[["Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a major advance in the field of biological mass spectrometry, providing high ion yields for low quantities of biopolymers, even in the presence of buffer and other biochemical components that would preclude analysis by other mass spectral techniques. Much of the research in the field to date has concentrated on peptides, proteins, and oligonucleotides, yet the technique also holds considerable promise for oligosaccharide analyses beyond the limited work that has already been demonstrated. This application seeks to develop methods to increase the applicability of MALDI-MS to challenging problems in glycobiology. The research will be directed towards four goals: (1) maximizing the sensitivity for biopolymers, particularly oligosaccharides and glycoconjugates and their derivatives; (2) increasing the information content of the spectra through sample pretreatments that induce mass shifts; (3) developing experimental methods to enhance fragmentation and obtain systematics to help determine the rules of post-source decay fragmentation; and (4) exploring approaches to the analysis of two-dimensional surfaces, both those that occur in biological or biomimetic systems, and those that result from analytical separations or the generation of synthetic product arrays."], ["The proposed research will continue investigations on: Alterations in the metabolic pool at the site of both aqueous humor and cerebrospinal fluid production in groups of unoperated animals and in animals in which bilateral superior cervical sympathetic ganglionectomy will be performed. In addition, a third group of sham-operated animals will be used. That supersensitivity to catecholamines takes place in sympathectomized animals has been shown previously in this and other laboratories. Ocular fluid dynamics in all animals will be determined by repeated weekly testing of ocular pressure (IOP) (with the Mackay-Marg tonometer), as well as measurement of pupil size. A base-line of measurements will have been made for a period of about two weeks prior to the performance of any surgery. Again, prior to surgery, this base-line will be followed by a period of testing of ocular responses to phenoxybenzamine (PBA), norepinephrine (NE) and prostaglandins (PGs). At the termination of a study, the animals will be anesthetized, the eyes cannulated and outflow resistance and IOP measured. The animals will then be sacrificed and tissues and fluids removed. The metabolism and/or concentrations of cyclic adenylic acid (cyclic-AMP) and the PGs will be of particular interest. We shall, in other words, look closely at the metabolic and physiological nature of ocular and non-ocular tissues in animals which would be made catecholamine-supersensitive (via the above surgical procedure) at sites of sympathetic innervation as compared with sham-operated and unoperated animals. BIBLIOGRAPHIC REFERNCES: Waitzman, M.B. and Law, M.L., \"Changes in Prostaglandin Concentration in Blood Subjected to Repetitive Freezing and Thawing\", Prostaglandins 10:949-962, 1975. Jackson R.T., Waitzman, M.B., Pickford, L. and Nathanson, S.E., \"Prostaglandins in Human Middle Ear Effusions\", Prostaglandins 10:365-371, 1975."], ["This loss of membrane-bound beta-adrenergic receptor recognition sites in frog erythrocytes during subsensitivity induced by exposure of cells to isoproterenol is due at least in part to the internalization of beta-receptor recognition sites. When the beta-adrenergic receptors are resensitized, the amounts of internalized receptor recognition sites are also returning to the normal. The internalized recognition sites of beta-adrenergic receptors have properties identical to those of membrane-bound receptors. Glycoprotein seems to play an important role in triggering this event. A series of tricyclic antidepressant drugs was found to inhibit the receptor internalization; these effects can be attributed to an inhibition of these drugs of the agonist binding to beta-receptor recognition sites in erythrocyte membranes. Inhibitors of transglutaminase diminish the extent of recognition site internalization with a concomitant prevention of the loss of membrane-bound receptors elicited by isoproterenol. There is an excellent correlation between the inhibition of recognition site internalization and the potency of these compounds to block the transglutaminase activity in vitro."], ["Activation of cellular tumor suppressor pathways is the cell's major defense against cancer induced by activated oncogenes. The ARF-p53 tumor suppressor pathway, which is one of the most important in mammalian cells, can be activated by a number of viral and cellular oncogenes. Activated ARF induces a p53-mediated block to cell division via a cell cycle arrest or apoptotic cell death. How oncogenic stress activates ARF remains to be elucidated. Polyoma virus middle T-antigen (PyMT) is a potent oncogene able to bind a number of key regulatory cellular proteins and activate a number of important cellular signaling pathways including the ARF-p53 tumor suppressor pathway. We will use PyMT as a model oncogene in order to better understand how ARF is being activated. We hypothesize that PyMT induces ARF by the inappropriate activation of one or more cellular signaling pathways that also mediate the ability of PyMT to transform cells. Our plan is to identify the cellular signaling pathways induced by PyMT that results in activation of ARF. REF52 cells differ from most other established cell lines in containing an intact ARF-p53 tumor suppressor pathway and are distinct in resembling primary cells in their requirement for oncogene cooperation for their transformation. PyMT activates the ARF-p53 pathway, blocking REF52 cell division and will not transform REF52 cells in the absence of a co-operating oncogene. We plan to take advantage of these unique properties of REF52 cells to isolate PyMT and cell mutants that are involved in the activation of ARF. Three interrelated aims will be pursued. Aim-1 will be to use previously isolated PyMT mutants to identify which domains of PyMT are required to activate ARF. Aim-2 will be to use mutagenized PyMT to identify sequences in both defined and undefined regions of PyMT that are required for the activation of the ARF-p53 pathway in REF52 cells. Aim-3 will be to isolate and define REF52 cellular mutants in which PyMT signaling fails to activate the ARF-p53 tumor suppressor surveillance pathway. We believe that such cell mutants will help to differentiate between normal and oncogene activation of important cellular signal transduction pathways. Understanding the mechanism(s) of oncogenic activation of the ARF-p53 tumor pathway will help in designing better drugs and therapies for the treatment of cancer."], ["Current studies have indicated that antibodies directed against the stalk region of CD23 cause enhancement of IgE synthesis in both the human in vitro and mouse in vivo systems. CD23 transgenic mice, which overexpress CD23 on all lymphocytes and FDCs, exhibit drastically reduced IgE production in both helminth and alum/ag models. The data suggest a model where the role of CD23 is initially to serve as a component of innate immunity to signal for IgE production by becoming destabilized and cleaved and later by overexpressing at the cell surface and modulating IgE production. This continuation application proposes to investigate the mechanism of these effects. Aim#1 examines the mouse system where the destabilizing mab 19G5 gives enhanced IgE synthesis in vivo. In the current funding, the metalloprotease, ADAM10 has been identified as the primary CD23 sheddase in mouse and humans. The role of ADAM10 in allergic disease will be modeled by making transgenic mouse that overexpress ADAM10 or dominant negative ADAM10. In addition, we will examine the mechanism for the 19G5-induced IgE production by investigating the association of CD23 with another negative signaling molecule, LAX, which has recently been shown to both modulate CD23 expression and regulate IgE levels. Aim#2 will investigate the affect of CD23 overexpression and CD23 destabilization on the mouse asthma model with respect to both modulation and exacerbation of disease. We will utilize both IgE and the new ADAM10 transgenics in order to evaluate the mechanism of the suppression of eosinophilia as well as the capacity of CD23 to modulate the asthma phenotype. Aim#3 will investigate the human in vitro IgE synthesis models with respect to the mechanisms involved in IgE synthesis enhancement, seen with anti-stalk antibodies and synthesis suppression, seen with certain anti-lectin mabs. The importance of ADAM10 in human CD23 cleavage and IgE production will also be explored as will the involvement of LAX. Finally, we will determine if IgE production by B cells obtained from normal and allergic subjects is affected differently by destabilization or stabilization of CD23. In summary, these studies examine the mechanism of action of a natural regulator of IgE production, CD23, with the objective of developing protocols to enhance CD23 expression and thereby regulate IgE, and by analogy, allergic disease in which IgE plays a dominant role.Project Narrative: This project examines mechanisms involved in control of IgE synthesis by a natural regulator. The latter is CD23, a low affinity receptor for IgE. Accumulated evidence indicates that cleavage of CD23 by the metalloprotease ADAM10 increases IgE production in both mouse and humans. This application proposes to study mechanisms involved in this regulation in order to develop new protocols to control allergic disease."], ["The population in the U.S. is aging. Data are sparse on cardiovascular disease in older adults, especially the oldest old, who are expected to number 18 million by 2050. The age at first myocardial infarction (MI) is increasing substantially, now to about age 71, and most of the cardiovascular disease (CVD) deaths in the U.S. occur in older individuals. CVD is the single most important cost of medical care in the US. In the early 1990s, the Cardiovascular Health Study (CHS), an NHLBI-supported cohort study of risk factors for coronary heart disease (CHD) and stroke in adults 65 years or older, recruited 5888 participants, who underwent extensive examinations at baseline and annually until 1999. Examinations included traditional risk factors as well as measures of subclinical disease. Since 1990, CHS has made major contributions to the understanding of CVD in older individuals, including the risks associated with subclinical disease, inflammation, diabetes, hypertension and renal disease. In 2008, CHS has the unique opportunity of evaluating clinical CHD in the oldest old-- MI, angina, heart failure (HF), atrial fibrillation-- and their contributions to both morbidity and mortality. In this competing continuation, the primary aim is to evaluate the incidence and determinants of cardiovascular disease and health in 1964 surviving participants aged 80 or older. The incidence of CVD in the oldest old will be related to demographic variables;measures of disability, physical functioning, and cognitive function;measures of subclinical disease;and traditional and novel clinical risk factors as well as their change over time. The data collection proposed in this application will increase the number of events in those 80 years and older by about 50% to 75% for stroke, CHD and HF. Power for assessing associations with stroke, CHD and HF is increased by 15 to 30%. The continued assessment of cardiovascular events is essential to identify determinants of successful cardiovascular aging among the oldest old. PUBLIC HEALTH RELEVANCE: Additional knowledge about the determinants of cardiovascular health and disease in older adults will help physicians and their patients make diagnostic, prognostic, and therapeutic decisions. PROJECT/"], ["Despite the detailed understanding of signals 1, 2, and 3 and the critical function of T cells and dendritic cells (DC), clinical transplantation tolerance is almost never achieved. In transplantation, models of immune function, and interventions designed to promote immune regulation, have been based on simplified receptor-ligand and cell-cell interaction models. There has been a lack of studies that place immunological recognition within anatomic contexts and evaluate the critical role of anatomic microdomains in the regulation of the immune response. Our preliminary data now demonstrate that during tolerization there is alloantigen specific clustering of T cells and plasmacytoid DC (pDC) in the T cell areas of the lymph node (LN) near the abluminal surface of the high endothelial venules (HEV). Within these clusters T cells undergo either priming or development into de novo CD4+CD25+ regulatory T cells (Treg). Importantly, B cells presenting specific alloantigen are present in these cell clusters and contribute to Treg development. We hypothesize these multicellular clustered interactions in the LN are key to the induction and maintenance of tolerance. Specifically, we hypothesize that the precise interaction of T-APC (pDC, B cells) determines the outcome of T cell migration, positioning, proliferation, and maturation, and ultimately whether rejection or tolerance are induced. This hypothesis integrates many of the known receptor-ligand and cell-cell interactions, and places these interactions in the context of secondary lymphoid organ structure. To investigate the role of these cellular and structural elements in LN clustered interactions, we propose the following specific aims: Specific Aim 1. What are the T-APC-LN interactions that are important for tolerance? Using a transplant model that allows the tracking of alloantigen specific T cells, specific alloantigen presenting APC, and the positioning of the cells with respect to HEV, we will characterize specific receptor-ligand interactions between T-HEV and pDC-HEV that regulate tolerance, and define the interactions between T-pDC and other DC that determine the generation of Treg and inhibition of effector T cells within the LN. Specific Aim 2. How is the priming of effector T cells altered in the LN during tolerance? This aim will study the specific interaction between effector T cells and Treg in the LN clusters during tolerization. We will determine how Treg-T effector interactions regulate effector T cell migration, proliferation, and differentiation. We will characterize specific receptor-ligand interactions between Treg and T effector cells that determine whether T cell priming and rejection versus suppression and tolerance predominate in the immune response. Specific Aim 3. Determine the role of B cells in tolerance in the LN We will investigate the roles of B cells in the LN during the T-APC clustered interaction that results in tolerization. Analyses will focus on B cell APC function, chemokine production, and immunoglobulin production. PUBLIC HEALTH RELEVANCE: The research will investigate and define the cellular and molecular interactions that are important for generating regulatory suppressor T cells and antigen specific tolerance. The ability to define these interactions and achieve tolerance is important for achieving good graft survival and patient survival for transplant recipients."], ["Population-based surveys estimate that the prevalence of methamphetamine (meth) use is 20 times higher among men who have sex with men (MSM) compared to the general population. Meth-associated sexual risk behavior is also a driving force in the MSM HIV epidemic: meth use is consistently associated with high-risk sexual behavior and sexually transmitted diseases, including HIV. Despite these alarming data, relatively few interventions have been tested among meth-using MSM, and no studies have tested the efficacy of pharmacologic interventions in reducing meth use in this population. Pilot studies demonstrate that aripiprazole (Abilify), an FDA-approved, well-tolerated antipsychotic and partial dopamine agonist, reduces the effects of meth in humans, decreases meth craving, and exhibits an excellent safety profile. Partial agonists - - ligands with target receptor affinity but low intrinsic activity - - have already been shown to be effective in treating opiate and nicotine dependence. In response to the compelling evidence supporting aripiprazole and the partial agonist approach, we propose conducting a randomized, double-blind, placebo- controlled trial of intermediate size (60 participants) and length (90 days of follow-up) to assess the efficacy of aripiprazole in reducing meth use among meth-dependent, sexually active MSM. Our specific aims are: 1) To test the hypothesis that aripiprazole 20 mg daily will reduce meth use significantly more than placebo among meth-dependent MSM, as determined by the proportion of meth-negative urines and by self- report of meth use in the aripiprazole versus placebo group. 2) To measure the acceptability of aripiprazole and placebo among meth-dependent MSM, by determining (via electronic pill caps and self-report) medication adherence to aripiprazole and placebo. 3) To measure the safety and tolerability of aripiprazole and placebo among meth-dependent MSM, as determined by the number of adverse clinical events in the aripiprazole and placebo arms. All participants will receive HIV risk-reduction counseling and brief substance use counseling. If promising, we anticipate that study results will be used to design a phase III study to determine if aripiprazole's effects on reducing meth use lead to significant reductions in meth-associated sexual risk and, if the trial sample size is appropriate, HIV incidence. [unreadable] [unreadable] [unreadable]"], ["DESCRIPTION (taken from the application: The concept of orthopaedic research encompasses a very broad area spanning the field of musculoskeletal research from the clinical aspect to both biological and biomechanical basic sciences. The Bioengineering and Orthopaedic Science Gordon Research Conference has been the premier conference for investigators to combine the three disciplines of orthopaedic sciences. The format of these conference, held in a small private school in New Hampshire every other summer, provides a unique environment for the exchange of new and unpublished information. The cross fertilization that can only come from a conference were clinicians, engineers and biologists meet, is more vital to this field than ever before. For the first time, biological principles are being used in the treatment of orthopaedic injuries and diseases and these treatments must confirm to and meet the biomechanical needs of the skeleton. For example, investigators and clinicians are now beginning to utilize molecules such as parathyroid hormone; parathyroid hormone related peptide, insulin-like growth factors, fibroblast growth factor and the hedgehogs. In addition, physical parameters such as ultrasounds and electrical stimulation are currently being used to stimulate bone growth. Bioengineering has moved from the mechanical design of joint implants to the problems associated with long-term use of implant materials and a general understanding of the biological effects of these materials in the living system. For the first time, a session will be held on skeletal tumors. Two noteworthy changes will be made in the format from previous meetings. In the comments on the 1996 Gordon Conference Program, many attendees felt that the junior members of the audience did not feel comfortable speaking out and that the discussion were dominated by only a few senior scientists. In an effort to increase the comfort level of the junior scientists and to increase their participation in general, the following changes were implemented: (1) five to ten scholarships will be designated for junior faculty and postdoctoral fellows and their availability will be announced in conference literature; (2) One evening will be dedicated to talks by junior scientists. Employing these options to encourage the participation of junior scientists has worked very well for other Gordon Conferences such as the Proteoglycan Gordon Conference which uses the scholarship mechanism and Molecular Genetics which uses the junior scientists evening option."], ["In the past year, effort in nuclear magnetic resonance imaging has been expanded on several research fronts. A. Following the failure of the magnet manufacturer to install successfully the clinical NMR imaging system in the Department of Radiology, Clinical Center, the research group took over the homogenizing of the magnetic field necessary for clinical imaging, and, using a new technique, achieved the specified homogeneity, thereby enabling the Radiology Department to produce high quality images. B. Considerable controversy exists within the NMR image community over the choice of an optimal field strength for imaging. Bench methods were developed for accurately assessing the signal-to-noise ratio from, and radio-frequency power deposition in, the human body at any frequency used for imaging. It is hoped these results will help resolve the matter. C. A pulse for highly selective spin population inversion has been discovered. The result is of considerable experimental and theoretical importance for it represents only the second known analytical solution of the non-linear differential equations governing the motion of an NMR spin system. Further, above a critical threshold, the inversion is independent of applied power. D. A so-called \"quadrature\" probe system has been invented which reduced radio-frequency power dissipation in the body by almost a factor of 2 while improving signal-to-noise ratio by almost 40%. E. The electronic upper frequency limit for adult head imaging has been pushed from 84 MHz to 130 MHz with the aid of a novel phased-array receiving coil."]]