Bcr/abl gene is derived from the t(9;22) reciprocal translocation and is present in most of chronic myelogenous leukemia (CML) and a cohort of acute lymphoblastic leukemia (ALL) patients. BCR/ABL oncogenic tyrosine kinase modulates response to DNA damage inducing resistance to genotoxic therapies. In addition BCR/ABL stimulates genomic instability, which may lead to mutations in BCR/ABL kinase causing resistance to imatinib mesylate. We hypothesize that: BCR/ABL elevates the levels of reactive oxygen species (ROS) which induce spontaneous DNA lesions (for example uracil residues), whose unfaithful repair introduces amino acid substitutions in the BCR/ABL kinase domain causing resistance to IM. The role of ROS in generation of oxidative DNA damage leading to mutagenesis and resistance to imatinib mesylate will be studied in CML hematopoietic stem cells (HSC), common myeloid progenitor cells (CMP), and granulocyte/macrophage progenitor cells (GMP) using anti-oxidant approaches and in vitro and in vivo models of BCR/ABL leukemogenesis. The efficiency and fidelity of the mechanisms processing ROS-dependent oxidative DNA damage in BCR/ABL leukemia cells will be determined by studying base excision repair (BER), focusing on UDG glycosylase removing uracil residues. These reactions will be examined using well-defined reporter/substrate systems and a combination of different approaches including immunofluorescence, phosphorylation-less and interaction- deprived mutants, transgenic mice, and sequencing. BCR/ABL-UDG functional interaction will be investigated by mutagenesis and targeted by aptamers to inhibit resistance to imatinib mesylate.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. It is becoming clear that regulation of protein function is often acheived via shifts in the populations of active and inactive conformers. X-ray crystallographic analysis gives only snapshots of possible configurations populated in solution. Taking advantage of our expertises in theoretical and experimental analysis of protein folding and assembly, we seek to use a combined SAXS and theoretical approach to define the structural ensembles present in the functional landscapes of multidomain proteins. This approach extends beyond current simulation analysis of SAXS data to include local and global folding/unfolding transitions that are important in function. Our initial choice in developing this methodology is the enzyme Csk as it is not only an important therapeutic target but also is a system we have characterized in terms of the optimal enzymatic and folding conditions. There is growing evidence that biases in the structural ensemble may play an important role in the regulation of Csk. The methodology developed here is broadly applicable to biological macromolecules and will provide useful information about what ensembles of conformations are consistent with the experimental data as well as the the ubiquitous dynamic reversible folding/assembly processes inherent in biology.


In this proposal we aim to investigate a role of the Activation-inducible isoform of Hypoxia-inducible Factor- 1alpha (aiHIF) in regulation of anti-bacterial response of T cells during sepsis. Our hypothesis indicate aiHIF as a potential target for improvement of anti-bacterial clearance in septic patients. This assumption is based on our findings that aiHIF plays inhibitory role in TCR-activated T cells, and we found that HIF-1alpha prevents T cells from fully contribute into anti-bacterial response during sepsis. Our preliminary studies support the hypothesis that aiHIF plays major role in the negative regulation of T cells during sepsis. Using our recently created aiHIF-deficient mice we will test whether the total or T-cell-specific deficiency of aiHIF enhances the pathogen destruction and survival in murine sepsis models. Using in vivo models of live bacterial sepsis in mice we will test whether aiHIF is a major negative regulator of activated T cells during sepsis. In addition, we will determine the mechanism of T-cell inhibition by aiHIF. The aiHIF-deficiency is expected to de-inhibit T cells by rendering them resistant to inhibition in hypoxic inflamed areas, and to allow T cells to fully participate in orchestrating the overall anti-pathogen response, which will improve sepsis survival. These studies will identify aiHIF as a novel molecular target and provide proof of a principle for a feasible strategy to improve therapy of sepsis, especially when combined with our recent discovery of previously unknown aiHIF isoform in human T cells that will allow our mouse sepsis studies of aiHIF to translate into humans. PUBLIC HEALTH RELEVANCE This proposal aims to provide proof of principle for a novel strategy of improving the therapy of sepsis by prevention of T cells inhibition by activation-inducible HIF-1alpha.


Various storage and retrieval algorithms have been studied. The development of flexible and efficient storage and retrieval algorithms is very useful, because such algorithms are used in almost all computer programs. Thus, biomedical computation in particular can benefit from improved storage and retrieval methods. In FY85, work continued on the analysis of trie data structures and the development of algorithms and analysis for a new type of doubly-chained trie capable of holding prefixes. This work is of particular import for text storage and retrieval, including dictionaries, indices to document collections, and various special thesari. The proposed data structure provides a general mechanism for storing arbitrary words and/or phrases without restriction on content. The storage scheme is fast and parsimonious, and the retrieval scheme is also fast. Prior to discovering how to handle prefix items, tries could not be as easily employed for arbitrary collections of words.


Thymocyte maturation and thymic architecture is dependent upon precise intrathymic cell migration. The orchestration of thymocyte trafficking, however, is not well understood at the molecular level. Recently, we described the highly-regulated plexinD1 expression on CD4+CD8+ double positive (DP) thymocytes which is further modified by T cell receptor/co-receptor engagement. Activation of plexinD1 via its ligand, semaphorin 3E (sema3E), represses CXCR4/CXCL12 signaling in pre-selection DP thymocytes and CCR9/CCL25 signaling in CD69+ post-selection DP thymocytes. Using Plxnd1-deficient fetal liver cell-transplanted mice, loss of plexinD1 causes CD69+ thymocytes to remain in the cortex, maturing in situ to form ectopic single positive (SP) thymocyte clusters. As a consequence, the boundary between DP and SP thymocytes at corticomedullary junctions is disrupted and medullary structures form under the thymic capsule. These results demonstrate the importance of plexinD1 in directing migration of maturing thymocytes via modulation of biological responses to chemokine gradients. The dysmorphic corticomedullary structures observed in Plxnd1-/- and Sema3e-/- knockout mice are further consistent with this view. To address the cellular interactions and functional consequences of Plxnd1 deficiency in thymic development, the present proposal will pursue three major aims. First, we will focus on the role of plexinD1 in regulation of CCR9/CCL25 and CXCR4/CXCL12 pathways and the directed migration of DP thymocytes using Ccr9-/- and Cxcr4 conditional knockout mice. Specifically, the links between these chemokine signaling pathways and Plxnd1 deficiency as well as the associated retarded thymocyte migration towards the medulla of pre-selected (CD69-) and post-selected (CD69+) DP thymocytes will be assessed. Second, we will analyze by multi-photon excitation microscopy the dynamics of GFP- expressing (green) Plxnd1-/- thymocytes in fetal liver-transplanted mice interacting with tdTomato (red) cortical thymic epithelial cells within renal subcapsular fetal thymic implants, using CFP-expressing (blue) wild type cells as an internal control. Third, we will analyze the impact of Plxnd1 deficiency on negative selection during T cell development using OT-I/OT-II TCR and RIP-OVA transgenic mice and fetal liver cell transplantation, in particular screening mice transplanted with Plxnd1-/- fetal liver cells for evidence of autoimmune phenomena. Collectively, our studies will offer insight into the molecular basis of plexinD1-mediated regulation of DP thymocyte movements within the thymus with consequential effects upon thymocyte differentiation and selection that may lead to autoimmune pathology in the mature immune system. PUBLIC HEALTH RELEVANCE: T cell function is critical for mammalian host protection against a wide variety of infectious disorders and cancers. T cells emanate from the thymus. Hence, understanding of the processes required for normal thymic architecture, differentiation and maturation will contribute to generation of more effective immunity.


The overarching goal of the BioRepository Center (BRC) is to facilitate translational research. Tight coordination between surgical staff, pathologists, and BRC enables collection of specimens to fulfill investigators' needs. Our protocols for rapid acquisition of tissue samples allows for preparation of high quality analytes. Patients' rights and privacy by our participation in obtaining and confirming informed consent for tissue collection; guidance with regard to preparation and submission of IRB documents is offered to help ensure compliance. The BRC also promotes collaborations between clinical and basic scientists, and can aid in the design and execution of prospective, hypothesis-driven studies to examine the impact of genetics or treatment on disease progression. By promoting investigators access to appropriate biospecimens, the BRC enhances the ability of NYULMC Cancer Institute members to perform meaningful translational research.


Traumatic brain injury (TBI) is a significant problem in the pediatric population. Ninety per cent of pediatric TBIs present to an emergency department, but only 8% are hospitalized. Since the majority of pediatric patients with mild TBI are therefore discharged home with the diagnosis of concussion, accurate assessment of the severity if concussion and consequent outpatient management and instructions are critical for ensuring safe recovery from injury. Without state-of-the-art knowledge and clinical tools, mild TBI (mTBI) may go undiagnosed and untreated, leaving individuals who have sustained a mTBI with an increased risk for functional problems. The ACE and ACE care plan were developed as part of the CDC's "Heads Up: Brain Injury in your Practice" toolkit for physicians to manage mTBI. Adapting the ACE for the ED and implementing a standardized clinical protocol by ED physicians systematically should improve management by ensuring accurate diagnosis and improving patient education and adherence with discharge recommendations. The goal of this research is to demonstrate the capacity to improve diagnosis and management of mTBI presenting to the Emergency Department (ED) by the feasible application of systematic procedures in the form of the ACE and the ACE Care Plan. This study will be conducted collaboratively by Children's National Medical Center and UPMC/ Children's Hospital of Pittsburgh with the specific aims to: (1) evaluate the feasibility of the ACE and ACE Care Plan for standardized implementation in the ED setting (2) determine if the ACE and ACE-ED Care Plan can be implemented by the ED staff and disseminated to the Primary Care Providers and (3) determine if routine use of the ACE-ED and ACE-ED Care Plan will improve patient/family follow-up behavior and patient recovery. We have designed the study to progress in two stages: Stage 1 proposes to develop expert consensus agreement regarding the importance and feasibility of using the ACE and ACE Care Plan in the ED setting, including an understanding of current concussion management care pathways. An outcome of Stage 1 will be the consensus-based adaptation of the ACE and ACE Care Plan for the ED, referred to as the ACE-ED and the ACE-ED Care Plan. Stage 2 applies these revised tools via a pilot implementation study for patients age 5-22 years old presenting with mTBI. The primary outcome will be patient/family follow-up behavior with the primary care/specialist. As secondary outcomes, we will examine clinician adherence to use of the ACE-ED and ACE-ED Care Plan, and its dissemination to primary care providers. Feasibility of implementation will be further evaluated by identifying the actual facilitative and barrier conditions to ACE-ED/ Care Plan use within the ED setting. We will also develop estimates of effect of this implementation on patient recovery. Upon study completion, key data will be available to support the development of


The p53 protein plays a critical role in tumor suppression, and represents a pivotal mediator of cellular response to physiological stress. One of the major regulators of p53 activity and abundance is the MDM2 oncoprotein. The long-term goals of this research are to better understand the complex regulation of the p53 tumor suppressor and the MDM2 oncogene, specifically regarding interacting factors that modulate p53 function. In normal cells, p53 has a short half-life and its abundance is low. In response to cellular stress, p53 becomes stabilized and activated as a transcription factor, and the expression levels of p53-target genes increase of decrease. The physiological outcome of such p53 activation is growth arrest or cell death (apoptosis). Thus, elucidation of the parameters that control p53 abundance and function in a cell remains an important area of study in cancer biology. They have identified two proteins that can regulate the activity and stability of p53 when bound. One of these factors, the co-repressor mSin3A (Sin3), was previously determined by them to be a critical mediator of p53-dependent trans-repression and apoptosis. Thus, p53 transcriptional repression activity and stabilization may be functionally linked. The other factor, MDMX, is a homologue of the MDM2 oncoprotein. While MDMX stabilizes p53 by interfering with MDM2-mediated degradation of p53, Sin3 appears to stabilize p53 by a novel mechanism, not dependent on MDM2. The broad aims of this proposal are to elucidate the mechanisms whereby interaction with MDMX and Sin3 influence p53 regulation. Significantly, they have found that Sin3 can bind the p53 homologue p73 as well. This indicates that transcriptional repression and stabilization by interaction with Sin3 may be conserved properties of the family of p53-related transcription factors, with implications for understanding pathways of apoptosis. Therefore, as a natural corollary to this work, this hypothesis will also be tested.


Our aim is to test the common assumption that aging in mammals involves a nonrandom decrease in neuron number and to consider the hypothesis that the decrease is preceded by changes in connections made and received by those neurons. The hypothesis derives from well-established data that at critical periods early in life, the number of neurons is adjusted downward by about 50% in many regions of the nervous system as a normal programmed event, and that axonal innervation of target cells also may be adjusted downward. Do such adjustments occur late in life and what controls them? Our analysis uses standard mice of the inbred C57BL/6 inbred strains, at ages from early adulthood to beyond 30 months of age, which is unusually old for mice. Complete serial section series through the whole brain are being prepared by a new embedding and sectioning technique. We have developed computer-assisted quantitative methods applicable at light and electron microscopic levels for scoring the numbers and positions of neurons of defined classes, counting and classifying axons with statistical reliability in central tracts and peripheral nerves, and measuring volumes and surface areas of brain regions and of individual cells. With the support of this grant, we are among the first to be able to measure fiber number and sizes automatically, directly from the electron microscope image, without photographic mediation. Further efforts will focus on the difficult micro-alignment and other problems inherent in three-dimensional quantitative reconstruction of synaptic organization at high magnification. We are concentrating overall on 3) numbers of cerebellar neurons in the major classes as a function of age, 2) the relation of retinal ganglion cell size, number, and distribution to the corresponding parameters in optic nerve, and 1) sampling and statistical methods for motor and sensory components in mixed peripheral nerves. Our objectives is to establish methods and a normative baseline against which to test experimentally in the future, whether long-term neuronal survival is influenced by quantitative synaptic relationships at an earlier period of life.


Experimental evidence is emerging that the gasotransmitter hydrogen sulfide serves an important role in the cardiovascular system especially during tissue ischemia. However, specific cellular sources, targets, and mechanisms of hydrogen sulfide within the vasculature remain largely unknown. Work from our lab and others demonstrate that hydrogen sulfide is protective against chronic tissue ischemia, which involves increased vascular remodeling responses such as angiogenesis. Importantly, very little mechanistic information exists regarding how hydrogen sulfide modulates ischemic vascular remodeling in vivo or in vitro. Data in this application reveals a novel finding that hydrogen sulfide emanating from cystathionine !-lyase (CSE) selectively augments ischemic tissue nitrite reduction to NO that mediates increased angiogenic activity involving HIF-1 activity and VEGF expression. Experiments in this proposal will determine molecular mechanisms for these novel results through the hypothesis that endothelial cell CSE dependent hydrogen sulfide generation augments ischemic nitrite reduction to NO that increases ischemic vascular remodeling. The hypothesis will be examined through the pursuit of three specific aims including: 1) determine how endothelial cell CSE expression regulates ischemic vascular remodeling responses, 2) determine the mechanisms by which hydrogen sulfide increases NO generation in ischemic tissue and how this regulates HIF-1 activity and VEGF expression, and 3) determine the effect of endothelial cell CSE dependent hydrogen sulfide on diabetic ischemic vascular remodeling responses. Successful completion of this project will significantly advance the fields understanding of hydrogen sulfide biology in the vascular system, provide clear mechanistic information on hydrogen sulfide-NO pathway interactions, and identify novel approaches for ischemic vascular disease therapy.


Indole-3-carbinol (I-3-C), a normal dietary constituent, has been shown to decrease chemical carcinogenesis in experimental animals. N-nitrosodimethylamine (NDMA) requires metabolic oxidation in order to express its hepatotoxic, mutagenic and carcinogenic potential. Our results show that treatment of mice by gavage with I-3-C decreases the covalent binding of (14C)NDMA metabolites to DNA or protein up to 90% with no change in the rate of NDMA demethylation of cytochrome P-450 levels. We found that ethanol or methylene chloride extracts of livers from mice treated with I-3-C decreased the covalent binding of NDMA metabolites to DNA and protein in liver cell fractions from untreated mice. In order to explain these observations, we developed the hypothesis that treatment of mice with I-3-C gives rise to lipophilic nucleophiles that protect tissue macromolecules from electrophilic attack by a scavenging mechanism. To test this hypothesis, we developed a procedure to assign a relative value of nucleophilicity to chemicals or chemical mixtures, such as tissue extracts. The nucleophilic index value (NIV) for liver tissue extracts is inversely proportional to the rate of covalent binding of MDMA metabolites to DNA and protein, and independent of the rate of NDMA demethylation. The Specific Aims of this project are: a) to find the I-3-C dose regimen to maximize tissue NIV; b) to correlate NIV with indices of hepatotoxicity and DNA damage; c) to fractionate solvent extracts of livers from I-3-C treated mice in order to purify and identify the active nucleophilic material(s), using the HPLC separation techniques in parallel with liquid scintillation counting and high resolution mass spectrometry compound identification procedures; d0 to establish the mechanism of protection by I-3-C. The objectives of this research plan are a) to establish and validate a model system which uses the NIV of the liver to predict biochemical and functional susceptibility of the liver to damage from the hepatotoxic and caracinogenic chemical NDMA; b) to identify the chemical species and the mechanisms involved in the I-3-C protection from molecular and cellular damage. This project offers a predictive model for tissue susceptibility to, and a molecular basis for dietary protection from certain environmental toxic and mutagenic/carcinogenic insults.


Recent evidence indicates that the ingestion of increased amounts of the trace mineral selenium (Se) during the developmental period of tooth formation increases dental caries in man and experimental animals. The long term goal of the research proposed in this application is to provide a biochemical understanding of how selenium causes an increased susceptibility to dental caries. Utilizing C14 labeled radioactive precursors, we plan to study the integrity of metabolic pathways for: (1) the oxidation of amino acids, glucose, and fatty acids; (2) the synthesis of proteins, glycogen and lipids; and (3) the synthesis of matrix proteins in developing teeth, in rats receiving drinking water containing inorganic and organic selenium. Because of the dental, as well as metabolic significance, of selenium- induced depressions in food intake, results will be compared to control animals receiving diet provided by a pellet dispensing device in the same pattern of food intake as the selenium animals.


Present cage assignments for primates are based solely on the animals' weight. Variation in shape between species of primates of the same weight indicate that the current weight-based standard may be inappropriate. A large number (410) of primates of four different species have been measured (arms, legs, chest, tail, crown to rump, crown to heel) in order to determine association of and variations in weight as functions of shape measurements. The results of this study provide for the assignment of cages based not only on animal weight, but also on allometric measurements and accounting for species differences. This project has been completed.


Type I diabetes is characterized by the permanent loss of pancreatic insulin-producing beta cells. Our goal is to understand the mechanisms determining pancreatic beta cell mass, as a first step toward the development of regenerative therapies for diabetes. In collaboration with the BCBC Coordinating Center and members of the consortium, we will use transgenic mouse technology to examine the hypothesis that beta cells have a significant regenerative capacity, which is modulated by specific signals. The specific aims are to: 1. Develop a system for regulated ablation of pancreatic beta cells. Our preliminary studies indicate that mice can recover from a pulse of beta cell ablation. We will characterize in detail the physiological and histological aspects of ablation and recovery, in order to understand the mechanisims of beta cell regeneration. 2. Determine the cellular origins of regenerating beta cells. We will employ genetic lineage tracing to definitely determine the contributions of stem cells and pre-existing beta cells to beta cell regeneration. 3. Characterize the signals that regulate beta cell mass. The relative importance of blood-borne signals will be determined, with emphasis on the role of insulin and glucose metabolism. In addition, we will assess the significance of beta cell dedifferentiation in vivo and in vitro. Insights from these studies will be applied to primary beta cell cultures, with the aim of improving yield, preventing dedifferentiation and minimizing cell death


Preliminary studies in our laboratory are consistent with a relationship between hyperglycosylation (additional antennae and fucose residues on N-linked oligosaccharides) and nicking or cleavage of hCG. Other preliminary studies indicate the structural instability and diminished biological activity of hyperglycosylated and of nicked hCG molecules, and how they may be the source of free Beta-subunit in the circulation and Beta-core fragment in urine samples. A large number of papers have been published showing raised hCG levels, and raised proportions of free Beta-subunit and Beta-core fragment levels in serum or urine samples from patients with Down syndrome pregnancies, preeclampsia, trophoblast disease, or hyperemesis gravidarum. It is our hypothesis, that all of these raised levels arise from the hyperglycosylation of hCG, through a pathway involving nicking of hyperglycosylated hCG, ineffective autocrine control of hCG production, and rapid dissociation to generate nicked free Beta-subunit, and degradation to Beta-core fragment. Five sets of experiments are proposed to test this hypothesis and investigate the clinical ramification of the observations in normal and abnormal pregnancies. Studies are proposed to confirm and compare the levels of hCG, free Beta-subunit and Beta-core fragment in 720 sets of parallel serum and urine samples from normal and abnormal pregnancies (Aim 1A); to purify hCG from normal first, second, and third trimester pregnancy urine samples and trophoblast disease, preeclampsia, hyperemesis gravidarum and Down syndrome pregnancy urine samples (3 each) and compare the extents and sites of nicking, and the type, amounts and locations of hyperglycosylated N-linked oligosaccharides (Aim 2); to investigate the hyperglycosylation-nicking relationship by comparing normal and hyperglycosylated hCG as substrates for nicking enzymes (Aim 3); to examine the biological activities of normal and abnormal pregnancy hCG and abilities to feedback through to the placenta and control hCG synthesis (Aim 4); to investigate the and compare the stabilities of hCG from normal and abnormal pregnancy, and determine whether dissociation could explain the raised proportion of free Beta- subunit (Aim 5); and to use the parallel serum and urine samples to investigate the clinical utility of new immunoassays specifically measuring nicked or hyperglycosylated hCG molecules (Aim 1B).


The over-all objective of this program is to test a theoretical model which attempts to account for the frequently-encountered phenomenon of increase longevity of irradiated insects. The major tenet of the model is that radiation damage to DNA induces higher levels of various DNA-repair enzymes which would otherwise decline in differentiated postmitotic tissues. It is generally accepted that many enzymes which repair DNA damaged by radiation also play a role in repair of DNA lesions produced by chemicals, including, presumably, some metabolic products. Thus, the model predicts that DNA-damaging chemicals would also increase longevity in insects (and other organisms whose somatic tissues are predominantly or entirely postmitotic); that nonrepairable damage, such as that produced by high LET radiations, would be effective, in the manner of gratuitous inducers; that there would by age-related declines in resistance to two types of deleterious agents: those which act by damaging DNA in differentiated tissues, and those whose effects must be overcome by transcribing the genetic information encoded in such tissues. The model also predicts that effects on adult longevity would not be correlated with decreased fecundity or reproductive activity.


HIV-positive adult male and female study subjects will be recruited from Moore Clinic patients who have positive HCV antibodies and positive HCV RNA on stable antiretroviral regimens (>4week). Thirty (30) subjects will be selected for study participation according to inclusion and exclusion criteria in the clinical protocol.


More than 29 million children have a dental restoration. The majority of restorations now placed are composites. Most resin-based composites placed in children contain a derivative of Bisphenol A (BPA) called BPA-glycidyl methacrylate (BisGMA). When BisGMA was developed in 1962, no studies were required by the FDA to evaluate biological toxicity. BPA is a widespread xenoestrogen that may affect brain, endocrine, and reproductive development. National dental organizations maintain the amount of BPA from BisGMA-based dental materials is too small to be relevant to human health. There is scant scientific evidence for or against this assertion. Contrary to this claim, our pilot data and data from the only other prospective study in children show urinary BPA (uBPA) concentrations are a magnitude of 2 to 6-fold higher post-treatment. Studies show exposure to medical products increase BPA concentrations, yet there are no studies in children that quantify BPA exposure from medical products used in anesthesia (tubes, syringes) for dental treatment. We need to determine the contribution of dental-related BPA exposure to overall BPA load, and distinguish between different sources of dental-related BPA exposure. The aims of this application are to: (1) Quantify the magnitude and duration of BPA exposure resulting from dental treatment; (2) Determine associations between number of BisGMA-based treated surfaces and BPA, overall and by type of material (composites, sealants); and (3) Determine the association between type of anesthesia and BPA. We will measure uBPA concentrations in 210 children 4 to 8 years old receiving BisGMA-based dental materials with different types of sedation at the University of Washington Center for Pediatric Dentistry 2 times before and 4 times after treatment from 24 hours to 16 weeks. To ensure we have sufficient numbers of highly exposed children we will employ stratified sampling. We will recruit children who are treated with <4 surfaces (n=105) and =4 surfaces (n=105) with BisGMA-based dental materials. Within each of these two groups, we will recruit 35 patients in three groups who receive: (1) no sedation, (2) nitrous oxide; or (3) general anesthesia. We will administer surveys to collect demographic data and data on food security and other sources of BPA. We will measure the height and weight of the child, and collect detailed treatment records. We will conduct the first large study in children to comprehensively examine multiple sources of dental-related BPA exposure. Distinguishing BPA from dental materials and medical products used in anesthesia may enable us to develop interventions to reduce dental-related BPA exposure. By oversampling children receiving treatment on =4 surfaces with BisGMA-based dental materials and children receiving GA, we will include high-risk children likely to have high baseline BPA who may receive among the largest amounts of dental-related BPA exposure from materials or anesthesia, and who may be most likely to experience adverse health effects from BPA.


Agents that inhibit the reaction of activated GPIIb/IIIa (alphallb/betaS integrin) with fibrinogen and other ligands are a promising family of anti-thrombotics now widely used to prevent adverse events following coronary angioplasty. Acute, severe thrombocytopenia, often occurring within hours of first exposure to one of these agents, is a recognized side effect of all drugs in this class. In the previous period of support, we obtained evidence that drug-specific antibodies, which can be naturally occurring (or at least pre-existing), are the major cause of this complication, characterized clinical and serologic aspects of this group of disorders and obtained evidence that the responsible antibodies recognize ligand (drug)-induced structural conformers of GPIIb/IIIa. We now propose to extend these observations with the following specific aims: 1) Characterize epitopes on GPIIb/IIIa recognized by antibodies causing thrombocytopenia in patients treated with GPIIb/IIIa inhibitors. Epitopes on ligand-occupied GPIIb/IIIa for which this apparently unique class of immunoglobulins is specific will be defined at the molecular level. 2) Develop new methods for identification of clinically significant antibodies not detected in conventional immunoassays. We hypothesize that platelet destruction in a significant subset of patients is caused by low affinity antibodies not detected in most immunoassays and propose to improve diagnostic yield with assays that a) detect antibody binding in real time and/or b) increase the Ka by preserving the structural integrity of the target. 3) Characterize the incidence, clinical significance and genetic origin of pre-existing ("naturally occurring") immunoglobulins (NA) that recognize GPIIb/IIIa-inhibitor complexes. The incidence of potentially "dangerous" NA specific for ligand-occupied GPIIb/IIIa in the general population, their genetic origin and their relationship to antibodies causing thrombocytopenia will be defined and their implications for platelet physiology and transfusion therapy and for the design of "safe" GPIIb/IIIa inhibitors will be explored. Findings made are expected to elucidate a previously unrecognized mechanism of disease resulting from the immune response to conformational changes induced in an integrin by its ligand or a ligand-mimetic drug and to advance understanding of the role of "natural" antibodies in health and disease.


1) Detailed laboratory procedures for isotachophoresis, "quantitative" PAGE and electrofocusing on polyacrylamide gel were written; 2) An apparatus for packing of multiple 6 and 18 mm Sephadex electrofocusing gel tubes was designed and constructed; 3) a zwitterionic monomer was synthesized, polymerized to a cross-linked gel and tested for its capacity of bridging conductance gaps in EF and its effect on protein mobilities; 4) Restacking of unstacked protein zones, postulated by the Jovin theory of multiphasic buffer systems, was demonstrated for the first time and applied to the simultaneous isolation of several hCG isohormones; 5) Purify of protein isolation by Steady-State Stacking was greatly improved by replacing polyacrylamide with agarose in the extraction and concentration step.


Project Abstract: Aging is associated with enhanced inflammation. An altered ratio between angiotensin receptors AT1R and AT2R results in induction of inflammation in animal models. The effects of aging on the expression of AT1R and AT2R in humans and the contribution of changes in AT1R and AT2R to increased inflammation in the older have not been previously studied. Our preliminary evidence suggests that frail older adults have up-regulation of AT1R, down-regulation of AT2R expression and implicate for IL6 in this imbalance. We hypothesize that human frail aging is associated with up-regulation of AT1R and down-regulation of AT2R expression and function in immune system cells. We hypothesize that these receptor changes contribute to decreased immune system cells phagocytic capacity and to the increased production of inflammatory cytokines in older individuals which will further heighten the divergence in AT1R and AT2R expression. In order to test these hypotheses, we propose a comprehensive study of AT1R and AT2R using immune system cells (lymphocytes and monocytes) from young, healthy adults (age 20-30) and four comparison groups that consist of (1) robust, older adults (age 70-90), (2) robust, older adults (age 70-90) treated with AT1R blockers, (3) frail, older adults (age 70-90), (4) frail, older adults (age 70-90) treated with AT1R blockers. From these subjects we will collect lymphocytes and monocytes that will be utilized for the following proposed studies: 1. Measure changes in gene expression, protein synthesis, and signaling pathways of AT1R and AT2R in immune system cells from young control (20-30Y) and the four comparison groups (N=33 in each group) using Q-PCR, western blot, confocal microscopy, flow cytometry and Bio-Plex Phosphoprotein Cellular Signaling Assays. 2. Evaluate age-related difference and contribution of Angiotensin receptors blockade to monocytes phagocytic function by incubating immune system cells from the same individuals with specific AT1R and/or AT2R blockers and measuring changes in monocytes phagocytic function with Phagocytosis Assay at baseline and in response to treatment(s). 3. Evaluate contribution of AT1R and AT2R to cytokine production in the older individuals by incubating immune system cells from the same individuals with specific AT1R and/or AT2R blockers and measuring cytokines with ELISA and Bio-Plex cytokine assays at baseline and in response to treatment(s). 4. Assess the feedback of inflammation on AT1R and AT2R expression and function by incubating immune system cells from the same subjects with IL-6. Q-PCR and western blot will be used to quantify the change in expression of AT1R and AT2R in response to IL-6 treatment.


This study is designed to examine the immune regulatory functions of monocytes and lymphocytes as a function of age of otherwise healthy subjects and to examine co-factors in the development of tuberculosis particularly as regards the immune status.


The research is designed to evaluate the relative roles of the cell mediated and humoral arms of the immune system in experimental candidiasis. Unmodified (normal) mice and mice immunosuppressed following adult thymectomy or treatment with cyclophosphamide will be studied from various perspectives. First, the development of cell-mediated immunity in such animals will be studied in vivo by footpad testing and in vitro by assays for lymphocyte transformation and migration inhibition factor. Three extracts of Candida albicans will be studied in the in vitro assays, but one extract, designated ppt-HEX, will be studied in more detail both in vivo and in vitro. Secondly, since normal mice develop resistance to reinfection following cutaneous infection, attempts will be made to passively transfer such resistance to naive normal and immunosuppressed mice with cells or serum from normal immune mice. If cells transfer the resistance, attempts will be made to abrogate the transfer by preincubation with anti-theta serum. Thirdly, cutaneous lesions developing in normal and immunosuppressed mice in response to one or two inoculations of viable C. albicans yeasts will be analyzed at selected intervals following inoculation for the number of colony forming units, and by immunofluorescence techniques for the infiltration of T and B lymphocytes into tissue surrounding the lesions. Finally, if time permits, additional studies are planned to assay for cell-mediated immunity in animals infected intravenously, to investigate the specificity of ppt-HEX, and to attempt to induce resistance to reinfection by artificial immunization with subcellular fractions of Candida.


Volunteers will have their sway measured on two separate outpatient visits using the Biomechanics platform.


An integrated approach has been taken to elucidate important factors contributing to the pathogenesis of microangiopathy. Dynamic studies measuring mean retinal circulation time by means of fluorescein retinography will be continued to be performed in populations of normal subjects, prediabetics, mild diabetics and severe diabetics. A simplified, but highly standardized system for obtaining consistent fluorescein retinograms including an automatic scanning microdensitometry system and a computerized format for the computation of circulation times has been perfected. A particular heavy focus will be placed on prospective studies of retinal blood flow in all of the above named populations. Relationships between the circulation time and metabolic factors such as the degree of carbohydrate intolerance, the magnitude of serum insulin and growth hormone responses during a variety of stimulation tests and plasma lipids, such as triglyceride and cholesterol will be sought in the milder diabetic patients. In the more severe diabetic patients the duration of the severity of diabetes and the degree of control will be placed in juxtaposition to the circulation time parameters.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have reported on experiments in vitro showing that in response to live Borrelia burgdorferi (Bb) and its purified outer surface lipoprotein A (L-OspA), IL-10 dampens the prototypic IL-6, IL-12, TNF-alpha and IL-1beta inflammatory responses in mouse J774 macrophages. We also showed that mouse macrophages incubated with IL-10 together with either live Bb or L-OspA additively augmented the expression of the suppressor of cytokine signaling (SOCS) 3 in these cells. In this study we first used a chemokine/cytokine multiplex approach to determine the extent of the IL-10-mediated inhibition of inflammatory mediators in macrophages in response to live Bb and L- OspA. RNA interference was then used to silence socs3 gene expression in macrophages to determine the effect of silencing on IL-10 inhibition of inflammatory mediators in response to live Bb and L-OspA. Multiplex analysis revealed that IL-10 significantly (P0.05) down-regulated the production of several cytokines (TNF-alpha, IL-12, IL-6, IL-1alpha, IL-1beta, IL-9) and chemokines (G-CSF, CXCL1, CXCL5, CXCL10 and CCL5) in macrophages stimulated with live Bb or L-OspA. In all instances live Bb induced higher levels of cytokines and chemokines than did L-OspA. All mediators induced by both stimulants in macrophages were down-modulated by IL-10 in a similar fashion and correlated with enhanced SOCS3 expression in these cells. Silencing of SOCS3 expression in macrophages abrogated the IL-10-anti-inflammatory actions against IL-6 production in response to live Bb and L-OspA. This study demonstrates that SOCS3 in part mediates the IL-10 anti-inflammatory effect in macrophages stimulated with live B. burgdorferi spirochetes and lipoproteins. Comparisons of macrophage genome-wide transcriptomics data before and after the silencing of SOCS3 will ultimately identify the extent of the SOCS3-mediated anti-IL-10 inhibitory effect in response to B. burgdorferi.


Experiments are proposed which will determine mechanisms governing synthesis and assembly of acetylcholine receptor. The nicotinic ACh-receptor is an integral membrane glycoprotein of muscle which binds ACh release the nerve. The mechanism by which ACh binding results in a change in membrane permeability culminating in muscle contraction is under intensive study in a number of laboratories. Mechanisms governing receptor synthesis and destruction also deserve intensive study because these are important to the overall function of AChR during development, disease, and regeneration. The synthesis of ACh-receptor as well as other specific proteins is regulated by nerve induced muscle activity. My goal is to study the regulation of synthesis and assembly of the ACh-receptor at the molecular and cellular level. Studies will include the development of immunochemical methods for detection of nascent receptor polypeptides and receptor synthesis in cell free protein syntheiszing systems. Combination of these techniques will provide a means for direct study of receptor synthesis at the translational and transcriptional level. Studies of ACh-receptor regulation should serve as a model for the study of other receptors. Control of receptor synthesis may be involved in the regulation of synapse formation, and thereby in the mechanism of establishment of synaptic specificity. Regulation of ACh-receptor content is perturbed in the experimental autoimmune animal model for Myasthenia Gravis. Studies of this experimental model may lead to improved therapeutic treatment of the human disease. They may also serve in understanding other receptor diseases.


Heart failure is the scourge of the elderly accounting for nearly $35 billion dollars and causing substantial morbidity and mortality; ~50% of all aged patients with CHF have heart failure with a preserved ejection fraction (HFpEF), a multi-factorial condition associated with several abnormalities in diastolic function. Stimulated by previous work from this research program the global objective of the next 5 years is to test novel strategies to prevent and reverse the impaired diastolic function associated with sedentary aging and HFpEF. These include: A) novel exercise training strategies implemented early enough in life while CV plasticity still exists; and B) pharmacologic probes of underlying pathophysiology targeted to improve relaxation in the elderly and HFpEF patients, and restore functional capacity. The aims of the program are: Specific Aim 1: to test the hypothesis that exercise training implemented 4-5 times/week for 2 yrs in sedentary middle aged men and women (45-64yr) will improve cardiac and vascular compliance to a degree equivalent to life-long exercisers (and sedentary young). We will perform invasive and non-invasive assessment of cardiovascular structure and function before and after an exercise program involving high intensity aerobic intervals, lower intensity endurance and strength training or yoga control. Specific Aim 2: to test the hypothesis that the stiff, slowly relaxing heart of patients with HFpEF causes a marked elevation in pulmonary capillary pressure during exercise which leads to premature fatigue prior to achieving maximal HR, thus causing apparent chronotropic incompetence. We further hypothesize that both sedentary aged and HFpEF patients have slowed relaxation due to down regulation of SERCA2a activity, the putative cellular mechanism underlying age related impaired lusitropic function. We plan to perform 2 sets of experiments for this aim: 1) to measure the HR response to two separate interventions: a) maximal activation of central cardiovascular pathways (central command) from static handgrip at 40% maximal voluntary contraction to fatigue; b) incremental doses of isoproterenol in the face of ganglionic blockade to isolate ? adrenergic responsiveness without reflex compensation; and 2) to perform invasive and non-invasive assessment of cardiac function at rest and during exercise in patients with HFpEF compared to age matched and young controls: a) after infusion of istaroxime an experimental drug with prominent lusitropic properties due to its ability to upregulate SERCA2a activity; and b) after Na+/K+ ATPase inhibition control (digoxin). After these aims are accomplished, we will have established a novel, practical exercise training strategy designed to prevent the cardiovascular stiffening with aging, and ultimately to prevent HFpEF. Such a determination would have enormous public health significance since this condition is quite difficult to treat once established. In addition, we will use novel physiological and pharmacological probes to determine the mechanism of the apparent chronotropic incompetence and impaired exercise capacity in patients with HFpEF which has the potential to open up new therapeutic options for this challenging syndrome.


The general purpose of the proposed research is to gain an understanding of the true dynamic picture of proteins and membranes rather than the more common static picture. The off-resonance rotating frame spin-lattice relaxation technique we have just developed will permit the investigation of the rotational motion of proteins and internal motions of different parts of proteins. The influence of substrates, inhibitors, and allosteric effectors on those motions will be explored. We have also demonstrated that spin-lattice relaxation in the rotating frame can be used to determine the lateral diffusion coefficient of a phospholipid in a model membrane bilayer. This technique will be employed to determine the lateral diffusion coefficient of lipids in model membranes and biological membranes as a quantitative parameter characterizing membrane "fluidity" which is an important factor for various membrane functions. In particular, the effect of the composition of the phospholipid, temperature, phase, and presence of other membrane constituents such as other phospholipids, cholesterol, and membrane proteins will be examined. The effect of general and local anesthetics on the lateral diffusion coefficient of the phospholipid in the bilayer will also be investigated.


Compare the safety and virologic efficacy of nelfinavir and DMP-266 when used separately or in combination with nucleoside-containing regimens for patients previously in ACTG 302 or 303.


We plan to continue our studies on the mechanisms of action of antitumor antibiotics. In particular, we are studying the nature and mechanism of the damage induced in DNA by the protein antibiotic, neocarzinostatin. Such damage can be induced in HeLa cells in culture or in vitro with isolated DNA. We are exploring the possible relationship between the two observations.


The overall objective of our study is to understand the manner in which the genetic information of mammalian cells is expressed and regulated and to determine how these processes are altered in cancerous cells. Our immediate goal is to describe the steps involved in the synthesis of mRNA in the nucleus, any modifications that it may undergo before being expressed in the cytoplasm, and finally its fate during and after translation. In addition we wish to characterize the early events associated with globin mRNA synthesis in erythroleukemia cells. These studies are in extension of our earlier work concerned with the identification, isolation and characterization of the rabbit and mouse globin mRNAs. During this time we have successfully translated these mRNAs in a variety of cell-free systems, described their translation efficiency, purification, poly(A) content, molecular weight, methylated base content, presence in ribonucleoprotein particles and their distribution between membrane bound and free polysomes. We have initiated studies concerning the synthesis of globin mRNA in the nucleus and described a specific poly(A) shortening mechanism. Our immediate aims are: (1) to continue our studies on the shortening of the poly(A) sequence of mouse globin mRNA, (2) to describe the location of the methylated nucleosides in globin mRNA, (3) to isolate and characterize the globin mRNA precursor, (4) to measure the synthesis and turnover of globin mRNA following the committed division in erythroleukemia cells and (5) to ascertain if post-transcriptional regulation occurs during differentiation of these cells. The ability to isolate molecules containing globin mRNA sequences using mRNA specific affinity column prepared by attaching globin cDNA to cellulose is an important technical advance which will aid these studies. We have prepared such a column and found it successfully isolates globin mRNA. The column will now be used to prepare precursors to globin mRNA.


The University of California, Berkeley School of Optometry (UCBSO) seeks 5 years of support to train clinician-scientists in accordance with the guidelines of the Institutional Clinical Scientist Development Program (K12). The Berkeley Clinical Scientist Development Program will provide interdisciplinary training to enable well-established clinicians to carry out high-level, independent, clinical (patient-based) vision research. The University of California, Berkeley (UCB) K12 program will draw upon the rich resources of world-renowned departments and groups on the Berkeley campus, including Vision Science, Optometry, Public Health, Neuroscience, Infectious Disease, Bioengineering, Health and Medical Science, and Molecular &Cell Biology. The Berkeley K12 Program will feature two unique elements: (1) The recruitment of established clinicians who wish to make a career shift from clinical practice and teaching to one centered on the combination of teaching and independent, federally funded, patient-based vision research. (2) The multidisciplinary composition of a distinguished and diverse mentoring group composed of faculty with expertise in vision research, plus faculty whose primary expertise is not in vision, but who have extensive experience in clinical research. The program will provide the awardees with focused, cutting-edge didactic and mentored clinical research training tailored specifically to the individual's career plan. The curriculum will consist of course work, seminars, research in campus-wide laboratory facilities and clinics, and closely mentored supervision from a group of nationally recognized scientists and clinicians. Trainees will conduct research in one of seven clinical research areas: Retinal Disease, Corneal Disease, Strabismus, Amblyopia, Visual Processing, Low Vision &Blindness Rehabilitation, and Lens &Cataract - all of which are consistent with the Programs and Research Priorities of the National Eye Institute. The Berkeley program is designed so that upon completion, the trainees will be able to compete successfully for research resources and maintain a productive and independent career in patient-based vision research. Several strategies will be used to recruit established clinicians (e.g., OD, MD, DVM, DDS, or equivalent professional degree) whose clinical expertise is either within or outside the ophthalmic community. Four trainees would be recruited during the first year, and then one additional trainee admitted in each of the following four years (total admitted by 5th year = 8).


It is proposed to investigate the use of somatic cell gene therapy for the correction of genetic diseases using domestic animals as test systems. A large number of models of human genetic disease have been discovered in domestic animals and characterized in detail at the Univ. of Pennsylvania School of Veterinary Medicine. Among these diseases are the mucopolysaccaridoses (MPS) which are recessive and result from defects in single genes encoding enzymes involved in the degradation of glycosaminoglycans (GAGs). Breeding colonies of MPS cats and dogs have been established. Thus, diseased animals are immediately available as test subjects. MPS cells can endocytose exogenous normal enzyme and degrade GAGs. Therefore, the experimental strategy will be to construct retrovirus vectors containing cDNA copies of normal genes, introduce the vectors by infection into bone marrow cells, and autologously transplant the cells to repopulate the donor/patient with cells producing normal enzyme. The sponsors, Drs. D. Patterson and M. Haskins, are members of the University-wide NIH Human Genetics Center and have extensive interactions with scientists working on genetic diseases in man. However, there is no one at the Veterinary School who has the expertise in molecular genetics who can concentrate on constructing and testing the vectors that are needed to perform the proposed experiments. Therefore, it is proposed that the candidate, Dr. John Wolfe, receive the necessary further training in molecular genetics to enable him to conduct these experiments. Therefore, it is proposed that the candidate, Dr. John Wolfe, receive the necessary further training in molecular genetics to enable him to conduct these experiments. He is a graduate of the NIH Veterinary Medical Scientist Training Program where he received training in immunogenetics, retrovirology and protein chemistry and is now an Exxon Fellow in Molecular Genetics at Sloan-Kettering Institute. Thus, he has training in most of the techniques needed to do the proposed experiments. A leading researcher on retroviral vectors, Dr. E. Gilboa of Princeton Univ., has agreed to collaborate with him on the construction and evaluation of the vectors. Dr. R. Schmikel of the Medical School who is the principle investigator of the Genetics Center and Dr. R. Brinster of the Veterinary School who is a leading investigator in the technology of gene transfer into embryo cells have agreed to participate in Dr. Wolfe's training. It is anticipated that the proposed training in recombinant DNA technology will enable him to establish a laboratory at the Veterinary School that will concentrate on the molecular aspects of genetic diseases and gene therapy using the domestic animal models at test systems.


This second revision of an R29 application seeks to advance our understanding of the molecular mechanisms by which the movement of water across the human cervical epithelial barrier. The applicant will use cultured human epithelial cell models developed in his laboratory to explore three hypotheses: 1) that estrogen increases transcervical permeability by means of an effect on cervical cell tight junctions, 2) that estrogen increases calcium mobilization in the cells, which in turn increases the permeability of the intercellular space, and 3) that the mechanism of Ca++ mobilization in the human cervical cell is a Ca++-dependent stimulation of cellular KCl transport, which results in a reduction in cell volume. It is proposed that the results of these studies will increase both our understanding of the basic biology of cervical fluid homeostasis, but also a better understanding of clinically relevant settings in which the cervical fluid status is abnormal.


The generally held view that the immobilization of ciliated cells by specific immune serum results from antibodies cross-linking adjacent cilia is only superficially correct. Our current study has shown that the ciliary agglutination occurs only in living cells and is characterized in its later stages by a fusion into a continuous unit membrane of those ciliary membranes that come into contact with one another. In a number of key aspects, this agglutination process resembles the "capping" observed in other systems more than it resembles microorganisms or cell suspensions treated with specific antiserum. Globular material is seen to accumulate at the ciliary tips by phase contrast and fluorescence microscopy. When cells are examined by SEM, the fused ciliary tips are seen to be distended, discoidal membranes. TEM often reveals several ciliary axonemes within a single, enlarged membrane that is oriented with the ferritin-labelled second antibody directed against the i-antigen antibody on the outer surface only. Fixed cells or living cells treated with immune Fab do not show membrane changes, but do bind antibody. Membrane fusion occurs only if cells are alive and the i-antigen is cross-linked by intact immune IgG. In addition, the redistribution of the immune complex appears uneffected in the presence of colchicine. In conjunction with these studies on the immobilization of cells, the surface i-antigen designated C was purified and characterized.


Early clinical reports of severe morbidity and mortality in infants born passively addicted to opiates are being reinterpreted in light of recent reports of an improved prognosis in children born to women on methadone maintenance programs who avail themselves of antenatal and well-baby clinics, and in whom illicit polydrug abuse and poor nutrition are kept to a minimum. Most of the preclinical animal literature dealing with effects of methadone during development would suggest that methadone should never have been released for clinical use by people of childbearing age or who are pregnant. However, we believe that there are many flaws in the design and interpretation of these experiments and have completed or are in the process of extending a series of experiments with the opiate 1-alpha-acetylmethadol (LAAM) or its active metabolites in which we can duplicate many effects reported for methadone, but which appear to be preventable or nonexistent, if careful attention is paid to animal modeling and potential epiphenomena. We propose to continue these studies as well as incorporate several new studies to determine the relevance (i.e. generality or specificity) of our observations with LAAM, to study the consequences of hypoxia and hypercapnia equivalent to that produced by (acutely toxic) doses of methadone typically used by other laboratories for such studies. Very little is known about the effects of cocaine on the developing child, when exposed in utero. The dearth of data is more recently being added to by anecdotal, small group or clinical case reports of neonates born to cocaine abusers who also invariably are polydrug abusers. This makes it difficult or impossible to separate direct from indirect effects, as well as effects due solely or primarily to cocaine exposure, except in those instances where clearcut acute toxic manifestations are responsible (e.g. subdural hematoma or other evidence of CVA or seizures). Even then, combinations of multiple drug exposure, poor nutritional or health status (i.e. infectious hepatitis due to i.v. drug self-administration) may be more responsible for morbidity/mortality of cocaine-exposed neonates. We propose to study the effects of cocaine, administered at different doses, to pregnant rats (prior to and throughout pregnancy or at different stages of pregnancy) or to breeder male rats in order to assess the neurobehavioral consequences in offspring of such exposure. Rats will be tested in adulthood, after exposure prior to fertilization (gametes) and during in utero development. Treatment of neonates with cocaine and drugs known to attenuate or block opiate withdrawal in rats and/or humans will be used to determine if cocaine-exposed neonates show morbidity or mortality as a result of residual cocaine or if toxicity and/or a withdrawal syndrome is treatable during the neonatal period.


PNU-142731 is a pyrrolopyrimidine, non-corticosteroid anti-inflammatory agent developed by the Pharmacia & Upjohn Pharmaceutical Company to be used in asthma patients. It is an oral, once daily agent proposed for use in the chronic treatment of asthma. Animal studies have shown that PNU-142731 can ameliorate both the early-phase and late-phase asthmatic response. Three human trials have shown that PNU-142731 is tolerated well by humans. The proposed study is a double-blind, randomized, placebo-conrolled trial of two doses of PNU-142731 to evaluate the efficacy and safety of PNU-142731 in subjects with moderate persistent asthma. The end-points of the study include direct measures of asthma including lung function, daily symptom logs, and beta-agonist use as well as indirect measures of asthma such as assessment of inflammatory markers in induced sputum and serum. A secondary purpose of this study is to validate the use fo asthma quality-of-life (QOL) questionnaires in clinical trials."


Plasma high density lipoprotein (HDL) elicit their anti-atherogenic effect by transporting cholesterol from arterial macrophages to the liver for degradation, a process known as reverse cholesterol transport (RCT). Current HDL therapies are inadequate and new interventions for increasing plasma HDL levels and RCT are needed. HDL contains exchangeable apolipoproteins (apos) that are potential targets for HDL therapy. These include apo A-l, the major protein, and apos A-ll and E, which contain cysteines that are the sites of apo homo/heterodimerization. This project will fill gaps in our knowledge about HDL assembly, structure, and properties by testing new hypotheses about a] the mechanistic coordination of HDL phospholipidation and the dimerization of apos E3 and A-ll, b] the configurations of apos on HDL subfractions sorted by size and apo composition, and c] the hepatic production and phospholipidation of HDL-apos. These hypotheses will be addressed by the three specific aims: Aim 1: To use chemical kinetics to identify physico-chemical determinants of apo dimerization. This aim addresses a fundamental question about intracellular oxidative folding that is applicable to other cysteine-containing lipophilic proteins, including apo B. Aim 2: To use peptide mapping with and without chemical cross-linking and mass spectrometry to determine the apo configurations on native and reconstituted HDL. The configurations of apos on various HDL species determine HDL interactions with cell surface lipid transporters and with hepatic HDL receptors. Aim 3: To identify the itinerary of nascent HDL speciation in hepatocytes with respect to apo phospholipidation and in the case of cysteine-containing apos, the homo/heterodimerization of apos A-ll and E, by steady state and pulse chase labeling methods. Fibrates, which increase apo A-ll synthesis, produce modest elevations of HDL-C, an effect that might occur through altered HDL biogenesis. Distinct mechanisms for assembly of each apo into HDL could provide distinct therapeutic targets for increasing plasma HDL-C and RCT. Obesity-linked diabetes, a serious and growing public health problem in the United States, is associated with a cluster of lipid risk factors that include low plasma HDL-C. Although niacin and the statin class of hypolipidemic drugs can increase HDL-C, their effects are modest and better HDL therapies are needed. The information derived from our research would permit a rational approach to the development of new interventions that could operate at the level of the three main components of RCT-hepatic HDL production, remodeling in the plasma compartment, and hepatic HDL-cholesterol uptake.


The study will investigate the pathophysiologic effects of both acute and chronic treatment of rats with various amount of cadmium. The principal focus wwll be on cardiovascular changes, and will include effects on venous and arterial pressure, on cardiac activity and upon neural control of these functions. Histologic examination of tissues will be correlated with altered functional activity observed during life. Different pharmacological agents, including other metallic radicals, will be used to modify or alter the primary responses with a view to gaining further sight into the mode of cadmium's action. The responses will be correlated with sex, size, and strain, and with concurrent changes in body and urinary electrolytes and hematology. There are circumstances under which cadmium treatment may induce changes in carbohydrate metabolism, either hypoglycemia or hyperglycemia. These will be explored in depth.


This project aims to investigate the unique regulation of ornithine decarboxylase (ODC), the key rate limiting enzyme of polyamine biosynthesis, involving a unique protein termed antizyme. Antizyme binds to ODC, inhibits ODC activity and targets the degradation of ODC by the 26S proteasome without poly-ubiquitination. In our current studies we have been studying the stoichiometry and characterization of this interaction. For this purpose we have purified both yeast ornithine decarboxylase and yeast antizyme to homogeneity both for attempts at crystallization and for biophysical studies on the interactions of the two proteins. Recently we have developed methods to purify the ODC:antizyme complex and studied their interactions by various biophysical and biochemical techniques (Gel filtration, size exclusion-static-light scattering, dynamic light scattering, equilibrium ultracentrifugation, CD spectra). The binding affinity for yeast AZ to yeast ODC is 5X10-11 M. Using purified His-tagged AZ as a binding partner, we have purified the AZ:ODC heterodimer, which has no ODC activity. The native molecular weight of the complex is 90 kDa, which suggests a 1:1 stoichiometric binding of AZ and ODC in vitro. Circular dichroism (CD) spectra of the two individual proteins and the ODC:AZ complex show no significant change in the secondary structure of either ODC or AZ after binding to each other. These results indicate that yeast antizyme and ornithine decarboxylase form a heterodimer, consisting of each monomer and that there is no change in secondary structure in the AZ:ODC complex.


We propose to investigate the role of circadian rhythms in photoperiodic time measurement. One of the goals is to establish the relation between biological clocks, photoperiodism and seasonal reproductive activity in representative mammals. The participation of the nervous and endocrine systems in these phenomena will be detailed. The overall goal is to describe the ways in which temporal integration of the animals' internal milieu is affected and the fashion in which behavior is synchronized with cyclical fluctuations in the external environment.


The goal of this R21 application is to refine an experimental design to examine the impact of commonly used pain medications on sleep and next-day fatigue. However, before such studies can be conducted in patients with acute or chronic pain associated with cancer or other severe chronic illness, crucial information is needed to optimize the study design. This proposal seeks to answer certain experimental methodological design questions that can be safely, quickly, and inexpensively addressed in healthy volunteers. This R21 application will generate data guiding the research design and power estimates for subsequent RO1 applications intended to address the impact of pain medication on sleep and fatigue in patients with acute or chronic pain. There are 5 specific aims: 1. Characterize the effect of a typical nighttime dose of MS-Contin and methadone on cytokines, polysomnographic sleep and on rest/activity patterns in healthy volunteers: 2. Characterize daytime fatigue the day after drug administration with self-reported measure of fatigue and mood 3. Characterize neuropsychological performance on the day after drug administration. 4. Estimate the covariance structure of sleep measures to assess the comparative advantages of a crossover or parallel groups design. 5. Evaluate the necessity of repeated first nights and whether this is the optimum design for follow-up studies. Over a two-year period, 50 healthy volunteer subjects will be examined with polysomnography, actigraphy, and neuropsychological tests. Data will be collected on self-reported mood and fatigue, as well as proinflammatory cytokine levels (IL-6, IL-1beta, and TNF-alpha. The study design involves a double-blind, placebo-controlled crossover. Each individual will be admitted to the UCSD GCRC on 3 pairs of nights. Each admission will feature an acclimation night in the sleep laboratory followed by a night providing placebo, MS-Contin (15 rag) or methadone (5 mg). On the morning after each dosing subjects will complete fatigue and mood ratings and will perform a brief neuropsychological test battery. They will also undergo actigraphy monitoring for 3 continuous days to examine whether there are subtle lingering effects of the drugs. Blood levels ofproinflammatory cytokines will be obtained at various points before and after drug dosing and the next morning to test the hypothesis that opioid-induced changes in these cytokines are associated with sleep disruption and increased fatigue.


The long term objective of this proposal is to increase the sensitivity of DNA concentration measurements by 1000X over the state of the art. In addition, the microfluidic platform we will develop to accomplish this will also enable, for the fist time, the targeted recovery of genome-size DNA fragments out of a heterogeneous sample. Nucleic acid analysis through technologies like microarrays and next-generation sequencing are transforming the way that many biological questions are addressed. The power of these approaches stems from their ability to obtain system-wide information with single molecule detail. However, a major technical barrier that often prevents their effective application is that many biological systems are too complex to be straightforwardly sequenced: Even with bioinformatic algorithms, the billions of short, sequence dreads are often too complex to piece together into useful information. To address this challenge, we will develop a technology that allows nucleic acids to be quantitated and sorted in a heterogeneous sample. Importantly, this method will utilize specific multiplexedTaqMan PCR assays performed in giant-unilamellarvesicles (GUVs); this will make it much more specific and targetable than methods that sort DNA based on size or staining properties. Moreover, by assaying individual molecules or cells in GUVs, we will be able to perform billions of PCR assays in parallel, enabling massive, heterogeneous samples to be screened to identify and recover extremely rare targets. This basic technology will be broadly useful throughout biological research and has immediate human health impacts, including for detecting and sequencing cancer DNA in the blood early in the disease, analyzing genetic heterogeneity in tumor cells, and identifying immune cells latently infected with HIV. The aims are: Specific Aim 1: Demonstrate microfluidic generation and FACS sorting of thermostable femtoliterGiant-Unilamellar Vesicles. We will develop the microfluidic hardware and processes for generating billions of monodisperse femtoliter GUVs. We will also optimize processes to FACS GUVs, both into positive and negative pools and, individually, into wells on a microliter plate. Specific Aim 2: Optimize and characterize GUV-PCR and benchmark against aqueous droplets. We will explore different PCR reagents to optimize the GUV-PCRs and benchmark the efficiency of these reactions against ones performed in aqueous-in-oil droplets generated with our own microfluidics and with the Bio-rad QX100 digital PCR machine. We will measure efficiency using endpoint fluorescence, fraction of positive reactors, and yield of DNA recovered out of the reactors. We will also optimize protocols for rupturing GUVs to access their contents. Specific Aim 3: Demonstrate 1000X greater sensitivity than competing platforms by performing over 1 billion digital GUV-PCRs, and recovery of positive molecules with FACS. We will demonstrate the superiority of GUVs for digital PCR by performing 1000X more reactions than is possible with existing droplet technologies. We will also demonstrate the ability to recover rare DNA molecules by FACS sorting the positive GUVs.


1. In vivo anti-tumor activity of CD3-AK cells. (A). CD3-AK cells were induced by activation of mouse splenocytes with alphaCD3-AK antibody. The conventional LAK cells were induced by activation of mouse splenocytes with purified human recombinant IL-2. We found that different tumors showed different degrees of susceptibility to the in vivo killing by CD3-AK or LAK cells and the results also correlated with the in vivo effect of the killer cells on these tumors. A plasmacytoma P815 was much-more susceptible to the killing by CD3-AK than by LAK cells, and the in vivo growth of P815 cells was inhibited by simultaneous inoculation of CD3-AK and was not affected by LAK cells. In contrast, a melanoma JBMS was susceptible to the killing by LAK but was resistant to - CD3-AK mediated killing, and the in vivo growth of JBMS was retarded by LAK cells and was not affected by CD3-AK. Due to the ability of CD3-AK to expand rapidly in vitro, CD3-AK may be an attractive candidate for immunotherapy of susceptible tumors. 2. Mechanisms for lymphocyte-mediated cytolysis. (B). Two subsets of killer cells were induced by alphaCD3. The killer cells that were maintained in IL2 and alpha-CD3 (CD3-AK+) induced fast lysis, whereas the killer cells that were maintained in IL-2 alone induced slow lysis. Protein kinase C (PKC) was not required in fast lysis but was required in slow lysis. Cytolytic granules might be involved in fast lysis but had very little effect in slow lysis. On the other hand, cytokines such as IL-2, IL-4 and TNF were involved in slow lysis but had no effect in fast lysis.


Our objective is to define structural events at the cellular level in intestinal antigen processing. Our investigations are focused on Peyer's patches where M cells in the epithelium take up soluble and particulate antigen, then transport it to lymphocytes. Giardia which are too large to be transported by the M cell penetrate the intestinal epithelium over Peyer's patches and are taken up by macrophages. We will determine by morphometric analysis whether lysosomal volume within the cytoplasm of M cells is reduced compared to adjacent columnar cells. This may explain how M cells can transport luminal material without degrading it. We will determine whether lymphocytes within M cell interstices are B or T cells using peroxidase labeled antibodies. The pathway of radiolabeled ferritin from Peyer's patch through the circulation to other peripheral lymphoid organs will be determined by autoradiography and scintillation counting. Traffic of lymphocytes to lymphoid follicles will be studied using scanning electron microscopy of fractured post capillary venules. The specificity of Giardia trophozoite uptake by macrophages will be studied in vitro to determine the effect of host and parasite strain and the influence of immune or non-immune serum on phagocytosis.


The aim is to gain information leading to a deeper and more coherent understanding of enzymatically catalyzed glycosylation, the process whereby complex saccharides of living organisms are synthesized from (and degraded to) their sugar components. A unique experimental approach, developed in our laboratory, is being used and has proved highly effective in providing fresh insight into the catalytic capabilities and mechanisms of a variety of enzymes considered to be well-characterized by traditional studies. This approach consists of the study of glycosylation reactions that occur without glycosidic bond cleavage. We have, for example, recently demonstrated that exo-alpha-glucanases are able to attack both alpha- and beta-forms of appropriate glycosyl fluorides, and that these enzymes, long considered strict hydrolases, have a second (nonhydrolytic) mode of action that is stereocomplementary to the well known (hydrolytic) mode. Studies in progress show, moreover, that exo-glucanases as well as alpha- and beta-glucosidases are able to create specific anomeric configuration anew from enolic substrates that lack alpha- or beta-configuration. From the results obtained with these several systems, a new mechanistic principle has emerged which may well be applicable to carbohydrases in general; namely, that the catalytic groups have functional flexibility beyond that expected from the principle of microscopic reversibility. A potential consequence of such functional flexibility is that a carbohydrase may not be limited to a single reaction mechanism with all substrates. This is already apparent with most of the enzymes being studied, and will be further investigated. We also plan to study the extent to which classic types of endo-glucanases (alpha-amylases, pullulanase, cellulases) can catalyze polymerative glycosylation reactions in which water is kept out of a succession of steps, using appropriate glycosyl fluorides as substrates.


The Dana-Farber Cancer Institute is an independent, federally dcsignated, Comprehensive Cancer Center affiliated with the Harvard Medical School. The aims of the Institute can be summarized as follows: (a) to develop and maintain clinical and laboratory research programs of high quality related to the biology, cause and prevention, diagnosis and treatment of cancer; (b) to provide total care for cancer patients on an interdisciplinary basis and in association with neighboring institutions; c) to serve as the major cancer center in the Harvard-Longwood medical area and to play a leadership role for cancer research and patient care in New England providing expertise in cancer diagnosis and treatment; (d) to add to the total oncology resources of the medical community in terms of available staff, facilities and expertise; (e) to participate in the teaching and training of medical students, house staff, fellows and graduate students and in the continuing education of practicing physicians and other health professionals, and to provide post-doctoral research training. To maintain these activities, the present application requests renewal of the Institute's Cancer Core Support Grant.


Many neurons of the cerebellum are spontaneously active, firing 10 to 100 action potentials per second even in the absence of synaptic input. This high basal activity correlates with information coding mechanisms that differ from those of cells in circuits that are generally quiescent until excited synaptically. For example, in the cerebellar nuclei, long-term changes in the strength of excitatory synaptic inputs are not generated by classical Hebbian rules of coincident synaptic excitation and postsynaptic firing. Instead, synaptic currents are potentiated by patterns of stimulation that combine inhibition and excitation, in a manner that resembles the activity of (inhibitory) Purkinje afferents and (excitatory) mossy fiber afferents predicted to occur during cerebellar associative learning tasks. Such results support the idea that cerebellar circuits have rules for information transfer and storage that distinguish them from other well studied brain regions. The present proposal is motivated by the question of how spontaneous firing sets the stage for plasticity that is independent of spike timing. In the proposed research, experiments will be performed on neurons of the cerebellar nuclei in cerebellar slices of mice. Voltage-clamp and current-clamp recordings of synaptic responses, ionic currents, and action potentials, as well as imaging of Ca signals in nuclear cell dendrites, will be directed toward identifying the mechanisms of potentiation of excitatory synaptic responses to mossy fiber input, as well as toward examining the influence of spontaneous activity in Purkinje afferents and nuclear cells on plasticity. The resulting data will provide general information about the fundamental properties of signal encoding across brain regions, as well as specific information about the ionic mechanisms underlying cerebellar synaptic plasticity under normal and pathophysiological conditions. PUBLIC HEALTH RELEVANCE: In the present work, we are studying cells in the cerebellum, a part of the brain that controls the learning and execution of coordinated muscle movements, and whose electrical and chemical signaling patterns are disrupted in ataxia, dystonia, dyslexia, and autism. Because signaling patterns by brain cells depend on specialized proteins called ion channels, we are studying the properties of these ion channels, with the goal of understanding how their activity leads to long-lasting changes in cerebellar signals that are important for motor learning. These data can be used to make comparisons to disrupted signals in the cerebella of animals that have ataxia as a result of genetic mutations of ion channels, with the goal of understanding what goes wrong under pathophysiological conditions and whether ion channels can serve as a target for therapeutic interventions.


Work under this contract includes: The establishment and maintenance of cancer reporting system in Pureto Rico (The system will utilize the standard abstracts of records of all patients with active disease seen in any of the Puerto Rican Hospitals.); The follow-up of patients to determine their vital status and end-results of cancer treatment; Providing National Cancer Institute with computer tapes containing cancer incidence and end results data; and Setting up a working relationship with other registries in Latin American countries regarding ad hoc studies and epidemiological investigations.


ACTG 303 - Influence of Risk Status for Disease Progression on the Response to Antiretroviral Interventions: A Follow-up Study to ACTG 175. The hypothesis of this trial was that HIV-infected patients who had received prolonged combination therapy with zidovudine (ZDV) plus didanosine (ddl) or ADV plus zalcitabine (ddC) would show virologic and immunologic improvement upon addition of lamivudine (3TC) or after changing to ADV plus 3TC. The virologic and immunologic responses of these patients was expected to be influenced by virus load, CD4+ cell count, biologic phenotype of the HIV-1 isolate (ability of HIV-1 to induce syncytium formation in vitro), and presence or absence of symptoms of HIV disease at the time the new treatment regimens are instituted. The primary goals outlined in this trial were to carefully characterize subjects who have a well defined nucleoside exposure history with respect to the symptomatic state, their CD4 count trajectory, and their virologic profile, and to relate this to the response to new antiretroviral interventions in HIV infected patients who had been treated either with monotherapy (ZDV or ddl) or dual combination therapy (ZDV/ddl or ZDV/ddC) while participating in ACTG 175. Results of this trial are not yet available.


Electron paramagnetic resonance (EPR) spectroscopy is being evaluated as a potential technique for the early detection of cancers and is being used to elucidate the nature of free radicals and macromolecular conformational changes involved in cancer. Detailed EPR measurements on ceruloplasmin obtained from blood samples of large numbers of patients with diagnosed cancer are being compared with same kinds of measurements on blood samples from selected groups of control volunteers to determine whether statistically reliable differences exist which might be used in screening patients for the early detection of cancers. The effects of surgery, chemotherapy, and radiation therapy on the EPR spectral propertis of plasma ceruloplasmin and certain spin labelled blood components are being determined for a large number of patients to evaluate statistically the use of these properties as reliable indicators of the success or failure of the therapy. A variety of techniques, including ENDOR and electron spin echo, are being employed to identify and characterize free radical changes and changes in ceruloplasmin molecules associated with cancer. The information provided by this study may verify that EPR can be successful as a screening technique in the early diagnosis of cancer and in the monitoring of cancer therapy.


The virtual slide scanner and quantitative software is absolutely essential in allowing pathology truth to define imaging truth. We are funded through NIH grants to undertake this important task for lung and prostate cancer in the human. The proposed instrumentation, the Olympus VS120 slide-scanning microscope with VisioMorph newCAST software is critical for the completion of several very important NIH funded research projects and NIH funded educational grants at the University of Iowa. Some of the immediate applications include distinguishing malignant from benign lung nodules, 3D reconstruction imaging of mouse, ferret and pig lungs as a baseline for animal models in cystic fibrosis animals, defining the development of cystic fibrosis phenotype in the pancreas. The successful completion of Dr. Henry's research will lead to identification of patients at risk for lethal prostte cancer at stages early enough for effective treatment. Dr. Hammond seeks clarification of the role of inflammation in chronic pain leading to enhanced therapies for a higher quality of life for chronic pain patients. Dr. Dunnwald and Dr. Murray are analyzing the role of the protein Irf6 in immune cells associated with wound healing. Dr. Bonthius will test the efficacy of gene therapy against Alexander's disease, a disease with devastating and lethal affects on infants and children. The virtual slide scanner will also support the microscopy component of a current National Library of Medicine funded educational research and resource proposal that is aimed at increasing the efficiency and cost effectiveness of laboratory test ordering in clinical medicin. Updating and expanding the Virtual Slidebox for medical, dental and graduate students. The Virtual Slidebox increases accessibility, student satisfaction and efficiency of learning. This database is also used to assess the diagnostic competency of resident physicians. Enhanced medical student and resident education will continue to improve the lives and health of countless patients.


Noninvasive prenatal genetic testing, which utilizes cell-free fetal DNA and advances in sequencing technology, is revolutionizing the practice of obstetrics. While currently used as a screen for a limited number of aneuploidies and genetic conditions, noninvasive testing is anticipated to employ whole fetal exome and genome sequencing to identify not only monogenic disorders but also microduplications, microdeletions, and variants of uncertain clinical significance. This will give rise to a vast and complex body of information that expectant parents and healthcare providers must be prepared to interpret. Given the impact of fetal genetic information on reproductive choices, it is critical that effectiv informed consent practices are in place to ensure pregnant women and their partners make informed and value-reflective decisions about incorporating genomic technologies into their prenatal care. However, there is a critical lack of knowledge about how to structure an informed consent process that effectively meets the needs and preferences of expectant parents and is responsive to the challenges posed by noninvasive prenatal genomic testing and the clinical practice of medicine. The primary goal of this study is to ensure patient-centered counseling and effective informed consent practices are in place for noninvasive fetal genomic testing. Our central hypothesis is that the current absence of data concerning the informed consent process for such tests will not only have serious clinical and ethical implications for the delivery of prenatal care but also interfere with 1) patients' informed access to emerging applications noninvasive testing and 2) the translational process of other new prenatal technologies from the bench to the bedside. This project has two aims: 1) to describe the components of an effective informed consent process for noninvasive fetal genomic testing from the perspectives of pregnant women and partners and 2) to determine obstetric and genetic healthcare providers' perspectives regarding approaches and barriers to an effective informed consent process for noninvasive fetal genomic testing. We will conduct this work in collaboration with a multidisciplinary team of leading experts in the field of obstetrics, genetics, ethics, law, and medical decision-making and use a combination of qualitative and quantitative methods. This project is innovative because it will provide a new framework for the informed consent process and bring in the perspectives of partners in decisions about the use of noninvasive fetal testing. This work is significant because it is the first step in a line of research expected to develop and implement clinically relevant strategies to support expectant parents' informed consent about the use of next generation sequencing technologies. These results are expected to have an important positive impact on public health, as informed consent is not only foundational to the ethical practice of medicine but also a key component of healthcare quality, access, and outcomes. We anticipate that this study's findings will ultimately contribute to empowering pregnant women and their partners to make informed choices that reflect their needs and preferences as individuals and parents.


The term pseudohypoparathyroidism (PHP) is used for three distinct disorders, PHP type Ia (PHP-Ia), pseudo-PHP (pPHP), and PHP type Ib (PHP-Ib). PHP-Ia is characterized by PTH-resistant hypocalcemia and hyperphosphatemia, impaired function of several other endocrine systems, and a variety of developmental/skeletal defects (Albright's hereditary osteodystrophy (AHO)), including short stature, obesity, impaired intelligence, and skeletal abnormalities. Patients with pPHP are also affected by AHO, but show no resistance toward PTH or other hormones. PHP-Ia and pPHP occur within the same family, and individuals affected by either form show, as a result of inactivating mutations, reduced activity of the stimulatory G protein (Gsa); the Gsa gene is located on chromosome 20q13.3. Recent findings provided strong evidence for paternal imprinting in PHP-Ia/pPHP (i.e. patients with AHO), and showed that the abnormal control of calcium homeostasis, occurs only if the disease is inherited from an obligate female carrier. PHP-Ib patients show only resistance toward PTH, but no AHO phenotype. Since PTH-resistance is the only discernible abnormality in PHP-Ib, the disorder was first thought to be caused by inactivating PTH/PTHrP receptor mutations, but these were excluded by us and others. To continue our search for the molecular defect in PHP-Ib without prior knowledge of its primary structure, we collected several large PHP-Ib kindreds, conducted a genome-wide search with greater than 350 polymorphic microsatellite markers, and obtained linkage with a maximal combined LOD score of 6.48 to chromosome 20q13.3 (the Gsa gene is in same region). Furthermore, we showed that PHP-Ib is paternally imprinted, as is PHP-Ia/pPHP. We now propose to further reduce the linked interval and to confirm the 20q13.3 locus with additional PHP-Ib kindreds (Aim 1), to determine whether Gsa mutations cause the disease, and, if this candidate gene can be excluded, to search for the PHP-Ib gene (Aim 2), to explore the role of the PHP-Ib gene with regard to PTH/PTHrP receptor function (Aim 3), and to explore the importance of the stimulatory G protein in endochondral bone formation using heterozygous Gsa gene ablated mice (Aim 4).


In the proposed study we intend to focus on the long term effects of increased cortisol activity in depressed patients. The major aims of the research are: 1) to Confirm that patients with psychotic major depression (PMD) exhibit greater cognitive impairment as measured by neuropsychological testing than nonpsychotic major depressed (NPMD) patients at baseline and to test the hypothesis that cognitive impairment at baseline correlates with plasma and urinary measures of cortisol in both PMD and NPMD patients; 2) to test the hypothesis that in comparison to NPHD patients, PMD patients have poorer outcome on measures of functioning, but not necessarily on measures of symptoms, at 15 months after the index episode; 3) to test the hypothesis that baseline measures of plasma and urinary cortisol do not predict outcome on measures of symptoms and functioning in PMD and NPMD patients at 4, 9, and 15 months after the index episode; 4) to test the hypothesis that at each of the three follow-up time points (4, 9, and 15 months), higher cortisol levels are associated with poorer outcome on measures of functioning, independent of depressive symptoms; 5) to test the hypothesis that at 15 month follow-up, plasma and urinary measures of cortisol in depressed patients correlate with cognitive impairment; and, 6) to explore whether in healthy control subjects there exists a relationship among urinary and plasma measures of cortisol, neuropsychological testing scores, and measures of social and occupational functioning. Eighty-one patients with diagnoses of unipolar major depression (42 psychotic and 39 nonpsychotic will be evaluated at baseline on measured of symptoms, social and occupational functioning, neuropsychological tenting, and plasma/urinary cortisol. Psychopharmacologic treatment will then be given according to a standardized protocol. Patients will be reevaluated at 4, 9, and 15 months on the same measures as at baseline except for the neuropsychological testing which will be repeated only at 9 and 15 months. Further understanding of the relationship between cortisol and long-term functioning in depression should help clinicians and researchers make more knowledgeable decisions regarding pharmacologic and psychological treatments during the first 15 months after the index episode.