The rapidly growing database of completely and nearly completely sequenced genomes of bacteria, archaea, eukaryotes and viruses (several thousand genomes already available and many more in progress) creates both extensive new opportunities and major new challenges for genome research. During the last year, we performed a variety of studies that took advantage of the genomic information to establish fundamental principles of genome evolution. We combined mathematical modeling of genome evolution with comparative analysis of prokaryotic genomes to estimate the relative contributions of selection and intrinsic loss bias to the evolution of different functional classes of genes and mobile genetic elements (MGE). An exact solution for the dynamics of gene family size was obtained under a linear duplication-transfer-loss model with selection. With the exception of genes involved in information processing, particularly translation, which are maintained by strong selection, the average selection coefficient for most nonparasitic genes is low albeit positive, compatible with observed positive correlation between genome size and effective population size. Free-living microbes evolve under stronger selection for gene retention than parasites. Different classes of MGE show a broad range of fitness effects, from the nearly neutral transposons to prophages, which are actively eliminated by selection. Genes involved in antiparasite defense, on average, incur a fitness cost to the host that is at least as high as the cost of plasmids. This cost is probably due to the adverse effects of autoimmunity and curtailment of horizontal gene transfer caused by the defense systems and selfish behavior of some of these systems, such as toxin-antitoxin and restriction modification modules. Transposons follow a biphasic dynamics, with bursts of gene proliferation followed by decay in the copy number that is quantitatively captured by the model. The horizontal gene transfer to loss ratio, but not duplication to loss ratio, correlates with genome size, potentially explaining increased abundance of neutral and costly elements in larger genomes. The evolution of bacterial and archaeal genomes is highly dynamic and involves extensive horizontal gene transfer and gene loss. Furthermore, many microbial species appear to have open pangenomes, where each newly sequenced genome contains more than 10% ORFans, that is, genes without detectable homologues in other species5,6. Here, we report a quantitative analysis of microbial genome evolution by fitting the parameters of a simple, steady-state evolutionary model to the comparative genomic data on the gene content and gene order similarity between archaeal genomes. The results reveal two sharply distinct classes of microbial genes, one of which is characterized by effectively instantaneous gene replacement, and the other consists of genes with finite, distributed replacement rates. These findings imply a conservative estimate of the size of the prokaryotic genomic universe, which appears to consist of at least a billion distinct genes. Furthermore, the same distribution of constraints is shown to govern the evolution of gene complement and gene order, without the need to invoke long-range conservation or the selfish operon concept. Much of our work aimed at understanding evolution of viruses and mobile elements. Among other developments in this area, a survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f, and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We showed that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesized that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNA-guided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element. The rapidly growing metagenomic databases have become a rich information source for discovery of new groups of viruses and microbes. We have performed several projects in this direction. One of these involved the discovery of a previously unknown but apparently abundant and ecologically important group of viruses. Marine group II Euryarchaeota (MG-II) are among the most abundant microbes in oceanic surface waters. So far, however, representatives of MG-II have not been cultivated, and no viruses infecting these organisms have been described. Here, we present complete genomes for three distinct groups of viruses assembled from metagenomic sequence datasets highly enriched for MG-II. These novel viruses, which we denote magroviruses, possess double-stranded DNA genomes of 65 to 100 kilobases in size that encode a structural module characteristic of head-tailed viruses and, unusually for archaeal and bacterial viruses, a nearly complete replication apparatus of apparent archaeal origin. The newly identified magroviruses are widespread and abundant and therefore are likely to be major ecological agents. Taken together, these studies advance the existing understanding of the general principles and specific aspects of genome evolution in diverse life forms, in particular viruses and mobile elements, and provide new insights into general principles of genome evolution.


DESCRIPTION (Investigator's Abstract): The principal aim of the proposal in this Phase I application is to develop a device with immobilized chelator of clinical capacity, for removal of excess iron from patients with iron overload. Dr. Ambrus and her colleagues have developed such a device, using polysulfon hollow fiber cartridges with immobilized chelators, for aluminum chelation in dialysis patients. Preliminary studies using this chelating device indicate that iron removal by such methodology is feasible. Other preliminary studies done in dogs using artridges with immobilized desferrioxamine showed that iron could be removed rapidly by such techniques. In Phase I of the proposed study, Dr. Ambrus plans to develop several types of immobilized chelators. Using activated agarose as a macromolecular support, she will immobilize some new oral chelators (EHPG, PIH) and alpha-aminoacyl hydroxamate, a chelator ligand with high iron affinity. She will prepare a series of carboxymethyl-dextrans with increase content of acid gropus for the binding of large amounts of the desferrioxamine for iron chelators. She will then measure iron binding capacity of each of the above chelating agents in batch experiments. Iron depletion by each of the chelators will be measured using saline solution with albumin which contains various concentrations of iron, between 50 and 500 mcg/dL. The strength of iron binding will be estimated by the eluted amount of iron by water soluble chelators, such as EDTA. Chelators that look promising in the batch experiments will be immobilized onto hollow fiber cartridges. Cartridges for immobilization will be those marked for hemodialysis with high ultrafiltration performance. Each cartridge contains about 12,000, asymmetric poly-sulfon hollow fibers consisting of a thin capillary membrane surrounded by a relative thick, macroporous shell which imparts mechanical strength to the membrane and serves as a support to the chelating agent which is deposited in the macropores and at the outside wall of the fibers. The nominal pore size of the membrane is less than 30 kilodaltons, and allows iron and other metals to pass through. Hollow fiber cartridges with the immobilized chelators will then be tested. Using aqueous solutions of iron, the device will be tested for kinetics of iron removal and iron binding capacity. Using blood from hemodialysis patients having iron overload, the devices will be tested for kinetics of iron removal. Identification of the sources of iron removed will be explored by measuring iron in individual serum fractions separated by HPLC in recirculating blood. The above Phase I studies will test the feasibility of this approach to iron chelation and identify the final chelator for the prototype to be developed in Phase II.


Project Summary/Abstract: Early biomarkers of Alzheimer's disease are urgently required for at least three reasons: (i) to determine which individuals are at greatest Alzheimer's disease risk, thereby (ii) offering preventative intervention, pre-disease onset, and (iii) further allowing nascent treatment intervention as early as possible in the disease process. All three goals demand a sensitive, non-invasive, affordable, accessible biomarker of Alzheimer's disease pathology/progression. Addressing these needs, we propose to test the hypothesis that a unique NREM sleep EEG signature provides a candidate early biomarker of A? pathology, one that may accurately track and forecast Alzheimer's disease risk and Alzheimer's disease pathophysiological progression. If correct, the proposal would establish a novel, inexpensive, and early diagnostic tool for determining Alzheimer's disease risk and pathology progression before clinical symptoms emerge, and one that is suitable for community settings. Moreover, such data would motivate an increased medical awareness regarding the importance of treating sleep difficulties across the lifespan, and further motivate the development (clinical or commercial) of sleep-based interventions that improve adult sleep and thus reduce Alzheimer's disease prevalence and its societal burden. More generally, such findings would argue for improved public health policies advocating for sufficient quality sleep throughout adulthood?a memorandum that may lower dementia risk and maintain cognitive health across the populous.


Objective: To determine a consensus on the criteria for pre-treatment workup and treatment delivery in five disease sites for which radiation therapy has a major curative impact. Develop method to present data to the specialty.


Hot flashes, the most common symptom of menopause, cause extreme discomfort and disrupt sleep patterns. Depression and mood changes during menopause also compromise quality of life. Hormone therapy, the primary treatment for menopausal symptoms, is associated with increased risk of breast and endometrial cancer. The majority of menopausal women refuses or fails to comply with hormone therapy, suggesting the need for alternative treatments. Black cohosh, a plant which has proven effective in reducing menopausal hot flashes in several small clinical trials, does not contain estrogenic compounds, but does contain agonists for serotonin receptors. Our hypothesis is that black cohosh alleviates hot flashes and depression in animal models through serotonergic action. Aim 1 will test the hypothesis that black cohosh extracts alleviate hot flashes using an ovariectomized rat model implanted with thermosensors. Aim 2 is designed to deliver solid behavioral data to support the antidepressant activity of black cohosh through the Forced Swim Test animal model of depression. Aim 3 will test the possibility that effects of estrogen therapy on menopausal complaints occur through modulation of serotonin action in a neuronal cell culture model. [unreadable] [unreadable]


Charges are produced in organic materials homogeneously by a multiphoton ionization mechanism. It is then possible to determine wavelength dependent ionization cross-sections at energies where the sample is opaque, but using light to which the sample is transparent. The transport of charges in organic materials is detected by a new interferometric technique. Finally the act of photoinduced charge-transfer is being probed by fluorescence lifetime measurements on the picosecond time scale.


The contractile properties of smooth muscle are broadly classified as phasic (fast) and tonic (slow). Phasic smooth muscle is characterized by relatively rapid rates of force activation, force relaxation and Vmax, whereas tonic smooth muscle is characterized by slower rates of force activation, force relaxation and Vmax However, the molecular mechanism that regulates the contractile properties of smooth muscle is unknown. Others and we have demonstrated that the mechanism for this diversity in contractile properties between phasic and tonic smooth muscle lies at the level of the contractile filaments, and differences in the kinetics of the actomyosin ATPase (AMATPase). The overall goal of this grant is to investigate the molecular mechanism for the differences in the kinetics of the AMATPase in tonic and phasic smooth muscle. Our hypothesis is that differences in the kinetics of the AMATPase are due to splice variant expression of the myosin heavy chain (MHC) and the 17-kDa myosin light chain (MLC17). The Specific Aims to test this hypothesis are to determine if splice variants of MHC and MLC17 are molecular determinants for the kinetics of the AMATPase of smooth muscle (Specific Aims 1-3). In addition, we will determine if non-muscle myosin is responsible for force maintenance in smooth muscle (Specific Aim 4). To test Specific Aims 1-3, we will force the expression of both splice variant isoforms of MHC and MLC17 in cultured embryonic aortic and gizzard smooth muscle cells. We will over-express both the endogenous and the alternative isoform of MHC and MLC17 in cultured aortic and gizzard smooth muscle cells and tissue strips. After forcing the expression of a single contractile protein, we will determine the effect of elevations of inorganic Pi and MgADP on steady state force and stiffness of single cultured smooth muscle cells, as well as in smooth muscle strips and compare the results to those obtained in the non-transfected controls. These experiments will elucidate the effects of the expression of a single contractile protein, in isolation, on the kinetics of the AMATPase of smooth muscle. In addition, to determine if non-muscle myosin participates in the molecular mechanism for force maintenance (Specific Aim 4), we will determine the properties of force maintenance in a transgenic mouse line, which lacks non-muscle myosin IIB. The results of these studies should elucidate the mechanism that determines the kinetics of the AMATPase of smooth muscle, and form a foundation for future investigation of how smooth muscle contractility is altered by disease states.


The long term objective of this proposal is to produce and accurate physical map and complete molecular clones of human chromosome segment 18q21.3 and to use this to characterize human diseases mapping to 18q21.3. Six probes known to map 18q21.3 provide the initial toehold in this segment. A long-range physical map of this segment using contour- clamped homogeneous electric field electrophoresis (CHEF) and rare cutting restriction endonucleases will order these loci. However, long-range restriction maps have technical limitations and emphasize the need to obtain large contiguous molecular clones. This approximately 2 megabase segment constitutes a reasonably sized test case to obtain overlapping clones from a yeast artificial chromosome (YAC) library of the human genome. YAC isolates obtained with the six 18q21.3 probes will be assessed for their authenticity and further mapped. Distal ends of YAC genomic inserts will be rescued and used to rescreen the library to link clones of 18q21.3. Molecular clones of this region provide the framework to localize new genes reveal associations between geography and the regulation of neural and immune related genes. Follicular lymphomas translocate a new proto-oncogene, Bc1-2 from 18q21.3, into the immunoglobulin locus (14q32) resulting in a marked overproduction of Bc1-2 mRNA. However, CHEF analysis reveals that the Bc1-2 gene possesses and enormous approximately 350 Kb Intron II and that the deregulatory effects of translocation are transmitted across this distance. YAC technology provides the capability to clone this intron as a single fragment. Other genes located within this intron would be identified, cloned, and their effects upon neoplasia or other cell types assessed. The 18q- deletion syndrome is a congenital developmental abnormality manifesting as craniofacial abnormalities, mental retardation, and humoral immunodeficiency. The deleted segment common to these patients is band 18q21.3 18q- syndrome patients with interstitial deletions or constitutional translocations at 18q21.3 will be used to identify distinct breakpoints. This should determine whether a single gene is always altered or whether multigenic loss is responsible. The physical map and YAC clones of 18q21.3 open new avenues for assessing normal and pathologic gene regulation and serve as a model for approaching human genetic disease.


Astrocytes are an important component of the neuropil and their dysfunction has been associated with a variety of idiopathic and genetic diseases including Alzheimer's and Parkinson's disease and in energy deprivation syndromes such as thiamine deficiency and antimetabolite poisoning. The prevailing model of healthy astrocyte-neuron interaction is one of continuous and intimate physical contact between adjacent membranes that promotes neuronal homeostasis. While the loss of astrocytes in the neuropil is generally viewed as a negative event, we propose here that in the acute phases of intoxication loss of astrocytes may protect neurons against further injury mediated by release of adenosine. Specifically, in this proposal we hypothesize that DNB-induced oxidative stress in astrocytes induces the release of adenosine which in turn activates A1 receptors in neurons via paracrine mechanisms and self-regulates A2 receptors on injured astrocytes. Since oxidative stress is the precipitating event, the corollary to this hypothesis is that in neurons, oxidative stress converges on PI3K/ERK to regulate the activity of BCL proteins that promote mitochondrial fusion and stabilization of the cell. Additional neuronal protection is achieved via A1-mediated activation of AKT with blockage of pro-death Bcl proteins and activation of survival Bcl-proteins. Conversely, in astrocytes, activation of the A2 receptor exacerbates loss of calcium control, swelling and cell death. This hypothesis for the role of astrocytes in the protection of neurons from oxidative stress-induced cell death will be tested by addressing the following specific questions. AIM 1: Can adenosine released by astrocytes silence neurons and protect them from the effects of exposure to 1,3-DNB? Aim 2: Does A1 receptor mediated signaling through PI3K, AKT and/or ERK block death- related members of the Bcl-family of proteins in neurons? Aim 3: Is the course of mitochondrial fusion or fission determined by or dependent upon binding of Mfn1/2, Bax/Bad/Bcl-XL and Drp 1? Aim 4: Does binding of proteins that alter mitochondrial morphology also alter membrane potential and function? Dinitrobenzene (DNB) provides and excellent model of energy deprivation syndromes with selective damage to astrocytes. This experimental approach will enable dissection of the role of BCL-proteins, mitofusins and Drp-1 in coordinating the loss of mitochondrial function and may provide new insights into neuronal/glial interactions that form the foundation for pathoclisis, or selective cellular susceptibility to environmental neurotoxicants. PUBLIC HEALTH RELEVANCE Astrocytes are supporting cells in the central nervous system. Data from our laboratories show that they are a primary target of many environmental chemicals that result in dysfunction of the central nervous system. As injury to the astrocyte progresses, ATP is converted to adenosine and is released into the extracellular space where it can interact with A1 receptors on neurons (protective) and A2 receptors on astrocytes (injurious). We propose here that in the acute phases of CNS injury, loss of neuronal function (silencing) is elicited by adenosine and may spare the neuron from the deleterious effects of oxidative stress and excitotoxicity.


VECTOR In order to understand the manner by which specific mutations lead to malignancy, the Vector Core provides Cancer Center members with several technological platforms that mediate overexpression or reduced expression of specific genes and the corresponding mutants for in vitro and in vivo studies. These platforms include both recombinant non-viral and viral products that facilitate the transfer of specific genes into either normal or cancer cells. The Vector Core also provides intellectual and technical advice to Cancer Center researchers regarding the optimal use and production of these systems. Despite the wide application of molecular biology to the study of cancer, many investigators do not possess a working knowledge of recombinant vectors. Many of the operational techniques required for optimal use of these gene transfer vectors are specialized and difficult to acquire without expert assistance. In addition, the manufacture of these reagents needs to be performed in laboratory space that has been specifically configured to comply with NIH biological containment guidelines. The requirements for compliance with these biosafety guidelines inhibit many investigators from pursuing the use of these valuable reagents. The Vector Core has been established to provide a cost-effective source of these valuable platforms while minimizing Cancer Center members'need to spend time and money on new laboratory space and hiring their own vector experts. The Specific Aims of the Vector Core are to: 1. Provide a Core laboratory for the development, construction, purification and characterization of recombinant vectors containing genes relevant to the study of cancer disease models for use as in vitro and in vivo gene transfer reagents. These systems include both non-viral (expression plasmid) and viral (recombinant retrovirus, recombinant adenovirus, and recombinant AAV) technologies. 2. Collaborate closely with Cancer Center researchers to ensure the Vector Core provides the platforms Cancer Center members require 3. Maintain qualified staff and monitor the competition to continually improve the Vector Core and provide high quality, cost effective products to Cancer Center members.


Ubiquitin-like modification is a conserved biochemical mechanism that is used by cells for various purposes. These include targeted degradation of proteins, endocytosis of receptors, regulation of protein localization, and signal transduction. Apart from a role of ubiquitin itself to modulate insulin receptors and IRS proteins, ubiquitin-like modifications have not been described to play a prominent role in insulin signaling. TUG was identified as a target of insulin signaling, and is implicated regulating GLUT4 glucose transporter trafficking and glucose uptake in adipocytes. According to the proposed model, TUG acts by "tethering" GLUT4 transporters intracellularly in the absence of insulin, excluding them from the plasma membrane and limiting glucose uptake. Insulin "untethers" the transporters to redistribute GLUT4 and to enhance glucose transport into cells. The mechanism by which insulin acts on TUG and GLUT4 is not well understood. The present proposal will test the hypothesis that insulin stimulates rapid and site-specific cleavage of TUG to liberate an amino terminal fragment, TUGUL, that is a new ubiquitin-like modifier. Using cultured 3T3-L1 adipocytes, several biochemical and cell biological approaches will be used in Aim 1 to test whether insulin stimulates TUG cleavage, and whether this is required for insulin to mobilize GLUT4. Aim 2 will test the hypothesis that TUGUL functions as a ubiquitin-like modifier, and will study the role of the putative TUG C terminal cleavage product. Aim 3 will study how the insulin signal may act on TUG to cause its processing. It is anticipated that, together, accomplishment of these aims will begin to define a novel pathway for insulin regulated ubiquitin-like modification. Furthermore, it is anticipated that the results will have importance for understanding how insulin stimulates glucose uptake. Type 2 diabetes is a major public health problem that results in part from a defect in the ability of insulin to stimulate glucose uptake. It is anticipated that the proposed studies of insulin action and glucose uptake will lead to a greater understanding of mechanisms that may contribute to the development of type 2 diabetes. [unreadable] [unreadable] [unreadable]


Thalamocortical afferents are the sole source of sensory input to the mammalian neocortex. Their role during cortical development is equally important. Recent work has established that thalamocortical afferents are required during development for the differentiation of the architectural, connectional and functional features that distinguish and define the functionally specialized "areas" in the adult neocortex. The four specific aims of this proposal are designed to test hypotheses on mechanisms postulated to control the development of area-specific thalamocortical connections. This work will be done principally in rats, an excellent model for studies of cortical development and plasticity. It will combine a variety of anatomical and cellular methods, using in vivo and in vitro experimental approaches, as well as time-lapse imaging of cultured tissue. The major experimental goals are: (1) to assess the specificity in the targeting of thalamocortical axons and their invasion of the cortical plate. (2) to investigate a role for putative position-dependent molecular cues hypothesized to control specificity in thalamocortical axon targeting. (3) to determine whether maturation-dependent changes in membrane- associate molecules on the surfaces of cortical plate neurons and/or their secretion of a diffusible chemoattractant promotes the invasion of the cortical plate by waiting thalamocortical axons. (4) to analyze the degree of dispersion of neuroepithelial cells and their progeny during the development of the neocortical preplate as a means to evaluate hypothesized mechanisms of establishing position-dependent information in the subplate. The long term objectives of this work are to elucidate critical early events in the development of the mammalian cerebral cortex, with a focus on the neocortex. It is expected that the information gathered will provide insights not only into normal developmental mechanisms employed in the cortex, but that it will contribute to our understanding of the basis of the many congenital cortical abnormalities that afflict humans, as well as later striking neurodegenerative diseases.


During fiscal year 2010 we accomplished the following: 1) We used Bim-deficient B cells to further distinguish transcriptional and post-transcriptional regulation of p100 protein production in response to acute and tonic BCR stimulation. These studies substantiated the idea of PI3K-dependent translational control of p100 production. 2) We used NFκB2-deficient mice to establish unequivocally that p100 is essential for BAFF-dependent survival of B cells in vitro. It is interesting to note that p100-deficient mice have reasonable numbers of mature B cells, whereas BAFF- or BAFF-R- deficient mice do not. Our results suggest that BAFF-dependent survival in vivo can be mediated by pathways other than p100. Since BCR is essential for survival of mature B cells in vivo, these other pathways must also be initiated at the BCR. We initiated studies of Mcl-1 regulation by BCR to determine if this anti-apoptotic protein is responsible in part for p100 independent B cell survival in vivo. 3) Downstream of PI3K activation we found that suppression of mTOR by rapamycin did not abolish p100 up-regulation in response to tonic or acute BCR signaling. However, rapamycin treatment of Pim2-deficient B cells abolished BCR-induced p100 up-regulation. We infer that Pim 2 and mTOR provide redundant pathways to p100 induction.


CORE D ? ENVIRONMENTAL ASSESSMENT CORE PROJECT SUMMARY The goals of the Dog Aging Project are to create a cohort of 10,000 companion dogs through which it will become possible to identify the genetic and environmental determinants of healthy aging, discover the underlying mechanisms through which genes and environment influence aging, and test a highly promising drug for its ability to increase lifespan and healthspan. Given that the majority of variation in aging phenotypes within mammalian species is likely to be explained by differences in environmental exposures ? either alone or via interactions with inherited genetic factors ? measurement of environmental variables for each dog in the cohort is essential. In order to meet the needs of the scientific projects for coordinated expertise in determining which aspects of the environment should and can be characterized, and through which approaches, an Environmental Assessment Core (EAC) will be established. The overall goal of the EAC is to support the scientific aims of the research projects by developing and implementing strategies for acquiring data that characterize various aspects of the companion dog environment. Specific aims of this Core are to: 1) Design and implement annual dog owner-administered questionnaires that elicit each dog's travel history, non- prescription medications, diet and feeding, household environment, physical activity, reproductive activity, and behavior, as well as key behavioral and health conditions for owners; 2) Collect, process, validate, and prepare primary and secondary data related to conditions of the dog's environment; and, 3) Collaborate with Project Investigators to develop analytic strategies to assess the role of environmental conditions, and their interactions with genetic factors, in processes of aging.


An estimated 1.5 percent to 7.5 percent of children suffer from encopresis (fecal soiling). One of the most effective ways of treating encopresis is with Enhanced Toilet Training (ETT). ETT is twice as effective as intensive medical management alone at 3, 6, and 12-month follow-up when delivered by skilled and knowledgeable clinicians. We have successfully transformed ETT into an interactive internet intervention, a web program accessible by anyone with a computer and internet access. This program has been shown to have significant additive value to standard clinical care, and is based on a theoretical model we developed for therapeutic behavior change achieved through internet interventions. To date, this Internet intervention for pediatric encopresis has only been offered to individuals who are currently in treatment with a health care provider. In order to significantly increase its potential for wide-spread dissemination, a new clinical trial is proposed in which families can self-refer to the intervention. In addition, as adherence is often a significant issue with internet interventions, an enhanced condition involving a stepped care approach with graduated levels of support (automated email, personalized email, phone support) for different levels of non-adherence will be evaluated as part of a large, national, randomized clinical trial. We propose a four year project in which we will experimentally test the short and long term benefits of the intervention as compared to use of the system with a stepped care approach to enhance adherence and ultimately symptom improvement. A static, educational website will be used with the control group. Cost-benefit analyses will be evaluated and documented. We hypothesize that the pediatric encopresis internet intervention will be more effective than a static website at 6 weeks and 12 months post treatment in terms of encopretic symptom reduction (including fewer accidents, increased BMs in toilet, and increased trips to the bathroom), behavior change (measured by acceptable cleanout, laxatives, straining, toileting, and routine), and reduced costs (doctor visits, medication usage, missed school/work days, diapers used). We also predict that the stepped care version of the program, as compared to the standard program, will produce greater website utilization and thus improved behavior change and symptom reduction. While overall cost will likely be higher with stepped care, we expect cost-benefit analyses to show that this increased cost is acceptable for a certain segment of the population studied. Additional testing of our model for internet interventions will also be conducted. Relevance: Internet interventions can provide sophisticated and personalized treatment at low cost. This application plans to take a program for children with encopresis and evaluate how effective it is when offered "direct-to-consumers." It also attempts to increase success by integrating methods to improve adherence to the program. Findings from this study will have far reaching implications for the management of encopresis in particular and internet interventions in general. [unreadable] [unreadable] [unreadable]


Malaria and diarrheal diseases are major barriers to improving maternal and child health in sub-Saharan Africa (SSA). Each year about 900,000 people in SSA die from malaria. Pregnant women and infants are particularly vulnerable. An estimated 10,000 women and 200,000 newborns, mostly in Africa, die each year as a result of malaria infection during pregnancy and severe malanal anemia (WHO, 2007a). Worldwide, diarrheal diseases kill almost two million children in poor countries each year (WHO, 2002, Kosek, et al., 2003), and 640,000 children under the age of five in SSA alone (Rao, et al., 2006). Overall, malaria and diarrhea account for 18 percent and 16 percent of under-five mortality, respectively (UNICEF, 2007). Many of these deaths could have been averted using available preventative measures. For instance, using an insecticide-treated net (ITN) during pregnancy reduces the incidence of severe maternal anemia by up to 47 percent (Marchant, et al., 2002; Ter Kuile, et al., 2003). Overall, ITNs can reduce the incidence of malaria by 50 percent and mortality by 20 percent at an estimated cost of $29 per disability-adjusted life year (DALY) averted. Point-of-use chlorination of drinking water reduces the incidence of endemic diarrhea by 37 percent at an estimated cost of $53 per DALY averted (Morel, et al., 2005; Clasen, 2007). However, ITN ownership and point-of-use water chlorination coverage are both under 10 percent in SSA (Miller, et al., 2007; Stockman et al., 2007). These low coverage rates suggest that these preventative products are not reaching the most vulnerable populations, raising two important questions: First, how can demand for these products be increased? Second, how can distribution systems be made more effective?


DESCRIPTION: The cri-du-chat syndrome is a well-described partial aneusomy resulting from the deletion of the short arm of chromosome 5. Patients with this syndrome present with microcephaly, a round face, hypertelorism, micrognathia, prominent nasal bridge, epicanthal folds, hypotonia, and severe psychomotor and mental retardation. Through the characterization of over 50 patients with 5p deletions, a chromosomal region that is 2 Mbp in 5p15.2 and the chromosomal segment involved in the cat-like cry has been mapped to a 750 region of 5p15.3 YAC contigs of both regions have been completed and nonchimeric YACS that span each region have been identified. While the long term goal of this proposal is to identify genes that, when present in one copy, cause the clinical features associated with the cri-du-chat syndrome, the initial goal will be to develop a transcriptional map of the region in order to identify candidate genes based on their location with respect to the cri-du-chat critical regions. The strategy to identify these genes is first to continue with the characterization of patients with small 5p deletions or partial cri-du-chat phenotypes such that the two critical regions can be further narrowed. Second, the construction of a cosmid contig which has been initiated will be completed. Third, several approaches to identify the genes mapping within the critical regions will be performed and include EST mapping, cDNA selection, exon-trapping and cloning HTF islands. After performing screening strategies to identify clones that are derived from the same gene, the pattern of expression of unique genes will be investigated. This investigation will include investigating the level of expression in human adult and fetal tissues as well as the level of expression in different murine gestational stages. The initial characterization of these genes will identify candidate genes that may be involved in the clinical etiology of the cri-du-chat syndrome based on whether a gene is expressed during fetal development.


Development and application of centrifugal precipitation chromatography has been continued. The system internally generates a concentration gradient of ammonium sulfate (AS) along a long channel to fractionate proteins according to their solubility in AS solution. The separation column consists of a pair of disks with mutually mirror-imaged spiral channels that are separated by a semipermeable membrane. The disk assembly is mounted on a sealless continuous flow centrifuge. Concentrated As solution is introduced into the upper channel while a water solution is passed through the lower channel in the opposite direction in a rotating column. A mixture of proteins injected into the water channel moves along an AS gradient of increasing concentration that has been established in the water solution. Each protein species precipitates at a different AS concentration along the gradient. The eluate is continuously monitored and collected using a fraction collector. A series of basic experiments was performed to study the rates of AS transfer and osmosis through the membrane, and the operational parameters including elution time,revolution speed, inclination of the gradient and sample size were optimized using stable protein samples. A new set of columns with different channel configuration was made to improve the efficiency of separation.Preliminary applications were successful in purification of monoclonal antibodies from cell culture supernatant and ascitic fluid, and affinity separation of recombinant ketosteroid isomerase from a crude E. coli lysate. The method was also successfully applied to fractionation of other biopolymers such as hyaluronic acid, chondroitin sulfate, and polymerized catechins using a gradient between suitable organic solvents and water.


This application to join the NICHD Maternal Fetal Medicine Units Network is a combined proposal from the Oregon Health & Science University (OHSU) and Kaiser Permanente Oregon/Washington (KPNW) region. It is the result of a long-standing collaboration between OHSU and KPNW in maternal fetal medicine and brings together university and community research capabilities, which will increase diversity in enrollment. The combined deliveries (4091 in 2003 and 4043 in 2004) concentrate high risk patients in principally two sites: the Perinatal Center at Doernbecher Children's hospital at OHSU and the KPNW Perinatal Center at St. Vincent's Hospital. Both centers serve to selectively funnel consultations for genetics, preconception counseling, high resolution ultrasonography, high risk obstetrics and fetal echocardiography and are staffed by Maternal Fetal Medicine Specialists. Both OHSU and KPNW have a long history of conducting respected clinical research independently and together. This collaboration is based upon shared laboratory services and maternal fetal medicine coverage, an integrated residency in Obstetrics & Gynecology, and the combined strengths in research of the KPNW Center for Health Research and the Oregon Evidence-based Practice Center. The Center for Health Research (KPNW) and the Women's Health Research Unit (OHSU) have extensive experience in clinical trials and the research staff to facilitate the enrollment of patients in clinical studies. Focusing patient recruitment in two sites staffed by maternal fetal medicine specialists committed to this proposal will enhance enrollment and participation by research staff as well as maximize the quality of data obtained. [unreadable] [unreadable] [unreadable] [unreadable]


In the United States over 6% of individuals greater than 12 years old have used marijuana in the past month and this substance accounts for approximately 55% of the illicit drug use in this country. The perception that cannabis is a "safe" illicit drug, without long-term adverse consequences, may contribute to the higher rates of use of marijuana when compared with more "hard" drugs. However, there has been relatively few brain imaging studies exploring the neurobiological effects of cannabis. Two main areas of recent interest with regards to cannabis use, both potentially related to the dopamine (DA) system, highlight the importance of further in vivo human brain imaging studies examining DA function in cannabis users. First, a relatively strong association (odds ratio > 2) between premorbid cannabis use and schizophrenia has been observed, particularly when cannabis use occurs in adolescence. In schizophrenia, abnormalities of the DA system have been observed in receptor imaging studies. Given the epidemiological link between cannabis use in adolescence and schizophrenia a neuroreceptor imaging study of this nature in individuals who use/abuse cannabis is of particular interest. Second, early use of cannabis has been considered a risk factor for the future use of hard drugs. This has been termed the "gateway" hypothesis and neurobiological factors such as sensitization may play a role. Sensitization refers to the enhanced DA response in the reward pathway of the nucleus accumbens (NAc) that occurs following repeated and chronic exposure to drugs of abuse. If exposure to cannabis can induce a cross- sensitization to other drugs of abuse (i.e. prior exposure to cannabis results in an increased DA release in the NAc on subsequent exposure to another class of drugs) this would be supportive of the gateway effect. This application seeks to examine the effects of repeated cannabis exposure, beginning in adolescent, on DA transmission in vivo within reward system of the ventral striatum (VST), an area which includes the NAc,. This will be accomplished with positron emission tomography (PET) by measuring amphetamine-induced displacement of [11C]raclopride in 8 adult individuals with a history of repeated cannabis use beginning in adolescence and 8 matched controls. If the hypothesis we seek to test is confirmed - that marijuana use beginning in adolescence alters dopamine signaling in the striatum later in life - it would provide a strong neurobiological basis for the epidemiological link between this drug and schizophrenia, have implications for the theory that marijuana serves as a gateway drug, and strengthen the argument that marijuana use cannot be considered a "safe" drug of abuse. In the United States over 6% of individuals greater than 12 years old have used marijuana in the past month and this substance accounts for approximately 55% of the illicit drug use in this country. However, there has been relatively few brain imaging studies exploring the neurobiological effects of cannabis. If the hypothesis we seek to test is confirmed - that marijuana use beginning in adolescence alters dopamine reward signaling in the brain later in life - it would provide a strong neurobiological basis for the epidemiological link between this drug and schizophrenia, have implications for the theory that marijuana serves as a gateway drug, and strengthen the argument that marijuana use cannot be considered a "safe" drug of abuse. [unreadable] [unreadable] [unreadable]


The long-term objective of this application is to elucidate the molecular mechanism(s) underlying specification of intestinal cell identity. We have identified a previously unrecognized patterning step that takes place at embryonic day 16.5 in the mouse. This step involves the upregulation of >1,000 genes in the intestinal epithelium, a process that creates the distinct epithelial border that defines intestinal vs. stomach identity. We call this process intestinalization. Our evidence also implicates the transcription factor Tcfec as a potential regulator of intestinalization. Thus, the Specific Aims of this application are to: 1) determine if Tcfec is an activator in intestinal epithelium, 2) characterize the downstream pathways affected by Tcfec during intestinal epithelial cell specification, and 3) analyze the contribution of a conserved non-coding element (CNE) near the Tcfec gene to the temporal regulation of its expression in intestinal epithelium. For Aim 1, we will clone intestinal forms of Tcfec by RLM-RACE and determine their transcriptional activity using cell-based, luciferase reporter assays. For Aim 2, Tcfec expression will be manipulated in Caco-2 cells, an in vitro system that recapitulates intestinal epithelial cell differentiation. We will also establish several mouse models to test the requirement for Tcfec in the intestine, as well as the effect of Tcfec misexpression in the stomach. For Aim 3, we will generate transgenic reporter mice to ascertain the pattern of expression directed by the Tcfec CNE. The requirement of specific transcription factor binding sites will then be tested by mutagenesis. In addition to their contribution to the understanding of intestinalization per se, these studies may well be relevant to the pathologic state known as intestinal metaplasia of the stomach, in which cells within the stomach take on intestinal character. Since intestinal metaplasia is thought to represent an early step in the development of some forms of gastric cancer, our findings could eventually impact diagnosis or early treatment of this often fatal cancer.


A.Protein kinase C project: The generation of novel chemical libraries of DAG-lactones using our new solid-phase method of synthesis continues to yield important lead compounds in various areas of potential therapeutic importance: 1) The results on our first C1 domain-selective DAG-lactone with exclusive activity for RasGRP was published recently. This compound has allowed us to dissect an ERK phosphorylation pathway via RasGRP, which is totally independent of PKC alpha. This DAG-lactone, identified as 130C037, is capable of exclusively translocating RasGRP to intracellular compartments while other PKC isozymes remain in the cytoplasm; 2) Optimization and structure-activity studies of DAG-lactones capable of stimulating alpha-secretase activity continues; 3) Research using the Balb/C mouse JB6 model has helped identify some DAG-lactones that retain AP-1 activation but are not tumor promoting agents; 4) A different set of DAG-lactones is capable of stimulating interferon production in combination with IL-12 to levels superior to those achieved with the paradigm PKC activator, phorbol myristate acetate (PMA) without inducing tumor promotion. Large-scale syntheses of some of these compounds are in progress to perform animal studies. B.A new azido-functionalized flavone acetic acid analogue was conjugated to a FLAG peptide phosphine derivative via the Staudinger reaction. When the conjugate was mixed with cell extracts from mouse macrophage cells, the FAA-FLAG conjugated proteins could be immunoprecipiated using an anti-FLAG antibody. This work was recently selected to be highlighted in the CCR bulletin. C.Zebularine [2(1H)-pyrimidinone riboside] has been the subject of numerous studies in the last 3 years (31 papers in major journals). Unfortunately its path towards the clinic was brought to an end by an unforeseen toxicity in rhesus monkeys, which was lethal when plasma levels reached 25 micromolar. This is in total contrast to the complete lack of toxicity in rodents (rats and mice), even at high doses. Efforts to circumvent toxicity and improve the activity of zebularine at lower doses in monkeys have been centered on preventing the high levels of a metabolizing enzyme, aldehyde oxidase, which neutralizes the action of zebularine. Despite the discovery of raloxifene, a clinically approved agent to inhibit this enzyme, our efforts have been directed to the development of pro-drugs of 2'-deoxyzebularine which were initiated last year with the intent to improve the incorporation of 2'-deoxyzebularine-5'-triphosphate into DNA. After studying an extensive series of pro-drugs, which resulted in inactive compounds, we have now identified two analogues that are 10-fold more potent than zebularine in the activation of silenced tumor suppressor genes, particularly in CF-Pac-1 pancreatic tumor cells. Securing intellectual protection of these findings is in progress. The selective incorporation of zebularine-5'-triphosphate opposite to G in was demonstrated using DNA pyrosequencing; whe zebularine is part of the template base paring to G is less selective.D.A stable NAD analogue, beta-methylene-TAD, synthesized at the LMC in years past was used for the high-resolution crystal structure of a mono-ADP -ribosylating toxin (similar to diphteria toxin). The structure has implications for the mechanism of translocation on the 80S ribosome.


Multiple inputs and controls participate in the maintenance of energy homeostasis, and numerous neural and endocrine systems are involved in the integrated responses that maintain energy stores in the body at a level. Since most stored energy is in the form of adipose tissue (fat), and since the brain is the major controller of energy homeostasis, a fundamental requirement is that the brain receive accurate and timely information about the amount and distribution of body fat. This information is then integrated with other factors to determine whether an individual seeks and eats food (i.e., energy intake), as well as the efficiency with which the ingested food is utilized (energy expenditure) or stored. Information regarding the amount of fat stored in the body reaches the brain via the circulating signals insulin and leptin. Each is secreted into the blood in direct proportion to total body fat, and each is transported from the blood into the brain where it interacts with neurons situated to influence energy intake and expenditure. Hence, insulin and leptin are considered to be adiposity signals, and the long-term goal of this project is to elucidate how adiposity signals interact with other factors to influence energy homeostasis. We anticipate that the coming decade will see considerable progress in this arena, as well as the translation of the new-found information into potential novel therapeutic strategies to tackle clinical problems of dysregulation of energy homeostasis (i.e., obesity, eating disorders). With this as an ultimate end point, the present proposal has three specific aims that address critical and as yet unanswered questions regarding the actions of adiposity signals in the brain. The first assesses the hypothesis that insulin and leptin act in the hypothalamus by enhancing the signal provided by local levels of nutrients, including glucose and fatty acids. The second assesses several hypotheses following from our observation that there are important gender differences in the actions of insulin and leptin in the central control of energy homeostasis, and that males and females utilize different strategies to regulate energy homeostasis and defend their body fat stores. The final aim assesses hypotheses regarding the effect of reducing insulin signaling in specific brain regions on food intake and body weight, and upon related levels of metabolic hormones and neuropeptides, utilizing lentiviral technology. PUBLIC HEALTH RELEVANCE: Hormones such as insulin and leptin that are secreted in direct proportion to body fat interact in the brain with nutrients such as glucose, fatty acids and amino acids to determine food intake and energy expenditure. Proposed research will determine how these signals interact within neurons in the hypothalamus and intervene by manipulating the ability of the brain of laboratory rats to detect and respond to insulin or leptin. Experiments will assess behavior as well as molecular signals within cells. [unreadable] [unreadable] [unreadable]


The purpose of this study is to demonstate the vagal nerve stimulation via a surgically implanted investigational device in the left vagas nerve will reduce the frequency of seizures in epileptic patients refactory to medical therapy.


The amiloride-sensitive epithelial sodium channel (ENaC) mediates the final event in the reabsorption of sodium by the kidney. The activity of these channels determines the amount of sodium retained by the kidneys hence, ENAaC has a fundamental role in the maintenance of extra- volume and blood pressure. Many stimuli modulate the activity and expression of ENaC in the kidney but among them, aldosterone is the single most important one. There are two aims in this research proposal. The first one is to study one of the mechanisms that mediate the aldosterone-induced activation of ENaC in the distal nephron by a novel serine/threonine kinase known as sgk. Specifically, we proposal to investigate whether sgk is i) necessary and sufficient to mediate the aldosterone response in epithelial cells, ii) to elucidate the mechanism(s) triggered by sgk that results in activation of ENaC, iii) to find proteins that associated with sgk that may constitute substrates of the kinase or regulatory proteins, and iv) to generate animal models (transgenic mice) that exhibit decreased or increased activity of sgk selectively in the principal cells of the distal nephron. The design of the research allows us to obtain and correlate data from cells in culture, in kidney, and in whole animals. In the second aim of the proposal we will investigate the molecular mechanisms of one human mutation in the gene of the gamma subunit of ENaC that produces pseudohypoaldosteronism type 1. These studies will contribute to further elucidate the structure-function of ENaC channels.


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. Staphylococcus aureus (Staph) is a germ that can cause skin infections and other serious and deadly infections. Community-Associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are types of Staph infections that are resistant to many antibiotics. CA-MRSA infections are suddenly very common, whereas 10 years ago they were unheard of. CA-MRSA are resistant to many antibiotics so doctors are not sure which antibiotics work best against skin infections, which are now caused mostly by CA-MRSA. To understand how to best treat skin infections, specifically ones called ?uncomplicated skin and soft tissue infections? (uSSTIs), we need to do a clinical trial to compare antibiotics that experts think are best. However, there is no proof about how good (efficacious) or safe these antibiotics are. We will perform a clinical trial of treatment of uSSTIs --specifically treatment of abscesses (boils) and/or cellulitis (bland skin infection)--in both children and adults in areas where CA-MRSA is common. The primary objective of this study is to compare the cure rates of two antibiotics, clindamycin and trimethoprim-sulfamethoxazole (TMP-SMX) for the treatment of uSSTIs. Treatment will be done in conjunction with the standard of care for treatment of abscesses, which is incision and drainage (i.e., lancing a boil). There is preliminary information that incision and drainage alone without antibiotics may be sufficient for patients with small abscesses. Therefore, subjects with small abscesses will be randomized to be treated with either clindamycin, TMP-SMX, or placebo.


The postnatal development of both the optical and neural visual systems is dependent on visual experience. Visual experience is defined by the information available in the retinal images in the two eyes. The goal of the proposed research is to extend our previous examination of retinal image quality in one eye during infancy to full binocular viewing conditions. We will examine human infants' visual experience in the context of image clarity and image alignment, which are primarily defined by accommodation and vergence responses and their interaction. These studies will document the emergence of the interaction between accommodation and vergence and their role in the development of refractive and accommodative strabismus. There are three specific aims: i) To understand the normal maturation of the relationship between accommodation and vergence with emmetropisation and growth of the distance between the eyes. ii) To determine the relative bias towards accommodation or vergence accuracy during the critical period of human development. iii) To understand the effects of accommodation and vergence behavior on visual experience of infants and children with high hyperopia or strabismus. PUBLIC HEALTH RELEVANCE: This project will determine how young infants and children manage apparently conflicting focusing and eye alignment demands during typical development. It will also investigate why some children develop refractive or accommodative strabismus while others, with apparently matching visual systems, do not. The goal is to develop intervention strategies to prevent this strabismus and associated amblyopia.


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. Total chemical synthesis was used to prepare a series of unique chemical analogues of HIV-1 protease, where we systematically substituted the residues Gly51, Gly51'at the tips of the mobile 'flaps'(residues 37-61 in each domain of the protein homodimer) with L-Ala, D-Ala or Aib (alpha-aminoisobutyric acid) in both symmetric and asymmetric fashion. Such substitutions, although in regions distant from the catalytic aspartates, led in most cases to reduction of catalytic activity. In contrast to this, a 'covalent dimer'with L-Ala51 in one flap and D-Ala51'in another flap has shown native-like enzyme activity. We used continuous-wave and pulse EPR to characterize dynamic properties of flaps in spin-labeled analogues and found that the thermodynamic balance of the closed, semi-open and open ensembles (with respect to flaps) in HIV-1 protease analogues is perturbed in comparison to that of wild-type enzyme.We interpreted our results on the basis of Northrop's 'kinetic isomechanism'for aspartic proteases which employs a low-barrier hydrogen bond (LBHB) as the central part of the concept. We have developed Northrop's concept further to take into account dynamic properties of the protein and the non-equivalence of the flaps in the ligand-bound enzyme. We proposed a mechanical coupling of motions of the flaps and the catalytic residues Asp25 and Asp25'. On a mechanistic level, opening of the flaps corresponds to shortening of the distance between catalytic residues Asp25 and Asp25'thus promoting formation of the LBHB, and vice versa.Our goal is to employ NMR methods to detect and unambiguously prove presence of LBHB. In addition we would like to determine ionization states of catalytic residues for chemical analogues which demonstrate distinctly different protein dynamics and thus attempt to correlate dynamic properties of the enzyme with its chemical mechanism.


To examine if oxandrolone can promote weight gain in HIV-infected men. To ex-amine the effects of oxandrolone on body composition by DEXA, muscle performance by IRM and other functional tests, oxandrolone pharmacokinetics. To examine if anabolic effects of oxandrolone occur at doses that do not result in suppression of LH and FSH; this will help determine whether anabolic and androgenic activi-ties of oxandrolone can be dissociated, as has been claimed in the literature. Thus although this is a multi-center study, several important aspects of the study will only be addressed in studies being done at this center.


The Short-term Mentored Career Enhancement Award in Basic Behavioral and Social Sciences: Cross-Training at the Intersection of Animal Models and Human Investigation (K18) is a unique opportunity to fund significant and innovative research while simultaneously building the skill set, and thereby future scientific discovery potential, of a scientist. The current application proposes to secure training in human investigation for Gretchen Neigh, Ph.D., a behavioral neuroendocrinologist currently adept at the use of animal models to address questions of the neurobiological basis of behavior. Dr. Neigh's research portfolio focuses on potential metabolic underpinnings of behavioral disorders such as Major Depressive Disorder (MDD) and Post-Traumatic Stress Disorder (PTSD). The work proposed serves the dual purpose of extending Dr. Neigh's innovative hypotheses regarding glucose transport and origin of neuropsychiatric disorders from her current work in rodent models to assessments using human investigation. This training experience will provide a foundation for translational reciprocity in Dr. Neigh's research program and address a question of fundamental importance: assessment of the potential role of metabolism in manifestation of MDD and/or PTSD. Over 23 million people in the United States suffer from MDD or PTSD and these disorders compromise quality of life and generate significant economic burden. Both human and animal studies have provided evidence of changes in cerebral metabolism in these conditions and related animal models. The alterations in metabolic activity are generally attributed to reduced glutamate release from neurons which thereby decreases regional glucose transport. Counter to this traditional dogma, it is possible that a primary change in facilitated glucose transport subsequently suppresses neuronal activity. Facilitated glucose transport is mediated by a family of transporters (GLUT) that are responsible for glucose transport across the endothelial cells of the blood brain barrier, and for uptake of glucose into astrocytes and neurons. Deficits in the expression and translocation of members of the GLUT family have been linked to neuropathological conditions including Alzheimer pathology, post-ischemic brain function, and post- traumatic brain injury deficits. Alterations in the expression o translocation of members of the GLUT family in either the endothelial cells of the blood brain barrier, astrocytes, or neurons, could alter neuronal energy supply and thereby neuronal function, subsequently altering behavior. A polymorphism in GLUT1 has been demonstrated in humans and linked to altered progression and prognosis in cancer and diabetic nephropathy. Alterations in the availability of GLUT1, due to genetic differences in expression, may alter the relative risk 0f development of aberrant behaviors following trauma, leading to the manifestation of MDD and/or PTSD. Given the crucial role of GLUT proteins in the transport of energy substrates into cerebral tissue, evidence of altered cerebral metabolism in neuropsychiatric disorders, and evidence that glucose transporters are altered after stress exposure in animal models, the proposed work tests the hypothesis that a polymorphism in GLUT 1 will decrease the incidence of MDD and/or PTSD following trauma exposure. This work challenges the standard paradigm because cerebral GLUT is a novel point of origin for consideration in the pathophysiology of MDD and PTSD as it is generally accepted that the changes in glucose transport that are documented in imaging of patients are the result of altered neuronal activity as opposed to the cause of altered neuronal activity. Specific Aim 1 will determine the extent to which a polymorphism in GLUT 1 decreases the manifestation of MDD following trauma exposure. DNA samples will be assessed for the rs710218 polymorphism in GLUT 1. Multivariate analyses will be conducted to determine if individuals that have been exposed to trauma and have the polymorphism are resistant to the manifestation of MDD. Specific Aim 2 will ascertain the degree to which a polymorphism in GLUT 1 decreases the incidence of PTSD following trauma exposure. Similar to work described in Aim 1, Aim 2 will again assess the influence of the GLUT 1 polymorphism on outcome from trauma, but in this case the focus will be on PTSD. Completion of the proposed work will provide novel and innovative insight into a potential metabolic susceptibility of genetic origin to trauma-induced mental health impairments. Appreciation for the role of metabolic factors in the manifestation of behavioral disorders will provide a new direction of consideration for novel therapeutic options. In addition, the proposed work will facilitate the cross- training of an established behavioral neuroscientist, Dr. Gretchen Neigh, currently specialized in investigation of the neural substrates of behavior using animal models, in human investigation. Enhancement of Dr. Neigh's training will lead to increased translational reciprocity in her research program and through her interactions with trainees and collaborators will improve interactions between clinical and basic researchers in the behavioral sciences.


Gram-positive bacterial pathogens inflict an enormous burden of human disease world-wide. This closely related group of microbes includes Bacillus anthracis, the most notorius bioterror agent, as well as Staphylococcus aureus, which, judged by human morbidity, is currently the single most important infectious agent in the United States. Broad dissemination of antibiotic (methicillin) resistant S. aureus (MRSA) strains in American communities implies the return of the pre-antibiotic era unless new therapies can reduce human mortality. B. anthracis has been weaponized and engineered to acquire antibiotic resistance traits that render currently available antibiotics ineffective and human populations defenseless, if they had been exposed to drug-resistant anthrax spores. The GLRCE Research Project 3 proposal addresses the need for new antibiotics by unraveling molecular mechanisms that lead to assembly of siderophores, proteins, capsules or teichoic acids in the cell wall envelope of B. anthracis and S. aureus. Biosynthesis of all four types of compounds is either essential for bacterial growth or absolutely required for the pathogenesis of infection. An interdisciplinary team of researchers at Argonne National Laboratory, the University of Michigan and the University of Chicago applies multiple different technological platforms to focus on these questions: bioinformatics, molecular genetics, biochemical purification and assay development, structure determination, organic chemistry and small molecule inhibition, as well as infectious disease modeling. Products of this research are the in depth molecular appreciation of envelope function and pathogenesis in B. anthracis and S. aureus and the identification of small molecule inhibitors that will be tested for their property of antiinfective or antibiotic therapies. The specific aims are: 1. Inhibition of capsular biosynthesis in Bacillus anthracis; 2. Inhibition of lipoteichoic acid biosynthesis in Bacillus anthracis and MRSA; 3. Inhibition of iron siderophore biosynthetic pathways in Bacillus anthracis and MRSA; 4. Inhibition of protein assembly pathways in the envelope of Bacillus anthracis and MRSA; 5. Inhibition of siderophore amide hydrolases in B. anthracis and MRSA.


In up to 26% surgical patients, subtle yet persistent deficits in learning and memory occur postoperatively, referred to as postoperative cognitive dysfunction (POCD). POCD has become a serious public health concern as it is associated with worse clinical outcomes including increased mortality. The pathogenesis underlying POCD remains unclear. Both modifiable and non-modifiable factors may contribute to POCD. To date, studies on POCD have primarily focused on direct influences of surgery and anesthesia on the central nervous system, which have identified age and genetics as major risk factors in POCD. Unfortunately, these are non-modifiable factors and difficult to be translated into clinical treatment. As such, there is an urgent need to identify modifiable factors underlying POCD. Among many modifiable factors, dietary influences and gut microbiota have been implicated in many neurological diseases with inflammatory features. Whether gut microbiota influences POCD has yet to be examined. In our preliminary studies, we observed a previously unrecognized role for gut microbiota in the development of POCD in mice post femoral artery exposure under isoflurane anesthesia. Specifically, we found: 1) mice with normal gut microbiota did not develop POCD while mice with gut dysbiosis developed POCD; 2) oral ampicillin treatment led to a status of gut dysbiosis, characterized by gut microbiota community structure changes and a dramatic decrease of indoles, particularly indoxyl-3-sulfate (IS) and indole-3-propionic acid (IPA); 3) oral administration of IPA, but not IS, deterred the POCD development; 4) mice with POCD displayed increased oxidation and impaired mitochondria function in the hippocampus, suggested by an enhanced production of reactive oxygen species (ROS), decreased production of NADH, and decreased protein levels of NDUFS4 (a critical mitochondria complex I component), when compared with mice without POCD; and 5) oral administration of IPA decreased ROS generation, increased NADH production and NDUFS4 protein levels in the hippocampus of ampicillin-treated mice. Based on these preliminary findings, we hypothesize that gut microbiota has a key influence on the development of POCD through IPA. In the research program proposed in this grant, we will examine the hypothesis by addressing three key questions: 1) Does the observed effect of gut dysbiosis on POCD represent an epiphenomenon or a ?permissive? effect? 2) What are the mechanisms underlying the IPA?s protective role in POCD? and 3) Can we develop a strategy based on gut microbiota and metabolites to prevent and treat POCD? This grant is built on our novel preliminary findings and our established research platform that combines cutting-edge metagenomics and metabolomics with immunological and neurobehavioral assays. Successful execution of this proposal will establish a novel conceptual framework linking modifiable factors such as diet and gut microbiota with POCD, and lead to new therapeutic strategies.


This study will compare 4 different study drug combinations for treatment of HIV infection. The purpose of this study is to see which of these study drug combinations does the best job of reducing HIV in the blood. Safety and tolerability of these drugs in children will be assessed.


To understand the mechanism of contractile process of the cleavage furrow, we propose to study (1) isolated cleavage furrow by biochemical methods, (2) immunohistochemical localization of contractile proteins in the furrow and (3) ultrastructural analysis of the contractile ring using cells which are frozen rapidly and freeze-substituted. Isolating cleavage furrow will be attempted by using micromanipulators on stabilized furrow. Immunohistochemical studies will be done at both light and electron microscopic levels, using antibodies against actin, myosin, alpha-actinin, actin binding protein and calmodulin. Rapid freezing is done using Heuser's apparatus and freeze-substitution will be done by cold acetone in the presence of OsO4.


The aims of this proposal are to develop flexible, semiparametric statistical models, methods, and inferences for longitudinal data, and to apply these models and methods to analyze AIDS data. The proposal consists of two aims. The first specific aim is to develop flexible models, methods, and inference for longitudinal data, which will involve (a) applying the penalized spline methods to longitudinal data analysis, comparing the methods with local kernel, regression spline methods to determine which is best in practice, developing statistical inference methods for penalized spline with longitudinal data; (b) developing flexible and efficient methods for time-varying coefficient mixed-effects models with longitudinal data, including investigation of local kernel regression and the penalized spline methods; and developing flexible methods for generalized varying-coefficient mixed-effects models with longitudinal data; (c) developing flexible methods for general two-stage semiparametric nonlinear mixed-effects models; (d) developing computer packages to implement the proposed methods. The second aim is to apply the proposed models and methods developed to study HIV dynamics by using data from AIDS clinical trials run by the AIDS Clinical Trial Group (ACTG) and data from AIDS clinical trials conducted at St. Jude Children's Research Hospital. We will focus on (a) characterizing long-term HIV/T-cell dynamics in HIV-l-infected patients treated with highly active antiretroviral therapy by using flexible, nonparametric or semiparametric methods for longitudinal data; and studying the relation between long-term HIV dynamics and T-lymphocyte kinetics during long-term antiretroviral drug exposure; (b) investigating which clinical and specific factors affect HIV dynamics; (c) investigating the relation between clinical endpoints and HIV dynamics; and (d) comparing the effects of different antiviral agents, and presenting statistical evidence to justify which of several treatments is the most effective. The proposed research is expected to benefit studies of the immune system in HIV-infected patients (one of the main focuses of future AIDS research).


Post-translational modifications (PTMs) of histone tails are major marks for transcription activation and silencing. Accordingly, detecting and capturing post-translationally modified histones represent a critical step in epigenetic research. A major technological bottleneck in this area of research is a paucity of high-quality affinity reagents. Polyclonal and monoclonal antibodies are the only widely available affinity reagents for histone tails, but they have fundamental limitations in reproducibility, scalability, storage and production throughput and expenses. The long-term goals of this project are to develop an innovative and powerful technology platform for facile production of high-quality affinity reagents for histone tails containing PTMs and to make a standard set of such affinity reagents broadly available to the epigenetics research community. This project is built on an innovative protein-engineering concept that our group has recently established. The concept, termed Affinity Clamping, harnesses the inherent specificity present in the so-called interaction domains and dramatically enhances their affinity and specificity by attaching an "enhancer domain" and subsequently optimizing its interaction interface by directed evolution of combinatorial libraries. The resulting affinity reagents with clamshell architecture, collectively termed "Affinity Clamps" thus "clamp" the target, leading to orders-of-magnitude higher affinity and specificity. Protein libraries made in this manner are predisposed to binding to a specific class of peptide motifs (e.g. histone tails with a methylated lysine), and they virtually guarantee successful engineering of high-performance affinity reagents for a predefined peptide motif. Affinity Clamping represents a paradigm shift in affinity reagent generation. Because Affinity Clamps are fully recombinant reagents produced in E. coli, they can be easily produced in large quantities and distributed. Also they can be reformatted into a variety of fusion proteins suitable for in vitro and in vivo applications. Our proof-of-concept experiments have successfully demonstrated the general feasibility of the Affinity Clamping concept and suggest its enormous potential. Because there exist a number of interaction domains that weakly bind to post-translationally modified histone tails, we are confident that we can apply the Affinity Clamping strategy to produce high-quality affinity reagents to a variety of histone motifs. The proposed project will critically evaluate the feasibility and potential of applying the Affinity Clamping technology to epigenetic histone marks. The specific aims of the initial project period are (i) to produce "Histone Clamps" for well-characterized histone lysine methylation sites and benchmark them against commercially available monoclonal antibodies for their performance in commonly used assays;and (ii) to produce Histone Clamps for histone methylation sites for which no high-quality antibodies exist. Such Histone Clamps will be provided to the epigenetic community, which will have a major impact on the quality, scale and types of epigenetics research. PUBLIC HEALTH RELEVANCE: Accurately measuring the type and amounts of chemically modified forms of histones and capturing them for downstream analysis are major technological challenges in epigenetic research. This project will establish a totally new approach to facile generation of high-performance reagents for these purposes. This innovative and powerful technology will fill a major void in the currently epigenetic research, and products from this project, termed "Histone Clamps", will make it feasible to establish a standard set of epigenetic capture reagents that can be distributed broadly to the community and open new avenues of epigenetics research.


We have continued our studies of chromatin structure in the neighborhood of expressed genes. The globin gene family in chicken erythroid cells serves as a model system in which it is possible to study the mechanisms associated with regulation of the cluster and individual members of the family during erythroid development. We have focused attention on the 1.2 kb insulator DNA sequence at the 5' end of the chicken beta-globin locus, which is capable of blocking enhancer-promoter interaction when it lies between them. We had earlier narrowed down this activity to a small DNA fragment within the 1.2 kb sequence. We also purified the protein responsible for this activity and identified it as CTCF, which is a known factor ubiquitous in vertebrates, with both stimulatory and inhibitory effects on expression of a variety of genes, and we showed that CTCF sites are present in other vertebrate gene loci, including the 3' end of the chicken beta globin locus. We were also led to investigate a potential insulator activity in the neighborhood of the Igf2 (Insulin-like growth factor 2)/H19 locus in mouse and human. This is an imprinted locus: In the maternally transmitted allele Igf2 is silent and H19 is expressed. The reverse is true in the paternally transmitted allele. A region between the two genes is methylated on the paternal allele only, and it had been suggested that the insulator was within this region. We have now shown that this region contains multiple binding sites for CTCF (four in mouse, seven in human). They serve as very strong enhancer blocking elements, preventing a downstream enhancer from activating Igf2 in the maternal allele. Deletion of these sites abolishes insulation. We also have shown that methylation of the sites prevents CTCF binding. Thus the selective activation of Igf2 on the paternal allele is explained by inactivation of the insulator, which allows the downstream enhancer to turn on this gene. Our results also show that insulator activity can be modulated during development by site methylation. This greatly widens the potential range of action of insulator elements. In other studies we have examined the pattern of chromatin structural changes over the globin locus and its neighborhood during development. We had shown earlier that an erythroid specific folate receptor (FR) gene lies upstream of the chicken beta-globin locus, separated from it by 16 kilobases of condensed non-expressed chromatin. Making use of high resolution immunoprecipitation methods, we have shown that there are major changes in histone acetylation levels over these genes in cells corresponding to various developmental stages, but that the condensed chromatin domain and certain regulatory elements maintain their low and high acetylation levels (respectively) throughout development.


A number of recent studies have found a schizophrenia-associated loss of cells or volume in the human thalamus. Unfortunately, these previous studies did not have the resolution necessary to precisely localize the abnormalities. The specific aims of the proposed research are to use efficient stereological methods to: 1) Survey the entire thalamus for schizophrenia-related loss of volume or cellularity, 2) Localize any detected abnormalities to particular cytoarchitectonic divisions, and 3) Investigate the specificity of any detected abnormalities to schizophrenia. The investigator will use well-characterized postmortem material from the Mount Sinai Medical Center/Bronx Veteran's Affairs Hospital Schizophrenia Brain Bank to test the following primary hypotheses: 1) Previously reported schizophrenia-associated abnormalities in the mediodorsal thalamic nucleus will primarily involve its parvocellular and caudal divisions which, in man, have a prefrontal cortical dependence. The magnocellular division which does not degenerate following prefrontal ablations may be relatively spared in schizophrenia. 2) Schizophrenia-associated loss of cells and volume will not be restricted to the mediodorsal nucleus. Other nuclei where abnormalities are likely to occur include those with predominately temporolimbic connections and those with patterns of connections suggesting the potential for a role in arousal or in gating signals to and from cortex. The more specific sensory and motor relay nuclei are likely to be spared. 3) The schizophrenia-associated thalamic abnormalities are not common to all chronic mental illnesses and will not be seen in subjects with affective disorders. The studies of the present proposal will lay the groundwork for more definitive future explorations that will more fully assess the main and interactive effects of age, chronic institutionalization, neuroleptic exposure, and gender on the anatomy of thalamic nuclei where schizophrenia-associated abnormalities are found.


Recently, we have successfully produced simian-human immunodeficiency virus-like particles (SHIV VLPs) which contain SIV Gag and HIV Env by using a baculovirus expression system. Furthermore, we have incorporated the influenza virus surface glycoprotein, hemagglutinin (HA), into SHIV VLPs. Taking advantage of HA having a high affinity to bind to the mucosa of the upper respiratory track, our central hypothesis is that use of SHIV VLPs containing influenza HA as a mucosal vaccine will enhance both systemic and mucosal immune responses against HIV infection. The major focus of this project is to investigate the efficiency and mechanisms of the built-in adjuvanticity of HA in SHIV VLPs for intranasal immunization in a mouse model. Specifically, we propose: 1). To determine the role of incorporation with influenza HA in SHIV VLPs as a mucosal vaccine in enhancement of immune responses against HIV. Proposed experiments will investigate: a) whether the built-in adjuvanticity of influenza HA in SHIV VLPs as a mucosal vaccine is more potent than soluble influenza HA in enhancement of both systemic and mucosal immunity; and b). whether the receptor binding or membrane fusion activity of influenza HA affects its adjuvanticity for SHIV VLPs. 2). To determine the role of incorporation with influenza HA in SHIV VLPs in dendritic cell (DC) binding, activation, cytokine production, and antigen presentation. We propose to investigate: a). whether HA/SHIV VLPs have an increased ability to DC binding, internalization, and subcellular localization; b). whether HA/SHIV VLPs have an increased effect on DC activation, and cytokine production; c). whether HA/SHIV VLP-activated DCs increase naive T cell proliferation; and d). whether HA/SHIV VLPs increase the efficiency of antigen cross-presentation of DCs to CD8+ T cells and what are the associated intracellular pathways. 3). To determine the role of incorporation with influenza HA in SHIV VLPs in specific B cell binding, activation, and antibody production without CD4+ T cell help. Experiments are designed to investigate: a). whether HA/SHIV VLPs increase their ability to bind to naive B cells; b). whether HA/SHIV VLPs have an increased effect on naive B cell proliferation; c). whether HA/SHIV VLP-activated DCs have an increased ability to adhere to naive B cells; d). whether HA/SHIV VLP-activated DCs increase naive B cell proliferation; and e). whether B cell activation and differentiation and cytotoxic CD8+ formation occur in nasal-associated lymphoid tissue (NALT), an inductive site after intranasal immunization with HA/SHIV VLPs in CD4+ T-cell-deficient mice. This project represents a novel approach to develop an effective and safe HIV vaccine. Understanding the cellular and molecular mechanisms of HA/SHIV VLP-enhanced immune responses in mice is critical for the future design and testing of a successful HIV vaccine in non-human primate models and in human trials.


The experiments outlined in this application are designed to address the role of the synaptic protein PSD-95 in the synaptic transmission and plasticity of excitatory synapses in the nucleus accumbens shell (NAc) and if these mechanisms underlie some of the effects of chronic drug exposure. Genetic manipulation via recombinant lentivirus will be used to acutely knock down or enhance expression of PSD-95 proteins in medium spiny neurons of the NAc shell in living wild type mice. Characteristics of basal excitatory synaptic transmission will be examined in infected vs uninfected neurons via whole cell patch clamp recording and pharmacological manipulation of acute NAc slices. The effects of PSD-95 depletion or enhancement upon synaptic plasticity in the NAc shell will be addressed by studying NMDAR dependant forms of LTD and LTP as well as endocannabinoid LTD in infected neurons. If time permits, in vivo manipulation of PSD-95 levels will also be combined with a chronic cocaine regimen in order to examine the contributions of this protein to the depression of synaptic strength seen in the NAc shell in response to long term drug exposure.


ABSTRACT: A significant need exists to address complications associated with the growing pandemic of diabetes mellitus and metabolic syndrome now estimated to affect >20 million people in the United States (~7% of the population!). Impaired wound repair, a major complication of poor glycemic control, costs >$10 billion annually. Our laboratory first identified and cloned rabbit (Larrick et al., 1991) and then human Cationic Anti- microbial Protein (hCAP18) based on its LPS binding and anti-microbial activities. Over the past 10 years hCAP18 has been shown to be an important component of the innate immune system with broad anti-microbial activity conferred by its C-terminal fragment LL-37. hCAP18 is constitutively produced in leukocytes and is induced in barrier organs (e.g. lung, GI, skin) upon inflammation and infection. Recent work demonstrated a key role of hCAP18 in vascularization and re-epithelialization of skin wounds. High levels of hCAP18 are produced in skin in vivo upon wounding with a peak at 48 h post-injury, declining to pre-injury levels upon wound closure. hCAP18 is detected in the inflammatory infiltrate and in the epithelium migrating over the wound bed. For example, using a noninflammatory ex vivo wound healing model, composed of organ-cultured human skin, Heilbron et al. (2003) showed that treatment with anti-LL-37 antibodies inhibits re-epithelialization. In chronic non-healing wounds, hCAP18 levels are low and immunoreactivity for hCAP18/LL-37 is absent in ulcer edge epithelium. We hypothesize that LL-37 plays a critical role in wound closure and that its absence in chronic wounds impairs re-epithelialization, promotes bacterial colonization and contributes to failure of the wounds to heal. Hence, the overall goal of this proposal is to develop a protease resistance form of LL-37 as a novel therapy for chronic, non-healing wounds. To this end in phase I we will prepare prLL-37, a protease resistant form comprised of D-amino acids, demonstrate resistance to degradation by proteases, and evaluate activity of prLL-37 in a wound healing model using diabetic mice. In phase II we will carry out preclinical studies to support submission of an IND. This therapy will address a major unmet need among patients suffering from burn injuries and chronic non-healing, diabetic foot, decubitus and venous stasis ulcers. ABSTRACT: A significant need exists to address complications associated with the growing pandemic of diabetes mellitus and metabolic syndrome now estimated to affect >20 million people in the United States (~7% of the population!). Impaired wound repair, a major complication of poor glycemic control, costs >$10 billion annually. Our laboratory first identified and cloned rabbit (Larrick et al., 1991) and then human Cationic Anti- microbial Protein (hCAP18) based on its LPS binding and anti-microbial activities. Over the past 10 years hCAP18 has been shown to be an important component of the innate immune system with broad anti-microbial activity conferred by its C-terminal fragment LL-37. hCAP18 is constitutively produced in leukocytes and is induced in barrier organs (e.g. lung, GI, skin) upon inflammation and infection. Recent work demonstrated a key role of hCAP18 in vascularization and re-epithelialization of skin wounds. High levels of hCAP18 are produced in skin in vivo upon wounding with a peak at 48 h post-injury, declining to pre-injury levels upon wound closure. hCAP18 is detected in the inflammatory infiltrate and in the epithelium migrating over the wound bed. For example, using a noninflammatory ex vivo wound healing model, composed of organ-cultured human skin, Heilbron et al. (2003) showed that treatment with anti-LL-37 antibodies inhibits re-epithelialization. In chronic non-healing wounds, hCAP18 levels are low and immunoreactivity for hCAP18/LL-37 is absent in ulcer edge epithelium. We hypothesize that LL-37 plays a critical role in wound closure and that its absence in chronic wounds impairs re-epithelialization, promotes bacterial colonization and contributes to failure of the wounds to heal. Hence, the overall goal of this proposal is to develop a protease resistance form of LL-37 as a novel therapy for chronic, non-healing wounds. To this end in phase I we will prepare prLL-37, a protease resistant form comprised of D-amino acids, demonstrate resistance to degradation by proteases, and evaluate activity of prLL-37 in a wound healing model using diabetic mice. In phase II we will carry out preclinical studies to support submission of an IND. This therapy will address a major unmet need among patients suffering from burn injuries and chronic non-healing, diabetic foot, decubitus and venous stasis ulcers. [unreadable] [unreadable] [unreadable]


This contract is to assess the new third-generation SERM, Bazedoxifene, alone and in combination with CE and/or an AI, to prevent mammary carcinogenesis induced in virgin female Sprague-Dawley rats by N-methyl-N-nitrosourea (MNU). The tasks will involve optimizing the dose and timing of BZA administration, and PK profile of BZA in combination with the AI.


Our microchemical facility at Caltech has exceeded all expectations in being able to sequence small quantities of protein and synthesize small or large fragments of genes. Several gas phase microsequenators are now functional and each has the capacity to sequence 20 or so residues a day. We have a DNA synthesizer with a three column system that has the capacity to synthesize simultaneously three oligonucleotides with a 14 min. cycle time. Thus, the microchemical facility has the considerable ability to sequence proteins and synthesize genes. These abilities have been turned to a variety of projects that are directly related to the cancer problem. Microsequencing done in this facility on platelet derived growth factor demonstrated for the first time the serum polypeptide hormone appears to be strikingly homologous to the primate oncogene. We are now in the process of analyzing the sequences of a series of other growth hormones and will have the opportunity to determine whether additional similar correlations can be made. We have also started to analyze the gene structure organization and rearrangements of genetic elements in coding the T cell receptor. Once again this receptor molecule plays a central role in facilitating the immune response against foreign invaders including tumors. The microchemical facility has been used to synthesize a wide range of DNA probes that have been used to clone the corresponding genes (partially documented in the Progress Report). Indeed, the DNA synthesizer has been used to synthesize an entire functional E. coli serine tRNA gene. The computer facility has added valuable backup and data analysis capacity to these and many other projects. The cell sorter is functional and has provided valuable data for a number of different laboratories. Within the next year we expect to have a fully functional automated peptide synthesizer and be well along in developing an automated DNA sequenator. Thus, this complement of instruments will allow us to both sequence and synthesize genes and DNA in a sensitive and rapid fashion. This year the microchemical facility has been used by more than 15 professors at Caltech and a much larger number of investigators outside the Institute.


This project is designed to investigate the roles of monocytes in limiting blood-borne metastasis of tumor cells, and to provide a valid basis for effective immunochemo prophylaxis (and therapy) of metastatic spread. Utilizing both metastasing and non-metastasing syngeneic tumor lines of DBA/2 mice, host immunity is semi-quantitatively assessed in normal and tumor-bearing animals by i.v. injection of radio-labeled tumor cells and by measuring the rate of tumor cell clearance in various target organs of these animals. Non-metastasing T1699 mammary adenocarcinoma is employed as the model in which immune suppression of the host is a prerequisite for successful development of metastatic growth. Immune suppression is induced by treatment with anti-thymocyte serum, antimacrophage serum, tilorone HCl, or by over dosage of chemotherapeutic agents. Restoration of therapeutic activity by in vivo (or in vitro) armed monocytes is attempted in this system. Spontaneously metastasing P1534 lymphatic leukemia or PP388D1 macrophage-like tumor line are used to study the effect of monocyte stimulants, e.g., pyran copolymers and beta-1,3-glucan on chemotherapeutic regimens and also on augmentative effect of in vitro armed monocytes on such chemotherapeutic effects. Results will be evaluated in relation to actual inhibition of metastatic growth of these tumors.


The proposed research seeks to understand how the spatial and temporal control of actin dynamics is regulated differentially across the developing vertebrate embryo. This will be investigated within the specific context of mesodermal cell behaviors during gastrulation and segmentation in the zebrafish embryo. The potential role of actin regulatory molecules (ARMs) that are expressed in specific areas of developing mesoderm will be investigated by loss of function analysis, using confocal imaging and a transgenic line of reporter fish expressing actin-GFP fusion protein that reveals cellular actin distribution. The signaling pathways controlling these ARMs will be characterized using mutagenesis screening and protein-protein interaction assays. The misregulation of actin and cell movements has been implicated in multiple human conditions such as Wiskott- Aldrich syndrome and Spina Bifida, in which compromised cell polarity causes a failure of neural tube closure in early development. An understanding of actin regulation during vertebrate development in the zebrafish may thus provide insights into the causes and treatment of certain human diseases.