PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human diseases. The focus of research, which had been placed mostly on development of therapeutic agents, has shifted gradually towards its prevention. In this context, many studies have linked obesity and long-standing type-2 diabetes mellitus with PDAC development. These metabolic diseases are characterized by peripheral insulin resistance, hyperinsulinemia, increased IGF-1 and chronic inflammation. Previously, crosstalk mechanisms between insulin/IGF-1 receptors, G protein-coupled receptor (GPCR) and EGF receptor (EGFR) signaling systems have been identified that potently stimulate proliferation of PDAC cells harboring a KRAS mutations. Mitogenic crosstalk depended on the function of mTORC1, ERK and PKD and opposed by AMPK, a target for the antidiabetic agent metformin. The identification of the key downstream targets of this signaling network is of fundamental significance and major translational interest. The YAP/TAZ transcriptional co-activators are emerging as points of integration in the action of KRAS, GPCRs and AMPK, all signaling pathways highly relevant in PDAC. New preliminary studies demonstrate that YAP/TAZ activation is a crucial point of convergence in the crosstalk between GPCR and insulin signaling in PDAC cells. Importantly, lipid-lowering drugs of the statin family potently blocked YAP/TAZ activity, including YAP/TAZ/TEAD-regulated genes, such as CTGF, Cyr61 and NUAK2. Lipophilic statins, including cerivastatin, simvastatin and atorvastatin, strikingly inhibited colony formation of human and mouse PDAC cells. Statins inhibited PDAC colony formation acting synergistically with metformin. Further preliminary results in vivo show that oral administration of simvastatin attenuated the loss of intact acini and the development of pre-neoplastic lesions in the pancreas promoted by an obesogenic diet in conditional KrasG12D (KC) mice. Accordingly, the central hypothesis to be explored in Project 2 of this P01 is that the well tolerated cholesterol-lowering drugs of the statin family inhibit obesity-induced promotion of PDAC via inhibition of PKD/YAP/TAZ. The Specific Aims of Project 2 have been designed to investigate important gaps in current knowledge: 1) Characterize the chemopreventive effects of statins on the progression of PanINs and development of PDAC using the conditional KrasG12D model (KC mice) subjected to control or diet-induced obesity (DIO) and in KC mice carrying a homozygous deletion of the ob gene. 2) Identify a novel molecular mechanism by which statins inhibit YAP/TEAD signaling in mouse and human pancreatic cells. 3) Characterize the inhibitory effect of a low-dose combination of statin and metformin on the development of PDAC: a novel chemopreventive strategy. The studies proposed in Project 2 of this P01 will provide mechanisms and rationale for novel chemo-preventive strategies in obesity-related PDAC. Since statins and metformin are widely used Food and Drug Administration (FDA)-approved drugs, the mechanistic and preclinical studies proposed have the potential for rapid translation to PDAC and other obesity-associated cancers.


[unreadable] [unreadable] Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardio-degenerative disorder for which there are currently no established effective treatments. The identification of the disease gene in 1996 and the subsequent elucidation of the function of the encoded protein, frataxin, have opened the door to possible therapeutic approaches, including conventional high-throughput drug screening. This proposal describes a novel, complementary approach to the development of therapeutics for FRDA. We devised a method to construct a library that encodes random, short-hairpin-loop RNAs (shRNAs). This library can be used to identify shRNA sequences of therapeutic, therapeutic-targeting, and/or biological interest. The identification is based on functional selection, with effective sequences retrieved by PCR from cells that survive a particular condition or exhibit a predetermined phenotype. The design allows for hit-optimization of effective sequences, with re-selection for sequences with improved effects. Using primary FRDA fibroblasts, we developed screening and selection assays based on the critical role of mitochondrial dysfunction in the signs and symptoms of FRDA and on the sensitivity of FRDA cells to oxidative stress. With our live/dead selection assays, we can use our random shRNA-encoding library to identify shRNA sequences of benefit to FRDA cells. With our screening assays of mitochondrial function, we can confirm and prioritize these sequences. The primary objective of this proposal is to identify potential shRNA therapeutics. A secondary objective is to begin to understand the mechanisms of action of the shRNAs we identify. The Specific Aims are: 1. To identify shRNA sequences that allow survival under conditions lethal to primary FRDA fibroblasts but non-lethal to normal control cells. 2. To optimize the shRNA sequences identified in Aim 1. 3. To confirm and prioritize optimized shRNAs and begin to understand mechanisms of action. This proposal describes an approach to develop novel therapeutics for Friedreich ataxia using a random shRNA library. This approach has potential implications for the development of therapeutics for other genetic diseases, as well as for infectious diseases, and is therefore highly relevant to public health. [unreadable] [unreadable] [unreadable] [unreadable]


It is proposed to investigate the immunological status of mice with mammary cancer and of mammary tumor-prone and -resistant mice of several inbred strains. Emphasis will be placed on the analysis of both cell- mediated and humoral immune response to viral and tumor antigens associated with mammary tumors as a model for immunological studies on human breast cancer. In addition, techniques and concepts developed in animal studies will be concomitantly applied to human breast cancer material in a study of antigens in cells derived from human breast cancer patients and antibodies to these antigens in the sera of these patients. Conventional immunological methods to be applied in the study include analysis of lymphocyte-mediated toxicity, blastogenesis determinations, lymphoid cell migration inhibition, cytotoxicity, immunofluorescence, immunoelectron microscopy, immunodiffusion, and radioimmunoassay. The goals of the proposed research are to determine the role of viral and tumor antigens in the immune response to breast cancer in mice and in women and to determine by these methods whether viruses similar or related to mouse mammary tumor virus may be associated with the human neoplasm.


A search for potential regulatory proteins among the components of mammalian cell chromatin is in progress, using high-resolution, two-dimensional polyacrylamide gel electrophoresis. Several hundred rare species (less than 10,000 copies/cell) have been found in HeLa chromatin, and it has been shown that these are not detectable in cytoplasm. Comparisons with the protein of other human cell types are underway. Similar studies are in progress with Friend leukemia (mouse) cells, comparing protein patterns of control cells and cells induced to form hemoglobin. Several possible regulators of globin gene transcription and/or post-transcriptional processing of globin mRNAs have been identified. Attempts to test these proteins for specificity of interaction with globin mRNAs and/or globin genes are planned.


The overarching goal of this proposal is to create and validate pragmatic tools to rapidly detect and define the mechanism of diuretic resistance (DR), allowing individualized therapy in patients with acute decompensated heart failure (ADHF). ADHF is the most common hospital discharge diagnosis among Medicare beneficiaries and accounts for more than half of all heart failure (HF) related expenditures. This epidemic of ADHF is primarily driven by fluid and sodium overload, leaving the loop diuretics as the most commonly used medications to treat and prevent ADHF. Unfortunately, a loss of response to loop diuretics, termed diuretic resistance (DR), is common and contributes to a vicious cycle of out of hospital fluid/sodium retention, incomplete in-hospital decongestion, followed by post-discharge re-accumulation of fluid/sodium and worsened outcomes. Despite its importance, the speed and fidelity with which we can diagnose DR is extremely limited. This results in potentially avoidable hospitalization of outpatients and wasted hospital days where effective diuresis does not occur in inpatients. Once recognized, tools to determine the mechanism for DR and thus individualize treatment are nonexistent. Although multiple mechanisms contribute to loop DR, two therapeutically distinct groups exist: (1) inadequate effect at the tubular site of action, whih requires treatment with increased dose or delivery of loop diuretic and (2) compensatory distal tubular sodium reabsorption, which requires treatment with sequential nephron blockade (i.e., thiazide diuretics). Our inability to differentiate these mechanisms leaves trial and error as the only method to treat DR, further delaying effective diuresis and exposing patients to medications with known toxicities when the empiric choice is incorrect. In the present study, we will enroll 200 ADHF patients and evaluate them longitudinally through key transitions in loop diuretic therapy (early into IV therapy, late IV therapy, after conversion to oral diuretics, and 5-7 days post discharge). Patients with significant DR during early IV therapy will be randomized to increased loop diuretic dose or add on thiazide diuretic stratified by the DR mechanism. The data generated through the above investigation will allow us to: (1) develop inexpensive and efficient tools to predict diuretic response in a reliable and timely manner; (2) understand the prevalence of therapeutically targetable mechanisms of DR using endogenous lithium clearance, a gold standard technique to query in vivo proximal tubular/loop of Henle sodium handling; (3) develop methodology to differentiate DR mechanisms using common/inexpensive laboratory tests; and (4) provide proof of concept that mechanistically tailored diuretic therapy can improve natriuresis. Our preliminary data suggests that diagnosis and phenotyping of DR can in fact be done with excellent accuracy (AUC =~0.9) using universally available urine/serum chemistries. At the conclusion of this research, our goal is to provide clinicians and researchers with a workable tool to accurately/rapidly diagnose and phenotype DR allowing individualized diuretic therapy for both inpatients and outpatients with heart failure.


Investigators who have achieved independent National Eye Institute (NEI) funding will be provided with additional shared support to enhance their own and University of Washington's capability for conducting vision research. Collaborative studies will be facilitated, and scientists will be attracted to research on the visual system by the presence of this shared support. A modular organizational structure will be maintained, with each module devoted to a specific activity that would be impractical or less efficient to support on an individual research grant. Each module will support a service or resource that enhances or facilitates the research efforts of a CORE group of investigators, each having independent funding. Some sharing of resources and services with non-NEI-funded collaborators and with investigators new to vision research will occur. Junior researchers will also have access to these facilities to improve their ability to attain independent NEI funding. Proposed modules include: Biochemistry/Immunology (B/l), Genotyping/Phenotyping (G/P), Morphological Imaging (M/l), and Psychophysics/Physiology (PIP). Areas of investigation include retinal and choroidal diseases, corneal wound healing, corneal diseases, lens and cataract, glaucoma, strabismus, amblyopia, visual processing, and ocular development. Specific disciplines that will be brought to bear on these problems include: behavioral studies, biochemistry, biostatistics, molecular biology, cell biology, proteomics, immunology, microscopy, microbiology, morphometry, neurophysiology, and pathology. This project will elucidate basic mechanisms that underlie the function of the eye and the visual system and apply this knowledge and other information to the solution of problems in vision and ophthalmology. Collaboration among investigators from the University of Washington and elsewhere will be promoted. This proposal will improve the effectiveness of funding available on individual research project grants.


At total body radiation exposures of 3-8 Gy, the predominant cause of death is the hematopoietic syndrome. Many of these deaths are preventable with rapid triage of victims for cytokine therapies and aggressive supportive care. Unfortunately current modalities for identifying patients at risk for the hematopoietic syndrome suffer from inaccuracy, high expense, long analysis times, and delayed diagnosis. Furthermore, none of these methods directly measures radiation damage to the bone marrow, nor do they indicate the existence of residual hematopoiesis, and most are not amenable to point-of-care in emergency conditions. Thus, there is a critical unmet need for a field-deployable diagnostic for use following a radiological incident to identify victims who will develop the potentially fatal ye often treatable hematopoietic syndrome. We propose to build upon a highly successful paradigm in clinical diagnostics, which is that injured tissues leak, shed, and/or secrete proteins into the plasma, where they are useful biomarkers indicating the extent of tissue injury. Precedents include the troponins in myocardial infarction, transaminases in liver injury, creatine kinase in muscle injury, and lipase in pancreatic processes. Accordingly, we hypothesize that following radiation-induced injury to the bone marrow, proteins are released from the marrow into the bloodstream, where they provide useful biomarker signals predictive of the onset and severity of the hematopoietic syndrome. We propose to identify these plasma biomarkers of radiation-induced marrow injury using an innovative approach incorporating targeted proteomic technologies that we have developed and validated in a biomarker discovery pipeline that will substantially increase our chances of success compared with traditional approaches by enabling the testing of a very large (unprecedented) number of plasma biomarker candidates. Aim 1. Using SILAC-labeled mice, identify proteins induced in the bone marrow in response to radiation, and subsequently test hundreds of these putative tissue injury markers to identify the subset that are stably elevated in the plasma post-exposure. Aim 2: Characterize candidate marrow injury biomarkers identified in Aim 1 with respect to their: a. stability in plasma over time, dose range, and dose rates b. correlation with clinical endpoints (indicators of hematopoietic syndrome) c. specificity for damage to the hematopoietic system and damage caused by radiation d. use across a heterogeneous population (pediatric, geriatric, gender, genetically susceptible) Aim 3. Determine which of the radiation biomarkers of marrow damage identified in the mouse are elevated in human blood following radiation exposure, and develop a point-of-care assay device that will form the basis of subsequent human clinical validation trials (beyond this proposal).


This study will examine patterns of care and management of mental illness among displaced persons in the Republic of Georgia, with a focus on decision-making about care- seeking and types of informal strategies and services used. The study will take a mixed methods approach with semi-structured interviews leading to instrument development. Background: There is limited research on mental health concerns or access to mental health care in low and middle income countries, including among displaced populations. Care-seeking to mental health services is impeded by limited access and stigma of mental illness. The Republic of Georgia is a country in political and economic transition that has experienced significant displacement within its borders. Available research on the displaced population in Georgia suggests that they experience higher rates of psychological disorders and chronic disease. It is not clear how mental health problems among the displaced are conceptualized, addressed, and managed. There is a need to study care-seeking behavior to formal services, as well as to informal services and within social support structures. Available literature on mental health services does not clearly delineate care-seeking and management processes to informal services. Understanding such care-seeking will inform the development of prevention and service delivery interventions. A mixed methods approach allows for in-depth exploration and the development of an instrument that can be adapted to different programmatic settings with migrant and displaced populations. Methods: The study site is Zugdidi, an urban center in northwestern Georgia. The study population is protracted, internally displaced persons (IDPs) ages 18 and over residing in the community. In-depth, qualitative interviews will be carried out with 30-45 IDPs and will include modules on migration history, problem recognition, perceived need, and care-seeking behavior. Interview data will be used to construct an instrument to measure patterns of care and management. The instrument will be pilot tested with 165 IDP households. Local service organizations will be partnered with for future validation studies and hypothesis testing.


Many important biological processes are regulated by tyrosyl phosphorylation, which is controlled by the opposing actions of protein-- tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). Abnormal regulation of these pathways can lead to diseases such as cancer. A complete understanding of cellular and whole organism regulation by tyrosyl phosphorylation requires defining how specific PTKs and PTPs interact. Such understanding may lead to the development of new, more specific therapeutic reagents for treating human disease. The long range goal of this research program is to understand the biological functions and mechanism of action of the SH2 domain-containing tyrosine phosphatase SHP2 in hematopoietic cells. Previous studies by several laboratories, including our own, established SHP2 as a vital component of signaling pathways downstream of cytokine receptors, hematopoietic growth factor receptors and multi- chain immune recognition receptors. These studies suggested that a 97kD phosphotyrosyl protein was a major target/regulator of SHP2 action, but the identity of this protein and its functions had remained unclear. During the initial funding period, we identified, purified and cloned p97. p97 is a relative of Gab1 and Drosophila Dos, a regulator of the fly homolog of SHP-2; thus, we renamed p97 as Gab2. Analyses of the effects of wild type and mutant versions of Gab2 suggest that it is a key regulator of cytokine signal transduction and also participates in signaling downstream of other types of hematopoietic cell receptors. Our studies establish that SHP2 acts at more than one step in cytokine receptor signal transduction: one requires Gab2 binding and the other is Gab-2 independent. The Ciab2-independent pathway is required for MAP kinase activation, whereas Gab2, acting via SHP2, regulates a novel signaling pathway downstream of/or parallel to MAP kinase activation but upstream of cytokine-induced gene expression. Our recent studies show that Gab2 is recruited to cytokine receptors via a novel complex involving Shc, and then provides the major route to PI-3 kinase activation for those receptors that do not bind PI3K directly. We will elucidate the details of the Shc/Gab2 pathway in cytokine signaling. and determine what other pathways are regulated by Gab2/PI3K complexes. Next, we will define how SHP2 regulates MAP kinase activation in a Gab-2- independent manner and how the Gab2/SHP2 complex regulates immediate early gene transcription. We will characterize the hematopoietic effects of "true-null" SHP2 knockout mice and Gab2-/- mice to determine the physiological function of these molecules in vivo. These studies should yield new insights into how tyrosyl phosphorylation is controlled. clarify how specific PTPs contribute to regulation, and identify a new signaling pathway regulating cytokine-induced gene expression. Since abnormal regulation of cytokine signaling is implicated in leukemogenesis and some carcinomas, our studies may also suggest new targets for therapeutic intervention in human neoplastic disease.


1. We are studying the transmembrane channel-like (TMC) genes 1 and 2. We have generated mice segregating knockout (null) alleles of Tmc1 and Tmc2. We are characterizing their mutant auditory and vestibular (balance) phenotypes. Mice that are homozygous for knockout alleles of both genes are deaf and have abnormal vestibular function. Mice that are homozygous for the Tmc1 knockout allele are deaf. Mice that are homozygous for the Tmc2 knockout allele have normal hearing and balance. These results indicate that both Tmc1 and Tmc2 are required for normal vestibular function (balance), whereas only Tmc1 is required for hearing. We use LacZ reporter genes in the knockout mice to study where the genetic interaction might be taking place. The Tmc1 reporter is expressed in hair cells of all of the vestibular end organs whereas the Tmc2 reporter is expressed in hair cells of the cristae ampullaris. We are now using hair cell markers to determine if these genes are expressed in type 1 or type 2 hair cells, or both. We are collaborating with Dr. Charley Della Santina to measure vestibulo-ocular responses in the mice to determine if their abnormal balance and gait is due to abnormal function of the semicircular canals (cristae) or other parts of the vestibular system. [unreadable] [unreadable] 2. We used a yeast two-hybrid screen to isolate genes encoding proteins that potentially interact with TMC1. We narrowed the list to a few candidate genes implicated in vesicular trafficking and in antiapoptosis and cell survival. We are using a combination of approaches to determine which interactions occur in situ in hair cells.[unreadable] [unreadable] 3. We generated knockout mice for Tmc6 and Tmc8 to better understand the function(s) of Tmc genes and proteins. Mutations in human TMC6 or TMC8 genes cause epidermodysplasia verruciformis, a recessive disease resulting in chronic cutaneous HPV infections (papillomas) with increased susceptibility to non-melanoma skin cancers. We have done extensive RNA expression analyses to show that Tmc6 and Tmc8 are primarily expressed in lymphoid cells and tissues and lung and skin, and primarily during development. The homozygous knockout mice have no obvious phenotypic abnormalities, so we are collaborating with Dr. Paul Lambert to determine if these mice have alterations in their susceptibility or response to papillomavirus infection.[unreadable] [unreadable] 4. We are continuing our work to identify the gene mutated in the mouse Twirler strain. Heterozygous Twirler mice have inner ear malformations and obesity, whereas homozygous mice are born with cleft palate and die at birth. The critical interval containing the Twirler gene is approximately 750 kilobases and contains one known gene and two predicted genes. We have identified a probable mutation. We have generated a knock-in mouse line with this mutation to confirm its pathogenicity. We have completed a comprehensive characterization of the Twirler obesity and inner ear phenotypes. The obesity phenotype is associated with hyperphagia of unknown etiology and insulin-resistant diabetes mellitus. The inner ear phenotype is characterized by hypoplasia or dysplasia of the semicircular canals; the lateral canals are most severely affected.[unreadable] [unreadable] 5. Enlargement of the vestibular aqueduct (EVA) is the most commonly detected radiologic malformation in temporal bones of individuals with hearing loss. A significant proportion of EVA cases have been reported to be associated with mutations of the SLC26A4 gene, in which mutations cause Pendred syndrome. PS is an autosomal recessive disorder comprised of bilateral sensorineural hearing loss and a defect in the ability of the thyroid gland to organify iodine. EVA is a universal finding in the ears of affected PS individuals. PS is correlated with two mutant SLC26A4 alleles, and nonsyndromic EVA is associated with one or zero mutant SLC26A4 alleles. Based upon our data, we hypothesize that one or more other genetic or environmental factors may act alone or in combination with a single SLC26A4 mutation to cause EVA. We have completed a comparative genome hybridization-microarray analysis to search for deletion and duplication mutations of SLC26A4 that might cause EVA. We have also PCR-amplified and sequenced all noncoding conserved sequences in and around SLC26A4. We found not evidence for such mutations. [unreadable] [unreadable] We have completed segregation analyses of EVA to gain insight into the causes of EVA in patients with one or zero mutant alleles. The results are consistent with inheritance of a second recessive allele in the patients with one detectable SLC26A4 mutation, whereas the results for patients with no mutant alleles of SLC26A4 suggest one or more of the following: autosomal recessive inheritance of alleles at another locus, non-genetic causes, a complex etiology of two or more genetic and/or non-genetic factors, or a variety of different etiologies among different patients. These results yield recurrence risk estimates, dependent upon the number of SLC26A4 mutant alleles, that are very helpful for counseling patients and families with EVA.[unreadable] [unreadable] We analyzed our genotype and phenotype data to identify clinical features that guide molecular diagnosis or clinical prognosis. We comprehensively and quantitatively analyzed the thyroid phenotypes in our EVA cohort. Our study is the first to rigorously analyze and describe the ultrasonographic phenotype of the thyroid gland in EVA patients. Our results show that thyroid gland volume (goiter) is dependent upon the number of mutant alleles in pre-pubescent subjects whereas it is dependent upon age in post-pubescent subjects. Therefore goiter is a more specific sign of biallelic SlC264A4 mutations (Pendred syndrome) in children than in adults. We continue to observe a strong correlation of an abnormal perchlorate discharge test with two mutant alleles of SLC26A4. Our data provides a basis for the surveillance and management of the thyroid gland in EVA subjects.[unreadable] [unreadable] We have completed functional studies of several missense substitutions of SLC26A4 that have always or usually been detected as the single SLC26A4 variant in nonsyndromic EVA patients and shown that they appear to have wild type trafficking and functional activity, indicating they are benign polymorphic variants. These results have important implications for molecular diagnosis of EVA patients, as well as for categorizing patients according to SLC26A4 genotype for studies to identify other causes of EVA.[unreadable] [unreadable] 6. We ascertained a large North American family segregating progressive, nonsyndromic sensorineural hearing loss in a matrilineal/maternal/mitochondrial pattern of inheritance. We sequenced the entire mitochondrial genome in several affected individuals and have identified a rare mutation in the tRNA-Ser(UCN) gene. We analyzed the mitochondrial haplogroup associated with this mutation and compared it to that associated with this same mutation in a previously reported family. Our results show the haplogroups are different, and we did not find the mutation in haplogroup-matched normal control samples. Our study confirms the pathogenicicty of the tRNA-Ser(UCN) mutation in sensorineural hearing loss.[unreadable] [unreadable] 7. The research laboratory of Dr. Guy Van Camp (University of Antwerp, Belgium) discovered a family with dominant progressive hearing loss caused by the D572N mutation of TMC1, the same mutation causing hearing loss in a family we studied. We compared the mutation-linked genetic marker genotypes and haplotypes, and found they were different between the two families. This indicates that the mutations probably arose independently, not from a common founder.[unreadable] [unreadable] 8. We performed a genotypic survey for DFNB7/B11 deafness caused by TMC1 mutations in Pakistan. We identified several novel mutations and showed that TMC1 mutations account for approximately 3.4% of severe to profound, prelingual-onset deafness in Pakistan.


Conventional methods for treating chronic diseases such as HIV/AIDS have proven to be relatively inefficient both in terms of administration of therapeutic molecules and eradication of the underlying diseases themselves; while conventional antiretroviral therapy (ART) suppresses plasma viremia, low-level viral replication persist. Our recent findings have indicated that pigtailed macaques transplanted with gene-modified CD34+ cells expressing a membrane-anchored fusion inhibitor (mC46) develop infection-resistant CD4+ T-cells, maintain normal CD4+ T-cell levels, and develop an enhanced immune response against the SHIV-challenge virus resulting in a 300- to 1400-fold decrease in plasma viremia. Despite these very encouraging results, additional methods to further reduce plasma viremia to undetectable levels and/or potentially eliminate viral reservoirs will be required. Hence, we propose the development of a novel delivery system based on modifying hematopoietic stem cells (HSCs) or CD4+ T-cells in order to directly target viral reservoirs in vivo. The use of primary cells to deliver therapeutic peptides represents an innovative and ideal mode for systemically delivering therapeutic molecules throughout a patient's body. Furthermore, the ability of HSC-derived lineages to transverse both physiological and anatomical barriers represents a unique potential of this therapy. Low levels of engraftment would likely yield sufficient quantities of secreted antiretroviral peptides, while the efficiency f uptake would be greatly enhanced as there is a continuous source of the therapeutic molecules at sites typically thought to maintain persistent viral reservoirs. In order to deliver proviral targeting endonucleases to latent reservoirs, terminally differentiated primary cell such as CD4+ T-cells that migrate throughout lymphoid tissue represents the ideal vehicle. Hence, the goal of this study is to examine the feasibility of using genetically modified HSCs and CD4+ T-cells to secrete antiretroviral molecules to control viral replication and directly target latent reservoirsin vivo.


The storage and mobilization of lipid are fundamental cellular processes that are conserved throughout metazoan evolution and are present in virtually all mammalian cells. Intracellular FFA (FFAi) are normally kept under tight control since excessive FFAi are toxic. Indeed, excessive accumulation of intracellular FFA and FFA metabolites leads to cellular dysfunction, termed 'lipotoxicity', which is thought to be a major means by which obesity contributes to diabetes and cardiovascular disease. Thus, a mechanistic understanding of how cells assimilate, mobilize and channel FFA is an important biological question with broad implications for health and disease. The uptake and mobilization of FFA within cells appear to have important temporal and spatial domains. Nonetheless, investigation of the "when and where" FFA metabolism occurs within live cells has not been possible because intracellular FFA levels cannot presently be imaged. Therefore, the aim of the R21 application is to develop and implement an optical sensor of FFAi having high spatial and temporal resolution for biological applications. This sensor is designed to be used as a general cytosolic sensor, or can be specifically targeted to proposed subcellular sites of FFA uptake, mobilization and oxidation. We anticipate that a validated sensor will have wide applications and will lead to new insights into biology of cellular lipid trafficking. PUBLIC HEALTH RELEVANCE: The storage and mobilization of lipid are fundamental cellular processes that are present in virtually all mammalian cells. Intracellular FFA (FFAi) are normally kept under tight control and since excessive FFAi are toxic and can contribute to obesity-related diabetes and cardiovascular disease. Thus, a mechanistic understanding of how cells assimilate, mobilize and channel FFA is an important biological question with broad implications for health and disease. Evidence indicates that the uptake and mobilization of FFA within cells has important temporal and spatial domains, however, analysis of these dynamics has not been difficult because intracellular FFA levels cannot presently be imaged. Therefore, the aim of the R21 application is to develop and implement an optical sensor of intracellular FFA with high spatial and temporal resolution for biological applications. This sensor can be used as a general cytosolic sensor, or can be specifically targeted to key subcellular sites of FFA uptake, mobilization and oxidation. We anticipate that a validated sensor will have wide applications and will lead to new insights into biology of cellular lipid trafficking.


The overall objective is to develop an organoselenium compound with lowest toxicity and yet maximal chemopreventative efficacy against colon cancer, and to understand the mechanisms of cancer inhibition by this agent. Our studies indicate that 1,4-phenylenebis(methylene)selenocyanate (p-XSC) is superior to other organoselenium compounds against colon carcinogenesis. We hypothesize that the metabolism of p-XSC by glutathione leading to the formation of putative glutathione conjugate (p-XSe-SG) and/or metabolite of p-XSC, tetraselenocyclophane (TSC) are responsible for its chemopreventative activity. Initially, 40 and 80% MTD levels of these two agents and 80% MTD of p-XSC (parent compound) will be evaluated for their chemopreventative efficacy during initiation and post-initiation phases of AOM-induced colon carcinogenesis. Five week old male F344 rats will be fed high-fat diet or 40 and 80% MTD levels of organoselenium compounds and at 7 weeks of age, all animals except vehicle-treated groups will be administered s.c., AOM (15 mg/kg b.w., once weekly for 2 weeks) and one week later, animals on control diet will be transferred to organoselenium diets and those on organoselenium diets will be transferred to control diet and continued on this regimen until 50 weeks and sacrificed. Histopathological evaluation of colon tumors will be performed and organoselenium exhibiting highest chemopreventative index will be evaluated during promotion/progression phase. Groups of 5-week old male F344 rats will be fed a high-fat diet. At 7 weeks of age groups of rats in each dietary group will be treated s.c. with AOM as above. Fourteen weeks later, groups of animals on high-fat diet will be transferred to high-fat diet with and without organoselenium compound and continued on this regimen until termination of this study. Animals will be sacrificed at 30 and 50 (termination) weeks after second AOM injection. Colon tumor incidence and multiplicity among the dietary groups will be compared by appropriate statistical methods. The mechanisms of colon tumor inhibition by this compound will also be examined. These aspects will include measurement of colonic mucosal and tumor (a) glutathione peroxidase (b) cyclooxygenase (COX-1 and COX-2) isozymes (c) apoptosis, and (d) F/2- isoprostane as an indicator of lipid peroxidation and oxidative damage; and (e) DNA cytosine methylation and DNA methyl transferase during different time periods.


Telomeres are made up of repeat sequences of nucleotides at the ends of human chromosomes. The function of telomeres is to protect chromosomes from degradation and to maintain their structural integrity. Telomeres are analogous to a molecular clock reflecting the number of divisions a cell has undergone and cells with critically short telomeres are predisposed to enter senescence. Previous research suggests that telomere length is reduced in association with lifestyle and clinical factors that are common in study spinal cord injury (lac of exercise, obesity, chronic or recurrent inflammation from skin ulcers, and urinary tract infections) as well as in persons with cardiovascular and pulmonary diseases. These latter diseases are the most common causes of death in chronic spinal cord injury. By using telomere length as a molecular biomarker, it is proposed to study spinal cord injury as a disease state that promotes accelerated aging at the cellular level. It is hypothesized that greater central obesity determined by DXA scan and greater systemic inflammation assessed by plasma C-reactive protein and interleukin-6 will be associated with shorter telomere length, and that persons with shorter telomere length will have reduced pulmonary function. This pilot project will obtain preliminary data regarding these associations in 350 persons with chronic SCI, and assess longitudinal associations between systemic inflammation and telomere loss in a subset. The study of telomere length is significant since it may serve a biomarker of persons with chronic spinal cord injury most likely to develop of chronic cardiopulmonary disease and premature mortality. PUBLIC HEALTH RELEVANCE: Telomeres are a part of human chromosomes that function to protect chromosomes from damage. It has been proposed that telomeres are analogous to a molecular clock reflecting the number of divisions a cell has undergone. Cells with critically short telomeres ultimately undergo cell death. The proposed pilot study will obtain preliminary information regarding associations between reduced telomere length with greater central obesity, reduced pulmonary function, and increased blood biomarkers of systemic inflammation. It is proposed that the measurement of telomere length in persons with spinal cord injury will be a molecular biomarker that can be used to detect persons at greatest risk to develop reduced pulmonary function and cardiopulmonary diseases. This study is consistent with the Veterans Health Administration's commitment to providing longitudinal care to persons with chronic spinal cord injury and supporting research directed at understanding causes of disability and death.


The Central Core Media Laboratory will be responsible for the production of stringent quality-controlled sterile tissue culture and bacteriological media and buffers necessary for in vitro research by all laboratory researchers at the University of Texas M.D. Anderson Cancer Center. Numerous pathogen-free chemically defined solutions will be available for immediate delivery at lower cost than commercial products. The Central Core Media Laboratory will be located in the new Bertner Clinical Research Building, in T5.3964 and consist of approximately 1,300 square feet of laboratory space and 200 square feet of cold-room space. The high trained staff will offer personalized service tailored to each researcher's needs. Two research technicians will prepare solutions and perform quality-control checks. A facility manager will be responsible for performance evaluations, accounting procedures, and general supervision of the laboratory. The facility manager will report regularly to the project investigator. An oversight committee will meet quarterly to assist with the commission of the facility and in determining policy changes. This centralized resource will alleviate the duplication of personnel and valuable laboratory space by concentrating these services into one dedicated location.


We propose an innovative video teleconference hosted by the Brown University Center for Primary Care and Prevention and the University of Oxford Department of Primary Healthcare with the goals of: 1. Promoting evidence-based best practice models of cancer prevention though applied genetics by primary care physicians and 2. Advancing the role of the primary care scholar in translational genetics research. Our specific aims are to translate emerging genetic epidemiological and pharmacogenetics research into evidence-based practice models for primary care physicians by: 1. Fostering a broad understanding of the emerging role of the new genetics in primary care. 2. Promoting a culturally-appropriate skill set to identify, screen, and counsel patients with an elevated heritable risk of cancer and other illnesses. 3. Addressing key policy and ethical implications of genetics for primary care. 4. Identifying strategies for organizing primary care office system to promote the use of genetic technology. 5. Envisioning the new frontier of primary care genetics research from "bench-to-bedside-to-society". Trans-disciplinary experts from primary care, genetic epidemiology, behavioral medicine, and health policy will present an interactive trans-Atlantic forum to promote the development of key knowledge, skills, and practice behavior among practicing, community-based primary care physicians. This half-day conference will take place in Providence, Rhode Island, USA, with video simulcast to London, United Kingdom, and will serve as the nidus for synergistic collaboration in translational genetics research and education.


Glomerulosclerosis is the common pathologic process responsible for 90% of End Stage Kidney Disease (ESKD) associated with diabetes, hypertension, IgA nephropathy, Focal Segmental Glomerulosclerosis (FSGS), lupus and other glomerular diseases. Together these diseases cost approximately $18 billion per year in the US, and carry high morbidity and mortality. They are increasing in prevalence and are commoner in minority populations. They affect both children and adults, although ESKD is particularly a disease of aging with average onset of ESKD treatment at 64 years. The central hypothesis underlying this work is that glomerulosclerosis is caused by depletion of one of the glomerular cell types, the podocyte. During the prior grant cycle we developed a new transgenic model system in the rat where we can deplete podocytes by an amount and at a time of prior choosing. We show that podocyte depletion does indeed cause all the features of FSGS in a dose-dependant manner. Second we have developed a new method for counting podocytes in glomeruli. Third, we have shown how glomerular enlargement during aging on a high calorie diet causes relative podocyte depletion that predisposes to glomerulosclerosis, and how this can be prevented by calorie restiction. Because we know that hypertension is commonly associated with glomerulosclerosis and progression of glomerulosclerosis to ESKD, these new tools and concepts will be used to test specific hypotheses relevant to human glomerulosclerosis: Aim 1: Podocyte depletion itself causes systemic hypertension and mesangial expansion, an important pathologic component of early glomerulosclerosis in man. Aim 2: Test the hypothesis that limitation of the ability of the podocyte to increase in size makes the glomerulus more susceptible to glomerulosclerosis, while enhancement of podocyte's ability to increase in size prevents glomerulosclerosis, by using transgenes of the mTOR pathway of cell size control to modulate podocyte size. Aim 3: Validate the novel concepts of podocyte alteration in phenotype in response to stress that we have termed "adaptation" and "decompensation" in two rat models of glomerulosclerosis, with the underlying concept that understanding the biology of this process will allow us to develop markers for human podocyte stress as well as new approaches to treat and prevent podocyte stress before it results in loss of podocytes and consequent glomerulosclerosis. [unreadable] [unreadable] [unreadable]


Corneal hydration and transparency are dependent on the ion and fluid transport properties of the corneal endothelium. Fluid transport from stroma to aqueous humor is apparently coupled to the net transport of Na and HCO3 and is sensitive to the pH of the bathing solution. A complete model for ion coupled fluid transport however, is not available. Since extra- and intracellular [HCO3] are determined by extra- and intracellular pH (pHo and pHi), it is reasonable to suggest that any model of HCO3 transport will require an understanding of pHi regulation. This notion is supported by work showing that additions of drugs which are known to influence pHi regulation (amiloride and DIDS) also inhibit endothelial fluid transport. This study proposes to identify and characterize the pHi regulatory and HCO3- transport mechanisms present in fresh and cultured bovine corneal endothelial cells by utilizing the pH sensitive intracellular fluorescent probe, BCECF. Cells will also be grown on filter supports to allow independent access to apical and basal surfaces in order to determine the locations of the HCO3 transport mechanisms and establish a complete model for transendothelial HCO3- transport. Transendothelial fluid secretion can also be measured across filter cultures so that the HCO3- transport mechanisms essential for fluid transport and the transport mechanism-that is rate limiting for fluid flux can be identified. A long term goal is to identify agents or conditions that stimulate HCO3- transport and test their ability to stimulate fluid secretion as well. Lastly, post-surgical specimens of Fuch's Endothelial Dystrophy will be obtained and the pH regulatory and HCO3- transport capacities will be tested in order to determine if and how pump function is deranged in this disease. Once the transport deficit is identified, the long term goal is to test drugs which stimulate it or drugs that inhibit an opposing transport mechanism in order to enhance fluid secretion in this diseased endothelia.


Defects within the insulin signaling pathways may lead to insulin resistance in peripheral tissues. The prevalence of obesity generally increases with age and is associated with insulin resistance characterized by an elevation in hepatic glucose production, impaired glucose tolerance and defects in the ability of the insulin receptor to transduce insulin signals. Cross-sectional studies have associated obesity and other components of the so-called metabolic syndrome with low-grade inflammation. Hence, better understanding of the complex interplay between proinflammatory cytokines and insulin signal transduction could lead to the development of effective strategies to reduce or delay the progression of complications associated with insulin resistance states, such as Type 2 diabetes. A strong positive correlation has been reported between adipose tissue levels of adipocytokines (e.g., interleukin (IL)-6, tumor necrosis factor (TNF)) and the increased secretion of acute phase proteins by the liver of obese subjects. IL-6 activates the Janus kinase/signal transducer(s) and activator(s) of transcription (JAK/STAT) pathway to elicit the synthesis and secretion of several hepatic inflammatory proteins. In response to IL-6, JAK induces tyrosine phosphorylation of STAT3 (pY-STAT3) at a single residue, followed by its nuclear translocation for the inducible expression of genes through binding to specific DNA responsive elements. In addition to tyrosine phosphorylation, STAT3 is serine phosphorylated at a single residue in response to IL-6, which enhances its transcriptional activation. We have recently completed a study showing that the antioxidant pyrolidine dithiocarbamate (PDTC) was protective against IL-6?induced JAK/STAT3 signaling in liver-derived human HepG2 cells and a primary culture of rat hepatocytes. We found that PDTC inhibited IL-6?stimulated phosphorylation and nuclear translocation of STAT3 by interfering with the inducible association of STAT3 with an adaptor protein. This reduction in STAT3 activity led to a sharp decrease in acute phase protein gene expression in response to IL-6 as determined by microarray analysis and Northern blot. Of significance, pretreatment of rat hepatocytes with PDTC selectively reduced the adverse effects of IL-6 on insulin-stimulated Akt phosphorylation. It is established that activation of Akt plays a major role in various biological processes triggered by insulin. It remains unknown whether PDTC can inhibit the development of insulin resistance in obesity and in aging, two chronic in vivo models of low grade inflammation. In a second project, we investigated the role of actin-binding proteins in the promotion of STAT3 signaling in response to IL-6. The IL-6-mediated increase in JAK/STAT activity is initiated at the level of raft plasma membrane microdomains of diverse cell types. Despite the fact that STAT3 has been shown to localize to the actin cytoskeleton, little is known about the involvement of actin-binding proteins in regulating the JAK/STAT signaling pathway. Therefore, we hypothesized that filamin A may be a component in STAT3 activation by IL-6. Human melanoma M2 cells that lack filamin A and filamin A-expressing M2A7 cells were used in a number of experiments to assess the kinetics of STAT3 phosphorylation, its nuclear translocation and transcriptional activity following IL-6 stimulation. Because Ser727 of STAT3 is located in a conserved Pro-Xaa-Ser/Thr-Pro site, which is recognized as a phosphorylation site for members of the mitogen-activated protein (MAP) kinase family, we have also defined which subgroup(s) of the MAP kinases is involved in regulating serine phosphorylation of STAT3. Our results show a functional role of filamin A in the modulation of IL-6-induced responses. While these cell lines are suitable for initial in vitro studies, they are surrogates for more physiologically relevant models. Toward this goal, functional studies in vivo will be carried out to address the potential relevance of filamin in the proinflammatory actions of IL-6.


The specific aims of this proposal are to investigate the mechanism of rejection of the murine ovarian teratocarcinoma that follows intraperitoneal injection of Corynebacterium parvum. We will test the hypothesis that the acute inflammatory response to the injection of microbial biologic response modifiers is capable of influencing tumor growth. The inflammatory neutrophil (PMN) is envisioned as initiating two distinct anti-tumor responses that occur sequentially. The first effect is direct tumor cell lysis induced through activation of the PMN by bacteria complexed to C'3b. Activation results in the release of reduced oxygen metabolites which lyse tumor cells within the peritoneal cavity. The second effect is the activation of tumor-cytostatic macrophages by inflammatory PMNs that have ingested bacteria. The long range goals of the proposal are to improve survival of patients with ovarian cancer. The use of the current animal model as a preclinical system for studying regional immunotherapy in human ovarian cancer is expected to lead to a better understanding of and offer insight into possible improvements in it for these patients. (TA)


Nanotechnology Materials Core: Project Summary/Abstract One of the primary areas of focus in the Koch Institute is the development of new materials systems and biomedical devices for cancer prevention, detection and treatment. These systems underpin development of new approaches for biomarker detection, imaging modalities, and targeted therapies and vaccines. Center Members are exploring a wide range of materials to achieve these goals, and employ combinatorial development approaches to facilitate the discovery of new biomaterials and nanoformulations. State-of-the-art capabilities for high resolution and surface imaging and chemical and physical analysis are key tools to enable these studies. To fully characterize new materials, establish structure-property-function relationships, and facilitate subsequent rational design, we established the Nanotechnology Materials Core in 2012. In the previous competing renewal, the Core was approved for CCSG funding as an established Shared Resource. This Core integrates a range of advanced technologies including a state-of-the-art cryo-electron microscopy (cryo-EM) imaging suite, which enables high resolution analyses of soft biomaterials and cellular ultrastructure. In the current funding period, the Core added a significant number of new capabilities and modified existing services, including: new EM sample preparation instruments; new EM and correlative light and EM imaging systems; new physical/chemical characterization capabilities; and upgrades to existing instruments. To best capitalize on these acquisitions, CCSG Developmental funds were used to support two Cryo-EM Specialists. Core service usage by Center Members has increased, from 14% at the time of the last renewal to 43% of Center Members, who account for 79% of Core service use and include investigators from all three Research Programs. Core staff has established cryo-EM workflows that are customized for Center Member research needs, and we seek continued, partial funding to support and stabilize the Research Specialists? efforts. In the upcoming period, the Nanotechnology Materials Core will continue to offer a wide range of state-of-the- art services to support Center Member research programs, and will evaluate emerging capabilities in the context of Center Member needs and interests. A primary focus will be on refining new workflows to take full advantage of newly acquired instrumentation. Other planned initiatives include: developing resources to support advanced image processing, visualization, quantitation and machine learning, and collaborating with other Cores to develop a monthly workshop series to support Center Members? access to imaging technology platforms and data analysis. This Shared Resource is essential to the success of the Koch Institute mission and provides exceptional value to the CCSG. The requested CCSG budget for Year 49 is decreased by 40.6% over the Core CCSG budget for the current period (Year 48), reflecting the increased fraction of support from user chargebacks as Core staff is moved from CCSG Developmental funds to CCSG Core funds.


This Program Project is concerned with three general areas of neurobiological investigation each of which combines studies of structure and function in attempts to provide correlations useful in defining the elementary processes underlying the organization of complex neural activities. Studies will be carried out on neuronal development, interaction and organization. A wide variety of species and preparations will be employed in order to examine: 1) the basis for electrotonic coupling between neurons in systems where coupling is demonstrable and 2) the contribution of synaptic vs. ephaptic interactions in neuronal organizations subserving highly synchronized activities. Morphophysiological studies of developing neurons and synapses in tissue culture, and in vivo systems will be carried out with emphasis on the role of hormonal influences in altering developmental features. Electron microscope as well as histochemical studies of synapses will be carried out in conjunction with electrophysiological investigations of synaptic activities and developing neural systems in different vertebrate species. Studies of the mechanism of hormone actions on developing neurons and synaptic complexes will also be pursued. Neuroendocrine relations will be studied in other aspects of this Program and investigations will be continued on the organization of subcortical systems regulating sensorimotor activities in the mammalian brain.


The aim of this proposal is to use personal digital assistants (PDAs) to integrate psychosocial assessments and interventions (based on Social Cognitive Theory) for disordered eating prevention for at-risk college women. PDAs have been used in two distinct capacities: (a) an assessment device employing Ecological Momentary Assessment (EMA); (b) a device for delivering interventions. The proposed work is innovative because it combines the assessment and intervention capacities of PDAs. Participants will complete EMA reports, and micro-interventions (Mis) will be administered on PDAs based on concurrent EMA responses. Mis are brief treatment extensions delivered on portable devices. The Ml will be targeted to three specific experiences (eating, media exposure, negative mood) when women are most vulnerable to body image dissatisfaction (identified in preliminary research). Undergraduate women at-risk of developing disordered eating (n=144) will be randomly assigned to a CD-ROM intervention and EMA reports (EMA+CD), CD, EMA and micro-interventions (EMA+CD+MI), or EMA only (EMA) groups. All participants will complete baseline assessments of body image dissatisfaction, disordered eating, and psychological well-being. Six times daily for the following 1 week, participants will complete EMA surveys (including body image dissatisfaction, eating behavior, media exposure, and mood items) on PDAs. The EMA+CD and EMA+CD+MI groups will receive the Food, Mood, and Attitude CD-ROM intervention. One week later all participants will receive the PDAs again and complete the same EMA survey. The EMA+CD+MI group will also receive targeted. Mis consist of reminders to use skills learned in the CD-ROM and will be administered on the PDA immediately following the survey, and then 10 to 20 minutes following the EMA. All participants will complete 1- and 3-month follow-up assessments of disordered eating, body image dissatisfaction, and psychological well-being. The EMA only group will receive the CD-ROM intervention after completing the 3-month assessment. All participants will be given referral information if they wish to seek additional care. The public health significance of this research includes the innovative development of a methodology to increase the efficacy of eating pathology prevention programs (i.e., micro-interventions). These prevention programs will prove useful in reducing the incidence of eating disorders and have implications for other treatments and prevention strategies, by informing the development and implementation of treatment extensions more broadly. [unreadable] [unreadable] [unreadable]


Epidemiological studies have consistently shown a linkage between individuals who chronically consume significant amounts of alcohol and hepatitis C viral (HCV) infection. The mechanism by which HCV is able to efficiently evade immune responses is still unknown. It is the purpose of this proposal to study the effects of chronic alcohol consumption on the expression of Toll-like receptors (TLR) and the function of hepatic plasmacytoid (p) dendritic cells (DC). This subset of DC has become of great interest as they are speculated to be primary responders in viral infection, producing large amounts of interferon-alpha upon stimulation. Studies on various TLR have shown that activation through these receptors can modulate the subsequent DC response in a distinct manner and thus influence the activation of T cells. We plan to investigate alterations in the expression and function of these receptors on pDC in established in vitro and in vivo mode s of culture-generated pDC and chronic alcohol consumption. If there are defects in the development of pDC or receptor expression as a result of chronic ethanol intake, it may be possible to promote an appropriate immune response through adoptive transfer of non-ethanol exposed culture-generated pDC. [unreadable] [unreadable]


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Pathologic processes affect the concentrations of certain metabolites or neurotransmitters as well as local surroundings such as pH and blood flow. Glycine is a neurotransmitter that serves as an important inhibitory neurotransmitter in the central nervous system. Glycine-dependent synapses are found to be highly concentrated in the brain stem, retina, and spinal cord. The presence and location of glycine receptors suggest that glycine signaling might be a key player in the neural pathology of many motor and cognitive diseases;some notable examples include Amyotrophic Lateral Sclerosis (ALS), Parkinson's disease, hyperekplexia, and paroxysmal movement. Measurement of these factors invasively is difficult and may pose more harm to the patient than benefit. While magnetic resonance spectroscopy (MRS) has previously been used to measure neurotransmitter concentrations in the central nervous system (CNS), this technique lacks the adequate spatial resolution to be used in routine clinical studies. The ability to measure such aspects non-invasively at high resolutions would be a major breakthrough in the diagnoses of these and many other disorders. Chemical exchange between labile protons of proteins and water protons can make Magnetic Resonance Imaging (MRI), utilized mainly for the detection of bulk water signal, sensitive to information about the concentrations of endogenous proteins and their environments. Recently, a technique called Chemical Exchange Dependent Saturation Transfer (CEST), which uses the attenuation of bulk water magnetization through magnetization exchange with saturated labile protons, has been used to characterize properties of dilute labile groups. While CEST studies have explored numerous metabolites, there have been no studies demonstrating the CEST effect in glycine. Spin-lattice relaxation in the rotating frame (T1[unreadable]) is another contrast technique that depends on chemical exchange. There have been no studies to date using T1[unreadable] for contrast in the chemical exchange of protons. T1[unreadable] Chemical exchange effects vary quadratically with the static magnetic field. Therefore, T1[unreadable] potentially offer higher sensitivity at higher fields in probing exchange mediated interactions in nuclear spin systems. This higher sensitivity of T1[unreadable] MRI may enable detection of metabolites with very low concentrations (~1 mM) in the brain. We hypothesize that it is possible to quantify the CEST effect in glycine at higher magnetic fields (e3T). Also, that T1[unreadable] MR imaging has higher sensitivity to proton chemical exchange than the CEST method at ultra-high static fields. Finally, we believe that it is feasible to measure glycine in vivo in the spinal cord. These hypotheses will be tested by accomplishing the following specific aims: Aim #1: To measure the chemical shift of glycine in-vitro under physiological conditions using CEST imaging. Aim #2: To determine the effect that concentration, pH, static magnetic field, B1 field strength and saturation time have on the chemical exchange of glycine. Aim #3: To demonstrate, at varying static magnetic field strengths, that the chemical shift of glycine can provide contrast on T1[unreadable][unreadable]weighted images and that at ultra-high static fields it shows greater sensitivity than the CEST effect. Aim #4: To measure glycine concentrations non-invasively in-vivo in rat models using CEST and T1[unreadable][unreadable]weighted images.


The overall goal of the Assays and Reagents Core is to provide state of the art assay methods, reagents, advice, training, assistance, and service in ways that will increase the cost effectiveness, quality, and timeliness of research performed by principal investigators of eligible projects. Specifically, the Core will: obtain, inventory, and maintain adequate stocks of analytical reagents needed by eligible projects (this includes developing, purchasing in bulk, requesting gifts or chemically synthesizing immunogens, first and second polyclonal and monoclonal antibodies and antibody fragments, standards, other reference and quality control preparations, and tracers labeled with radioiodide, biotin or enzymes); characterize and test acquired reagents and make them available to eligible projects; meet the needs of low volume Users, individuals with inadequate laboratory support, and Users with other needs by providing high quality, cost-effective service assays on a samples-in, results-out basis; incorporate advanced analytical quality control procedures in all activities; maintain performance records, inventories, and documentation for all reagents and assay systems provided by the Core; provide timely information, assistance, and advice in utilizing the methods effectively; develop or otherwise implement improved, state of the art assay methods and analytical procedures that reduce the use of radioactive isotopes and provide increased specificity, sensitivity, reliability and ease of use; provide quality control assessments for assays and assay kits provided to eligible projects and; teach assay technology and immunoassay data processing, and provide training and instruction in radioactive and hazardous materials safety, in universal precautions for biohazards, in laboratory safety and sanitation, and in applicable regulatory compliance.


Cholesterol homeostasis is maintained through coordinate regulation of cholesterol uptake, esterification, synthesis, secretion, and its degradation to bile acids account for approximately half of daily net cholesterol elimination, while remaining 50% takes place almost entirely via biliary cholesterol secretion. Bile acid synthesis from cholesterol occurs via two pathways. The initial/rate-determining enzyme in the major "neural" pathway of bile acid biosynthesis is microsomal cholesterol 7alpha-hydroxylase (C7alphaH. The "acidic" pathway of bile acid synthesis is initiated by sterol 27-hydroxylase (S27H). Whereas C7alphaH is present almost exclusively in the liver, S27H is also present in many extrahepatic tissues, where its function remains unclear. The overall objective of the proposed studies is to better define the physiologic role of these key enzymes in the maintenance of hepatic and total body cholesterol homeostasis. More specifically, we propose a systematic series of in vivo and in vitro studies, in both liver and non-liver cells, using recombinant adenovirus to deliver CMV driven genes encoding C7alphaH and S27H. These studies attempt to combine the techniques of molecular biology with recombinant adenoviral gene transfer to determine the kinetic and molecular changes in the pathways of cholesterol homeostasis which follow selective over expression of the genes encoding C7alphaH and S27H . A clearer understanding of the mechanisms which maintain cholesterol homeostasis is needed to continue to lay the groundwork for the development of better therapies for cholesterol related disorders.


The major objective of the proposed research is to determine the residual effects of low level lead exposure on the fetal and infant rhesus monkey. Pregnant rhesus monkeys will be fed low levels of lead throughout gestation and their infants evaluated biologically and behaviorally during infancy, adolescence, and early adulthood. Similar studies will also be conducted on infant monkeys that are exposed to low levels of lead during the initial year of life. These studies will help to clarify the effects of lead exposure during early life on the subsequent development of the infant. In addition, maximum tolerable levels of lead administered over a given period to fetal (via the mother) and infant monkeys will be established. The methods that will be employed in evaluating these aminals will include behavioral studies that will emphasize social interaction and learning capabilities of these animals from birth to adulthood. The clinical studies conducted will be systematic hematological, radiological, and biochemical determinations throughout infancy, adolescence, and adulthood. After the completion of the clinical and behavioral studies histological and ultrastructural studies will be conducted on all of the major organs. In addition, total concentrations of lead in various tissues of the body will be determined.


Quantitative cytochemistry and morphometry will be used to characterize the ultrastructural and enzymic configuration of pseudostratified granulosa cells of preovulatory follicles in rats. The enzyme complexes studied will include delta 5-3 beta hydroxysteroid dehydrogenase, glucose-6-phosphate dehydrogenase and cytochrome P-450. Acid phosphatase will be studied to localize organelles associated with lysosomal activity and the naphthylamidase reaction will be used to measure lysosomal membrane permeability. The development of cytochemical characteristics unique to pseudostratified granulosa cells of preovulatory follicles will be followed throughout the estrous cycle. The ultrastructural characteristics that distinguish the pseudostratified granulosa cells of the preovulatory follicle will be established. Since the granulosa cells form an essential component of the preovulatory follicle as well as of the corpus luteum formed from it, the information from these studies could provide a basis for evaluating abnormal development and the formation of inadequate corpora lutea--conditions associated with infertility.


Antibodies play a critical role in the immune system for recognition of foreign intruders. Because of their excellent affinity and specificity, they hav also been exploited as therapeutic molecules and biotechnological components for sensing and assembly. Structures of antibodies in complex with their antigens can yield insight into biological phenomena or drug and disease mechanisms. However, structures of antibodies and antibody-antigen complexes can be difficult, time consuming, and expensive to determine. The proposed research focuses on the computational prediction of the structure of antibodies and antibody-antigen complexes. Computational approaches are particularly important because the repertoire of antibodies in a human patient is far too large for complete structural characterization by experiment. Prior work has isolated the most critical challenges: most of the antibodies in the human repertoire have hypervariable CDR H3 loops longer than that which is predictable using current loop methods; backbone conformational uncertainty and flexibility confound current docking methods; and no current method can quantitatively predict antibody-antigen binding affinities from structure. Thus, the first three aims of the project are to (1) develop new methods to predict the structure of long CDR H3 loops using statistics to identify likely turns, (2) develop flexible backbone docking routines using an expanded ensemble approach with a conformational web, and (3) develop methods to quantitatively predict protein-protein binding affinity using improved electrostatics treatments. Finally, the fourth aim will be to (4) use existng and proposed methods to predict structures of antibodies and antibody-antigen complexes for entire polyclonal antibody repertoires. Structures will be predicted for antibody repertoires determined from bone marrow plasma cells of mice immunized against ovalbumin (a food allergen) and enzyme C1s (a therapeutic target for autoimmune diseases and transplant tolerance). Ultimately, these studies will yield insights into immunology, molecular recognition, and design of protein-protein interfaces and vaccines. PUBLIC HEALTH RELEVANCE: Antibodies play a critical role in the immune system for recognition of foreign intruders, and are excellent therapeutic molecules because of their high affinity and specificity. This project investigates computational approaches to determine the three-dimensional structures of antibodies alone and in complex with their target antigens, including specific applications relevant to food allergies and a treatment for transplant rejection autoimmune disorders and inflammatory diseases.


These investigations are devoted to the development of noninvasive methods of accessing tissue structure and function. Two general techniques are being developed: nuclear magnetic resonance (NMR) and optical spectroscopy/imaging. Over the last year we have made the following developments in NMR technology: (1) We have further developed the theory and application of magnetization transfer contrast (MTC) to the study of biological tissues. This approach permits the imaging of the interaction rate between water and macromolecules in the body resulting in a unique form of high resolution image contrast and tissue characterization. The quantitative aspects of this approach, defining its frequency and power dependence, were established out-lining the methods of obtaining quantitative rate constant maps in vivo. (2) 3-H NMR studies have revealed that the interaction of water with macromolecules, the dominant source of contrast in tissues, is due to spin diffusion between the water and the macromolecules. (3) The molecular basis with macromolecules was evaluated which of the dipoler interaction of water demonstrated a unique role for hydroxyl groups in lipid bilayers. (4) The first human images using MTC were collected by adding a second radiofrequency channel to a standard clinical scanner. These images were 3-dimensional images of the knee revealing excellent soft tissue contrast. (5) A quantitative relationship between 3-fluorsorbitol production from 3-fluorodeoxyglucose and aldose reductase activity was established in the isolated lens of the eye, suggesting that this approach is a viable method of monitoring aldose reductase activity in vivo. Using optical spectroscopy the following advancements were made: (1)The effects of inner filters on the optical behavior of calcium indicating dyes and NAD(P)H fluorescence in the intact heart were defined and methods developed to correct for these serious artifacts. (2) Methods were further developed for the monitoring of tissue oxygenation in vivo using optical spectroscopy in the 500 to 650 nm range.


The overall objective of this work is to analyze the genetic relationships of the various populations of the Common Hose Mosquito present in the Boston area. By comparing the electrophoretic mobility of selected isozymes, we shall determine (1) whether the autogenous, anautogenous and rural forms may be characterized, 2) whether these populations may be genetically isolated, 3) if so, whether isolation is absolute, and 4) whether one population may possess more genetic variability than others. We shall study the blood-feeding habits of each population and determine whether man-biting may be associated with certain populations or combinations of populations. A manual will be prepared describing procedures for applying the polyacetate strip method of electrophoresis to populations of mosquitoes.


The proposed research-will use the technique of single-unit recording to examine the characteristics of neurons within the postsubiculum and limbic thalamic nuclei. Specifically, the properties of "Head Direction" cells will be examined. These cells respond only when an animal orients it's head in a specific direction (e.g., a cell may only fire when an animal points its head to the northeast) regardless of the animals absolute position in an apparatus. It is hypothesized that these cells play a significant role in spatial abilities. The experiments in this proposal will examine two basic issues with regard to head-direction cells: First, what type of navigational information determines head- direction cell activity? Second, do head-direction cells exhibit additional behavioral correlates (e.g., responses to stimulus presentation, goal approach, or reward) when examined in various behavioral paradigms? The experiments designed to answer these questions will provide a further understanding specifically of the determinants of head-direction cells activity, and in general, of neural bases for spatial abilities.


Studies on the cause of congenital cataracts in the progeny of female rats fed a vegetable protein diet during pregnancy and lactation have shown that supplements of either tryptophan or Vitamin E were effective in prevention of the opacities. An interaction was confirmed by preparation of a semi-purified diet using L-amino acids. With this diet, low levels of tryptophan (0.075 mg%) or Vitamin E (0.1 mg%) could be tolerated singly but caused 30% incidence of F1 cataract when fed simultaneously. Current studies are aimed at elucidation of the biochemical mechanism of this effect.


The antimetabolites have been used for the treatment of childhood cancer for more than 40 years. This class of agent continues to play a critical role in the treatment of childhood leukemia. The primary objective of this project is to study the clinical pharmacology of antimetabolites and other anti-leukemic agents to determine if they are being utilized optimally, and to develop rational, pharmacological- based alternative therapeutic approaches to the treatment of childhood leukemia. In our current lower risk ALL trial, we are applying observations made in our preclinical studies showing that thioguanine (TG) is 10-fold more potent than mercaptopurine (MP) against leukemic cell lines and lymphoblasts from patients with ALL. Pharmacodynamic correlation between plasma TG and RBC TG-nucleotide levels, and measures of drug toxicity and disease outcome, are being investigated. Results of this trial and the clinical pharmacologic assessment of TG has guided the design of the current CCG phase 3 randomized trial comparing TG to MP in the lower risk ALL population. We are also exploring a new class of antimetabolite specific for thymidylate synthetase (TS). ZD1694, a classical antifol which is a potent inhibitor of TS, is currently being evaluated in a pediatric phase 1 trial and pharmacokinetic study. A novel and sensitive enzyme inhibition assay for TS inhibitors has been developed and is being evaluated. Methotrexate is an antimetabolite widely used in pediatric cancer, and when administered in high doses must be followed by the rescue agent leucovorin. We are studying carboxypeptidase-G2, (CPDG2) a recombinant enzyme capable of hydrolyzing MTX into inactive metabolites. CPDG2 may offer distinct advantages over leucovorin in certain clinical situations. Based on our pre-clinical investigations, nationwide protocols have been developed to treat patients with severe renal dysfunction following HDMTX with CPDG2. To date, more than 70 patients with renal failure have been treated, with the enzyme producing a > 98% decrease in MTX concentrations within 15 minutes of administration. We have prepared the data for presentation to the FDA as part of an NDA for CPDG2 - Leukemia, Antimetabolites, Methotrexate, Thioguanine, - Human Subjects: Minor under 18 Years Old


To maintain genomic integrity, eukaryotic cells duplicate their DNA precisely once before each cell division. In the G1 phase, pre-replication complexes (pre-RCs) are assembled at origins via the sequential recruitment of ORC, Cdc6, Cdt1, and MCM2-7. In S phase, when DNA replication initiates, pre-RCs are disassembled, and new pre-RC assembly is strictly prohibited. As a result, only one initiation event occurs at each origin, and DNA replication is limited to a single round. In vertebrates, pre-RC assembly is blocked in S phase due to Geminin and ubiquitin-mediated proteolysis of Cdt1. Using a cell-free system derived from Xenopus egg extracts, this laboratory made the surprising discovery that the destruction of Cdt1 in S phase is intimately coupled to DNA replication. Cdt1 is ubiquitylated on chromatin by the E3 ubiquitin ligase Cul4-Ddb1Cdt2, and this ubiquitylation event requires the interaction of Cdt1 with the processivity factor PCNA at the DNA replication fork. Studies from other laboratories suggest that PCNA-dependent Cdt1 destruction is conserved in humans, flies, worms, and possibly fission yeast. This pathway represents a new paradigm in which temporal control of proteolysis involves activation of a substrate (Cdt1) via its docking onto a cell-cycle regulated structure (chromatin-bound PCNA). This proposal contains experiments that will elucidate the mechanism by which PCNA-dependent Cdt1 destruction is temporally controlled. In Specific Aim 1, we address how Cdt1 interacts selectively with chromatin-bound PCNA to insure that Cdt1 is normally never destroyed in G1. In Specific Aim 2, we characterize the degron motif of Cdt1 and examine how it mediates binding to PCNA and Cul4-Ddb1Cdt2. In Specific Aim 3, we reconstitute Cdt1 ubiquitylation in a purified system so that we may distinguish between different mechanisms for how PCNA stimulates this process. Finally, in Specific Aim 4, we use real-time fluorescence microscopy to address how rapidly Cdt1 is destroyed at S phase onset, and we test a new model for local control of proteolysis by the PCNA/Cul4-Ddb1Cdt2 pathway. Together, these studies will illuminate the molecular mechanism underlying a novel proteolysis pathway that maintains genome stability. Narrative To maintain the integrity of our genomes and prevent diseases such as cancer, it is crucial that cells make one precise copy of their DNA before each cell division. This laboratory has discovered a new mechanism, which insures the accuracy of genome duplication in all higher organisms, and it involves the destruction of a DNA replication factor called Cdt1 after the first round of chromosome duplication has been initiated. The work is highly relevant for human health because failure to destroy Cdt1 is correlated with human cancer, and has been shown to cause cancer in mice. [unreadable] [unreadable] [unreadable]


When individuals with Tourette syndrome (TS) are treated with medications to suppress tics, some improve, others have little or no response, and several may develop significant side effects. In order to explain this variability, pharmacogenetic research has focused on two major areas, drug metabolism and drug site of action. In this study, genetic variations of metabolizing enzymes (CYP2D6), dopamine & serotonin transporters (DAT, 5-HTT) dopamine receptors (D2, D3, D4) and serotonin receptors (5HT2a and 5HT2c) will be determined with the goal of predicting a TS patients response to treatment and a diagnostic category (TS or control). Fifteen established polymorphisms will be investigated in individuals with the diagnosis of TS using DNA obtained by buccal swabs. The first major objectives is to determine whether the presence of specific patterns of neurotransmitter polymorphisms predict the tic-suppressing pharmacologic effect of the atypical neuroleptic, risperidone. Patient response to pharmacotherapy will be based on a prospective evaluation of tic reduction, as measured by the Total Tic score of the Yale Global Tic Severity Scale, in 200 individuals with TS. This approach is similar to one that has been successfully used in schizophrenia to predict, with nearly 80% success, the response to the atypical neuroleptic, clozapine. Our second goal is to determine whether there are associations between genetic variations in dopamine or serotonin receptors and the etiology of TS. This question will be addressed in a cohort of 500 individuals with TS & compared with an equal number of age and sex-matched controls. This application, defines a study (to my knowledge, the first of its kind in TS), which will use DNA obtained from buccal mucosa, to establish a predictive therapeutic index in TS patients. The identification of genetic polymorphisms that are associated with clinical disease or treatment success, has obvious benefits.


In the developing mammalian brain, directed cell migrations establish the neuronal layers of the cerebellum and the neocortex. While a number of receptor systems have been studied in vertebrate neuronal migrations, an important avenue of discovery of genes that potentially regulate CNS migrations is to search for homologues of genes that function in neuroblast migration in C. elegans and Drosophila. During the prior funding period of this grant, we have begun studies with the laboratory of Dr. Cynthia Kenyon to explore the role of homologues of C. elegans neuroblast migration genes in mammalian brain development. Toward that end, we have cloned homologues of the C. elegans gene mig-13 and initiated studies on the role of Mmig13 in brain development. The overall goal of this Research Plan is to define the function of murine homologues of the C. elegans gene mig-13 in the formation of murine cerebellar and neocortical neural laminae. Our preliminary studies suggest that Mmig13 functions in the formation of the EGL in the embryonic cerebellar anlagen, prior to migration of postmitotic precursors along the Bergmann glial fibers, and in the formation of the cortical plate, the region where young neurons in the neocortex halt their movements along radial glial fibers and assemble into specific layers. The proposed research is aimed at testing and refining this general hypothesis by providing more detailed studies on the timing of expression of Mmig13 in these two brain structures, by imaging living neurons in cells and tissue from Mmig13 BAC transgenic mice, where the Mmig13 gene is marked by EGFP, and by evaluating the effects of either gain of function or loss of function on granule cell migration and/or cortical neuron migration. To better understand the mechanism of action of the gene(s), we will define their extracellular ligands and their intracellular signaling pathways. This information should broaden our view of the mechanisms that control CNS migrations and of the downstream events important to neuronal differentiation within the neural layers.


This proposal studies the behavioral pharmacology of oral cocaine self-administration. The optimal conditions for the induction of cocaine overindulgence in rats will be established by varying drug concentration and the food-schedule induction parameters which are known to produce excessive behaviors. The effect commonly- used and abuse licit agents (nicotine and caffeine) might have in increasing schedule-induced cocaine intake will be determined, as well as possible attenuating effects of promising therapeutic (desipramine) and blocking agents (e.g., chlorpromazine and pimozide). A number of commodities and activities will be tested as behavioral alternatives to available cocaine under the excessive-behavior-generating conditions. Extensive exposure to prior experiences with non-drug, highly-preferred substances such as saccharin (which are then made unavailable) under conditions that should induce cocaine abuse may block its development, as may pre-adaptation to the inducing conditions. The possibility that prior overindulgence of nicotine or caffeine may predispose to cocaine abuse development will be explored. The effect of parenteral cocaine injection on a fine motor control task will be determined, as well as the interactive effects of caffeine and of nicotine with chronic cocaine on this kind of performance. Serum cocaine and metabolite levels will be correlated with behavioral effects.


Project Summary/Abstract Prostate cancer is listed as a primary diagnosis for approximately 16% of older male VHA users, and contributes significantly to morbidity and mortality rates in these individuals. The majority of VA patients with metastatic prostate cancer, similar to most non-VA patients in the United States, receives hormonal treatment which is initially effective, but frequently fail indicative of the development of castration resistant prostate cancer (CRPC). The treatment options for patients who fail hormonal therapy are limited, despite numerous clinical trials testing multiple drugs. Hence, our laboratory has undertaken the innovative overall strategy of identifying therapeutic targets that can prevent prostate cancer progression to castration resistance, rather than trying to cure it after it has already developed. Studies performed during the previous funding period, which resulted in 14 publications in related topics, contributed to understanding the importance of the cell survival regulator Akt in prostate cancer development and progression. While a number of Akt inhibitors have been developed commercially, their utility has been compromised by high levels of toxicity when used in human patients, caused by the induction of apoptosis in normal tissue including non-tumor prostate regions. As a result, a goal of the project was to identify natural antagonists of Akt that selectively counteract the effects of the increase in Akt phosphorylation in the tumor without affecting basal levels of Akt activation in other cells. Two of our recent publications demonstrate that Filamin A (FlnA) may be one such protein. In vitro studies showed that FlnA localization to the nucleus resulted in castration sensitive growth of prostate cancer cells which was mediated by antagonism of Akt phosphorylation and its downstream effectors. FlnA nuclear localization is regulated by its phosphorylation, which in turn is regulated by PKA activity. The effects of FlnA nuclear localization are likely caused by its interaction with the androgen receptor (AR), a nuclear steroid receptor mediating the effects of the androgens on cell mechanisms. Therefore, in the current application, we will investigate the role nuclear FlnA plays in regulating castration sensitive cell behavior. We hypothesize that FlnA counters the effects of Akt phosphorylation and its downstream effectors, and that its localization is in turn regulated by its phosphorylation. The following aims will test this hypothesis. Specific Aim 1: To test the hypothesis that FlnA 16-24 nuclear localization sensitizes prostate cancer cells to androgen withdrawal by preventing the effects of Akt on AR activity and cell survival. We will determine whether FlnA16-24 induces sensitivity to androgen blockade in CRPC cells by antagonizing an Akt- dependent signaling pathway and preventing AR/TIF2 interactions. We will also investigate whether nuclear localization of FlnA 16-24 induces apoptosis by repressing NF-kB activation and stimulating AR-dependent p21Cip1 transcription. Specific Aim 2: To test the hypothesis that FlnA phosphorylation is a key factor responsible for preventing FlnA nuclear localization in castrate resistant prostate cancer. We will examine whether FlnA phosphorylation prevents FlnA nuclear localization and causes castration resistance in prostate cancer cell lines expressing a functional AR. We will also determine whether inhibition of FlnA phosphorylation prevent the development of castration resistance in a mouse model of recurrent prostate cancer. Specific Aim 3: To test the hypothesis that increased FlnA phosphorylation and decreased FlnA nuclear localization predict time to treatment failure in VA patients with metastatic prostate cancer on androgen withdrawal therapy who did not undergo radical prostatectomy. We have identified patients within the VA Northern California System who presented with metastatic prostate cancer. We will determine in biopsies of the prostates of these patients the localization of Filamin A, FlnA phosphorylation, AR levels and Akt phosphorylation and determine whether they respond to androgen withdrawal therapy.


Local BBB changes in drug-induced seizures and in acute hypertension were correlated in the same animals with rCBF changes. Our findings indicate that BBB changes are associated with marked regional elevations of the CBF. However, this relationship is relative and it does not indicate existence of any absolute threshold for rCBF elevation to be associated with BBB breakdown.


This project investigates the roles of the family in facilitating disease management among Navajo individuals with type 2 diabetes, specifically: 1) the support given to diabetics by their families; and 2) how family support and Navajo philosophical concepts interact with two current diabetes interventions conducted by the Indian Health Service. In the previous MBRS research project, family support emerged as a critical factor in determining the extent to which Navajos with diabetes adhere to prescribed self-care behaviors such as diet change, weight control, and exercise. Certain traditional Navajo philosophical concepts also were associated with use of self-management techniques. The project will involve two distinct groups of respondents. The "Family Study" will involve ethnographic interviews and focus groups with members of 12 families (both diabetics and non-diabetics) who have one or more diabetic relatives to provide a depth of understanding of the dynamics of family support and use of traditional philosophical concepts in assisting diabetic family members. The "Intervention" will follow three groups of clinic patients with regard to change in their health status either following participation in one of two ongoing diabetes interventions, or after a similar period of time with non intervention. The interventions vary in the degree to which family involvement and tenets of Navajo philosophy are incorporated. In both parts of the study, the outcomes considered will include objective measures of diabetes management taken from the medical record, as well as self-management behaviors as reported by the Navajo diabetics. Hypotheses are: 1) that individuals whose families offer consistent, active and varied support will manage their diabetes better than those lacking such family support; 2) that families expressing traditional Navajo philosophical concepts such as those described above will be more likely to provide support for their diabetic relative; and 3) that interventions which target family-wide change, and involve active learning will have a greater impact on management behaviors and ultimately blood glucose than interventions which target individual change and stress didactic presentation. The findings in each of these areas of study will have significant ramifications for improving disease management and health care interventions for Navajos with diabetes.


The mechanism of lysosomal localization and function of cholesteryl ester hydrolase will be studied in cultured vascular smooth muscle and endothelial cells, and in fibroblasts. The concepts of the secretion-recapture model for lysosome formation (Neufeld, Sando, Garvin and Rome), which were developed from studies of mucopolysaccharide-metabolizing enzymes in cultured fibroblasts, will be applied in these studies. Hormonal and nutritional factors thought to be associated with vascular disease will be studied for effects on the secretion and internalization of the hydrolase. The relationship of secretion and internalization of cholesteryl ester hydrolase to the regulation of cholesteryl ester metabolism will be compared in the above cell types. The relationship of low density lipoprotein uptake, internalization and subsequent regulation of sterol metabolism to lysosomal localization and function of the hydrolase will be examined.


The objective of this study is to gain operational and mechanistic understanding of mechanical and electrical effects on bone and cartilage growth and remodeling. Cells from chick epiphyseal cartilage, from rat embryo calvaria and from rat osteosarcoma are exposed to controlled mechanical or electrical perturbations in culture. The effect of the perturbation on cyclic AMP accumulation, ornithine decarboxylase induction and DNA synthesis are measured. Hypotheses on the mechanism of action of the stimuli are tested through manipulation of the composition of the medium. Results obtained so far show that mechanical and electrical stimuli modulate DNA synthesis via effects on cell membranes involving changes in cyclic nucleotides and Ca ion and Na ion fluxes. The response is cell specific and depends on the state of cellular differentiation.


In this application, we propose to test two new hypotheses concerning the MBP gene. These hypotheses are based upon data derived from the identification of two classes of unusual myelin basic protein (MBP)-related transcripts isolated from screens of mouse brain and human fetal spinal cord cDNA libraries. The first hypothesis is that the structure of the MBP gene is presently incomplete and contains at least one more exon upstream of what is currently believed to be exon 1 and an additional promoter. The second hypothesis is that portions of the MBP gene (including parts of at least one exon and one intron) are transcribed into mRNAs encoding proteins other than the MBP variants characterized thus far. The location of any additional exon sequences will be determined by conventional procedures of mapping and sequencing genomic clones using an MBP cDNA that contains sequence corresponding to the additional exon region. Sites of transcription initiation at this new upstream exon will be determined by S-1 nuclease and primer extension analysis. Experiments will be performed to determine if initiation of transcription at this start site and the one previously identified in the MBP gene varies during development or in different regions of the CNS. Experiments will also be performed to determine if all MBP variant forms are produced when transcription begins at this new transcription site. Full length cDNAs corresponding to the second class of MBP-gene related transcripts will be isolated, sequenced, and characterized. The structural relationship between this second class of transcripts and the MBP gene will be determined. The intron/exon arrangement of the gene coding for these MBP-related cDNAs will be elucidated and it will be determined if all the exons of this gene are encoded by genomic segments completely included within the MBP gene or if some exons lie outside the MBP gene. To determine if these transcripts are expressed in tissues other than brain or in cell types other than oligodendrocytes, Northern blot analyses of mRNA from a variety of tissues and cultured cells will be performed. The developmental appearance of the transcripts in the brains of normal mice and dysmyelinating mutants will be compared with the expression of the MBP mRNAs. In situ hybridization studies will be performed to determine the cellular and regional localization of the transcripts.