Growth factors signaling through the cell-surface receptor tyrosine kinases (RTKs) represent one of the most critical pathways in the cellular and inter-cellular regulation of vertebrate biological systems. Defects/malfunctions of the class III RTKs, such as the Stem Cell Factor (SCF) receptor KIT, the Macrophage Colony Stimulating Factor (MCSF) receptor FMS, and the FLT3 Ligand (FLT3L) receptor FLT3, contribute to the genesis and development of many types of cancers such as leukemia, gastrointestinal stromal tumor (GIST), and others. These receptors are ideal targets for cancer therapies, but the mechanistic basis underlying their ligand recognition and activation is incompletely understood. Our long-term goal is to elucidate the structural mechanisms used by these receptors in cellular regulation and malignancies. The project will perform a thorough analysis of the whole activation process of class III RTKs: how these receptors maintain static states when the ligands are not present, how their extra cellular domains recognize ligands, and what are the conformational changes required for receptor activation. The specific aims are: (1) Biochemical reconstitution and structural analysis of the complex between KIT and its ligand SCF. Crystals of the SCF/KIT complex that diffract X-rays to 3.2 Angstroms resolution have been obtained. The structure will be solved by multi-wavelength anomalous diffraction (MAD). (2) Biochemical reconstitution and structural analysis of the complex between FMS and its ligand MCSF. Crystals of the MCSF/FMS complex that diffract to 2.4 Angstroms resolution have been obtained. The structure was determined by a combination of partial molecular replacement and single isomorphous replacement with anomalous scattering (SIRAS);refinement is in progress. (3) Biochemical characterization and structural analysis of the complex between FLT3 and its ligand FLT3L. Small crystals of the FLT3L/FLT3 complex have been obtained recently. (4) Investigation of the binding specificity and energetics of the receptor/ligand interactions through protein engineering of the receptor/ligand interfaces. These studies will elucidate novel structural mechanisms that will lay the groundwork for therapeutic development to treat class III-RTK-related cancers.


The retinoblastoma family of proteins (also designated as pocket proteins) comprises the product of the retinoblastoma tumor suppressor gene and the structurally and functionally related proteins p107 and p130. This project focuses on the regulation of the retinoblastoma family member p130 by CDKs. This project seeks to determine those phosphorylation events that modulate p 130 function in a unique manner, which obviously is not shared by pRB and/or p107. The project leader's goal is to link phosphorylation of certain clusters of phosphorylatable sites with changes in p130 protein stability, which deplete p130 from cells as they enter S phase. It is important to detemaine how this occurs and its potential role in maintaining cell cycle check-point activity and suppressing tumorigenesis. The phosphorylation status of the three pocket proteins is regulated in a cell cycle-dependent manner. Among the pocket proteins, p130 is the one that exhibits the most conserved pattern of phosphorylated forms in normal cells. While a variety of differently phosphorylated pRB forms are detected when comparing different cell types under similar physiological conditions, the patterns of p 130 forms detected by SDS/PAGE followed by Western blot analysis are coupled to cell cycle phases as well as to the quiescent stage. In addition, differently from pRB and p107, the levels of p130 appear to be linked also to the phosphorylation status of p 130 and thus, to the cell cycle stage. Dr. Grafia's team has studied in some detail how particular changes in the phosphorylation status and protein levels of p130 modulate its association with members of the E2F family of transcription factors. Their studies show that while certain changes on p130 phosphorylation status are catalyzed by the concerted action of at least two different types of G1/S cyclin/CDK complexes, other phosphorylation events do not involve these kinase complexes. Their recent data demonstrate that E1A blocks hyperphosphorylation of p130 and p107 without affecting phosphorylation of pRB. These studies have shed light on the regulation of p130 by phosphorylation during the cell cycle in normal and E1Aexpressing cells. The aims of this project are as follow: Aim 1. To identify the regulatory mechanism(s) that tag p130 for cellular depletion as cells progress through the mid-late G1 and S phases of the cell cycle. Aim 2. To determine the biochemical pathway that mediates cell cycle dependent degradation of the p130 protein. Aim 3. To characterize p130 /unctions other than its ability to repress E2F-dependent transcription by using a genetic annmach. This project will utilize core B for Aims 1-3. and core A for Aims 3.


(Supported by NIH GMS 43264 to R. Palazzo and GMS 40198 to C. Rieder) This is an ongoing project in which IVEM and HVEM tomography are being used to analyze the structure of centrosomes isolated from Spisula (surf clam) oocytes prior to and after various experimental manipulations. This year we characterized the high-resolution 3-D structure of control and KI extracted centrosomes, and also extracted centrosomes that had regained their function by incubation in high speed extracts from oocytes. We found that heavily KI extracted centrosomes are non-functional and consist of an insoluble 15-nm filamentous "centromatrix" material. When this centromatrix is incubated in high speed extracts of activated egg oocytes, the extracted centrosome regains its functional abilities. The loss of centrosome function is correlated, at the IVEM tomographic level, with the loss of gamma tubulin containing 20-nm diameter ring-shaped structures, while regain of function is correla ted with the reappearance of these structures. The data reveal that the centromatrix serves as a scaffold for binding those proteins involved in microtubule nucleation. Because centrosomes are extremely small (0.25 by 0.5 um) and complex, the only way their morphology and replication can be studied is with 3-D EM. Over the past 12 years we have shown that IVEM/HVEM tomography of serial thick sections greatly facilitates such studies, and have published numerous papers on this topic-many recently with the Palazzo laboratory. We are now exploring the utility of combining high resolution antibody labeling with IVEM tomography of high pressure frozen and freeze substituted samples. Schnackenberg, B.J., A. Khodjakov, C. L. Rieder and R. E. Palazzo (1998). The disassembly and reassembly of functional centrosomes in vitro. Proc. Natl. Acad. Sci. USA 95:9295-9300.


Aim: Acute rejection remains a major problem for pancreas transplant recipients and accounts for 30 percent of graft failures in the first year after transplantation, in spite of improved surgical techniques and advances in immuno suppression. There are no universally accepted clinical indicators of rejection for pancreas allografts. Biopsy proven rejection is the standard for diagnosis; however, pancreas biopsies may be technically difficult to perform and associated with risks to the recipient. Recently, increased cytotoxic lymphocytic expression of perforin, granzyme B, and Fas ligand in peripheral blood lymphocytes (PBL) has been associated with renal allograft rejection and may be indicative of subclinical acute rejection. Because of the increased incidence of acute rejection and the difficulty in establishing a clinical diagnosis, a similar technique to evaluate early markers of acute rejection is uniquely important in pancreas transplantation. We hypothesize that by measuring expression levels of 3 genes, perform, granzyme B, and Fas ligand, using a newly developed molecular technique (real-time reverse transcriptase polymerase chain reaction [RT-PCR]), we can noninvasively detect acute allograft rejection at the time or prior to development of biopsy proven rejection. The ultimate goal is to develop a reliable, non-invasive method to detect acute rejection, thus allowing for earlier therapeutic intervention and better outcomes in solitary pancreas recipients. Long-term, we aim to expand the use of this technology to identify other genetic markers for acute rejection and develop microarray chip technology to clinically monitor the immunological status of transplant recipients. Methodology: Over a 2-year period, 25 solitary pancreas transplant recipients will be entered into the study. At least 40 milliliters of blood will be obtained pretranspiant and at time of surveillance biopsies or clinic visits (10 time points). Peripheral blood lymphocytes (PBL) will be separated for mRNA extraction. State-of-the-art real-time RT-PCR (ABI PRISM 7700) will be used to quantify gene expression levels of perforin, granzyme B, and Fas ligand. T-tests will be used to examine differences in gene expression levels between those patients with and without hiopsv proven acute reiection.


This project is concerned with (a) the genetic diversity of Trypanosoma cruzi and its implications to the epidemiology, course and diagnosis of Chagas' disease, (b) the development of high resolution flow cytometry instrumentation for analyses of cell populations and, (c) the utilization of flow cytometry and low light level video microscopy for the analyses of infectious agents. Flow cytometry was used to analyze the population dynamics of T. cruzi clone mixtures. The relative numbers of each clone in the population changed rapidly and the results are in quantitative agreement with mathematical models of competitive population growth; no dynamic equilibrium was found. These data emphasize the importance of working with well-defined clones in the laboratory and stress the importance of rapid isolation of single clones from clinical specimens. A similar situation occurs in Dipetalogaster maximus except that clones can coexist for longer periods of time in the bug. Electrocardiographic analyses of inbred mice infected with T. cruzi clones demonstrate that several known aspects of chronic Chagas' disease can be mimiced with the model. Electron bean x-ray microchemical analyses of T. cruzi clones demonstrate marked inter-clonal differences in Fe, Zn, S, Mg, K, Ca and P.


The murine lymphocyte receptor for IgE, with regard to both structure and function is the focus of this application. Intact FceR levels increase during B cell activation in the presence of interleukin 4 (IL-4) suggesting a role for the FceR in B cell activation. Anti-FceR both in the soluble and sepharose bound form will be tested for the capacity to either directly activate B lymphocytes or to influence B cell activation by anti- Immunoqlobulin. Parameters to be measured include calcium influx, cell size changes, phosphatidyl inositol levels and Ig production. The FceR spontaneously releases lower molecular fragments via proteolysis at the cell surface and preliminary evidence suggests that these fragments enhance the level of IgE synthesis in the presence of IL-4. This phenomena will be further studied with regard to isotype specificity, role of FceR carbohydrate in the biologic activity and molecular size of the FceR fragment involved. In addition, it will be determined if the fragment interacts with a specific component on the B cell membrane. The site of interaction of IgE with the FceR is important with regard to receptor degradation and the location of this site will be explored in detail, both by using anti-peptide antibodies, mimicking specific areas of the FceR and by using crosslinking reagents that will label the site(s) of close proximity with IgE. Antibodies against the site of the FceR that interacts with IgE will also be tested for their capacity to induce FceR upregulation, analogous to IgE and their effect on IgE synthesis will be determined. The low affinity FceR on macrophages and T cells will be further investigated with regard to physiochemical relationship to the B cell FceR, association with other membrane components and regulation at the cell surface. The overall aim is to gain further insight into the structure and regulation of the low affinity FceR and to determine if FceR/anti-FceR related components are useful in the regulation of IgE synthesis.


Summary Osteoblasts are emerging as a key component of tumor microenvironment in breast cancer bone metastasis. It has been demonstrated recently that disseminated breast cancer cells interact with the osteoblastic niche to survive and colonize the bone. However, the detailed mechanism for the interaction between tumor cells and osteoblastic niche in bone colonization remains mostly uncharacterized. Furthermore, almost no studies have been done to elucidate the functions of osteoblasts in the resistance of bone metastasis to traditional therapies (i.e., chemotherapy and radiation therapy). In our preliminary studies, we discovered that chemotherapy significantly increases Jagged1 expression level in osteoblasts and in mesenchymal stem/stromal cells (MSCs), the precursors of osteoblasts. Transgenic expression of Jagged1 in osteoblasts in our newly created Col1a1-Jagged1 transgenic mouse model significantly increases the formation of bone metastasis. Importantly, we have developed a humanized monoclonal antibody against Jagged1 (15D11), which demonstrated promising therapeutic effects against bone metastasis in preliminary studies. We hypothesize that the increased expression of Jagged1 in MSCs and osteoblasts promote bone colonization of breast cancer and contribute to chemoresistance and radioresistance by providing survival signaling to metastatic tumor cells. Neutralizing antibodies against Jagged1 may block bone metastasis formation and outgrowth, and sensitize them to traditional therapy. Col1a1-Jagged1 and a series of well establish mouse models will be used for bone metastasis allograft and xenograft studies to evaluate the functional importance of osteoblast Jagged1 in bone metastasis formation and treatment resistance. We will use Jagged1 neutralizing antibody 15D11 in vivo to test its synergistic effect with chemotherapy, radiation therapy and osteoclast-targeting therapy. Furthermore, we will perform experiments to understand the exact molecular pathways leading to elevated expression of Jagged1 in osteoblast in response to chemotherapy, as well as Jagged1-dependent osteoblast-tumor interaction in promoting metastatic colonization and treatment resistance in bone. Our proposed studies will confirm the importance of osteoblast Jagged1 in bone metastasis colonization and treatment resistance. We will also reveal molecular signaling/pathways responsible for the Jagged1-dependent function of the osteoblast niche in bone colonization and treatment resistance. Successful pre-clinical testing of Jagged1 neutralizing antibody will also pave the way toward their application in human patients in the near future. Therefore, we believe our study will likely have a significant impact on improving the treatment of metastatic breast cancer.


Mitochondria from young and mature BHE and Wistar rats were studied. Strain and age affected respiration, shuttel activity, ATP production, ATPase activity, and adenylate kinase activity. Mitochondria were less active in BHE rats than in Wistar rats and with age became even less active. The slower shuttle activities and Ca ions Mg ions ATPase activity in BHE rats could be made more active with thyroid hormone treatment suggesting that the genetic aberration in metabolic control which results in maturity onset lipemia and glycemia, and in a shortened lifespan, is not due to an aberration in the mitochondrial shuttle systems or of the ATPases. Studies of glucose production by isolated hepatocytes showed that gluconeogenesis was more active in BHE rats than in Wistar rats but that in BHE rats, thyroid treatment was without effect as a stimulant of glucose production.


DESCRIPTION: (Provided by the Applicant) Most patient safety improvement occurs incrementally within single institutions. A new method to develop comprehensive statewide risk models that could be exported to large segments of the health care industry is evaluated. Working hypothesis: Sociotechnical probabilistic risk assessment (ST-PRA) can create risk models identifying common medication system and behavioral elements that raise the risk of serious errors and these risk models can be used to design statewide risk reduction programs for nursing and community based care (CBD) long term care facilities. These facilities need robust, well-designed medication systems because they serve a growing and often frail population, administer highly toxic drugs, and must perform to high standards using an unstable and sometimes minimally skilled labor force. Design: Developmental study. Methods: This project uses four tools--process mapping, control system mapping, failure modes and effects analysis (FMEA), and socio-technical probabilistic risk assessment (ST-PRA)--to create two comprehensive probability risk assessment (ST-PRA) models, one for nursing facilities (NFs) and one for CBC (residential care/assisted living) facilities, to identify processes and behaviors that increase the risk of wrong drug, wrong dose, wrong patient medication delivery errors in LTC facilities. The NF risk assessment model is created by focus groups of staff, pharmacists, and physicians from nine randomly selected facilities in a stratified sample of three large, volunteer LTC chains. The CBC process is similar, with nine randomly drawn CBC facilities. Focus groups of CBC residents and their families will be invited to provide input into the CBC model. Appropriate human subjects and privacy protections will be in place. Models are validated in stratified, random samples of nursing and community-based care facilities to determine whether each model is representative of medication delivery systems in the respective types of facilities, using a combination of structured focus groups and direct observation. Recommendations for interventions to address the systems and behavioral risks identified will be made and lessons learned while undertaking this large-scale, multi-facility ST-PRA project will be reported.


The melanotropic neuropeptide, alpha-MSH, is known to inhibit interleukin 1 (IL1)-induced fever in the brain and systemic immune responses. The long-term objectives of this proposal, in continuation of studies underway, are to determine the mechanism of alpha-MSH modulation of IL1 action within the brain and to characterize the melanotropin receptor(s) of the brain and of lacrimal gland, a peripheral melanotropin target organ and potential tissue source for purification of brain-type melanotropin receptors. Two separate hypotheses will be tested that may explain the mechanism of action of alpha-MSH-mediated inhibition of IL1 action within the brain: A) First, that alpha-MSH inhibits IL1-induced release of prostaglandins E and/or F2- alpha, and/or interleukin-6 (IL-6), in hypothalamic cells; B) Second, that alpha-MSH inhibits IL1-induced secretion of corticotropin releasing factor (CRF), recently implicated as a mediator of fever and other IL1 actions in brain. This will be tested in vivo, by measuring febrile responses to IL1 and alpha-MSH in normal and CRF-deficient Lewis rats, and in vitro, by measuring the effects of alpha-MSH on IL1-induced CRF secretion in cultured brain cells. Based on the observed biphasic nature of alpha-MSH actions including modulation of IL1 effects, the hypothesis will also be tested that multiple melanotropin receptor forms exist in brain and in lacrimal glands, which may mediate different aspects of melanotropin-IL1 interactions: A) By determining whether different structure-function relationships exist for binding of a series of melanotropins in the septal- preoptic area, hypothalamus, midbrain and lacrimal gland; B) by determining the relationship between melanotropin receptor occupancy and stimulation of adenylate cyclase in diencephalic cells; C) by determining whether melanotropin receptors are differentially down-regulated by exposure to alpha-MSH, ACTH, or melanotropin receptor class-selective ligands; D) by determining whether melanotropin receptors exhibit differential binding kinetics or regulation by cations and guanine nucleotides; and E) by isolating and characterizing the melanotropin receptor(s) of the brain and lacrimal gland. Endogenous melanotropic neuropeptides may form part of an endogenous counterregulatory system, protecting against the damaging effects of excessive cytokine activity. By determining the mechanisms of neuropeptide-cytokine interactions and characterizing the receptors for this model class of peptides, the proposed research will advance our understanding of the coordinated response to infection and trauma, and the basis of autoimmune and inflammatory diseases.


A study of electrical activity, how it is affected by ischemia and edema, and how recovery of activity is related to temporary ischemic insults of graded durations. During this period, instrumentation has been procured and or constructed, optimized and familiarized. Preliminary experiments have begun.


Abstract LSUHSC CARC Administrative Core The purpose of the Louisiana State University Health Sciences Center (LSUHSC) New Orleans Comprehensive Alcohol-HIV/AIDS Research Center (CARC) is to conduct cutting-edge translational research on the causes and biomedical consequences of alcohol use and abuse and their impact on biomedical and psychosocial comorbid conditions of persons living with HIV/AIDS. The goal of the CARC is to generate evidence-based knowledge on the interaction of alcohol and HIV disease that will inform health care providers and will lead to effective primary care-based interventions to decrease risky alcohol use, HIV transmission and improve health outcomes. The Administrative Core of the CARC will provide oversight and the organizational framework for the direction, management, and coordination of all activities. Specifically, the Administrative Core will (1) monitor the performance and productivity of the individual research components and cores; (2) manage the budget and fiscal activities of the CARC; (3) supervise the CARC personnel; (4) provide data collection, management, and analysis support, and (5) provide oversight for the educational, enrichment and dissemination functions of the CARC. In essence, the Administrative Core is responsible for centralizing, coordinating, and integrating research and administrative components of the CARC. Dr. Molina, Principal Investigator and Director, and Dr. Nelson, Co- Director, will direct all CARC-related activities in consultation with the Intramural Center Committee. The Administrative Core will facilitate logistics for synergistic, productive, and seamless interaction among the participants to substantially enhance the success of CARC activities. The Administrative Core will foster formal and informal exchanges among members of the CARC and the intra- and extramural scientific community to allow for peer-review, idea cross-fertilization, and development of new collaborative projects focused on the biomedical health consequences of alcohol abuse. The Administrative Core is a mature, integrated component of our CARC with a successful record in supervising, promoting, and developing comprehensive, thematically focused research programs that are both productive and cost-effective in an area of great clinical relevance.


In eukaryotic cells, heterochromatin governs diverse cellular processes such as silencing transcription and maintaining genomic integrity. Once formed, heterochromatin must be transmitted to daughter cells during cell division. Inappropriate gain or loss of heterochromatic structure plays a causal role in cancer. Therefore, studies on how heterochromatin is inherited during S phase or epigenetic inheritance will not only increase our understanding of this basic biological process, but also shed light on how this process goes awry in cancer cells. The yeast S. cerevisiae offers an excellent model system to study epigenetic inheritance. Yeast heterochromatin is marked by the presence of hypoacetylated histories and four silent information regulator (Sir) proteins. SirSp and Sir4p associate with hypoacetylated histones H3 and H4 for establishment and maintenance of yeast silent chromatin. During DNA synthesis, much of the epigenetic information such as modifications on histones as well as Sir proteins is temporarily disrupted. Immediately after DNA synthesis, the epigenetic information must be reestablished. Chromatin assembly factor 1 (CAF-1) is one of the factors involved in inheritance of epigenetic information. However, the molecular mechanisms by which CAF-1 functions in this process is not clear. Our long-term goal is to understand how chromatin structures are inherited during the S phase of the cell cycle. The objective of this proposal is to study molecular mechanisms whereby CAF-1 functions with its modulator and effector proteins to inherit silent chromatin structure in yeast. These studies will address how heterochromatin is inherited in yeast. Moreover, knowledge gained from these studies will also lead to a better understanding of epigenetic inheritance in mammalian cells and cancer caused by malfunction of this process.


To study the safety, effectiveness and pharmacokinetics of temozolomide in patients with advanced cancer.


Project Summary Approximately 85-90% of those with serious mental illness are unemployed (9, 10). This level of unemployment exists despite the finding that, among people who have a psychiatric disability and are unemployed, 55-61% desire employment, with the total reaching 75% when concern about losing benefits was taken into account (1, 2). The rewards of employment for adults with SMI are greater than simply an increase in financial resources. Studies have found that people with serious mental illness who worked competitively scored higher on measures of self-esteem, satisfaction with finances, leisure, and overall life satisfaction compared to those worked little or not at all (11). Supported employment (SE) has been more effective than any other vocational rehabilitation approach for this population (3, 4) and the VA has mandated that SE be integrated into all current VHA Compensated Work Therapy (CWT) programs (VHA Directive 2007-005). Two areas of concern include (a) national utilization of SE services is low (13), and (b) the percentage of SE participants who obtain at least one job during the studies averages 56% even though everyone who enters the program expressed an interest in working (13). Motivational interviewing (MI) is an evidence-based practice that has been effective in enhancing a range of clinical services (6), but has just recently been applied to employment and vocational services by Drebing et al. (7) and Glynn et al. (8). In the Drebing et al. study, a single session of motivational interviewing was associated with a 43% increase in entry and doubling the length of participation in VR, raising the likelihood that motivational enhancement interventions may be effective for addressing a range of VR outcomes. The proposed project is a randomized trial of Adapted Motivational Interviewing for Supported Employment (AMI-SE) provided to veterans and their significant others (family members or key friends) in order to address the internal and external barriers to the veteran enrolling in supported employment and returning to work. The intervention consists of 6 sessions that (a) identify and resolve a veteran's and the significant other's ambivalence about working, (b) provide information about the SE program to both parties, and (c) assist the dyad to develop a change plan to address the veteran's and significant other's emotional and practical concerns regarding SE enrollment and employment. One hundred and fifty veterans from the Bedford VA Hospital will be randomly assigned to AMI-SE or a control condition. The groups will be compared on SE entry rate, CE entry rate, number of weeks working in CE, and SE participation. In addition, the study will examine whether variables related to the Health Belief Model are predictive of vocational rehabilitation entry and outcome. PUBLIC HEALTH RELEVANCE: Project Narrative At completion, results will provide VHA policymakers, investigators, and vocational rehabilitation specialist with findings and recommendations regarding (a) the usefulness of an extended motivational interviewing protocol for veterans and their significant others, and (b) topic areas for development of additional strategies to improve access and outcomes in supported employment. If effective, the motivational intervention could be conducted by case managers of clinicians of veterans with serious mental illness to increase rates of employment. The proposed study seeks to ensure full utilization of SE, including the provision of services specifically designed for those who are unsure about work and employment, moving VA SE services into the cutting edge of access, utilization, and success.


This proposal was prepared in response to the announcement of a Research Program on Breastfeeding and Human Milk. We are specifically addressing th section of the program announcement headed Processes Involved in Milk Formation and Secretion, proposing studies which will determine how choline influences milk formation and secretion. Choline is necessary for normal growth and development. It is a precursor for the biosynthesis of phospholipids - essential components of all membranes, it is an important methyl donor, and it is needed to make acetylcholine. Most mammals derive much of their daily choline requirements from their diets, and milk is an important dietary source of choline. Choline moiety is needed to make the phosphatidylcholine which coats the secretory vesicles and milk fat globule made within the mammary epithelial cell. In hepatocytes, choline is absolutely required for the export of a fat globule (VLDL). It is possible that varying the availability of choline might alter the excretion of casei and lipid by the mammary epithelial cell. Our laboratory has made the following observations: 1) Neonatal rats, ferrets and humans have extremely high blood choline concentrations - making more choline available to tissues; 2) The above is, in part, due to ingestion of milk which is high in choline content; 3) There is a large increase (6-fold) in the choline concentration of human milk just after the start of lactation, followed by an equally large decrease in choline content 4-5 days later; 4) Mammary epithelial cells are capable of accumulating choline via mediated transport so that milk choline concentration can be 70-fold higher than is maternal blood choline concentration; 5) Mammary epithelial cells are capable of de novo biosynthesis of choline moiety. We propose to culture rat and human mammary epithelial cells on a matrix. We will characterize choline transport and metabolism, de novo biosynthesis of choline, as well as formation and secretion of milk fat globules and casein using this cell culture system. We will examine the changes that occur during differentiation of the cells, and during exposure to varying levels of prolactin, insulin hydrocortisone, progesterone, estradiol-17beta or oxytocin. We will also examine the effects of varying the availability of choline upon each of these parameters. In some tissues, carnitine transpor depends upon choline availability, we plan to determine whether this is tru in the mammary epithelial cell.


Summary of Work: The purpose of this agreement was to support the collection and analysis of data on cause of death and characteristics of the last year of life in the planning of the 1992 Pretest and 1993 Main Survey of the 1993 National Mortality Followback Survey (NMFS), conducted by NCHS, CDC. This survey supplements information from death certificates in the vital statistics file with information on characteristics of the decedent. The pretest examined approximately 800 deaths of individuals aged 15 years and over who died in 1992. The main survey examined approximately 22,951 deaths of individuals aged 15 years and over who died in 1993. This includes 884 deaths to centenarians. A revised death certificate/informant(DC/I)file was delivered to the NIA/EDB by the NCHS in 1998 and has been extensively examined by EDB staff and the SYTEL computing support contractor. This file contains information keyed from the death certificate file and the informant interviews. Data from the final 1993 mortality file has been merged with the DC/I file and stripped of personal identifiers to form a working file. Post-stratification adjustment factors and non-response adjustments have been developed to form the final sample weights for each file record.Some additional problems remain with the revised data set, which will be reissued by NCHS in the fall of 1999. Meanwhile, data analyses have been initiated by EDB staff.At the Kentucky conference on Statistical Methods in Alzheimers Disease Research in 1998, EDB staff held discussions with a neurologist who had completed an analysis of data from the 1986 NMFS on dementia in the last year of life. A preliminary analysis of the 1993 data to explore dementia trends over the 7-year period between the two surveys has shown an almost three-fold increase in the recognition of dementia mortality both by physicians completing the death certificates and by informants responding for the decedents in the survey. Another preliminary analysis has shown a high rate of receipt of surgeries for the implantation of hip and knee joints and other devices related to lower limb mobility problems in the decedents. Future analytic plans will look at trends in reported lifetime history of other conditions between the 1986 and 1993 NMFS, examination of lifestyle factors related to death at very old ages, hypotheses regarding decreased use of health services in the last years of life among the very old, and possible studies of compression ofmorbidity among the oldest old. - death certificate, morbidity, oldest old - Human Subjects & Human Subjects: Interview, Questionaires, or Surveys Only


A new X-ray facility will enable the full potential of 19 NIH sponsored grants to be realized. An existing X-ray generator is 22 years old and no longer functional;the existing area detector and low temperature device is 10 years old and no longer cost-effective to maintain. The equipment requested is: a Rigaku MSC, Inc., MicroMax-002+ sealed tube microfocus X-ray source coupled with a high performance Confocal Max-Flux optic that produces a monochromatic, focused X-ray beam with flux 3X greater than the existing RU200 rotating anode generator;a Saturn 944+ CCD X-ray area detector with 94 mm x 94 mm active image area that provides 8 MHz readout, 15100:1 dynamic range, and high sensitivity;a AFC-11 computer controlled 4-axis goniometer;and a X-STREAM-2000 low temperature system. This equipment will provide high throughput instrumental capacity for all crystallographic experiments with biological macromolecules, including screening for crystal quality and cryoprotection conditions with membrane proteins and protein-DNA complexes, and high resolution data collection from many hundreds of protein crystals. A reliable, in-house X-ray facility will accelerate prescreening of many crystals, optimize the use of synchrotron beam time for membrane proteins, and enable structure determination for drug discovery and protein engineering experiments. Equipment items are also requested to upgrade a specially designed single crystal microspectrophotometer for evaluation of protein oxidation states and active site chemistry in experiments that probe mechanism. The microspectrophotometer is essential to correlate biochemical properties with crystal structures, and will be used in direct conjunction with the new X-ray facility. The new equipment will benefit the projects of all seven principle investigators, who have a long and productive track record of collaboration and shared use of the existing X-ray equipment. The new equipment is cost-effective to maintain, and is manufactured in the United States. The new X-ray facility will provide hands-on training opportunities for graduate students and postdoctoral fellows in crystallographic methods, and will stimulate scientific interactions, enhance the quality of learning and research, and help to retain and create jobs in the academic and biomedical sectors. The new equipment is a long term investment, given that the existing X-ray generator was in use over 20 years, and the existing detector and low temperature device have been in use for 10 years. HEALTH RELEVANCE: Crystallographic experiments require a reliable in-house X-ray facility for high throughput screening of crystal quality and cryoprotection conditions, and for high resolution data collection from many hundreds of protein crystals. The new equipment is required to support these experiments for 19 active NIH grants being directed by seven principle investigators.


According to NCI's Strategic Plan, community-engaged research is vital for understanding cancer-related health disparifies and devising effective, culturally-appropriate strategies for reducing these disparities.^ In the first funding cycle (2001-2006), the MMC-VICC Cancer Partnership lacked a community outreach component and primarily focused on basic and clinical cancer research, with only one clinic-based behavioral research project. In 2006, the Partnership created a new Community Outreach Core (COC) guided by a framework of CBPR'*'^ to further the aims ofthe Cancer Partnership in reducing cancer disparities. Given its strong history of community-based research with minority communifies, TSU was brought in as a subcontractor to co-lead the new COC, under the leadership of Dr. Baqar Husaini (TSU), Dr. Elizabeth Williams (VICC) and Dr. Paul Juarez (MMC), and with Dr. Pamela Hull (TSU). In August 2010, Dr. Wujcik took over as VICC Co-Leader when Dr. Williams left VICC to join the TSU public health faculty.


The long-term objective of this program is to develop an automatic, non- contacting method of irrigating dry eyes. The device will be mounted on eyeglass frames, cosmetically acceptable, and capable of replacing the human tear volume. The specific aims of this first year project are to construct a working prototype with sufficient functionality to test its effectiveness on a small number of severe dry eye patients for up to 30 days. The system components consist of a miniaturized pump, a reservoir for solutions, and an electronic control circuit and battery for programming wetting solution applications.


UCLA and UCSD and their affiliated campuses at Cedars-Sinai and Salk, respectfully, have integrated, highly productive and well-funded programs in diabetes and its complications and in endocrinology. Because of growing interaction and collaboration between Los Angeles-based and San Diego-based investigators, the Southern California Diabetes Endocrinology Consortium (SCDEC) was conceived as a means to formalyze, enhance, and continue to fertilize these relationships. By joining forces and aligning common interests, basic and clinical scientists at both Centers have recognized the enormous potential to strengthen and expand their research opportunities and resources. Thus, the SCDEC DERC represents a critical foundation to consolidate diabetes and endocrinology investigation at two premier academic centers. The objectives of the SCDEC DERC are to: 1) Unite investigators from relevant disciplines in a manner that will enhance and extend the effectiveness of their research in diabetes/endocrinology and the complications of diabetes, 2) Foster collaborations between diabetes/endocrinology researchers at UCLA and UCSD so the whole is greater than the sum of its parts, 3) Enhance, support and develop Cores to facilitate diabetes/endocrinology research at UCLA and UCSD, and 4) Develop new areas of research and foster young investigators focused in diabetes and its complications through pilot and feasibility grants. Herein, the SCDEC DERC presents six exceptionally strong Research Bases comprised of 93 talented investigators supported by six Research Cores. The Research Bases include 1. Nuclear (n=10), 2. Signaling (16), 3. Metabolism (21), 4. Macrovascular (23), 5. Microvascular (14), and 6. Beta Cell (10). The Cores are: A. Transgenic and Knockout Mouse, B. Mouse Phenotyping, C. Transcriptional Genomics, d. Human Genetics, and E. Biochemistry and Molecular Assay. These Cores are designed to specifically meet the greatest needs of DERC investigators. They represent high quality, state-of-the-art and novel technology, which if provided to the SCDEC DERC will bring diabetes and endocrinology research in Southern California to a new level.


Whole body hyperthermia is being studied at NIH as a possible means of treatment for cancer. This project includes development of an instrumentation and control system based on utilization of a Tektronix 31 programmable calculator, digital plotter, and interface for data acquisition. The esophageal temperature of the patient is regulated to 0.1 degree C accuracy by feedback control of the temperature of water circulating in a set of hyperthermia blankets. This project was completed August 1980, however additional publications have been made.


As a result of the advocacy of the benefits of vegetable oil/polyunsaturated fatty acid rich diets in the prevention and amelioration of coronary heart disease and atherosclerosis the consumption of processed fats and oils has increased significantly. These hydrogenated fats contain unusual fatty acid isomers which may, over the long term have deleterious effects on normal lipid metabolism. The primary objective of this initial study is to determine if mixtures of trans isomers of linoleic acid when fed to male rats over a long (6 month) period results in an imbalance of prostaglandins and of their precursors in specific tissues. Initially the effects of linoleic isomers on the relative concentrations of different prostaglandin species, gamma-homolinolenic, and arachidonic acid in serum, stomach and heart tissue will be determined. The stomach is selected because the levels of prostalglandins are higher, which facilitates quantification. It also is sensitive to precursor availability which is helpful in this proposed dietary study. In addition, heart tissue will be carefully monitored to obtain more information on its prostaglandin complement, the effects of dietary fat on the precursors, the activities of prostaglandin synthetase and phospholipase A which may regulate the availability of prostaglandin precursors. Serum will be monitored for PGF2 levels. Male weanling rats (10 per treatment) will be fed increasing levels (0,25,50,100 percent) isomerized safflower oil, replacement in a normal diet containing 20 percent fresh safflower oil. Tissue will be removed at 2,4 and 6 months and analyzed for prostaglandins, their precursors, prostaglandin synthetase and phospholipase A activity to determine the effects of the trans isomers.


Pain remains an important cause of morbidity for frail, older persons in nursing homes. As high as 83% of nursing home residents voice complaints of pain. Often, nursing home residents' complaints of pain are not recognized or not appropriately treated. A substantial scientific evidence base attests to readily available pharmacological and non-pharmacological modalities to treat pain. Research utilizing continuous quality improvement, including a recently completed study by Center for Gerontology and Health Care Research in cooperation with RI Quality Partners, has impacted on nurses' behavior in the assessment, documentation, and the use of non-pharmacological treatment of pain. This study resulted in 33% reduction in pain. However, our intervention did not significantly impact on physician proscribing behavior. Based on debriefing of nurse coordinators and others research, nurse-physician communication and physicians' lack of knowledge are two important barriers to appropriate pain management. In this R21 application, we propose to develop and pilot test a Personal Digital Assistant (PDA) based decision support intervention for pain management in long term care. This decision support will assist the nurses in conducting a pain assessment, provide treatment recommendation(s), and print a summary report to be fixed to the physician office. Existing guide lines from the American Geriatric Society, American Medical Director Association, and AHRQ pain management guidelines as well as the advice of expert panel will be utilized to formulate decision support tool. In the development of this intervention, we will conduct focus groups with end users (long term care nurses and physicians), utilize cognitive testing, and conduct a six month pilot test that will examine the use of the decision support application in two nursing homes in RI (one academic base NH and one community base NH). An evaluation will collect information on the time to decision recommendation, acceptability of the PDA application, whether recommendations were utilized, and the reported level of pain prior to and after the use of decision support. If we are successful in development of this intervention, a R01 research application will be prepared to conduct a block randomized trial of whether this intervention when combined with the rapid cycle continuous quality improvement intervention improves quality of pain management. [unreadable] [unreadable]


The ocular lens provides an ideal system to study cell cycle regulation during terminal differentiation. The lens is composed of two cell types: the epithelial cells, which are capable of cellular proliferation, and the fiber cells, which are post-mitotic. I hypothesize that the differentiation of epithelial cells into fiber cells will cause alterations in activity of the genes that regulate the cell cycle. In order to help test this hypothesis, the alphaA-crystallin promoter will be used to direct lens-specific alterations in gene expression in transgenic mice. Transgenic and non-transgenic mice will be used to study two general classes of genes that are likely to be critical for cell cycle control during differentiation: tumor suppressors and cyclin-dependent kinases. The Specific Aims of this grant application are: l) to assess the role of the retinoblastoma (rb) protein in cell cycle control and terminal differentiation of lens fiber cells; 2) to assess the role of p53 in the induction of lens tumors by SV4O large T antigen; 3) to assay for changes in cyclin dependent kinases that accompany fiber cell differentiation; 4) to characterize the cell death that is induced by rb inactivation in fiber cells; and 5) to genetically reverse lens tumorigenesis induced by full-length T antigen. Our preliminary experiments have shown that expression of viral proteins that bind to rb and/or p53 can induce lens cell tumorigenesis or programmed cell death. Therefore, the cell cycle can be altered in lens cells with fascinating and unexpected consequences. The proposed studies should provide insights into cell cycle regulation in vivo, not only for the lens, but for other cells undergoing terminal differentiation or neoplastic transformation.


This work is designed to detect changes in gene expression in mouse fibroblasts or lymphocytes transfected with human leukemia cell DNA. Using a combination of 2-D electrophoresis, differential labeling and immunochemical techniques, we will detect gene products in the transfectants that differ qualitatively or quantitatively from products in the parental mouse cells. We will attempt to determine the relationship between these changes and the transforming event - i.e., Are they a direct effect of the incorporation of "oncogenic DNA" into the cells, or do they represent changes secondary to the initial transforming event? Are they produced by the human DNA transfected into the mouse cells or by the mouse genome? Do any of these "new" gene products produce pleiotropic effects which can account for the variety of phenotypic changes in the transformed cells? What is the relationship of these changes to the differentiated state of the transformed cell? Does the same DNA transfect fibroblasts and lymphocytes, and are the effects the same?


The long-term objective of this project is to study the components of the ternary complex of antigen, T cell receptor (TCR) and MHC class I molecule whose interaction leads to T helper cell activation structure/function relationships between components of this ternary complex are currently being analyzed using murine T cell clones specific for well defined epitopes on the model protein antigen sperm whale myoglobin (Spw Mb). A panel of T cell clones has been generated from DBA/2 mice, reactive with Spw mb epitopes characterized with overlapping sets of synthetic peptides. The specific aims are as follows: 1) To further characterize the antigen and MHC specificity of these clones and of additional mb clones generated in the mouse strains B10.D2 BALB/c and D2.GD. These studies will include the use of horse myoglobin and substituted peptides to identify residues within the epitope important of interaction with the TCR or restricting Class II molecules 2) To sequences alpha and beta chains from matched sets of clones that shave either epitope specificity or MHC restriction in common, and to compare these sequences with those already derived from independent clones that share epitope specificity and MHC restriction. The relationship between TCR and antigen specificity/MHC restriction will be further addressed by transfection of chosen TCR alpha or beta chain genes into Mb- reactive T cell hybridomas expressing endogenous TCR genes of known sequence; 3) To make use of class II (I-Ed) variants generated by site-directed mutagenesis to analyze the interaction of TCR and antigen with the restricting class II molecule. These experiments should determine whether the same set of TCR genes are used to respond to a particular epitope in association with an MHC class II gene product regardless of the non-MHC background if they do, then this result would support the concept that HLA associated diseases might result form the formation of a particular ternary complex involving a shared MHC restriction molecule, a common "disease-associated" epitope, and a shared T cell receptor.


Developmental funds have played an important role in the development of the Cancer Center, particularly in recruiting new investigators and in establishing new shared resources. During the current funding period developmental funds were instrumental in the recruitment of six new investigators and in the development of two new shared resources (Bioinformatics and Proteomics). We propose to continue our recruitment efforts to strengthen the scientific programs of the Cancer Center, and to develop a new shared resource devoted to Chemical and Systems Biology.


The normal function of the brain relies on precise patterns of neuronal connections, and aberrant connectivity leads to human neurological and psychiatric disorders. The human brain consists of approximately100 billion neurons with trillions of synapses. Because of the enormous neuronal diversity and staggering synaptic complexity, very little is known about the molecular mechanisms that lead to the assembly of specific synapses in different neuronal types. Protocadherin (Pcdh) genes (14 Pcdh-a, 22 Pcdh-fi and 22 Pcdh-y in mouse) are attractive candidates for such a role because they can potentially generate a significant number of cell-surface "codes" through a combination of cell-specific promoter activation andcis- alternative splicing. It has been suggested that the distinct combinatorial Pcdh expression patterns might specify neuronal types and their connectivity. To evaluate their roles in neural development, we initiated functional analyses of these genes using genetically modified mice. Our analyses on Pcdh-j mutant mice provide the first in vivo evidence that protocadherins are essential for vertebrate CNS development and play an important role in establishing neuronal connectivity. However, Pcdh-y's function during synaptic development is not well defined and its molecular mechanisms of action are completely unknown. We plan to combine molecular and genetic approaches to further our understandingof Pcdh-y's functions.Specifically, we propose 1) to investigate the rules of expression for individual isoforms of Pcdh-y;2) to define the role of Pcdh-y's diversity;and 3) to identify and characterize the signaling components of Pcdh-y. The attainment of these goals will shed light on our understandingof the molecular basis for the precision and complexity of neuronal circuitry in the brain.


The effects of normal aging on cerebral metabolic processes have been studied with methods developed in this Laboratory: the [C-14] leucine method for local rates of protein synthesis and the [ C-14] deoxyglucose method for local rates of glucose utilization. We have found that rates of glucose utilization and protein synthesis both decrease with age in the components of the primary auditory and visual pathways. These changes may be the consequences of a chronic lack of sensory input due to age-related degenerative changes which are known to occur in both retina and inner ear. Our studies also show that glucose utilization is particularly and significantly decreased in the striatum in aged rats. In order to examine the functional consequences of the senescent changes in the striatum we are studying the effects of age on the metabolic responsiveness to apomorphine, a dopaminergic agonist. Aluminum toxicity has been proposed as an etiological factor in Alzheimer's Disease. Aluminum has been shown to affect numerous biochemical processes including several enzymes in the metabolic pathway for glucose. Experiments have been carried out on the effects of prolonged (2 years) intake of low levels of aluminum in drinking water on local cerebral glucose utilization in aged rats. The results of these studies show decreased glucose utilization in the treated rats in 2 out of 26 regions, ventral pallidum and temporal cortex.


The purpose of this study is to identify factors associated with maintenance and change of mental health in the poor, urban, Black elderly using a short-term longitudinal approach. A panel of 150 Black men and women, over 60 years of age, will be contacted at short, regular intervals for repeated psychological, social physiological measurements for a period of three years. The project will be carried out in Newark, N.J. In order to optimize the longitudinal nature of the project, the sample will partly consist of individuals who took part in a recently terminated study on the mental health of the Black aged by the authors of this proposal. Personal and social data on this group are available for 1972 and 1974, and these contain some of the measurements expected to be used with the current population. Variables considered will include physical and mental health, life styles, self concept, life satisfaction and morale, depression, social interaction and isolation, use of available services, environmental conditions, family and friendship networks, need and use of short term and long term care facilities. Analysis of the data will pay particular attention to changes that occur with time. A review panel consisting of both expert and lay members will be consulted with regard to interpretation and health care implications.


The human peptidase clan CE is defined by seven proteases with a common fold and catalytic mechanism, commonly known as SENPs (sentrin specific proteases - sentrin being an alternative name for SUMO). Current information suggests that most SENPs are specific for processing of SUMO precursors and removing SUMO conjugated to protein substrates. Small-molecule compounds that target SENPs in a selective manner will provide an important toolset to study the biology of these enzymes, to aid in the discovery of their role in disease etiology and progression, and to facilitate the subsequent development of therapeutics against disease-relevant targets. The availability of simple kinetic assays that are applicable the SENPs would significantly enhance the discovery of chemical probes for members of the family, and for .orphan. SENPs with lesser known natural substrate specificity. Our application squarely addresses this [unreadable] important unmet need for a development of high-throughput substrate-based assays. We propose to develop small peptide based substrates with broad specificity for all seven human members of the SENP family, and generate simple assay procedures. The assays are expected to provide a tool for the rapid development of chemical probes for members of the family. [unreadable] [unreadable] [unreadable]


There is a general concern that many environmental chemicals to which humans are exposed to are mutagenic. Several metal compounds have been identified through occupational exposure and laboratory testing as potential carcinogens. Therefore, screening of these agents is of paramount importance. In the proposed study, the ongoing investigations on the influence of heavy metal salts on the induction of chromosome aberrations, sister-chromatid exchange and micronuclei in CHO cells will be performed. Additionally, a detailed study will be conducted on the effect of various arsenic compounds on the above parameters. the student participants will be involved in all phases of the investigation. They will be introduced to the techniques of mammalian cell culture, cytogenetics, and genetic toxicology. Through involvement in MBRS research, the student participants will be motivated to pursue advanced degrees in health-related sciences.


The goal of this study is to elucidate the molecular mechanisms by which lac repressor interacts with lac operator. The lactose operon of E. coli is chosen as a system in which the general principles of regulation of transcription can most easily be studied. Genetic methods will be used to isolate and characterize specific repressor mutants (a) which specifically overcome oc mutations and (b) which are defective in subunit aggregation. One class of repressor mutations which overcome oc mutations and of which some have a largely increased affinity for the operator has been obtained. These repressors will be further investigated. Operator mutations which increase the symmetry of the operator will also be selected, and the effect of these mutations on the repressor-operator interaction will be studied. A secondary binding site for the repressor will be mutated to increase its affinity for the repressor and make it function as an effective operator. My long-range concern is to identify the amino aicd residues of the lac repressor and the base pairs of the lac operator which contribute to the repressor-operator interaction.


Determine the relative importance of different T cell activation components in the induction of HIV replication, and the influence of viral early gene product, Nef, on these required activation events.


Marburg virus (MARV) belongs to the filovirus family and is highly pathogenic in humans. Despite being classified as Category A Priority Pathogen by NIAID, and its potential to cause large-scale outbreaks, similar to the recent Ebola virus outbreak, research on MARV lags significantly behind that on other non-segmented negative sense (NNS) RNA viruses. Here, we propose to perform in-depth analyses of MARV transcription and gene expression. Dissecting the mechanisms of MARV gene expression will not only be instrumental for the targeted development of antiviral drugs, it will also reveal unifying paradigms and distinctions between the NNS RNA viruses. The filovirus genome is transcribed by a virally encoded RNA-dependent RNA polymerase complex, which is capable of generating capped and polyadenylated mRNAs. This process occurs in the cell cytoplasm, close to ribosomes and cellular RNA binding proteins. This project will examine three different stages of MARV gene expression. In Aim 1, we will elucidate the mechanism of transcription initiation at the MARV promoter and investigate the role of structural features of the polymerase in this process. Notably, the MARV promoter sequence has some unusual features, and we intend to explore the functional relevance of these characteristics. In Aim 2, we will determine the function of conserved hairpin loops that are formed at the 5 end of each MARV mRNA. We will explore the effect of these structures on transcription, RNA stability, trafficking and translation. In Aim 3, we will focus on mRNA polyadenylation and release. The mechanism of mRNA release is not well understood for any NNS RNA virus, and the results obtained in this aim will help to broaden our understanding of NNS RNA virus transcription strategies. This proposal brings together expertise in studying NNS RNA polymerases, MARV molecular biology, and mRNA-protein interactions. Together, the research team has established a unique tool set to achieve the goals of this proposal, including a MARV in vitro polymerase assay, various MARV reverse genetics systems, and highly innovative single molecule mRNA-protein binding assays. These studies will shine new light on a crucial aspect of MARV infection and enhance our understanding of NNS RNA virus biology.


This project is being proposed in light of the continuing increase in reported AIDS cases among the Latino population of the United States. In the past two years the number of reported AIDS cases among Latinos has nearly doubled, going from 15,271 in June of 1989 to 29,586 in June of 1991. As HIV/AIDS continues to impact this population it is imperative that we develop a national AIDS research agenda to deal with this particular population. The goal of this project is to establish an organization and planning framework for the development of a national Latino focused HIV/AIDS research consortium. The objectives of this project are: 1) to begin to develop both national and regional networks of Latino AIDS researchers to facilitate networking, information sharing and research collaboration; 2) to identify potential institutions with the capabilities to implement the research activities of the consortium; and 3) to identify individuals to serve on a National Latino AIDS Advisory Committee to provide direction in planning the research consortium. To achieve these objectives the project will establish a Central Planning and Coordination office responsible for: 1) establishing an Executive Committee of Latino AIDS Researchers, 2) identifying institutions, 3) establishing regional linkages in areas with the highest concentration of Latino AIDS cases, 4) producing a national directory of Latino AIDS researchers, 5) hold regional meetings to establish regional networks of Latino AIDS researchers and to provide input on the development of a P20 planning grant proposal for Latino focused HIV/AIDS research.


The mechanism by which cell therapy improves cardiac function remains unclear. Although injected cells minimally differentiate into cardiomyocytes or vessels, and only a small fraction survive long term in the recipient, common beneficial effects are observed regardless of injected cell type: improvement in pump function, reduction of fibrosis, and enhanced angiogenesis. These results are consistent with the idea that cell therapy recruits endogenous repair mechanisms which, however, have not been identified. The immune system has been implicated in a variety of sterile diseases, including processes regulating myocardial damage and repair. Although unresolved inflammatory processes controlled by infiltrating and tissue-resident immune cells worsen heart failure, depletion of macrophages in the infarcted myocardium leads to LV rupture and death. Thus, immune cells appear to play diametrically opposite roles in the heart. Macrophages have been shown to be required for spontaneous regeneration of neonatal mammalian myocardium after injury, and recent findings implicate them as direct contributors to cell therapy-mediated myocardial repair. Nevertheless, how immune cells regulate myocardial repair and what determines their harmful versus salutary actions remains unknown. Furthermore, the impact of cell therapy on reparative immune cells has not yet been studied in the heart. Our preliminary data show that injection of cardiac mesenchymal cells (CMCs) into the infarcted heart promotes accumulation of reparative macrophages. Thus, the central hypothesis of this proposal is that CMCs facilitate recruitment of monocytes and activation of reparative macrophages, which are essential endogenous mediators of repair. By generating detailed flow cytometric analyses of immune cell populations following CMC administration, we will not only resolve the time course of immune cell recruitment, but also determine how inflammation is eventually extinguished in the heart after cell therapy. To elucidate the mechanism whereby CMCs regulate inflammatory processes in monocyte-derived macrophages, we will determine how these cells regulate NF?B-p65 subunit expression, with emphasis on horizontal transfer of miRNAs to macrophages through CMC-derived EVs. Finally, using macrophage genetic fate mapping and genetically modified CMCs, we will elucidate the role of monocyte-derived macrophages in CMC-induced myocardial repair. This project will be the first systematic analysis of how cell therapy modulates immune cells ? a mechanism that has been relatively understudied. The results will provide novel insights not only into the mechanisms regulating cell therapy-mediated myocardial repair, but also into how endogenous reparative activities of macrophages are recruited. We will also determine whether EVs recapitulate the salutary effects of CMCs on immune cells. Thus, these studies have far-reaching implications for our understanding of how the immune system regulates myocardial homeostasis in general.


This revised proposal to develop a mathematical model for the stability of pulmonary airways is a basic inquiry into the mechanisms that lead to airway closure of the lung in health, disease, and its modifications by therapies. Closure results from a surface tension instability that tends to pull the airway to a smaller diameter while potentially driving the liquid lining into the formation of a plug, given the right conditions. A plug blocks the lumen, stopping gas exchange and delivery of aerosol medications. It can be the terminal event in an asthmatic attack, for example. From purely the fluid mechanical perspective, the plug may or may not form depending on the liquid physical properties. Significant non-Newtonian fluid properties in disease (asthma, emphysema, cystic fibrosis) include viscoelasticity, shear-thinning viscosity, and a yield stress, all of which can alter the fluid movement. Surfactants at the air-liquid interface also influence the instability by reducing surface tension and creating surface tension gradients. The lung volume at which closure occurs is the closing volume, and it is a common pulmonary function test that is often difficult to interpret, due largely to inadequate models. This surface-tension/fluid system couples to the airway and lung elastic properties. New to this revision is the inclusion of non-linear airway elasticity, airway smooth muscle dynamics, and effects of a surrounding parenchymal tethering. This novel combination of effects will allow the development of a theory which can incorporate the simultaneous issues presented by diseases such as asthma and emphysema where bronchoconstriction, parenchymal tissue elasticity changes, and fluid property modifications (non-Newtonian fluids) acting together present a complex mechanical situation; The proposed model will provide a platform for predicting the impact of these interactions on closing volumes, as well as predict the effectiveness of therapies which may include those aimed at surfactants, or liquid material properties, or bronchoconstriction or the parenchyma. This proposal outlines a set of mathematical models, building from the simplest to the more complex, that examine the stability of a liquid-lined airway tube as it depends on (1) Newtonian vs non-Newtonian fluids, (2) rigid vs flexible tubes with parenchyma and smooth muscle tone, (3) axisymmetric vs non-axisymmetric deformations, (4) effects of surfactants, (5) effects of a forced, oscillatory core flow. [unreadable] [unreadable] [unreadable]


We and others have demonstrated that induction of antibodies to the beta-amyloid (AB) peptide endows therapeutic effects in Alzheimer's disease both in pre-clinical and clinical settings. This approach is currently hampered by elicitation of pathological autoreactivity. In order to overcome this problem we have developed several strategies to limit pathological immunity. For example, we generated an epitope vaccine composed of small immunodominant self B cell peptide of AB42 fused with a foreign CD4+Th cell epitope and demonstrated that such vaccine induced high titers of anti-AB antibodies without generation of potentially harmful autoreactive T cells specific to AB. Importantly, these antibodies were therapeutically active, as we showed in two different mouse models of AD (APP/Tg 2576 & 3xTg-AD). After having demonstrated feasibility of selectively inducing a beneficial antibody response to AB in absence of pathological autoreactivity, we decided to expand these studies to a more clinically applicable system. In collaboration with our co-investigator, we have used the influenza virus platform for delivery of immunodominant B cell epitopes of AB42 (AB1-10) into the host. In our preliminary data we have generated a flu-AB1-10 vaccine that induces robust anti-AB and anti-influenza antibodies and reduces AB-deposits in the brains of immune 3xTg-AD mice. Thus, in the first three translational Aims of this proposal we plan to learn about (i) immunogenicity and efficacy of recombinant flu- AB1-10 vaccine in 3xTg-AD mice without AD-like pathology (Aim 1), as well as with early (Aim 2) and late (Aim 3) AD-like pathology. The last Aim 4 of this study is designed to explore immunological mechanism/s of generation of anti-AB antibodies by this vaccine and identify specificity of anti-viral memory Th cells involved in this process. Thus, at the end of this study we will learn about cellular and molecular mechanisms governing the generation of antibodies specific to both AB1-10 and influenza. The long-term goal of this proposal will be a generation of the safe and effective dual (flu-AB) vaccine that may prevent development of AD pathology in pre-symptomatic people, protecting them from the flu infection at the same time. PUBLIC HEALTH RELEVANCE: Alzheimer's Disease is the major cause of dementia in the US and is characterized by an insidious onset and progressive cognitive decline that impacts memory, language, judgment, orientation to time and place, etc. Pathologically there is an increase in the presence in amyloid plaques, neurofibrillary tangles, dystrophic neurites and a general loss of neurons. It was demonstrated that induction of antibodies to the beta-amyloid peptide endows therapeutic effects in Alzheimer's disease both in pre-clinical and clinical settings. This approach is currently hampered by elicitation of pathological autoreactive T helper cells. In order to overcome this problem we and others are developing several strategies to limit pathological immunity. In current project we are proposing to generate the safe and effective dual vaccine, based on influenza viral vector and immunodominant B cell epitope from beta-amyloid peptide that may prevent/reduce development of Alzheimer's disease pathology in pre-symptomatic people diagnosed with early-stage AD, protecting them from the flu infection at the same time.


The longer-term goal of our research effort is to understand why persons with addictions change their social networks in either positive or negative ways, and how important health outcomes and health behaviors affect are influenced by social changes. Our research focuses on persons with addictive disorders because addiction can have strong, even catastrophic, effects on our strongest social ties. On the other hand, there is substantial evidence, including new analyses we present, that social relationships are a strong determinant of addictions outcome. Since addictions are chronic, relapsing disorders, if we were able to promote durable healthy relationships for addicts, these relationships might reduce the need for costly and not always accessible professional services. Unfortunately, we lack a basic understanding of why people change their social relationships, for better or for worse. Our research will illuminate the processes of change in addicts' social networks. We will recruit 300 persons from addictions treatment facilities and follow them naturalistically for two years, gathering intensive longitudinal data on social relationships, substance use, and other variables. Our study has three aims: (1) To determine how a person's relationships are affected by other relationships, and by substance use, co-varying for relationship type and history; (2) To replicate and extend prior research on the effects of social network characteristics on substance use outcome; and (3) To determine social network influences on entry into substance abuse treatment, and mutual-help participation. Cox proportional hazards regressions and hierarchical linear models with time varying covariates will be used to test the a priori predictions laid out i the proposal.


Veterans are disproportionately affected by peripheral artery disease (PAD) and coronary artery disease (CAD) because of high rates of tobacco use in the veteran population. Up to one-third of the saphenous vein bypass grafts performed for either PAD or CAD fail secondary to a process called neointimal hyperplasia. We have established a new model to study saphenous vein grafts by implanting human saphenous veins into immunodeficient nude rats. This model develops significant neointimal hyperplasia, unlike prior animal models using animal vein grafts. Our previous work on human saphenous vein grafts suggest that adventitial cells may prevent neointimal hyperplasia and graft failure. We have also been testing a novel therapeutic agent SB-030, which when administered luminally, decreases the neointimal hyperplasia reaction to angioplasty-induced arterial injury in humans presumably by inhibiting smooth muscle cell proliferation. This proposal will test whether adventitial cells can be used therapeutically to decrease neointimal hyperplasia. In addition, we will test whether inhibiting adventitial cell proliferation by applying SB-030 to the adventitia causes increased neointimal hyperplasia. In contrast, we expect luminal application of SB-030 will decrease neointimal hyperplasia by inhibiting smooth muscle cells. These results will be relevant for future treatment of vein grafts with SB- 030 or other therapeutics that target both adventitial cells and medial smooth muscle cells. Successful completion of this proposal will better establish the new model, demonstrate whether adventitial cells reduce neointimal hyperplasia, and determine whether adventitial or luminal application of a novel therapeutic will best inhibit neointimal hyperplasia. This will lead to new therapies to improve the success of vein bypass grafts for patients.


Osteoporosis is a leading cause of mortality and morbidity in aging women. The mechanisms of phosphate and calcium balance were studied as part of a major effort to understand the pathophysiological bases of senile osteoporosis and osteomalacia. This project represents a continuation and extension of previous projects - Z01 AG 00041-07 LMA and Z01 AG 00042-07 LMA. Notable scientific achievements include: (1) The active form of Vitamin D, 1, 25-dihydroxy Vitamin D3, was found to enhance specifically the sodium gradient-dependent phosphate uptake system in the kidney; (2) Vitamin D was found to have a direct effect on renal membrane phospholipids, resulting in an altered permeability towards calcium; (3) Evidence for a novel parathyroid hormone-independent mechanisms for regulating phosphate reabsorption by the kidney was obtained; and (4) The mechanism by which calcium is actively transported in the kidney was described.


The etiology of atrophic gastritis and the reason for the increased prevalence of gastric carcinoma with atrophic gastritis are not known. It is also not known why certain patients with atrophic gastritis, but not other, develop gastric carcinoma. There is a high prevalence of autoantibody and cell-mediated autoimmunity in patients with pernicious anemia (PA) suggesting that immunologic perturbation may contribute to the pathogenesis of the underlying atrophic gastritis. This possibility has been challenged, at least as it applies to gastric autoantibody, by our earlier work which showed that patients with severe impairment of humoral immunity have a greatly increased prevalence of PA. In the present study, tests for humoral and cell-mediated autoimmunity against gastric antigens will be performed on patients with classical PA, gastric carcinoma and controls with peptic duodenal ulcers. A group of at least 5 patients permits us a unique opportunity to study cell-mediated autoimmunity in patients with severe impairment of antibody responsiveness. Both homologous and hog gastric antigens will be used. Results using normal human gastric juice and gastric juice from patients with PA will be compared. The objectives are to compare manifestations of humoral and cell- mediated gastric autoimmunity. We wish to see if findings recorded with atrophic gastritis differ from those recorded with gastric carcinoma. We also wish to see if severe immunoglobulin deficiency and lack of gastric autoantibody changes results recorded with PA.


The objective of this project is to improve the clinical management of cancer patients in North Carolina. To accomplish this objective, the proposal combines basic research on the patterns of care provided by physicians in different type practice settings for selected disease sites (breast, cervical and endometrial) and the use of that data to encourage the acceptance of current technology in the clinical care provided cancer patients. The project is considered a demonstration cancer control effort involving a limited geographic area in North Carolina and involving only three disease sites. The project will provide the basis for requesting state funding to develop and maintain an onocologic medical education program. Moreover, the project will provide the basis for developing further studies of the patterns of care involving community physicians throughout the state in cooperation with the Cancer Centers at Duke, Bowman Gray and East Carolina University.


Functional inactivation of menin, encoded by the MEN1 gene, causes the inherited multiple endocrine neoplasia type 1 (MEN1) syndrome and some but not all sporadic pancreatic endocrine tumors. Therefore, unraveling molecular events upstream or downstream of menin could point to other causative genes and/or regulatory events responsible for such tumor types. Menin resides in a histone methylating protein complex that trimethylates histone H3 at lysine-4 (H3K4me3), an epigenetic mark for active gene expression. Therefore, we have determined a genome-wide map of menin-dependent H3K4me3 (using ChIP-Seq) and menin-dependent gene-expression program in wild-type (WT) and menin-null mouse embryonic stem cells (ESCs) and in pancreatic islet-like endocrine cells (PILECs), which we derived from WT and menin-null mouse ESCs through in vitro differentiation. We found menin-dependent H3K4me3 specifically targeting the Meg3 gene in mouse ESCs, and all four Hox loci in differentiated PILECs. Gene expression from the Meg3 locus and from all of the four Hox loci was abolished in menin-null cells. Both Meg3 and Hox loci have been implicated in MEN1-like sporadic tumors: MEG3 in pituitary tumors, and HOX in parathyroid tumors. Our data suggest that these genes with menin-dependent H3K4me3 could be relevant players in the tumorigenesis of endocrine cell types associated with MEN1. Furthermore, our work shows that menin-null mouse ESCs could also be differentiated in vitro into islet-like endocrine cells, underscoring the utility of menin-null ESC-derived specialized cell types for genome-wide analyses studies. Our current efforts are directed towards understanding the regulation and activity of genes at the MEG3 and HOX loci. We have shown that cyclin-dependent kinase inhibitors (CDKIs) of the INK4 family (4 genes) and the Cip/Kip family (3 genes) that negatively regulate cell cycle progression and cell proliferation have rare germline or somatic mutations in endocrine tumor states related to MEN1. Also, mouse models show an endocrine neoplasia phenotype in 'knock-in' mice homozygous for the CDK4-R24C mutation, or by the combined loss of two different CDKIs, p18 and p27. Therefore, understanding the molecular basis of CDK and CDKI regulation could provide insights into their contribution to endocrine tumorigenesis. We have investigated the contribution of cell cycle regulators in endocrine tumorigenesis, particularly mutations in CDKI genes. We are interested in investigating the molecular basis of cell cycle regulation in endocrine cells.


The long-term objective of this project is to characterize the molecular machinery responsible for eliminating misfolded, neurotoxic proteins so that they may be exploited for therapeutic intervention for neurodegenerative diseases such as spinobulbar muscular atrophy (SBMA). SBMA is a hereditary disease characterized by progressive loss of motor neurons in the brainstem and spinal cord. SBMA is caused by trinucleotide (CAG) repeat expansion in the first exon of the androgen receptor gene leading to polyglutamine expansion in AR protein. Like other polyglutamine diseases, SBMA is characterized by accumulation of misfolded protein aggregates in degenerating neurons. Histone deacetylase 6 (HDAC6) is a microtubule-associated deacetylase with intrinsic polyubiquitin-binding activity. We recently determined that over-expression of HDAC6 rescues degeneration in SBMA flies, flies with proteasome mutations, and other fly models of neurodegenerative disease. HDAC6 rescues degeneration by facilitating the degradation of aberrant, ubiquitinated protein by the autophagy-lysosomal system. In this application, we will (1) test specific hypotheses regarding the molecular mechanism whereby HDAC6 facilitates autophagic degradation of aberrant protein, (2) determine the therapeutic potential of HDAC6 in mammals, and (3) use the power of fly genetics to gain unanticipated insights into the role of HDAC6 in cellular management of misfolded protein stress. PUBLIC HEALTH RELEVANCE: Histone deacetylase 6 contributes to elimination of misfolded proteins from neurons and protects against neurodegeneration. It is not known how HDAC6 functions. This application seeks to understand the mechanism of HDAC6 function so that it may be exploited for therapeutics development. [unreadable] [unreadable]


Dementia, with its most prevalent subforms Alzheimer's disease (AD), dementia with Lewy bodies (DLB), and vascular dementia, is a very substantial medical and societal problem. The emergence of many drugs and drug candidates that interfere with the disease mechanism of specific types of dementia makes it crucial to distinguish early between the various forms. Useful diagnostic tools will make it possible to direct patients to the most appropriate drug regimen, to avoid exposure to potentially harmful drugs, and to monitor the efficacy of disease-modifying drugs. Brain imaging techniques provide a non-invasive tool for differential diagnosis and monitoring of disease progression of dementia. We and others have been developing radiopharmaceuticals that allow detection of A amyloid deposits typical for AD. Our efforts have focused on 18F-labeled compounds for PET imaging, with the goal that the imaging diagnostics can be made widely available at minimal costs. We now propose the clinical development of a new radioligand for the differential diagnosis of DLB from AD as well as the monitoring of DLB disease progression. 18F-AV-133 is a highly selective ligand for the vesicular monoamine transporter VMAT2, believed to be the best marker for the functional integrity of dopaminergic neurons which are known to degenerate in DLB. Our proposal tests that hypothesis that imaging of VMAT2 provides a useful biomarker for dopaminergic neuron degeneration, which can be synergistically combined with amyloid plaque imaging to generate a powerful diagnostic work up for classification of dementia patients. We propose to conduct two clinical studies to establish safety and proof-of-concept for 18F-AV-133 as a diagnostic imaging agent for DLB. PUBLIC HEALTH RELEVANCE: Successful completion of the proposed phase I studies will provide a key step towards the provision of diagnostic tools for the clinical diagnosis and monitoring of disease progression of dementia with Lewy bodies (DLB), Alzheimer's disease (AD) and dementias in general. The studies will identify a diagnostic tool to allow the physicians to direct patients towards the most appropriate drug regimen and prevent that the patients are exposed to potentially harmful drugs. Furthermore, the resulting diagnostic tools will help significantly in the development of disease-modifying drugs, by making it possible to monitor treatment efficacy at the molecular level in vivo. Such a diagnostic tool will represent a significant technological and medical benefit to society.


The candidate has a primary interest in investigating the associations between neighborhood socioeconomic (SES) environments, behavioral and psychosocial risk factors, and cardiovascular disease (CVD). The mentored phase will include coursework in Geographic Information Systems (GIS) and complex multilevel modeling, as well as data linkages prior to the independent phase project. The independent phase will establish the candidate's independence while enabling skill development in GIS and multilevel modeling. Data on nearly 90,000 women from the Harvard-based Nurses'Health Study, a well-established cohort, and nationally-representative data on approximately 262,000 men and 314,000 women from the National Health Interview Survey, will be analyzed using multilevel discrete-time survival analysis models to estimate the relations between neighborhood SES and risks of non-fatal and fatal coronary heart disease (CHD), and to test for the presence of behavioral and psychosocial mediators. Furthermore, GIS methods will be used to assess whether some of these mediators may be determined by particular neighborhood services and amenities. By contributing to the knowledge base on the neighborhood determinants of and pathways to CHD, the project's efforts may ultimately translate through interventions into more effective reductions in CVD burden among Americans. This award should enable the candidate to pursue a successful career in the study of the neighborhood determinants of CVD.


The Center for Botanical Lipids and Inflammatory Disease Prevention is a new version of a P50 at Wake Forest University Health Sciences that has been continuously funded since 2005. Research over the past 50 years has shown that both systemic and localized inflammation play an important role in the onset and progression of destructive diseases such as cardiovascular disease, metabolic syndrome/diabetes and allergic asthma. The overall goal of this Center is to delineate the molecular mechanisms by which botanical oils prevent or impact these diseases with a particular focus on immunity and inflammation. The central hypothesis of the Center is that medium chain poly-unsaturated fatty acids (PUFAs) in botanical oils and their metabolites impact several key mechanisms (including altering pro-and anti-inflammatory mediator production and blocking inflammatory gene expression) that inhibit inflammatory processes. A secondary hypothesis is that that the metabolism and effectiveness of PUFA-based botanical dietary supplements is strongly associated with genetic polymorphisms in the fatty acid desaturase (FADS 1-3) cluster on chromosome 11 in a region known as 11q12-q13. This chromosomal region has repeatedly been linked to pro-inflammatory conditions. This Center brings together investigators from five internationally-recognized lipid groups and a world-renowned human genomics center to examine cellular and molecular mechanisms by which PUFAs within botanical oils impact human health. Selected botanical oils and combinations (including olive, flax seed, borage seed and echium seed oils) found in supplements are used to test key hypotheses. Projects 1 and 2 examine the mechanisms leading to the pleiotropic effects of botanical PUFAs on macrophage/ monocyte activation, inflammatory states and eicosanoid generation related to atherosclerosis and asthmatic inflammation, respectively. Project 3 examines the critical role genetic variations in the FADS cluster plays in determining PUFA levels and investigates how specific variations in that cluster are associated with the effectiveness of PUFA-based botanicals supplements in metabolic syndrome/diabetes. The interactive and synergistic Projects and Cores have a strong, contemporary and translational scientific basis and should allow this scientific team to identify additional mechanisms and identify human individuals and populations that are most likely to be affected by PUFA- based botanical supplements. PUBLIC HEALTH RELEVANCE: Thirty-eight percent of adult Americans are using complementary and alternative medicine modalities. The most commonly used modalities are natural products enriched with PUFAs (37 % of natural products). The key purpose of this Center is determine the molecular mechanisms by which medium chain PUFAs in botanical oils prevent of complex diseases such as CVD, asthma or metabolic syndrome and to determine the populations where they are most likely to be effective.


The contact of whole mammalian blood with polymeric material results in rapid deposition of a film of plasma proteins. Protein deposition is followed by a complex series of events involving platelet activation and aggregation and blood transport of clotting factors and additional cellular elements to growing thrombi. Recently we have developed a sensitive in-vivo technique by which dynamic initial deposition profiles of radio-labeled platelets and protein fractions on polymeric arteriovenous shunt surfaces can be measured. Small amounts of platelets and proteins of interest are tagged with radio-isotopes and injected into canine subjects. The radiolabeled platelets and proteins adsorbed to the walls of femoral A-V shunts are detected in order to determine the platelet deposition profile and the composition of the adsorbed protein layer. Experiments using electron microscopy and x-ray photoelectron spectroscopy and immunolabeling of proteins in the adsorbed layer are planned to characterize the nature of protein-formed element interactions in thrombosis. We propose to study conventional medical polymers and additional polymeric substances thought to be superior in thromboresistance. In addition to the major protein fractions of blood consisting of albumin, gamma-globulin and fibrinogen, the thrombogenic characteristics of fibronectin, von Willebrand factor, alpha2 macroglobulin and high molecular weight kininogen pre-adsorbed on test surfaces will be studied. Adsorption profiles of each protein fraction will be determined. These measurements will provide insight into the mechanism of blood response to a variety of surfaces in-vivo, how the adsorbed protein layer interacts with the formed elements in blood and whether other minor protein fractions play a role in the blood-material interaction.


Protein drugs have been approved for many therapeutic indications and represent a rapidly growing segment of the pharmaceutical industry. However, many approved protein drugs and candidates in development fail to reach their potential efficacy due to suboptimal pharmacokinetic properties and immunogenicity concerns. These properties include short circulating half-lives, short shelf lives, low solubility, rapid kidney clearance and susceptibility to proteolytic degradation. The modification of proteins with hydrophilic chemical polymers like polyethylene glycol (PEG) is a clinically validated approach to addressing these limitations. However, the challenges associated with chemical modification procedures required for the attachment of these polymers present significant challenges. Our ultimate goal is to generate amino acid sequences that mimic the physiochemical properties of hydrophilic chemical polymers like PEG. Our proposal is based on the observation that glycine rich sequences (GRS) which contain few hydrophobic amino acids will not fold into compact 3-dimensional structures but will adopt random conformations with large hydrodynamic radii similar to PEG. We hypothesize that they will confer similar pharmacokinetic improvements when attached to therapeutic proteins. These sequences can be attached to proteins using conventional recombinant technology and thus completely obviates the need for chemical modifications steps. We have synthesized a 198 amino acid glycine rich sequence based on sequences that occur in human proteins. We aim to express this protein and systematically test its physiochemical and biological properties relevant to pharmacokinetic enhancement. Our specific aims are: 1) Produce 5 mg of a purified GRS protein in E. coli expression system for downstream characterization and studies. 2) Characterize serum stability, protease resistance and biophysical properties of the GRS protein. 3) Characterize plasma pharmacokinetics and immunogenic potential of the GRS protein in animal models. In Phase II, we will perform detailed optimization of GRS for high-level expression, plasma half-life extension and reduced immunogenicity. We aim to advance optimized GRS which are fused to pharmaceutically active proteins like interferon-alpha or G-CSF into animal and clinical studies. Our ultimate goal is to validate and make this method readily applicable to therapeutic proteins. Our project aims to generate glycine rich sequences that can be attached recombinantly to therapeutic proteins to improve their pharmacokinetic properties. This approach would circumvent the difficulties associated with the modifying proteins with hydrophilic polymers like PEG. [unreadable] [unreadable] [unreadable]


This research proposal, submitted in response to NIH RFA HG-03-004 (Technologies to Find Functional Elements in Genomic DNA), describes the development of a novel assay technology to begin to unravel the complexities of alternative RNA splicing. The proposal seeks to develop a means of measuring, in parallel, all predicted exon-specific mRNA sequences coded by the 30MB ENCODE targeted genomic sequences for their presence in a given population of mRNA and their contextual linkage to other exons. These studies are critical to understanding how the human genome is transcribed and translated into an enormous repertoire of molecular diversity from an unexpectedly small set of identified human genes. [unreadable] [unreadable] The proposed assay, termed an "Exon-Linkage Assay" will combine a variety of technological innovations to the high-throughput characterization of the human spliceome through the use of a strategy of dual, internal-primer mediated double-stranded cDNA synthesis on a spatially resolved solid phase. The assay leverages a highly adaptable DNA microarray synthesis technology to provide an RNA splicing analysis tool that would be labor or cost-prohibitive by other means. [unreadable] [unreadable] The described method has the potential to quickly identify differential pre-mRNA splicing patterns in different tissues as well as shifts in splicing (caused either by regulatory defects or genomic mutations) associated with human disease in a rapid, scalable assay platform. [unreadable] [unreadable]


The broad objective of this proposal is to identify effective culturally sensitive behavioral interventions by nurses to reduce the risk of sexually transmitted HIV infection and AIDS among inner-city Black women. AIDS is the leading cause of death among Black women of childbearing age in New Jersey. A related problem is pediatric AIDS cases. More than one-half of the reported cases of pediatric AIDS in New Jersey have occurred among Black children. To reduce the spread of HIV infection among women of childbearing age and the associated perinatally acquired infections f children, efficient and cost-effective theoretically based behavioral interventions are needed. Nurses are key health professionals in the effort to combat AIDS, and interventions by nurses in the health-care setting may maximize resources. There are no published studies that tested AIDS risk-reduction interventions on women of childbearing age. The subjects in the proposed experiment will be 720 Black women of childbearing age attending the Family Planning Clinic of a Women's Health Center serving a low-income community in Newark, New Jersey. The approach draws on Bandura's social cognitive theory, Fishbein and Ajzen's theory of reasoned action, and the applicant's previous AIDS risk-reduction research with inner-city Black populations. The subjects in the proposed research will be randomly assigned to a social cognitive intervention designed to increase perceived self- efficacy and favorable outcome expectancies regarding condom use or to one of two control conditions: an informational intervention designed to increase AIDS knowledge or the standard clinic treatment of brief one- to-one AIDS prevention counseling. All interventions will be implemented by nurses. Repeated measures ANOVA, planned contrasts, and multiple regression will be used to test experimental effects immediately postintervention and at 3, 6, and 12 month follow-ups. The outcome variables include self-reported sexual behavior, condom-coupon redemption, and clinically documented sexually transmitted infections. Other measures include theoretically relevant variables hypothesized to mediate intervention effects, including AIDS knowledge, self-efficacy, outcome expectancies, and intentions. Analyses on these variables will address the important theoretical question of why the interventions have effects on outcome measures. Analyses will also examine intervention effects are systematically different depending on participant characteristics, including age, relationship status, and self-esteem. The results of this project will contribute to the development of efficient and effective risk reduction programs for inner-city Black women of childbearing age.