaluations. This is a paradigm changing event in the evaluation of drugs for P. jirovecii infection that up until now has utilized the surrogate P. carinii for DHFR as well as in vivo models. The Specific Aims are to: 1) synthesize proposed compounds in Series I-XII; 2) evaluate the compounds as inhibitors of hDHFR and wild type and resistant mutant pjDHFR; 3) evaluate selected analogs from Aim 2 in human embryonic lung fibroblasts for toxicity; 4) evaluate selected analogs (5) from Aims 2 and 3 for serum binding, metabolic stability and pharmacokinetics and develop and evaluate in a mouse model of P. jirovecii infection. X-ray crystal structure determination of 1, 3 and 5 and selected compounds from Series I-XII with pjDHFR and hDHFR will be done by Dr. Cody to afford a molecular understanding of the selectivity and potency of the analogs. This study will determine the structural requirements for potent and selective inhibition of pjDHFR and will assist in future drug design and pharmacophore generation. In addition it will provide, for the first time, the evaluation of compounds in a P. jirovecii animal model based on DHFR from the human pathogen (P. jirovecii) rather than a surrogate (P. carinii). The study should identify potential compounds for clinical evaluation against PCP and resistant PCP to be used alone or in combination.
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{
"pile_set_name": "NIH ExPorter"
}
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Recent advances in gene therapy of solid cancers have focused attention on the "bystander-effect" in which sensitivity to a therapeutic agent is transferred from genetically sensitive to insensitive cancer cells within a mixed, heterogeneous cancer. Our laboratory has been studying the mechanisms by which clonal populations of tumor cells interact with each other to confer sensitivity to chemotherapeutic agents. Using a mouse mammary cancer model, we have demonstrated significant bystander effects with drugs as pharmacologically diverse as methotrexate, melphalan, thioguanine and cyclophosphamide. We have also demonstrated a diversity of bystander mechanisms, including transfer of drug metabolites via gap junctions, transfer of sensitivity by soluble mediators, cell-cell interactions that alter proliferation rate (hence, sensitivity to chemotherapy) and interactions that utilize host responses. We have prepared genetically engineered mammary tumor cells carrying HSV-TK gene which are sensitive to ganciclovir ((GCV) and have shown that they exhibit a bystander effect in vivo. This proposal is designed to test the hypothesis that (1) similar interactions between genetically engineered and wild type tumor populations are responsible for bystander effects and contribute significantly to chemotherapeutic response and (2) these interactions and their mechanisms are dependent upon tumor stage, site and specific drug regimen. Our aims are:(1) to introduce the GCV-sensitivity conferring, herpes simplex kinase (HSV-TK) gene into others in a series of sister subpopulation mouse mammary tumor cell lines that (a) exhibit bystander effects that work by different mechanisms, (b) differ in immunological and metastatic phenotypes, and (c) differ in intrinsic sensitivity to conventional drugs; (2) to test the mechanisms by which sensitivity to GCV is transmitted to bystander cells, in vivo and in vitro and (3) to develop a strategy for combination chemotherapy of primary tumors and of metastatic disease based on utilization of different bystander mechanisms.
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{
"pile_set_name": "NIH ExPorter"
}
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The project deals with three main areas: A) Transumbilical portal vein catheterization will be performed on subjects requiring surgical exploration. The peripheral and portal vein glucagon response to glucose and arginine in diabetic and non-diabetic subjects will be compared. B) Certain aspects of the control mechanism of glucagon secretion will be examined during this grant period. The effect of lipids on glucagon secretion will be accessed as well as an attempt to characterize the adrenergic control mechanism involved in glucagon secretion. C) A perfusion apparatus has been set up to study dynamic aspects of insulin and glucagon secretion.
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{
"pile_set_name": "NIH ExPorter"
}
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The project involves the determination of the crystal structures of metal chelates which are of relevance to the treatment of metal poisoning. The metals being studied include those which are of known high toxicity and with which people come into routine contact. The chelating agents involved in the project are cysteine and its derivatives (including Penicillamine), BAL, and derivatives of EDTA, which are currently used in the treatment of individuals who have been subjected to metal poisoning. The purpose of these structural studies is to determine which ligand sites are involved in the binding of the chelates to specific metals, and to see which bonds are in need of strengthening. This information will enable us to propose chemical modification of the ligands for application in the treatment of exposure to individual metals, and eventually should produce one or more effective antidotes for each of the metals under study.
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{
"pile_set_name": "NIH ExPorter"
}
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The microsomal metabolism of m-AMSA (NSC-141549) was studied and the products identified. The principal biliary metabolite of m-AMSA was isolated from rat bile and identified as m-AMSA-5'-glutathione. The distribution of AZQ (NSC-182986) and m-AMSA in monkey CSF was studied. Disposition studies in laboratory animals were carried out for Dihydroxyanthracenedione (NSC-279836) and Desmethyl-misonidazole (NSC-261036). The clinical pharmacokinetics of Misonidazole (NSC-261037) was studied in conjunction with the Phase I clinical evaluation of this agent. Development of analytical methodology for quantitating the radioprotector agent WR-2721 has begun in preparation for clinical pharmacokinetic studies. Specially formulated heat-labile liposomes with encapsulated Methotrexate were used to enhance the local accumulation of Methotrexate in tumors by combination with local tumor hyperthermia.
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{
"pile_set_name": "NIH ExPorter"
}
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Numerous studies in laboratory animals have demonstrated the importance of respiratory epithelial cells as targets for both inhaled and ingested chemicals. There are, however, significant uncertainties in estimating the risks to humans from exposure to chemicals which are respiratory cytotoxicants in animals. These uncertainties are both species- and dose- related. The overall goals of the work proposed in this application are to develop markers of exposure, effect and susceptibility for chemicals which produce lung toxicity in animals. This work is based on the premise that the development of markers capable of signalling that an exposure has been at a level sufficient to result in cytotoxicity is dependent upon a complete understanding of essential biochemical and metabolic steps involved in toxicity in animals. Accordingly, this work will define the importance of specific protein and nonprotein targets of electrophilic intermediates to cytotoxic injury for three chemicals that produce focal injury to the respiratory epithelium by virtue of cytochrome P450 dependent metabolism. These chemicals are naphthalene, nitronaphthalene and trichloroethylene. The well established difference in sensitivity of various species and the highly focal nature of the injury within the respiratory system will be used as an experimental tool to indicate which protein adducts are important to toxicity. This will then guide the development of biomarkers which are closely related to the mechanism of toxicity. The specific aims of the work are to: (l) validate immunochemical methodology for determination of diastereomeric mercapturic acids of naphthalene as an index of the rate and stereochemistry of formation of naphthalene epoxides, (2) identify critical protein targets for reactive metabolites generated from naphthalene, nitronaphthalene, and trichloroethylene, (3) use the information obtained in (2) to develop markers which can be monitored in nasal lavage, bronchiolar lavage, blood or urine as indices of exposure, effect and susceptibility and (4) refine new systems capable of evaluating samples obtained from hazardous waste sites for the possible presence of pulmonary toxic chemicals. This work is intended to reduce the uncertainty inherent in current assessments of risks associated with human exposure to pulmonary toxic chemicals by developing methodologies that are capable of distinguishing exposures that occur at levels sufficient to cause toxicity from those that are without significant effect. These studies should also provide the tools necessary for determining whether the metabolic and biochemical factors which lead to toxicity in animals are operative in human lungs and whether there are significant interindividual differences in these metabolic and biochemical pathways with human populations.
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{
"pile_set_name": "NIH ExPorter"
}
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The accomplishments from the two main projects of the section are: 1) Structure and function of the salivary protein LJM11, a protein from the sand fly Lutzomyia longipalpis that confers protective immunity against Leishmania major infection. LJM11, an abundant salivary protein from the sand fly Lutzomyia longipalpis, belongs to the insect yellow family of proteins. We demonstrated that DNA immunization with LJM11 confers protective immunity against Leishmania major infection. This protection correlates with a strong induction of a delayed-type hypersensitivity (DTH) response following exposure to Lu. longipalpis saliva. Additionally, splenocytes of exposed mice produce IFN- upon stimulation with LJM11 demonstrating the systemic induction of Th1 immunity by this protein. In contrast to LJM11, LJM111, another yellow protein from Lu. longipalpis saliva, does not produce a DTH response in these mice, suggesting that structural or functional features specific to LJM11 are important for the induction of a robust DTH response. To examine these features, we used calorimetric analysis to probe a possible ligand binding function for the salivary yellow proteins. Both LJM11 and LJM111 acted as high affinity binders of prohemostatic and proinflammatory biogenic amines, particularly serotonin, catecholamines and histamine. We also determined the crystal structure of LJM11, revealing a six-bladed -propeller fold with a single ligand binding pocket located in the central part of the propeller structure on one face of the molecule. A hypothetical model of LJM11 suggests a positive electrostatic potential on the face containing entry to the ligand binding pocket while LJM111 is negative to neutral over its entire surface. This may be the reason for differences in antigenicity between the two proteins. 2) Successful use in field conditions of two sand fly salivary recombinant proteins (LJM17 and LJM11) as markers of sand fly exposure. We have previously shown that two sand fly salivary proteins (LJM17 and LJM11) induce the production of anti-saliva antibodies, which can then be used as markers for insect (vector) biting or exposure. Epidemiological studies using sand fly salivary gland sonicate as antigens are hampered by the difficulty of obtaining large amounts of salivary glands. We investigated the use of the salivary recombinant proteins LJM17 and LJM11 as an alternative method for screening of exposure to the sand fly. We primarily tested the suitability of using the recombinant proteins to estimate positive anti-saliva ELISA test in small sets of serum samples. Further, we validated the assay in a large sample of 1,077 individuals from an epidemiological survey in a second area endemic for visceral leishmaniasis. Our findings indicate that these proteins represent a promising epidemiological tool that can aid in implementing control measures against leishmaniasis, a vector-borne neglected disease. 3) Seasonality and incrimination of Phlebotomus duboscqi as the vector of Leishmania major infection in two neighboring villages in central Mali. Phlebotomus duboscqi is the principle vector of Leishmania major, the causative agent of cutaneous leishmaniasis (CL), in West Africa and is the suspected vector in Mali. Although found throughout the country the seasonality and infection prevalence of P. duboscqi has not been established in Mali. We conducted a three year study in two neighboring villages, Kemena and Sougoula, in Central Mali, an area with a leishmanin skin test positivity of up to 45%. During the first year, we evaluated the overall diversity of sand flies. Of 18,595 flies collected, 12,952 (69%) belonged to 12 species of Sergentomyia and 5,643 (31%) to two species of the genus Phlebotomus, P. duboscqi and P. rodhaini. Of those, P. duboscqi was the most abundant, representing 99% of the collected Phlebotomus species. P. duboscqi was the primary sand fly collected inside dwellings, mostly by resting site collection. The seasonality and infection prevalence of P. duboscqi was monitored over two consecutive years. P. dubsocqi were collected throughout the year. Using a quasi-Poisson model we observed a significant annual (year 1 to year 2), seasonal (monthly) and village effect (Kemena versus Sougoula) on the number of collected P. duboscqi. The significant seasonal effect of the quasi-Poisson model reflects two seasonal collection peaks in May-July and October-November. The infection status of pooled P. duboscqi females was determined by PCR. The infection prevalence of pooled females, estimated using the maximum likelihood estimate of prevalence, was 2.7% in Kemena and Sougoula. Based on the PCR product size, L. major was identified as the only species found in flies from the two villages. This was confirmed by sequence alignment of a subset of PCR products from infected flies to known Leishmania species, incriminating P. duboscqi as the vector of CL in Mali. 4) Characterization of midgut proteins from the sand fly Phlebotomus perniciosus, a vector for Leishmania infantum Parasite-vector interactions are fundamental in the transmission of vector-borne diseases such as leishmaniasis. Leishmania development in the vector sand fly is confined to the digestive tract, where sand fly midgut molecules interact with the parasites. We sequenced and analyzed two midgut-specific cDNA libraries from sugar fed and blood fed female Phlebotomus perniciosus and compared the transcript expression profiles. A total of 4111 high quality sequences were obtained from the two libraries and assembled into 370 contigs and 1085 singletons. Molecules with putative roles in blood meal digestion, peritrophic matrix formation, immunity and response to oxidative stress were identified, including proteins that were not previously reported in sand flies. Comparative analysis of the two libraries revealed transcripts differentially expressed in response to blood feeding. Molecules up regulated by blood feeding include a putative peritrophin (PperPer1), two chymotrypsin-like proteins (PperChym1 and PperChym2), a putative trypsin (PperTryp3) and four putative microvillar proteins (PperMVP1, 2, 4 and 5). Additionally, several transcripts were more abundant in the sugar fed midgut, such as two putative trypsins (PperTryp1 and PperTryp2), a chymotrypsin (PperChym3) and a microvillar protein (PperMVP3). We performed a detailed temporal expression profile analysis of the putative trypsin transcripts using qPCR and confirmed the expression of blood-induced and blood-repressed trypsins. Trypsin expression was measured in Leishmania infantum-infected and uninfected sand flies, which identified the L. infantum-induced down regulation of PperTryp3 at 24 hours post-blood meal. This midgut tissue-specific transcriptome provides insight into the molecules expressed in the midgut of P. perniciosus, an important vector of visceral leishmaniasis in the Old World. Through the comparative analysis of the libraries we identified molecules differentially expressed during blood meal digestion. Additionally, this study provides a detailed comparison to transcripts of other sand flies. Moreover, our analysis of putative trypsins demonstrated that L. infantum infection can reduce the transcript abundance of trypsin PperTryp3 in the midgut of P. perniciosus.
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{
"pile_set_name": "NIH ExPorter"
}
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The Clinical Core of the UAB CFAR was established in 1988 to provide a bridge between patients attending the AIDS Outpatient Clinics (1917 Clinic and the Family Clinic) and the basic scientists working in the laboratories of the Center. The Center provides support to investigators through a clinical database, a clinical specimen repository, clinical trials, and ongoing programs in clinical pharmacology. The clinical database maintains quality assured clinical data on all outpatients seen in both the adult and children's HIV Outpatient Clinics. Baseline demographics along with critical clinical information is stored in a centralized computer. Blood specimens are collected on a periodic basis and stored in a central specimen repository. This repository has a computerized record of every specimen collected over the five years of clinical core's existence, yielding easy retrievability of specimens desired for a specific basic science or clinical science study. The specific objectives of the Core are to: (1) establish and maintain effective communication between clinical and basic scientists in order to develop multidisciplinary collaborative projects which study the nature and pathogenesis of HIV disease; (2) provide clinical specimens from well-characterized patients to basic science investigators through the establishment of a clinical specimen repository which is linked to a computerized database; (3) conduct state-of-the-art clinical trials on novel therapeutic agents and regimens for HIV-related disorders; (4)link the clinical trials effort with development of novel markers of anti-retroviral activity, new approaches in investigative pharmacology, and the discovery of new agents through an ongoing drug discovery program and experimental therapeutics program. The Clinical Core has been extremely successful, providing service to 46 CFAR investigators on over 100 specific projects. The Center has conducted 46 clinical trials, focusing on primary infection (369 patients enrolled), opportunistic infections (222 patients enrolled), and nutritional/cytokine studies (66 patients enrolled). These studies have encompassed the full range of trial designs from Phase I early pharmacokinetic studies to Phase III multicentered clinical efficacy trials. Future directions include the establishment of a Vaccine Evaluation Unit, an Outpatient Dental Facility for the Study of HIV-related oral disease, to modify the clinical database to provide a foundation for studies in behavioral science, and to develop a Women's Clinic for studies of HIV- related disease occurring in this population.
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{
"pile_set_name": "NIH ExPorter"
}
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There is little doubt that we are in the midst of a worldwide epidemic of diabetes. Insulin resistance is recognized as a characteristic trait of the disease, defined by the inability to respond to normal circulating levels of insulin, and is usuall closely associated with obesity. Recent data suggest an inflammatory link between obesity and insulin resistance. However, the teleological reasons or these findings, and the manner in which energy storage is preserved in the absence of insulin action remain a mystery. We hypothesize that the induction of a counter-inflammatory program plays a key role in preserving energy storage, reducing energy expenditure and ensuring that insulin resistance is maintained during obesity. We will explore this hypothesis with three aims: 1) we will elucidate the temporal and spatial aspect of counter-inflammation, paying particular attention to the induction of the kinases IKK? and TBK1. We will knock these out in tissue-specific manner to determine the primary sites at which these events occur relevant to changes in metabolism; 2) we will deeply explore the molecular targets of these kinases, focusing on their ability to repress sympathetic activation of adipocytes though changes in cAMP levels, and 3) we will evaluate the role of these counterinflammatory kinases in the generation and sustaining of insulin resistance by examining insulin receptor pathways in cells.
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{
"pile_set_name": "NIH ExPorter"
}
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Ttiis research focuses on the molecular mechanisms of neuromodulation. Neuromodulators are typically neuropeptides, or monoamines such as dopamine, noradrenaline, and serotonin. Defects in neuromodulatory pathways do not usually lead to death, but can cause mental disorders such as autism, depression, schizophrenia, and attention deficit and hyperactivity disorder, as well as eating disorders and drug addiction. Neuromodulators are released from dense-core vesicles (DCVs). Little is known about DCV biogenesis, transport, and release, in part because it has not been possible to biochemically purify proteins specific to DCVs as it has for synaptic vesicles. Instead, a genetic screen in the nematode C. elegans was performed and successfully identified a set of novel molecules that act in a dense-core vesicle trafficking pathway. These include the small GTPase RAB-2 and two novel effectors, RUND-1 and CCCP-1. These molecules physically interact and are colocalized at the trans-Golgi network where DCVs are generated. Loss of these molecules leads to defects in sorting DCV cargos. This proposal aims to identify additional molecules acting In DCV trafficking and determine their mechanism of action. Aim 3 will identify more molecules acting in the RAB-2 pathway using genetic and biochemical screens. These new molecules will be characterized for their effects on DCV trafficking, their physical and genetic interactions with known molecules, and their cellular localization.'Using similar methods. Aim 4 will identify and characterize new molecules acting in a pathway with HID-1, in parallel to the RAB-2 pathway to regulate DCV trafficking. This research directly relates to the mission of NIMH, in particular to the first objective of the NIMH Strategic Plan: to investigate the causes of mental disorders. Mental illness can be caused either by too little or too much of certain neuromodulators. Thus, a better understanding ofthe mechanisms by which neuromodulators are released could lead to the development of drugs that either increase or reduce release, to compensate for the defect. In particular, molecules such as the ones I have identified that are necessary for release would be good targets for drugs to treat disorders due to too much release. Additionally, as dense-core vesicle pathways are modulatory rather than essential for neurotransmission, humans with mutations in these pathways would be expected to be viable, but mentally ill. Thus, the new genes identified in my work are good candidates for genes linked to mental health disease in humans.
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{
"pile_set_name": "NIH ExPorter"
}
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The overall goal of this study is to determine whether RNA virus infection and its associated diseases can be prevented and/or terminated by the presence of virus-specific ribozymes. Viral diseases exact an enormous worldwide toll in morbidity and, increasingly, in mortality. However, the list of effective antiviral treatments remains distressingly short. One problem which has contributed to this is specificity; most substances which exert an antiviral effect are toxic also to the host cells in which the virus resides. Thus the development of a general method to prevent and/or treat viral diseases without harm to the host would be of great benefit. Specificity can be achieved by targeting the nucleic acid sequence of the virus: the experiments proposed herein study the feasibility of using ribozymes for this purpose. A ribozyme is an RNA molecule which cleaves RNA in a sequence-specific manner; ribozymes can be designed to cleave at a specific site in essentially any chosen RNA. Therefore by employing these molecules to cleave virus RNAs, while avoiding host sequences, we can potentially overcome the problem of specificity; furthermore the present burgeoning sequence information not only about many viruses, but also about the human genome, can only aid us in the search for specificity. The effects of ribozymes will be studied in a model system using lymphocytic choriomeningitis virus (LCMV). The advantages of this system are severalfold. (1) LCMV is an RNA virus with no DNA stage; all stages of the viral lifecycle are, therefore, vulnerable to ribozyme attack. Hopefully, results obtained from the study of this agent may be successfully extrapolated to other RNA viruses. (2) The complete sequence of the virus genome is known, allowing specific ribozymes to be readily designed. (3) The interactions between the virus and its natural host, the mouse, have been extensively studied and documented; we thus have access to an excellent animal system in which to study in vivo the effects of ribozymes on viral pathogenicity. For example, LCMV can establish both acute and persistent infections: thus the effects of ribozymes on both types of virus infection can be studied. Furthermore, the diseases associated with LCMV infection are well-characterized, and therefore the therapeutic effects of ribozymes can be readily determined. The effects of ribozymes will be studied at three levels. (i) In vitro. What are the criteria for maximal ribozyme effectiveness? Can a system be designed to simultaneously deliver ribozymes of multiple specificities? (ii) In cell culture. Several expression systems will be evaluated. Are ribozymes selected for their efficacy in vitro able to markedly depress viral replication/maturation in cell culture? If so, what is the mechanism underlying this reduction in virus? (iii) In vivo. Ribozymes will be expressed in all mouse tissues, or in selected cell types. What effects do such ribozymes have, on both viral titers, and on virus-induced diseases? The effects of ribozymes upon the virus also will be studied. When a virus grows in the presence of antiviral ribozymes, do ribozyme-resistant viruses emerge? If so, what is the mechanism? Do these resistant viruses have altered pathogenicity? How can the production of resistance be minimized?
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{
"pile_set_name": "NIH ExPorter"
}
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Converging data from our individual laboratories support the notion that brain cancers (gliomas) are complex and dynamic ecosystems composed of several non-neoplastic cell types critical for glioma formation and maintenance. Specifically, we have shown that one of these non-neoplastic cell types (microglia) in the tumor microenvironment is a critical determinant of glioma cell growth and invasion. To more completely define the molecular mechanisms underlying tumor microenvironment regulation of glioma behavior, we have assembled a highly interactive team of researchers, including one early stage investigator focused on the tumor microenvironment (Dolores Hambardzumyan), one senior scientist expert in microglia-glioma interactions (Helmut Kettenmann), and two established physician-scientists (Eric Holland and David Gutmann) whose laboratories employ genetically engineered mouse models (GEMMs) to evaluate the dynamic interactions between non-neoplastic and neoplastic cells in glioma. Based on experimental findings from each of our individual laboratories, we hypothesize neoplastic glia (glioma cells) recruit and alter the function of resident brain microglia to create specialized tumor-associated microglia, that elaborate molecules that both create a permissive stroma (stromagenesis) (Aim 1) and activate astrocytes (Aim 2). These reactive astrocytes support the creation and maintenance of the perivascular niche (gliomagens) (Aim 3), which provides the proper microenvironment for cancer stem cells - the treatment-resistant population of glioma cells (Aim 4). Collectively, this cross-disciplinary initiative capitalizes on converging lines of evidence that conceptualize gliomas as dynamic ecosystems composed of neoplastic and non-neoplastic cells and leverages expertise in mouse modeling, glioma biology, tumor microenvironment, microglia function, and translational medicine to identify new therapeutic targets for treating these deadly brain cancers.
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{
"pile_set_name": "NIH ExPorter"
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The genes for Histidine UTilization (hut) in Klebsiella aerogenes are typical of a large number of genes whose expression is strictly regulated by the available supply of nitrogen. Thus nitrogen regulation of hut serves as a model system for this kind of pleiotropic control. The goal of the research proposed here is to develop an in vitro transcription system for the hut operon of K. aerogenes that is responsive to regulation by the nitrogen regulatory factor(s) and to study the molecular basis for that regulation. The specific objectives of the five-year proposal period are: (1) To determine the mechanism of carbon regulation at the promoter of the hutUH operon, (2) To begin an analysis of the nitrogen regulation at the promoter of the hutUH operon, and (3) To extend our understanding of the hut operons themselves.
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{
"pile_set_name": "NIH ExPorter"
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This work is built on the hypothesis that molecular phenotyping (transcriptome and proteorhe characterization) will produce new insights into the pathogenesis of autoimmune diabetes in the Non Obese Diabetic (NOD) mouse at age 2-4 weeks. The very early pathogenic events may be particularly well suited for molecular characterization, because they may not be prominent enough to manifest themselves at the cellular/histological level. The initial work on this project has revealed promising new discoveries both regarding unique defects in the islet tissue (reduced oxidative stress defenses and increases in cell turn- over) and more universal defects present in both islets and immune cells (alterations in ribosomes and mitochondria). In this competitive renewal application we propose to shift the focus from islets of Langerhans to lymphocytes. We will characterize lymphocytes in great details. We believe it is important to conduct molecular characterization of a number of leukocyte subsets individually, to gain a detailed insight into the very early molecular signatures and immune system defects associated with the initiation of autoimmunity in NOD mice. The specific aims of this proposal are: Specific Aim 1. To compare the transcriptomes of spleen leukocyte subsets in NOD, NOR, and C57BL/6 female mice between 2 and 4 weeks of age (Specifically the B-lymphocytes, CD4 positive T-lymphocytes, CDSpositive T-lymphocytes, and NK cells. Specific Aim 2. To compare the proteomes of spleen leukocyte subsets in NOD, NOR, and C57BL/6 female mice between 2 & 4 weeks of age (looking at whole cells and subcellular fractions with a variety of proteome characterization approaches). Specific Aim 3. To reveal molecular network associations between specific proteins and gene expression by conducting cluster analysis on combined molecular phenotype data files (using and further developing some novel analysis and mining approaches). Specific Aim 4. To develop a public database for molecular phenotypes (to providing an easy-to-access and searchable portal to all of our data). Relevance to Public Health: This project aims to define the molcular defects responsible for the process that causes type 1 diabetes (also called insulin dependent or juvenile onset diabetes). With a better understanding of these processes it will be possible to develop interventions that prevents this disease.
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{
"pile_set_name": "NIH ExPorter"
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One of the key mediators of cardiovascular risk in obesity is the development of hypertension. Obesity is the principal modifiable risk factor for hypertension, and one of the major contributors to its very high prevalence. Nonetheless, the mechanisms underlying the dysregulation of blood pressure in obesity are incompletely understood. An impaired ability to handle a salt load, or salt-sensitivity, is regarded as an important mechanism for obesity-related hypertension. Salt-sensitivity is determined by the balance of salt-retaining and salt-excreting systems. One of the best-studied salt-retaining systems is the renin-angiotensin-aldosterone system (RAAS). Excess RAAS activation in obesity is well-established. The principal counter-regulatory system to the RAAS is the natriuretic peptide (NP) system. The NPs are natriuretic and vasodilatory molecules produced by the heart in response to increased chamber stress. However, little is known regarding changes in the NP axis in obesity. In preliminary work, we have shown that obese individuals have decreased circulating NP concentrations, which reverse with weight loss. However, resting NP levels may not adequately reflect the ability of the NP axis to respond to stimuli, emphasizing the need for more detailed physiologic studies, under controlled salt conditions and with standardized assessment of the NP and RAAS axes, and related target organs (kidney, heart, and vasculature). We postulate that obesity promotes a state of relative NP deficiency, which leads to impaired NP responses to salt loading, increased salt-sensitivity and elevated blood pressure. In Specific Aim 1, we will assess the NP and target organ responses to acute and chronic salt loading, in lean versus obese individuals. In Aim 2, we will examine the effect of weight loss on the NP and target organ responses to acute and chronic salt loading. The proposed research represents a systematic effort to build upon the investigators' prior clinical investigations into the novel interactions between the NP axis, obesity, and cardiometabolic risk. These studies have the potential to provide important insight into the causes of hypertension in obesity. Furthermore, because the NP system is easily accessible to pharmacologic manipulation, establishing that NP deficiency exists and defining its physiologic consequences could suggest novel approaches to the treatment and prevention of obesity-related cardiovascular disease.
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{
"pile_set_name": "NIH ExPorter"
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We propose to develop a model-driven, evidence-based pain item bank for use in pain assessment that is specific to geriatric oncology (gero-oncology) patients. This will build on our Novel Pain Assessment and Intervention Network (NoPAIN) project (Chang, PI, 1R21CA113191-01), and continue its Clinical Infometrics approach. This proposed study is designed in response to PA-03-152 (Biobehavioral Pain Research) to "refine existing techniques for measuring pain and develop new techniques that are disease- and outcome- specific for different populations." We have chosen the condition of pain in this population because: pain is a complex multidimensional experience; a critical mass of pain instruments now exist and there is an urgent need to refine and implement them; and pain management in cancer among older adults is a pressing issue given societal demographics. We also aim to construct a computerized adaptive testing platform for this item bank that: 1) can reliably measure the multidimensional pain experience of heterogeneous cancer patients; and 2) is sensitive to change so that it can assess the effectiveness of treatments over time. Our four distinct but related specific aims are: 1) To identify and refine the domains of pain assessment for a gero-oncology population, using existing theoretical frameworks in biopsychosocial medicine and palliative care as a guide; 2) To compile the items for a multidimensional pain item bank, drawing from existing pain questionnaires and supplementing with newly written items; 3) To develop empirically a pain item bank applicable to a gero-oncology population; and 4) To pilot test a computerized adaptive testing (CAT) platform to administer individualized pain assessments in clinical settings. At the end of this R21 project, we expect that (1) the conceptual model of the gero-oncology pain measurement can be improved; (2) the initial sets of pain items can be constructed; (3) items displaying differential item functioning (DIF) can be identified and revised; and (4) the CAT prototype will demonstrate how the system should be implemented and tested in clinical settings. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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The receptor cells for acoustic stimuli are particularly vulnerable to reduced oxygen tension because of the limited characteristics of blood supply to the inner ear. Electrophysiological data have demonstrated that asphyxiation disrupts audition, but the consequences of mild, prolonged hyposia upon the auditory system have not been investigated. One aim of this investigation is to determine whether subchronic exposure to chemical asphyxiants at doses relevant to levels widely encountered in occupational and environmental settings produces ototoxicity. Chronic and impulse noise can induce autitory damage, but whether this occurs through mechanical trauma or by metabolic exhaustion is uncertain. Because there are data which show that noise reduces oxygen tension in the endolymph fluid of the scala media and produces vascular damage to the inner ear, it has been hypothesized that noise-induced auditory loss may result from depressed oxygen availability. If noise does disrupt hearing by restricting oxygen to the Corti Organ or if it damages inner ear vascular structures it is logical to assume that noise would potentiate the ototoxic effects of chemical asphyxiant exposure. The final aim of this proposal will be to specify the nature of the interaction between noise and chemical asphyxiant exposure. The methods to be used include measures of modulation of the acoustic startle reflex by low intensity test stimuli and subsequent histological examination of the inner ear.
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{
"pile_set_name": "NIH ExPorter"
}
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Crying is the primary means through which an infant communicates with a caregiver and is therefore essential to infant survival and wellbeing. Crying, however, can also put a strain on parent-child relationships and place a child at risk. Excessive crying in early infancy is a common complaint brought to pediatricians, a frequent reason for giving up breastfeeding, and the most often cited trigger for infant abuse and Shaken Baby Syndrome. In addition to its important role in parent-child dynamics, infant crying serves as a diagnostic tool for clinicians. Recent research suggests that abnormalities in infant cry sounds may be used to detect infant brain injury and vulnerability for autism. Infant crying has been the focus of a great deal of research because of its centrality to infant health and the development of the parent-child bond. The International Infant Cry Workshop is a scientific meeting convened every two to three years devoted entirely to research on infant crying from multidisciplinary perspectives. Scientists from diverse fields gather to share findings on the a wide array of topics related to infant crying and health, including: crying's diagnostic value and value in predicting infant outcomes; how crying is affected by parental care and other environmental factors; relations between infant crying, physiology and emotional health; and characteristics of caregivers related to differences in optimal, sensitive and abusive responses to crying. There has been substantial progress in cry research since the last meeting in 2011, for example, using fMRI technology to document parents' responses to crying and identifying abnormalities in cry sounds associated with autism. The next cry workshop will be held in Warwick, England from July 7th through July 9th 2014. The theme of the workshop will be Infant Crying: Biological Bases, Developmental Consequences, and Clinical Issues. The workshop will examine infant crying from different basic research and applied/clinical perspectives with the aim of translating basic research findings into useful clinical interventions and identifying best practices in infant health care. This conference is unique because it brings basic scientists and clinical researchers together in an intimate setting that is ideally suited fo suggesting new avenues of research and sparking effective collaborations. Funding from the NIH is being sought to underwrite the participation costs of three invited speakers who will greatly enrich the scientific program of the meeting.
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{
"pile_set_name": "NIH ExPorter"
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Emerging evidence indicates that oxidative stress, a state of excessive reactive oxygen species (ROS) activity, is associated with cardiovascular diseases, such as hypertension. ROS, including superoxide and H202, have been implicated in several key events in the early process of hypertension and medial hypertrophy. The NADPH oxidase system of vascular cells, whose central component is Nox1, is the major source of ROS in vascular tissues. The Rho family GTP triphosphatase Rac1 is a key activator of NADPH oxidase, leading to the production of ROS, including superoxide and H202. We have engineered a transgenic mouse model that overexpresses the constitutively activated mutant of the human Rac1 protein in the smooth muscle cells in FVB/N mice (Rac-CA), using mouse smooth muscle a-actin promoter. The arterial vessels of these Rac-CA mice exhibit impaired vasodilatory activity of nitric oxide (NO), enhanced expression of superoxide dismutase (SOD) isoforms, indicative of superoxide activity, and a marked increase in H202 level. In addition, these mice develop hypertension that is mediated via a ROS-dependent, but angiotensin/renin-independent pathway, as the constitutively active state of the Rac1 transgene product does not require signaling reactions upstream of Rac (angiotensin receptor-1 engagement, CED-5IDockl 80, etc.). Our central hypothesis is that overproduction of superoxide by NADPH oxidase in response to Rac1 activation within the arterial vessel wall, antagonizes the vasodilatory effects of nitric oxide, resulting in hypertension and arterial media hypertrophy. We propose to characterize the molecular events associated with Rac-CA overexpression in the vessel wall leading to the observed phenotype. We will study the contribution of ROS to the development of hypertension and arterial media hypertrophy in Rac-CA mice in various genetic backgrounds (FVB/N and C57BL/6). Furthermore, through the use of strategic crosses between Rac-CA mice and other established models, we will study the role of NADPH oxidase, the manganese type superoxide dismutase, catalase and glutathione peroxidase in hypertension and vascular hypertrophy. The use of potent inhibitors will complement our work with transgenic/knockout mice, and provide further proof of concept to the role of Rac1 and resultant ROS in hypertension and arterial media hypertrophy. This program should substantially improve our understanding of the role of superoxide and derived ROS in the control of blood pressure homeostasis. Novel targets for therapeutic strategies will ensue, and mouse models created in the context of this program will be invaluable to many investigators in this field of research
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{
"pile_set_name": "NIH ExPorter"
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Understanding the mechanisms of regulation of cellular proliferation and differentiation is basic to understanding development of multicellular organisms. One approach to investigating these regulatory mechanisms is to study the behavior of transformed cells. Through the use of Ad2- (non-oncogenic) and SV40- (highly oncogenic) transformed hamster cells, we have identified the phenotypic characteristics of these cells (e.g., Ad2 sensitivity and SV40 resistance to in vitro lysis by non-specific immune effector cells) that correlate with their ability to form tumors in immunocompetent hamsters. We also find that while Ad2-and SV40-transformed cells are equally active in production of transforming growth factors, only SV40-transformed cells secrete a powerful anti-mitogen, which may inhibit host defenses in vivo. We are also using SV40 to study the genetic basis of viral issue tropism. We find that subcutaneously injected small t-antigen mutants of SV40 often induce abdominal B-cell lymphomas in hamsters, rather than the subcutaneous fibrosarcomas induced by wild-type SV40. The mutants may fail to produce a growth factor required for the in vivo transformation of non-proliferating cells. In another project, we are studying the mechanisms of mutagenesis, using an SV40-based shuttle vector as a probe to investigate the molecular mechanisms by which agents that damage DNA induce mutations in mammalian cells. Our studies on replication of UV-damaged SV40 DNA in vivo have led to a well-defined model of how the mammalian cell replication machinery responds to DNA damage, and at what steps in the replication process mutations become irreversibly established. Through use of the shuttle vector, we have extensively characterized the types of mutations that occur in mammalian cells either spontaneously or in response to DNA damage. Analysis of the sequence specificity of these mutations has led to a model which explains how the mammalian DNA polymerase introduces errors during DNA synthesis, causing mutations. Further studies with the vector in an in vitro DNA replication system should allow a correlation with the in vivo replication defects we observed.
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{
"pile_set_name": "NIH ExPorter"
}
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The purpose of this K01 application is to foster the candidate's career development in clinical and translational research on aging. His long term career goal is to become an independent translational investigator in functional decline and rehabilitation of older adults. After postdoctoral training in basic human skeletal muscle research, the applicant became interested in translational research on aging through his interactions with the UTMB Claude D. Pepper Older Americans Independence Center (OAIC). He now seeks additional training to gather a better understanding of the issues underlying functional decline and rehabilitation in older adults. The career development plan includes both formal and informal training combined with apprentice-style learning activities. A diverse mentoring team comprised of Drs. Volpi, Ottenbacher and Paddon-Jones will guide this development. UTMB is exceptionally well suited to foster the candidate's career development, as it provides a very collaborative environment for multidisciplinary research. Specifically, the candidate will continue to receive educational and core resource support from the UTMB OAIC and CTSA. The goal of the research project is to determine how aging and inactivity reduce the muscle anabolic effect of nutrients and lead to muscle and functional loss. The central hypothesis is that aging reduces mTORC1 signaling and the expression of skeletal muscle amino acid transporters in response to anabolic stimulation leading to reduced muscle adaptation to increased intracellular amino acid requirements. We further hypothesize that inactivity exacerbates this effect with significant muscle and functional loss, and rehabilitation restores muscle signaling, metabolism and function to baseline values. Controlled bed rest is a very powerful model of accelerated muscle loss and dysfunction via a reduction in muscle protein synthesis. The degree of muscle and functional loss achieved with bed rest significantly increases with aging. We will test in healthy subjects the following specific aims: 1) To determine if aging blunts the physiological upregulation of mTORC1 signaling and skeletal muscle amino acid transporters to amino acid ingestion and decreases intracellular amino acid availability and muscle protein anabolism. 2) To determine if physical inactivity (bed rest) further exacerbates the age-induced reduction in mTORC1 signaling and amino acid transporter expression in response to amino acid ingestion and induces a larger muscle and functional loss in older as compared to younger subjects. 3) To determine if physical rehabilitation can restore muscle signaling, metabolism and function to levels not different from the pre-bed rest condition. The pilot data collected will be used for future R01 submissions to identify mechanisms and novel targets for interventions to improve muscle function and preserve physical independence in older adults.
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{
"pile_set_name": "NIH ExPorter"
}
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The University of California, Los Angeles' Jonsson Comprehensive Cancer Center (JCCC) is an NCI-designated matrix center conducting a wide range of translational research in the areas of laboratory, clinical and population sciences integrating the activities of 244 members. Based on the JCCC Strategic Plan, the goals of the JCCC are the integration of clinical activities to provide patient-centered care; research infrastructure improvement through technology and shared resources; increased communication within JCCC and to our many audiences; faculty support and development at all levels; and the advancement of emerging areas of research. Over the past funding cycle 70% of patients enrolled in interventional clinical trials participated in investigator-initiated studies. The total funding base for the JCCC is $163 million with $40 million in NCI funding. Our NCI funding includes subcontracts from other institutions, of which a significant amount is derived from our partnership with California Institute of Technology. In addition, of 3,489 total publications in the reporting period, 35% were published in high-impact journals, 15% were inter-programmatic and 21% were intra-programmatic. Continuing support is requested for the following eight Programs Areas and six Shared Resources. Programs Cancer and Stem Cell Biology* Cancer Molecular Imaging Cancer Nanotechnology* Gene Regulation Healthy and At-Risk Populations Patients and Survivors Signal Transduction and Therapeutics Tumor Immunology *New Program Area Shared Resources Biostatistics, Analytical Support & Evaluation Genomics Flow Cytometry Translational Pathology Molecular Screening Shared Resource Small Animal Imaging Shared Resource
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{
"pile_set_name": "NIH ExPorter"
}
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The recent success with the Stargardt disease gene shows how a rare macular disease can lead to insight into the common age-related macular degeneration (AMD). This promise that continued progress and success will also lead to a better understanding of macular function and dysfunction as well as AMD. North Carolina macular dystrophy (MCDR1) is a congenital macular dystrophy that was mapped by linkage to chromosome 6 in 1992 by Small et al. The major goals from the original (previous) proposal were directed towards the fine mapping and cloning of the MCDR1 gene. All of these previous goals have been achieved except for the actual identification of the MCDR1 gene and identification of the mutations(s). The current proposal will accomplish this by continuing the ongoing efforts with positional cloning strategies. Specific aim 1 is to continue to ascertain North Carolina macular dystrophy (MCDR1) families in order to try to narrow the genetic interval even further. The current interval has been narrowed to 600 kb by the investigator's laboratory. Specific aim 2 is to continue the identification and characterization of the MCDR1 candidate genes. The group has recently identified 27 new candidate genes from their PACs (P1 artificial chromosomes) within the genomic region. Specific aim 3 is to perform mutations screening of these genes in MCDR1 families. This will be accomplished primarily by protein truncation testing (PTT) if the fragments are 300-1,000 bp in size or by single strand conformation polymorphism (SSCP) if the fragment is less than 300 bp. If no mutations are found in these candidate genes, additional ones will be identified and prioritized. Specific aim 4: Once the MCDR1 gene is identified, AMD patients will be screened for mutations in the MCDR1 gene in order to evaluate its role in AMD and the prevalence of the mutation in the general population. It is expected that the above specific aims will be accomplished within three years. However, because the timing of the identification of the MCDR1 gene is difficult to predict, the future aims of this proposal are discussed in case the gene is found sooner. These future aims and directions are to determine the function of the MCDR1 and to identify other proteins that interact with the MCDR1 gene product since they may also be involved in macular diseases including AMD. Finally, a transgenic, a knockout, and a knock in mouse will be created to better understand MCDR1 function and to create an animal model for future therapeutic attempts.
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{
"pile_set_name": "NIH ExPorter"
}
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This project is a comprehensive, multidisciplinary effort to understand the natural history and modes of transmission of viruses and other infectious agents that are associated with cancer. With numerous intramural and extramural laboratory, clinical, and epidemiologic collaborators, and a core of prospective cohort and case-control studies, the effort is focused on human immunodeficiency virus (HIV), hepatitis C virus (HCV), and Kaposi's sarcoma-associated herpesvirus (KSHV, also called human herpesvirus 8 or HHV -8). In our large Multicenter Hemophilia Cohort Study (MHCS), longitudinal patterns of HIV viral load among hemophilic children were found to resemble those previously noted in adults. Hemophilic participants who had escaped HIV infection, despite susceptible (wild-type) chemokine receptor geneotypes and intensive HIV exposure, were found to have diminished immune responses, suggesting that immune activation may be required for establishment of HIV infection. Polymorphisms in three more genes (CX3CR1, IL10, and HLA *B35-Px) were found to affect the rate ofHIV progression to AIDS. Injection drug users were found to have markedly increased mortality due to HIV and to drug overdose but not to infection .with the transforming human retrovirus, HTLV-II. MHCS participants and injection drug users with HCV infection were found to have a higher risk of progression to end-stage liver disease (ESLD) with particular HLA class II alleles. ESLD risk among MHCS participants with both HIV and HCV infections was related to atypical antibody patterns against HCV. A Second Multicenter Hemophilia Cohort Study (MHCS-II) was launched to improve understanding ofHCV natural history. New antibody assays for the diagnosis of KSHV infection were developed, and an enzyme immunoassay against the KSHV K8.1 recombinant protein was found to have high sensitivity and specificity in African and Maltese populations. In the elderly populations of three Mediterranean islands known to be endemic for Kaposi's sarcoma, KSHV seroprevalence and risk of Kaposi's sarcoma were quantified. A case-control study to identify risk factors for classical (non-AIDS) Kaposi's sarcoma is in progress. In a vase-control study of childhood leukemia, antibody seroprevalence against Haemophilus influenzae type b was found to be reduced among cases under age 6 and increased among older cases. This supports the hypothesis that a delay in the timing of early childhood infections(or other antigenic challenges)increases the risk of leukemia.
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{
"pile_set_name": "NIH ExPorter"
}
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Investigate differences in central serotonergic functioning in patients with social phobia, persons with extreme shyness, and non-shy control subjects through the use of pharmacological probes. The study will use the strategy employed to understand the psychobiology of other mood and anxiety disorders, the administration of a 5HT-selective pharmacologic probe, to examine the functioning of central 5HT systems in social phobia.
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{
"pile_set_name": "NIH ExPorter"
}
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The purpose of this project is to develop leads for the construction of clinically effective inhibitors of the HIV proteases, and consequently of the AIDS virus. Computer-based analyses of the active site of the HIV protease are being used as the target for an algorithm that matches the crystal structures of known compounds to that of the active site. A non-peptidic lead compound, haloperidol, has led to an effort to synthesize analogues that are more effective inhibitors of the enzyme, as well as to develop other lead structures. Mass spectrometry plays an important role in elucidation of the structures of the synthetic analogues and chemical intermediates in their synthesis. Irreversible inhibitors have also been developed. Mass spectrometry is being used with these agents to determine the nature of the alkylation process and the site of the alkylation.
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{
"pile_set_name": "NIH ExPorter"
}
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This project has two purposes: to develop the chemical basis for establishing sediment quality criteria for arsenic and chromium for arsenic and chromium; and to construct coupled water column-sediment fate and transport models. Arsenic and chromium in aquatic sediments are currently listed as contaminants at 113 and 142 Superfund sites, respectively. It is of critical importance, therefore, to have reliable methods for determining the sediment concentrations at which the metals pose an environmental and human health risk. Currently available methods are based on the concentration of total arsenic and chromium that ignore bioavailability that are known not to be predictive of toxicity. We intend to use the Equilibrium Partitioning (EqP) model, that is currently being used by EPA, in order to generate these criteria. This approach involves first: determining the solid phase or phases that regulate pore water concentrations; and third: determining the potential for remobilization (and future exposure) of sediment bound metal. For water column animals and their human consumers, the extent to which metals are released from sediments to the overlying water, and the extent to which the reverse process occurs, are critical components in a comprehensive analysis of the risk environmental and human health posed by these metals. We intend to develop sediment models first and then coupled water column-sediment models for arsenic and chromium. The purpose of the sediment model is to computer the flux of metal from the sediment to pore water, and ultimately, to the overlying water. This release is determined in large measure by the rate of oxidation of the reduced solid phase metal species. Once sediments are judged to pose an environmental or human health risk, it is necessary to project future exposure concentrations in the sediments and overlying water and to evaluate the efficacy of remedial actions. This step requires the use of mathematical models to described the combined effects of transport and chemical/biochemical reactions. We intend to construct these models and to apply them to data sets that are available in the literature.
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{
"pile_set_name": "NIH ExPorter"
}
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The broad, long-term objectives of this research project are to study RNA metabolism and gene expression. Areas such as RNA processing , RNA transport, and RNA stability and degradation will eventually be explored. Initially, the process of pre-mRNA splicing (i.e., the precise excision of introns from messenger RNA precursors) will be studied. Specifically, the in vitro pre-mRNA splicing system that we developed in the yeast, Saccharomyces cerevisiae, will be utilized to identify and characterize the components in the splicing machinery (i.e., the extrinsic splicing factors and the functional spliceosomes). The results of this study should provide clue to health-related problems which are resulted from abnormal gene expression due to defects in the RNA splicing process. Three extrinsic protein factors required to convert the pre-mRNA on a functional spliceosome to spliced products have been identified by analyzing extracts isolated from several temperature-sensitive rna mutants. Efforts will be made to purify these extrinsic factors by biochemical fractionation of wild-type or overproducer extracts. Antibodies against purified proteins will be raised. The corresponding genes will be isolated by screening yeast DNA library with the antibodies or synthetic oligonucleotides. DNA sequences will be determined, the homology with other genes searched, and the derived protein sequences analyzed. The purified extrinsic factors will be used to examine and to define the functional spliceosomes isolated under different conditions. The roles of ATP in the splicing pathway will be investigated. Attempt will be made to identify splicing factors which may be phosphorylated or adenylated during the conversion of the spliceosome. The interactions among the spliceosome, the extrinsic factors, and the RNA will be investigated by using immunoprecipitation and RNase protection assays. Ultimately, the machinery for nuclear pre-mRNA splicing may be revealed.
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{
"pile_set_name": "NIH ExPorter"
}
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The goal of this project is to elucidate the role of opioid peptides in the physiology and pathophysiology of posterior pituitary function. Specifically, the proposed studies will test the hypothesis that opioid interneurons are an integral part of the afferent pathways which mediates the effect of blood volume on vasopressin secretion. To this end, opioid antagonists specific for mu, delta or kappa receptors will be given to rats to determine if the antagonists alter the vasopressin response to osmotic, hemodynamic or glucopenic influences. To locate the receptors that mediate these effects, the antagonists also will be injected stereotaxically into brain ventricles to determine if blockage of opiate receptors in these areas reproduces the inhibition of specific vasopressin responses produced by systemic administration of the antagonists. The same opioid antagonists will be radiolabelled and used with autoradiography to determine specific in vivo binding sites. The effect of blood volume and other stimuli on the synthesis and/or secretion of individual opioid peptides in these regions will also be investigated using incorporation of radiolabelled aminoacids and radioimmunoassays specifically developed for this purpose. In these experiments, tissue will be obtained by punch biopsies of discrete brain areas and the peptides in questions will be extracted and identified by immunoassay as well as HPLC and Sephadex chromatography. Finally, to clarify their therapeutic potential, these antagonists will be given acutely and chronically to determine if they correct abnormalities in vasopressin secretion in animal models of diuretic abuse, cardiac and liver failure. The information obtained from these studies will help to clarify the role of endogenous opioids in brain function and may also provide a powerful new tool to treat s well as to define the pathogenesis of many clinical disorders of salt and water balance.
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{
"pile_set_name": "NIH ExPorter"
}
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DESCRIPTION: This proposal is designed to use the unique advantages of NMR spectroscopy for paramagnetic molecules, in conjunction with other complementary techniques, to examine hemes, skeletally modified hemes (verdoheme, meso-hydroxylated heme, isoporphyrins), heme enzymes (peroxidases, heme oxygenase), and reactive chemical models for these enzymes. 1D and 2D NMR studies offer the opportunity to examine geometric and electronic structure of the heme and its immediate environment (axial ligation, protein pocket) in intact, functioning enzymes and in reacting model systems to investigate critical factors that are responsible for the binding and activation of substrates, especially small molecules (dioxygen, peroxides), and in the case of heme oxygenase (HO), heme itself. One portion of the project will focus on NMR strategies for the use of 1D and 2D methods to enlarge the experimental capabilities and quantify the interpretive bases of hyperfine shifts. For proteins these will examine the second layer of protein residues near the heme (now that the first layer can be studied), evaluate methodologies for proteins larger than horseradish peroxidase (HRP), extract information from cross correlation peaks in COSY maps, and extract structural information from high-spin as well as low-spin states of heme enzymes. For models these techniques will focus on understanding the electronic effects of heme modification by introduction of functionalities on the heme periphery as occurs during heme degradation that accompanies heme enzyme operation. The methodological advances are expected to contribute not only to effective pursuit of the our goals, but to contribute significantly to the NMR investigation of a large variety of other paramagnetic metalloproteins. Studies on peroxidases will focus on horseradish peroxidase where the geometry of the active site, the substrate binding sites relevant to peroxidase vs oxygenase activity, as well as the effects of selective mutations on each of these, will be examined. Studies on HO will focus on establishing the identity, sequence origin and geometry of the components of the active site of the heme (substrate)/HO complex, with the goal of eventually understanding factors that account for the a- specificity of meso-site attack that occurs during heme catabolism. This process initiates bile pigment (especially bilirubin) and carbon monoxide (a putative neurotransmitter) formation. The complex series of events that result in heme catabolism will be elucidated at the molecular level through studies of fundamental, insoluble iron compounds that are likely intermediates in the process and through their structural characterization, examination of reactive models (coupled oxidation of heme with 02) for this process, and attempts to trap or prepare reactive intermediates with intact heme oxygenase itself. Throughout this work the goal will be to define the oxidation/ligation/spin profanation state of the heme or modified heme as it passes step-wise through the intricate process of heme catabolism in order to understand the role of the enzyme in guiding and hastening this process.
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{
"pile_set_name": "NIH ExPorter"
}
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DESCRIPTION (Investigator's Abstract): While it is generally accepted that quantitative EEG analysis is less subjective and can extract information that would be difficult or impossible to obtain by visual means, it remains uncertain whether it can enable the clinician to make more informed decisions about individual patients. This project will address this question in the contex of selecting patients for epilepsy surgery based on non-invasive EEG findings. The hypothesis is that optimal computerized scoring of scalp- recorded ictal EEGs with allow as accurate or more accurate lateralization and localization of seizure activity than expert visual reading. Two existing computerized seizure analysis techniques with be rigorously evaluated for their lateralization and localization capabilities and compared with visual scoring by expert raters. To do so, we will retrospectively analyze the scalp recorded icta EEGs of a large group of well-documented patients that have been evaluated for surgery will be analyzed retrospectively. Blinded visual and computerized scoring will be applied to the seizures of those patients whose seizure lateralization and localization was verified by longterm surgical follow up and/or the results of invasive EEG recording. Each technique will be applied independently to the same EEG data and the analysis will be converted to lateralizations and localizations, and two-by-two contingency tables of true- and false- positive and negative lateralization/localization with be tabulated. Plots of true-positive proportion vs. false-positive proportion constructed from this analysis will assess the potential accuracy of each technique in the absence of fixed decision criteria and then be used to identify optimal selection criteria for specific applications.
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{
"pile_set_name": "NIH ExPorter"
}
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The aim of the proposed research is to obtain a better understanding of the development and regulation of muscarinic acetylcholine receptor complexes (mAChR) in cardiac tissues, and in particular the influences of parasympathetic innervation on receptor development. Normal developmental patterns of mAChR will be studied in chick hearts to take advantage of the natural temporal separation of neural and myocardial elements. The influence of innervation on the development of mAChR will be assessed following cauterization of the areas of neural crest which give rise to the cardiac ganglia. Similarly, the influence of preganglionic parasympathectomy on cholinergic receptor systems will be studied after removal of the area of the rhombencephalon which gives rise to preganglionic cell bodies. Combined lesions will remove both pre- and postganglionic branches of parasympathetic innervation of the heart. The effectiveness of these procedures will be assessed through measures of ACh content and high affinity choline uptake in cardiac tissues. Histological examination will reveal the number of ganglion cells in the conotruncus during normal development and in the various experimental conditions. Receptor complex properties will be assessed using radiolabelled probes: The number of receptors, their agonist and antagonist binding affinities, their regulation by guanine nucleotides, and their ability to down regulate in response to exposure to muscarinic agonists will be measured. These experiments will provide a plethora of basic information concerning the relationship of the heart and its nerves, as well as the biochemical organization and regulation of cholinergic receptors in cardiac tissue.
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{
"pile_set_name": "NIH ExPorter"
}
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Tissue homeostasis is a highly dynamic process, which requires a strict regulation of cell growth, cell proliferation, and cell death within the tissue. During development and adult life of the organism, cell proliferation and cell death are precisely coordinated to ensure the integrity of the epithelium. However, due to genetic alteration(s) some cells can uncouple their proliferation and death cues from the rest of the epithelium and thus acquire an autonomous ability to proliferate uncontrollably. If not corrected, this deregulation of cell proliferation and/or cell death can in turn lead to tumor formation within the tissue, potentially causing tissue/organ malfunction and subsequent death of the individual/animal. For tumor cells to effectively grow they must overcome several challenges, including anti-cell proliferation and/or pro-apoptotic signals within their environment. However, our understanding of how tumors interact with the host, specifically with neighboring wild type cells is incomplete. Furthermore, deregulation of additional signaling pathway(s) in primary benign tumors can transform these tumors into malignant tumors, which have the ability to spread to distant organs and precipitate the patient<s death. Carcinomas account for ~90% of human malignancies and metastasis is associated with poor patients prognosis. Our understanding of the nature of these malignant transforming signaling pathways and how these cues are internalized in tumor cells to cause such transformation is unclear. Moreover, it is now evident that tumors are functionally heterogeneous. For example, in any given tumor, cells have differing potential to initiate tumor metastasis. A characterization of these metastasis-initiating tumor cells is lacking. Using a Drosophila tumor model, the objectives of this research proposal are 1) to investigate how cell proliferation and cell death cues are regulated in benign versus malignant tumors, 2) to test a role for TGF2 signaling in the benign to malignant tumor transformation, and 3) To characterize the cytokinetic properties of metastasis initiating tumor cells. Aim1. To examine cell death and cell proliferation of benign versus malignant tumor cells Using live imaging techniques in a Drosophila fly tumor model, I will monitor tumor cell proliferation and cell death in benign or malignant tumors. Similarly, the behavior of wild type cells neighboring tumor cells will be concomitantly analyzed. Aim2. To investigate a role for TGF2 signaling in the benign to malignant tumor transformation, A role for TGF2 signaling in converting benign tumors into malignant tumors will be examined using immuno- staining and somatic genetic approaches. Aim3. To characterize the cytokinetic properties of metastasis initiating tumor cells. Tracking experiments will be performed to trace metastatic tumor cells back to their position within the primary tumor relative to neighboring wild type cells. Their cell proliferation behaviors will subsequently be analyzed. PUBLIC HEALTH RELEVANCE: Carcinomas account for ~90% of human malignancies and metastatic tumors are often associated with poor patient prognosis. Understanding how tumors interact with host cells to facilitate tumor growth and metastasis could potentially provide novel insights for clinical interventions
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{
"pile_set_name": "NIH ExPorter"
}
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The objective of the proposal is to study the role of HIV-1 specific cytotoxic T cells in pathogenesis of vertical HIV infection. The hypothesis is that the role of HIV-1 specific CTL in controlling viremia and protecting against disease progression following vertical infection is dependent not only on the timing of detection, magnitude, and breadth of HIV-1 specific CTL responses, but also on their functional properties in rapid and slow progressors, and that HIV-1 specific CTL will be detected later and at lower frequencies in RP than in SP. The preliminary results suggest, however, that some HIV-1 infected infants may experience RP despite an apparently vigorous and broad CTL response. The principal investigator will therefore also examine the functional properties of CTL in RP and SP. Using a cohort of infants with well-defined laboratory parameters (viral load, CD4 counts), the principal investigator aims to serially quantify peripheral blood HIV-specific CTL precursors (CTLp) frequencies. The time to detection of CTLp, changes of CTLp over time, and the relationship between CTLp frequencies and blood viral load will be examined and compared in infants with rapid or slow disease progression. Next, the investigators will generate HIV-specific CTL clones from selected RP and SP in order to identify epitopes recognized and HLA restricting elements. Finally, functional properties of the HIV-specific clones from RP and SP (ability to recognize variant peptides, cytokine secretion patterns, and ability to lyse or to control HIV replication in naturally-infected CD4 T cells) will be examined. The proposed studies are important in understanding the pathogenesis of vertical HIV-1 infection and for the development of a vaccine to interrupt vertical infection
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{
"pile_set_name": "NIH ExPorter"
}
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The working hypothesis of this proposal is that enamel proteins play a central role in the process of amelogenesis and that determination of the structure and expression of these proteins is essential to an understanding of that role. With presently available techniques, solubilized enamel proteins can be divided into two classes of proteins, amelogenins and enamelins, containing a total of 15 to 25 components. It is not known how many of these components represent the products of individual genes, whether the multiple components are derived from the physiologic breakdown of a limited number of proteins or are artefactually produced. The overall objective of this proposal is to characterize the structure and expression of the enamelin protein, tuftelin, and to relate the structure of the protein to its potential function. To achieve this objective, 4 Aims are proposed: (1) To complete the characterization of the bovine tuftelin gene. (2) To identify the functional elements of the tuftelin gene promoter, the site(s) of transcription initiation and whether the primary transcript is alternatively spliced. (3) To determine the temporal and spatial expression of tuftelin-related polypeptides in the developing bovine enamel organ. (4) To investigate potential structure/function relationships of tuftelin using recombinantly expressed protein. Identification of functionally significant promoter elements is essential to an understanding of control of expression of the tuftelin gene, and it is important to determine whether alternative splicing contributes to the observed hetgerogeneity of enamelins. Because of the difficulty involved in purifying native tuftelin, recombinant synthesis is the method of choice for obtaining it in quantity. The proposed studies will use state of the art molecular and cellular biology, immunologic and physical techniques. The information gained from these studies may ultimately (i) lead to better understanding of the functions of enamel proteins, (ii) lead to an explanation of some genetic defects affecting the enamel matrix, (iii) lead to the development of more effective and biologically compatible restorative materials.
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{
"pile_set_name": "NIH ExPorter"
}
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This proposal to continue the Harvard Cancer Prevention Education and Career Development Program builds on the existing expertise in cancer prevention and control activities within the Harvard School of Public Health (HSPH) and the Dana Farber/Harvard Cancer Center (DF/HCC). The first ten years of this education program provide a solid base for this renewal. To date, this Program has trained 55 trainees in 57 slots, including 27 pre- doctoral fellows and 12 physicians earning a degree, 7 physicians in post-doctoral research positions, and 11 non- MD post-doctoral fellows. The curriculum and related seminars are now well-established, and the structure for mentored research experiences has been tested and refined. The specific objectives of this program are to: (1) provide an effective training program in cancer prevention and control through the provision of mentorship from primary and secondary mentors from different disciplines, a refined and flexible curriculum that provides the best balance of common shared training and individual instructional opportunities, and provision of opportunities to participate in research opportunities across the DF/HCC; (2) recruit strong candidates for the pre- and post- doctoral fellowship opportunities from a range of disciplines with adequate representations from ethnic minorities and women; (3) evaluate the effectiveness of the Program following a well-def'med set of criteria. As a result, the Program aims to create a cadre of cancer prevention researchers who will be: a) knowledgeable about the current state of the science of cancer prevention; b) expert in a specialized research area in which they will be positioned to generate new knowledge and public health practice to advance cancer prevention; c) skilled in policy analysis; and d) sufficiently well anchored in professional networks that they will be able to monitor future developments in the field of cancer prevention and translate their knowledge into cancer control programs at a variety of institutional levels. In this renewal, each year we propose to fund six pre-doctoral positions, and four post-doctoral fellowships, including one post-doctoral fellowship for physicians seeking a degree.
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{
"pile_set_name": "NIH ExPorter"
}
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Endurance exercise training has been suggested to alter the autonomic nervous system resulting in an apparent increase in parasympathetic efferent activity and a decrease in sympathetic efferent activity. In this context, it is interesting to speculate that exercise training may result in an altered neurohumoral regulation of the cardiovascular system by changing the interaction of systems directly involved in controlling the circulation. Changes in autonomic neural activity could be beneficial to patients at high risk for sudden cardiac death. For example, a reduction in the incidence of sudden cardiac death among postinfarction patients participating in a multifactorial intervention program that included exercise has been reported. This proposal is designed to examine the effects of endurance exercise training on the cardiopulmonary baroreflex regulation of the cardiovascular system. Specifically, the direct inhibitory influence of the cardiopulmonary baroreflex and the interaction of the cardiopulmonary and arterial baroreflex on the regulation of sympathetic nerve activity (lumbar and renal), regional blood flow, arterial pressure, heart rate and hormonal regulation (renin and arginine vasopressin) at rest and during exercise will be studied both before and after exercise training. The influence of peripheral (afferent activity) and central mechanisms in mediating the autonomic adaptations associated with exercise training will also be examined. The underlying hypothesis of this proposal is that the tonic inhibitory influence of cardiopulmonary baroreflex on the sympathetic nervous system, arterial baroreflex and hormonal regulation is enhanced as a result of endurance exercise training and that this response is mediated by both peripheral and central mechanisms. An enhanced cardiopulmonary reflex may significantly alter the integrative regulation of the cardiovascular system by initiating reflex alterations in the neurohumoral drive to the circulation as well as interacting with the arterial baroreflexes. These studies will provide a better understanding of the regulation of the neurohumoral drive to the circulation, the effect of exercise training on this regulation and the influence of central and peripheral mechanisms in mediating the autonomic adaptations associated with exercise training.
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{
"pile_set_name": "NIH ExPorter"
}
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Type II DM confers a markedly increased risk of cardiovascular disease (CVD) in premenopausal women. One potentially significant contributor to increased CVD risk may be impaired fibrinolysis. Healthy premenopausal women appear to have the greatest fibrinolytic potential. Yet studies that have demonstrated impaired fibrinolysis in diabetic patients have included few women and have not controlled for hormonal status. We postulated that the fibrinolytic response to stasis in premenopausal diabetic women would be more impaired than that of age and weight matched diabetic men. Healthy, sedentary, non-smoking diabetic women, diabetic men and control women with no evidence of CVD were studied. Baseline euglobulin lysis time (ELT) was drawn prior to inflation of a blood pressure cuff to mean arterial pressure. At the end of the cuff induced 15-minute stasis period a repeat ELT was drawn. Women with diabetes demonstrated an impaired response to stasis. Specifically there was a smaller decrease in ELT in diabetic women compared to both diabetic men and control women*, P<0.05 difference between diabetic women and other groups, +, P<0.05 difference pre to post stasis. Data suggests that fibrinolytic impairment is more pronounced in diabetic women than diabetic men. The changes may indicate that premenopausal diabetic women have fibrinolytic potential similar to that of postmenopausal women. Future studies should explore the mechanisms of impairment and the theory that increased CVD risk in premenopausal women with diabetes may be related to this significant impairment fibrinolytic response to stasis.
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{
"pile_set_name": "NIH ExPorter"
}
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Epidural clonidine will be administered in conjunction with epidural morphine in order to determine if there is an observable improvement in analgesia or reduction in epidural morphine requirement; safety and therapeutic efficacy will be monitored.
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{
"pile_set_name": "NIH ExPorter"
}
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The HIV-1 integrase is one three enzymes of HIV and is essential for integration of the viral DNA into the host chromosome, a step required for the continued expression of new virus. It has no human counterpart and is therefore an attractive target for drug design. However, despite the enormous successes with the reverse transcriptase and with the protease inhibitors, there are still no inhibitors of integrase that qualify as therapeutic agents. Now, however, many potential lead compounds exist and are being investigated. We have been able to bind an inhibitor to the active site of the enzyme. This inhibitor binds centrally in the site and provides a new lead compound for antiviral drug design. During the last year we have attempted to crystallize two constructs of integrase, full length and core plus C-terminal domain, bound to various DNA substrates. The integrase carries out two reactions during the integration process, 3' end processing and strand transfer. The molecule can also carry out a third reaction which is essentially the reverse of strand transfer and is known as the disintegration reaction. We have attempted to interact these two integrase constructs with a variety of oligonucleotides representing the U5 end of viral DNA, as well as with various disintegration substrates. For the latter, the length of both the U5 arm and target arms were varied. Having obtained no single diffracting crystals to date, we are now extending the range of variable in this investigation. The goal is find a mutation or combination of mutations which will yield single crystals of either the full length protein or any construct of integrase bound to DNA. We are also continuing our efforts to crystallize the catalytic core domain with various active site inhibitors. Large quantities of the core domain have been obtained by expression in E. coli, and crystals at both pH 7.0 (the original condition) and 5.6 have been obtained. We are currently soaking these crystals with inhibitors as well as cocrystallizing the protein with the inhibitors.
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{
"pile_set_name": "NIH ExPorter"
}
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Equine infectious anemia is a persistent viral infection with a number of immunologically mediated lesions. Infected horses have circulating antibodies measurable by several techniques, yet the blood is still infectious for horses. The research proposed here will be directed toward evaluation of the humoral and cellular immune processes and their interaction with the virus. The pathogenesis of the lesions will be investigated in detail. The virus and associated antigens will be characterized. The data obtained will be evaluated to delineate the mechanisms of viral persistence and of the pathogenesis of the lesions.
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{
"pile_set_name": "NIH ExPorter"
}
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The basic goal of this research program is to contribute to the understanding of the molecular basis of the biological activities of the liver microsomal membrane proteins. The following strategy will be used to approach these goals. Proteins present in the microsomal membranes are isolated and sequenced. Amino acid sequenced data, together with the methodology for isolating peptide fragments is used to define their topology in the membrane and to identify the residues involved in the function of these enzymes. Microsomal membranes, under controlled conditions, are treated with proteolytic enzymes and group specific reagents. The peptides and the proteins of the digests re characterized by means of electrophoresis and high performance liquid chromatography. Micro- sequence analysis of the isolated fragments and the transblottead proteins identifies the cleavage site or the position of the label in the particular protein. The orientation studies will be focused on the microsomal preparations enriched with cytochromes, deacylases, and epoxide hydrolase. Proteins present in microsomal membranes will be separated by high resolution two-dimensional electrophoresis. N-terminal sequences of these proteins will be obtained by direct blotting of the separated proteins from electrophoresis gels to chemically inert supports followed by excision of the protein band and sequencing. Attempts will be made to isolate and characterize some of the enzymes, involved in the posttranslational processing reactions that occur in the microsomal membranes. The health-relatedness of this project is that a preponderance of xenobiotics including chemical carcinogens and drugs as well as physiological compounds such as steriods and prostaglandins are metabolized by the microsomal cytochromes and the luminal esterases. Many intrinsic, as well as, viral proteins pass through the microsomal membrane to reach their destination. Many undergo proteolysis, glycosylation, acylation and deacylation reactions. Thus, the basic studies addressed in this proposal provide fundamental knowledge relevant to the clinical problems of atherosclerosis (cytochrome b5-desaturase system), and cancer (cytochrome P-450 system and esterases, chemical carcinogenesis). Elucidation of the mechanism of acylation, glycosylation and proteolysis steps. Many of these are provided by the enzymes of endoplasmic reticulum and all are essential for the assembly of active viral particles.
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{
"pile_set_name": "NIH ExPorter"
}
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Brain tumors are the cause of death in approximately 13,000 people in the U.S. every year, and these tumors represent the most common type of solid neoplasms in children. Recent studies have demonstrated that inducible tryptophan oxidation is an important mechanism of modulation of tumor cell proliferation and immuno-resistance, mainly via the immuno-modulatory enzyme indoleamine 2,3-dioxygenase (IDO), the rate-limiting step of the kynurenine pathway. Our preliminary studies using positron emission tomography (PET) with the tracer 1-[11C]methyl-L-tryptophan (AMT) showed differential increase of uptake and metabolism of AMT in various brain tumors, and expression of IDO in resected tumor tissue. These data suggest that accumulation of AMT in these tumors is related to increased metabolism of tryptophan via the kynurenine pathway. This application will address both basic and clinical aspects of the role of tryptophan in brain tumors in children and adults. We will perform quantitative PET studies to measure in vivo uptake and metabolism of AMT in various brain tumors and correlate these findings with tumor proliferative index, IDO immunoreactivity, and multidrug-resistance (MDR) proteins in resected tumor tissues. Three aims are proposed: (i) To establish that AMT PET can differentiate brain tumors from non-tumorous lesions preoperatively, and detect residual or recurrent tumors after initial treatment, as well as differentiate between recurrent tumors and radiation necrosis. (ii) To differentiate among various types of brain tumors based on measures of transport and metabolic trapping of AMT. (iii) To determine the relationship between kinetic parameters of AMT derived from PET imaging, and tumor proliferative index, IDO, and multidrug-resistance protein expression derived from histological assay of tumor tissue. These studies will elucidate mechanisms of abnormal uptake and metabolism of tryptophan and their role in the biological behavior of brain tumors using an innovative approach of combination of in vivo molecular imaging with AMT PET and in vitro tumor tissue studies. From a clinical perspective, our studies will establish the use of AMT PET imaging of primary and residual/recurrent brain tumors, which continue to pose a major challenge for accurate diagnosis. The findings will provide new insight into mechanisms of abnormal tryptophan uptake and metabolism in brain tumors with the potential of developing new strategies using pharmacological treatment of brain tumors by targeting tryptophan metabolism and MDR proteins. This project combines PET imaging of brain tumors with the tracer 1-[11C]methyl-L-tryptophan and in vitro analysis of the expression of the immuno-modulatory enzyme indoleamine 2,3-dioxygenase (IDO) as well as various multidrug-resistance (MDR) proteins in resected brain tumor tissues. The findings will provide improved diagnosis of primary and recurrent brain tumors and also will give new insights into mechanisms of abnormal tryptophan metabolism in brain tumors with the potential of developing new strategies using pharmacological treatment of brain tumors by targeting tryptophan metabolism and MDR proteins. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
}
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The long-term objective of this project is to understand the mechanisms of intracellular protein folding. Recent findings suggest that protein folding in vivo may generally involve assistance or catalysis by certain constitutively-expressed stress proteins termed molecular chaperones. This proposal focuses on the functions of Hsp70 and Hsp60 family members in this process. By studying protein folding reactions in mitochondria and with pure chaperone components, we have discovered the role of the mitochondrial Hsp60 and its bacterial homolog GroEL in mediating the folding of monomeric proteins. We have now reconstituted an ordered folding reaction in which the Hsp70 DnaK and GroEL act successively on folding polypeptides. The stress proteins DnaJ and GrpE function as coupling factors. The in vivo significance of this reaction is underscored by the finding that folding of proteins imported into mitochondria involves interaction with Hsp70 and subsequent transfer to Hsp60. Building upon this foundation, we propose to definitively establish the sequence, structural specificities and functions. of the interactions with chaperone proteins required for the correct folding of nascent and newly-synthesized polypeptides. We will test the hypothesis that there is a general protein folding pathway involving molecular chaperones of the Hsp70 and Hsp60 classes, or functional equivalents, in different cellular compartments. The following Specific Aims will be addressed in a multi-disciplinary experimental approach: 1.) Define the interactions between folding polypeptides and chaperone proteins in a reconstituted system containing pure DnaK, DnaJ, GrpE and GroEL/ES. The differential binding specificities of stress proteins for a series of intermediates of protein folding will be established. 2.) Define the sequential action of chaperones with folding polypeptides imported into intact mitochondria and translated in a cell-free system that can be depleted of endogenous chaperones. Luciferase will be used as a model protein whose activity can be assayed following in vitro synthesis. Protein folding intermediates and modified stress proteins will serve as tools. 3). Characterize the functions of stress proteins in the renaturation of thermally-denatured proteins in a reconstituted system and in intact mitochondria. 4). Discover novel molecular chaperones in eukaryotes, including DnaJ and GrpE homologs. The cytosolic protein complex Tcp1 will be isolated and assayed for an Hsp60-like activity in protein folding.
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{
"pile_set_name": "NIH ExPorter"
}
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The objective is to evaluate "neighborhood effects" in community trials for cardiovascular disease (CVD) prevention. During the 1980s, the NHLBI/NIH funded three community trials to test programs aimed at reducing CVD risks. While each study reported favorable risk factor changes, many overall net differences between treatment and control sites were modest and/or statistically insignificant. New research suggests that these analyses may mask important within-site neighborhood-wise endpoint differences. It may be that (1) "place," above and beyond individual level measures, influences CVD-related behaviors and (2) overall program outcomes may be confounded by neighborhood level influences. This study employs rich and existing data from the (1) Pawtucket Heart Health Program (PHHP) and (2) the 1980 and 1990 decennial censuses. Multilevel statistical methods and a geographic information system (GIS) are used for analyses. Briefly, the specific aims are to: Determine the extent to which individual-level CVD related variables vary by neighborhood; Identify neighborhood-level predictors of CVD risk after adjusting for individual-level measures; Examine neighborhood-wise endpoint trends; Analyze dose effects (i.e., intervention process) in terms of neighborhoods; Evaluate the effect of different neighborhood definitions on findings; Find best methods for handling demographic trends when census data are gathered decennially; Advance multilevel and GIS methodologies in cardiovascular epidemiology; Contribute to an epidemiologic theory of "place." This cost-effective three year study is significant, innovative and timely. It will advance the multidisciplinary substantive understanding and methodological tools necessary for public health practice in the twenty-first century.
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{
"pile_set_name": "NIH ExPorter"
}
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A Career Development Program will be a major and essential part of this proposed SPORE. Its goal will be to develop physicians, basic and population scientists for lifelong productive careers in translational prostate cancer research. The program will strive to make these individuals scientifically productive, academically successful and influential nationally, and good role models for recruitment of individuals to similar careers. The program will involve all of the institutions within the SPORE: in Seattle, the Fred Hutchinson Cancer Research Center (FHCRC), and the University of Washington (UW);in Vancouver, the University of British Columbia (UBC) and Prostate Center at the Vancouver General Hospital (VGH);and, in Portland the Oregon Health Sciences University (OHSU). The coordination between the three areas will involve recruitment, development and educational opportunities, and the interchange of mentors and research opportunities. The specific goals of this program are to: 1. Recruit and train four to seven post-doctoral fellows in prostate cancer translational research per year; 2. Recruit and nurture one to two faculty members at each institution whose main interest is prostate cancer translational research every 2-3 years; 3. Enhance and develop a broad program of education and mentoring within the SPORE environment. This educational program will involve not only a large spectrum of individual research opportunities, but also a more formal educational experience of didactic courses and a large number of scheduled conferences and seminars. Recruitment of fellows and junior faculty will occur through the planned educational program and by systematic recruitment efforts. These individuals will be monitored and mentored by a specific Career Development committee, and by 47 multidisciplinary investigators at these institutions both within and outside the SPORE whose research interests and accomplishments reflect prostate cancer research concerns. In addition to SPORE grant support, this program will be supported by extensive institutional resources.
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{
"pile_set_name": "NIH ExPorter"
}
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