[unreadable] [unreadable] Epilepsy affects over 2.5 million people in the US, and over 180,000 new cases of the disease are diagnosed each year. Nearly half of the people suffering from epilepsy are not effectively treated. Moreover, currently used anticonvulsants can cause significant side effects, which often interfere with compliance. Clearly, there is a need for new, safer drugs to treat epilepsy. Glutamate, by stimulating NMDA receptors, has been implicated in the neuropathology and clinical symptoms of epilepsy, and NMDA receptor antagonists are potent anticonvulsants. Antagonists at the GlyB co-agonist site inhibit NMDA receptor function and are also anticonvulsant. Importantly, GlyB antagonists have fewer side effects than classic NMDA receptor antagonists and other antiepileptic agents, making them a safer alternative to available anticonvulsant medications. 7-Chlorokynurenic acid (7-C1-KYNA) is one of the most potent and specific GlyB antagonists currently known and is a powerful anticonvulsant when injected into the brain. However, like almost all GlyB antagonists developed so far, 7-C1-KYNA crosses the blood-brain barrier very poorly and is therefore ineffective following peripheral administration. Its pro-drug, L-4-chlorokynurenine (4-C1-KYN), on the other hand, readily gains access to the brain. Following systemic administration, 4-C1-KYN is efficiently converted to 7-C1-KYNA and prevents seizures in animal models of epilepsy. 7-C1-KYNA formation occurs preferentially in brain areas that suffer seizure-related brain injury, thereby reducing the risk for side effects with chronic use. Furthermore, 4-C1-KYN forms a second metabolite, which blocks the synthesis of the proconvulsant quinolinic acid in brain. These unique properties make 4-C1-KYN a highly innovative candidate for the treatment of epilepsy. The project proposed here by Vistagen will advance the preclinical development of 4-C1-KYN as a treatment for adult and childhood epilepsy. We will conduct extensive pharmacokinetic and ADME analyses to further delineate the oral bioavailability of the drug observed in pilot studies, will subject 4-C1-KYN to a battery of safety and toxicology studies and will test anticonvulsant efficacy of the compound in two chronic animal models of epilepsy, CLE and kindling. Finally, we will begin to develop the protocols needed to submit an IND for FDA approval to conduct clinical testing of the drug in humans as a new and improved anti-epileptic agent. [unreadable] [unreadable] [unreadable]


Abstract for Coordinating Center Core (CCC) The UPCI-CMCR-CCC will provide three critical services to NIAID and the other components of the 5 ? 7 CMCR Programs. The CCC will cooperatively develop a program for archiving and distributing data from 5 ? 7 CMCR sites and will maintain electronic data storage and develop security passcodes and validations for retrieval of data. The CCC will develop a program for timely submission of data and data analysis at NIAID/NIH and maintain rapid availability of electronic exchange of records to CMCR Programs as well as the general scientific community. The CCC will coordinate administrative activities for the scientists organizing national meetings, coordinating conference calls, establishing three levels of firewalls to distribute information at levels of CMCR Centers locally, CMCR Programmatic Centers for all 5 ? 7 programs, and to the general scientific community. Finally, the CCC will administer funds for the development of web-based training programs in four key areas: 1) Principles of Radiobiology, 2) Use of assays, methods, reagents, and animal models, 3) Technologies to study radiobiology and develop new product regulatory process, and, finally, 4) A course on triaging radiation casualties in the event of a radiation terrorist event.


Diadenosine tetraphosphate (AP4A), two adenosine moieties bridged by 4 phosphates, is an endogenous purinergic ligand found in brain. Previous studies have shown that AP4A reduced neurodegeneration caused by dopaminergic neurotoxin 6-hydroxydopamine in rat striatum and substantia nigra. The purpose of this study was to determine whether AP4A is protective against methamphetamine (MA) mediated toxicity in dopaminergic neurons. Primary neuronal cultures were prepared from rat embryonic (E15) ventral mesencephalic tissue. On DIV8, cultures were treated with 2 mM MA for 48 hours. Application of MA increased LDH levels, decreased TH immunoreactivity, and increased TUNEL. All these changes were reduced by pretreatment with AP4A. The protective effect of AP4A was further examined in vivo. Adult Sprague Dawley rats were injected with AP4A or vehicle intracerebroventricularly followed by 4 doses of MA (5mg/kg), given subcutaneously every two hours. Administration of MA increased caspase-3 immunoreactivity in striatum and cortex. Pretreatment with AP4A significantly reduced the density of caspase-3 cells. Using microdialysis, dopamine (DA) release was monitored in dorsal striatum in freely moving rats. AP4A did not acutely alter MA-evoked DA release, suggesting that AP4A -induced protection is not directly mediated through a change in DA overflow. Taken together, these data show that AP4A has protective effects against MA-mediated neuronal injury both in vitro and in vivo. The mechanism of action may involve suppression of MA -induced apoptosis.[unreadable] [unreadable] Astaxanthin (ATX) is a carotenoid antioxidant found in crustaceans. Other carotenoids, such as beta-carotene, lutein and lycopene have been shown to have beneficial effects against oxidative stress. The purpose of this study was to examine whether ATX has protective effects against ischemic brain injury. Adult male Sprague-Dawley rats were anesthetized with chloral hydrate. ATX or vehicle was administered intracerebroventricularly 10-20 minutes prior to a 60-min middle cerebral artery ligation. At 2 days after MCA occlusion (MCAo), rats that received ATX had an increase in locomotor activity. ATX significantly reduced cerebral infarction and TUNEL labeling in ischemic brain. At 8-hour after MCAo, ATX antagonized ischemia/reperfusion induced loss of aconitase activity, an indirect marker for the production of reactive oxygen species, and translocation of cytochrome C from mitochondria. Ischemia or free radical -induced glutamate release was examined using microdialysis and HPLC. Pretreatment with ATX reduced ischemia or H2O2 mediated glutamate release in cerebral cortex. ATX did not alter blood gases, blood pH, blood pressure, body temperature, brain temperature and cerebral blood flow. Collectively, our data suggest that astaxanthin has protective effects against free radical toxicity and ischemia-related injury in vivo through the inhibition of oxidative stress, inhibition of glutamate release, and anti-apoptosis.


It is proposed to study the binding affinities and behavioral effects of several classes of hallucinogenic (psychotomimetic) agents, i.e. tryptamines, phenethylamines, phenylisopropylamines. The serotonin receptor binding affinities will be determined using the isolated rat fundus preparation, as we have previously described. The behavioral studies will be performed by investigating the discriminative stimulus properties of these agents, and their ability to generalize with a 5-methoxy-N,N-dimethyltryptamine (5-OMe DMT) stimulus. In this manner, information can be obtained regarding mechanism of action, structure-activity relationships, structural requirements of the serotonin receptor, etc... Agents which produce similar interoceptive cues (in the generalization studies) and which possess similar binding affinity characteristics will lend support to the hypothesis that certain of these agents may produce their effects via a common mechanism. Using rats, cannulae will be implanted in various brain regions so that 5-OMe DMT can be administered directly into the brain. Employing the discriminative stimulus paradigm, generalization between centrally and peripherally administered 5-OMe DMT will yield information on the brain site of action. Dopaminergic, as well as serotonergic sites will be investigated. Bufotenine esters and other novel compounds which possess, within the same molecule, features related to more than one class of hallucinogens, are proposed for synthesis. The behavioral effects and receptor affinities of such compounds should aid in determining whether various hallucinogens act via a common mechanism.


Abstract Hedgehog signals are the key regulators of embryonic patterning and adult tissue homeostasis. Consequently, defects in Hedgehog signaling can cause developmental diseases, congenital heart disease, and cancers such as basal cell carcinoma. There is an urgent need to understand the molecular mechanisms that underlie the modulation of Hedgehog signaling pathway for its potential preventative and therapeutic value. It is known ver- tebrate Hedgehog signaling relies on the ciliary trafficking of Hedgehog signaling receptors, among which smoothened (SMO) is the central positive mediator of Hedgehog signaling. If mutations occur in either intrafla- gellar transporters (IFTs), or in the ciliary transition zone, SMO activities can be severely disrupted. However, the molecular mechanism of how IFT particles and transition zone regulate trafficking of SMO is currently un- known. Determination of the molecular regulation mechanisms is the objective of this application. Our prelimi- nary data acquired by Stochastic Optical Reconstruction Microscopy (STORM) showed the colocalization of transition zone proteins with SMO and IFT88, suggesting that transition zone proteins and IFT particles interact with SMO. Based on previous studies and our own primary data, my central hypothesis is that the transition zone serves as a checkpoint for Hedgehog signaling receptors, and IFTs help Hedgehog signaling receptors cross the transition zone. This transition zone checkpoint model represents a novel mechanism for the control of cilium trafficking and the cross-interaction between different ciliary cargos. It could potentially allow new ap- proaches to manipulate Hedgehog signals, and underlie the foundation for treatments of diseases caused by defects in Hedgehog signaling. To approach to the project, I plan to map SMO molecules and IFT particles in the transition zone using multicolor 3D STORM. It will reveal the spatial relationship between SMO molecules and these ciliary components at a resolution of ~15 nm. Algorithms will be developed to reduce the uncertainty of the spatial easements caused by structural heterogeneity and immunostaining, providing a ~ 5nm precision of the distance between investigated proteins, indicating protein-protein interaction. Equally important as the static structural study, I also plan to detect the interactions among SMO molecules, IFT particles, and transition zone proteins dynamically using single-particle tracking and photoconversion imaging. The proposed project will not only offer new insights into the molecular mechanisms of Hedgehog signaling regulation, but also ad- vance a suite of microscopy-based technologies and algorithms that can be broadly applied to the fields of cell signaling and structural biology. Furthermore, the results are expected to have broad impact, because the reg- ulatory components to be identified by this project will provide new mechanisms and new drug screen for pre- ventive and therapeutic interventions. 1


This proposal addresses a fundamental topic relative to Complementary and Alternative Medicine (CAM) treatments: How do we measure the context of an encounter? Encounters are defined as what patients experience between when they arrive at a CAM practice site and when they leave. The encounter can be divided into two parts: (a) the experience of the main treatment intervention (the intervention) and (b) all other experiences before, during, and after the intervention itself (the context). Contextual effects play two critical roles in assessing the efficacy and effectiveness of CAM interventions. First, contextual effects may mediate how well a treatment works (context as mediator). Second, context effects may actually contribute to outcomes directly (context-as-intervention). In a classic RCT, investigators typically want to control for context-as-mediator effects and measure context-as-intervention effects to disentangle what portion of the results are due to the intervention and what are due to the context. In CER, investigators are less concerned about controlling for context-as-mediator effects, but they would like to understand what part of the encounter accounts for any positive results. In either case, investigators MUST know how to measure the context. Without such measures, it is impossible to assess either type of context effect. This proposal has 3 specific aims: 1. to understand what kinds of contextual factors patients are exposed to during CAM encounters? 2. To determine how to measure such contextual factors reliably via observation and/or patient and provider recall; and 3. To assess the degree to which contextual factors might vary within and across (a) CAM modalities (i.e., chiropractic versus osteopath); (b) practice sites, (c) providers, and (d) individual patient encounters This study will use the chiropractic encounter as an exemplar for CAM and develop a rapid ethnographic observation method to study the context of the encounter. The objective of this project is to develop a reliable, valid, and rigorous methodology for collecting data about the context. The proposed study addresses a fundamental issue of all outcome studies in health: how to measure the effect of the context on the health outcomes.


Activated TGF-beta family factors signal via dual Type II (TbetaRII) and dual Type I (TbetaRI/Activin-like kinase 5/ALK5) transmembrane serine/threonine kinases receptors and effector Smad transcription factors. While TGF-beta signaling is critical during endocrine pancreas development, whether TGF-beta plays a role in maintaining adult beta-cell function is unclear. Interestingly, TGF-beta levels are elevated in diabetes, diabetes-associated complications, and obesity. [unreadable] [unreadable] Using primary islets and established beta-cell lines, we are examining the role of various TGF-beta isoforms and their respective receptors on the regulation of key pancreatic specific genes. Smad proteins are the established intracellular effectors of TGF-beta signaling and upon TGF-beta ligand binding, the activated TbetaRI/ALK-5 receptors recruit and phosphorylate receptor-Smads, i.e., Smad2 and Smad3. We are examining the roles of various Smad proteins, in the regulation of gene expression specific to the pancreas. [unreadable] [unreadable] TGF-beta signalling is known to suppress proliferation and promote growth arrest, differentiation/apoptosis of cells of the epithelial lineage. However, the role of this pathway in the growth, proliferation, differentiation and death of constituent pancreatic epithelial cells is unknowm. Using primary islets and established pancreatic ductal and beta cell lines, we are examining the role of TGF-beta signaling in these processes. [unreadable] [unreadable] Of particular interest will be the contribution of TGF-beta signaling in regulation of beta cell mass amd beta cell function. We are using primary islets, and established beta cell lines to examine the effects of TGF-beta isoforms, its receptor activation/inactivation, on beta cell mass and beta cell fucntion. In addition the role of downstream Smad proteins is also being investigated. Several parallel approaches will be taken to explore this issue, which will hopefully lead to an improved understanding of the role of this complex signaling pathway in pancreatic development and specifically in regulation of beta cell mass and function. The information gained will be useful to delineate the role of this pathway in diseases of the pancreas, primarily diabetes, and hold promise to allow design and development of novel rational therapeutics for these diseases.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The purpose of this study is to understand the effects of body fat, particularly fat locked within the abdomen, on metabolism. Patients who are scheduled to undergo surgery, will be consented to donate a sample of their abdominal fat (omental adipose tissue), a small amount of abdominal wall muscle, and some of the fluid in their belly. We are interested in ways in which abdominal fat can mimic the immune system in causing inflammation.


The objective of the proposed Drug Abuse Research Center is to determine the extent and magnitude of the effects of narcotics on brain biochemistry and ultrastructure during the development of tolerance and physical dependence. The specific goal of the proposed research is to lay a foundation of basic information on the neurochemical and morphological effects of the narcotics from which studies of the actual "mechanisms" involved in tolerance and dependence can logically progress. We shall use a variety of micro-analytic chemical and immunochemical techniques, radioassays and quantitative-histochemistry to determine the effects of narcotics on: 1) The biogenic amines; 2) Brain protein synthesis; 3) Brain metabolism; 4) Representative cerebral enzymes; 5) Brain morphology and ultrastructure; and 6) Additional studies will be conducted to examine the neuroanatomical correlates of tolerance to and dependence on narcotics. Our approach will be highly integrated and interdisciplinary and we shall correlate, whenever possible, pharmacologic, biochemical, and morphological observations within a given region of brain during various phases of morphine treatment and subsequent withdrawal. All of these determinations will be made in histologically well-defined regions of brain, including the major nuclei of the hypothalamus, thalamus, caudate, midbrain, medulla, cerebellum and cerebral cortex.


PROJECT SUMMARY/ABSTRACT The cerebral cortex mediates all of human and animal cognition, encompassing a diverse set of abilities including sensation, perception, decision making, and motor planning. Dysfunctions of the cerebral cortex are thought to underlie numerous neurological and psychiatric disorders. A major obstacle both to understanding normal behaving and to treating pathology is the high degree of complexity of cortical circuitry, which has remained largely enigmatic. The conventional view of neocortex has been that sensory processing begins in layer 4 (L4), which was identified a century ago as the principal target of thalamic axons carrying information from our sensory organs. Sensory transforms are widely believed to occur as excitation spreads serially along the densest axonal pathways (thalamus?L4?L2/3?L5/6). Recently we discovered that the cerebral cortex, rather than being a monolithic structure, may contain two entirely separate processing systems, activated by the same signals arising from the thalamus. L4 is thus not an obligatory distribution hub for cortical activity, and thalamus activates two distinct ?strata? of cortex in parallel. This proposal's goal is to identify the behavioral and computational roles of the upper (L2-4) and lower strata (L5/6) as well as the interactions between them. We will investigate the behavioral roles of these layers in the mouse whisker system. Specific layers will be optogenetically disrupted in a series of tactile behavioral tasks, in which task complexity is progressively increased. Interlaminar interactions will also be studied by recording electrophysiologically from specific layers during behavior and using novel machine learning techniques designed to identify the type of computation performed in different levels of ?deep networks?. The dimensionality of the representation in a layer will be estimated under normal behaviors and when specific layers are inactivated. Identifying fundamental functions of upper versus cortical layers will likely pave the way for future studies in other neocortical systems and in higher-order species. Moreover, as the different layers contain molecularly and biophysically distinct cell types and project to distinct downstream targets, specific neurological disorders may involve dysfunction of specific pathways, cell types, and layers. Establishing the behavioral and computational roles of these elements may contribute to development of targeted therapies.


This proposal describes an investigation of the effects of aluminum and other trivalent cations on cholinergic activity and other metabolic pathways in rat brain. These findings are crucial because aluminum toxicity has been proposed to play a major role in the development of senile dementia of the Alzheimer type (SDAT) and selective impairmant of the cholinergic system has been established in patients with SDAT. Both in vitro and in vivo effects of aluminum will be sought. Brain slices will be incubated with (U-14C) glucose to test the effects of aluminum on carbohydrate utilization, lipid synthesis and acetylcholine (ACh) synthesis. Synaptosomes will be incubated with deuterium labelled choline to test the effects of aluminum on high affinity choline transport, ACh synthesis, spontaneous and K+ induced ACh release and the release of choline from lipids. (3H)-Choline will be used to measure the rate of incorporation of choline into lipids in synaptosomes in the presence and absence of aluminun. The effects of several other trivalent cations on these processes will be compared to those of aluminum to establish their relative potencies and to obtain information about the mechanism of the toxic effects of aluminum. Rats will be treated in vivo with aluminum followed by determination of the rate of turnover of ACh in rat brain regions using pulse labelling techniques with deuteriumzlabelled choline. Endogenous and deuterium labelled choline and ACh will be measured using combined gas chromatography mass spectrometry. In other rats treated with aluminum we will (i) measure the activity of ChAT, (ii) prepare synaptosomes and compare the rates of Ch transport and ACh synthesis and release to those observed in synaptosomes from control rats, and (iii) measure the rate of utilization of (U-14C) glucose in brain slices. This investigation will establish whether or not aluminum causes impairment of cholinergic activity or several other metabolic processes, will indicate how specific for aluminum these toxic effects are and will allow a comparison of the biochemical effects of aluminum toxicity and SDAT.


This research proposal tests the hypothesis that identification of the requirements for in situ activation of tumor-infiltrating lymphocytes (TIL) will provide the biological basis for immunotherapy of human solid tumors without infusion of ex vivo- activated lymphocytes. Specific aims are as follows: 1. Characterization of TIL from melanoma, renal carcinoma and sarcoma. There are at least six different TIL subsets as follows: CD4-CD8+ T cells with (i) autologous tumor-specific or (ii) MHC- nonrestricted (LAK) cytotoxicity; CD4+CD8- T cells with (iii) autologous tumor-specific The activity or (iv) LAK activity, (v) CD3-CD16+ NK cells with LAK activity and (vi) CD4-CD8- T cells. We will (i) purify each of these subsets before and after incubation with rIL2; (ii) establish cloned cells; (iii) investigate their functions; (iv) elucidate requirements for activation of each TIL subset (IL-2,other cytokines, autologous tumor cells or autologous macrophages), and (v) investigate molecules involved in the TIL activation (TCR, CD3, CD2, CD4, CD8, CD16 and LFA antigens on TIL). 2. MHC-restriction of antitumor activity. We will investigate if autologous tumor-specific CTL or The activity in melanoma TIL is restricted by MHC-Class I or Class II antigen expression on tumor targets respectively by determining MHC-haplotypes of tumors. 3 and 4. Determination of requirements for CD4 CD8- TIL to produce IL-2, and differentiation of resting blood lymphocytes into TIL. Role of macrophages. Requirements tested include: IL-l, IL-2, IL2R-inducing factor, anti-CD3 mAb., hybridomas expressing anti-CD3 mAb, autologous monocytes, tumor cells, CD4+CD8- or CD4-CD8+ cloned TIL. 5. Antitumor activity of lymphocytes from LN with melanoma metastasis. LN-lymphocyte subsets will be incubated in the presence of IL-2, anti-CD3 mAb, IL-l and/or autologous monocytes, followed by testing their proli- feration and antitumor activity. 6. Identification and purification of IL-2 receptor- inducing factor (IL2R-IF). Culture supernatants from the established CD4+ TIL line (TILh-Ll) and PBMC stimulated with PHA and PMA will be used for this study. Knowledge of immunological properties of TIL and understanding the mechanisms involved in TIL activation and the biology of host-tumor relationships will provide important findings for development of in situ lymphocyte activation and new strategies in the immunotherapy of human solid cancers.


Vibrational Optical Activity studies consisting of vibrational circular dichroism (VCD) and Raman circular intensity differential (CID) will be applied to several biochemical systems. These techniques are exceptionally sensitive to structural features of the molecules being studied and offer distinct advantages over currently used spectroscopic techniques. The data obtained will be used to give new insight into the secondary structure of polypeptides and nucleic acids, into the conformations of cyclic peptides, and into the stereochemistry of phosphorylation reactions. In particular, the sensitivity of VCD to coupling of strong electric dipole moment transitions will be exploited to study amide linkages in poly- and cyclic peptides, base stacking arrangements in oligo- and poly-nucleotides, drug induced conformation changes in poly-nucleotides, phosphate coupling in DNA and RNA, and deoxy-ribose ring conformations. These interactions and their resultant VCD are straight-forward to model with the coupled oscillator and fixed partial charge theories that we have previously tested on small molecules.


Hypothetical examples of dental clinical trial data were developed to demonstrate the utility of a statistical technique of blocking on baseline DMFS. In the six trials studied, crude means ranged from one extreme of highly significant caries reduction in the fluoride group for one trial to the opposite extreme of highly significant caries reduction in the control group for another trial. Blocking on baseline DMFS, however, revealed that the trials were essentially identical.


PROJECT SUMMARY Direct cell reprogramming has the potential to facilitate the development of safer and more effective patient- specific cell-based therapies. However, current methodologies that induce direct reprogramming face major translational hurdles, including heavy reliance on viral transfection. While the results are promising, biosafety concerns, capsid size constraints and/or the stochastic nature of conventional methods (viral and non-viral) pose significant limitations. We developed and novel 3D nanochannel electroporation (3D NEP) platform technology that overcomes these barriers by enabling deterministic transduction of reprogramming factors with single-cell resolution, and without the need for viral vectors. This nanotechnology-based approach promotes remarkably fast and efficient direct cellular reprogramming, as demonstrated with well-established and newly-developed models of induced neurons and endothelium, respectively. Non-viral direct derivation of induced endothelial cells, in particular, could find applications in the treatment of a number of disorders, including critical limb ischemia and stroke. Ischemic strokes, for example, result in significant cellular deficiencies (e.g., vascular, neuronal) that could lead to death or major morbidity. Nevertheless, currently no study has looked into developing methods for virus-free direct reprograming of endothelial cells. Moreover, the regenerative potential of these cells has not been investigated within the context of stroke. Here we are proposing to develop an optimized method for direct derivation of endothelial cells by 3D NEP, and to study the extent to which these cells induce functional recovery in a middle cerebral artery occlusion (MCAO) mouse stroke model. In the US, strokes occur every ~40 s and have a death rate of ~42%. With an estimated cost of >70 billion dollars, strokes represent a substantial burden to the health care system. 1


The proposed NRSA Short-Term Training Grant at the University of Michigan (UM) builds upon a strong foundation and its long-standing commitment to support medical student research in aging. The UM has served as a training site for 90 medical students who participated in its Hartford Foundation supported program since 1989. Its research and training environment provides an ideal institutional setting to support medical student research in aging. A partnership with Wayne State University Medical School and Institute of Gerontology will expand opportunities for medical student and faculty participation. Fifty-five qualified faculty mentors with active aging-related research programs have been identified as potential mentors for medical student trainees. The overall goal of the proposed program is to provide quality experiences in aging-related research for up to 18 medical students each year. The program's specific objectives are: 1. To provide medical students exposure to and participation in aging-related research; 2. To give students the opportunity to learn research concepts and methodology from experienced investigators, and assess whether they wish to pursue careers in aging-related research; 3. To expand the scholarly and research experiences of medical students as part of an overall career development strategy aimed to increase the number choosing aging-related research careers in basic science, health services or clinical research; 4. To increase the number of underrepresented minority medical students who participate in a research experience, and ultimately choose careers as physician scientists; 5. To promote research experiences that can develop into longer-term student-faculty mentoring relationships; 6. To provide trainees with experience in scientific presentation and publication of research; 7. To provide students with a high quality didactic program in research with an emphasis on unique aspects of aging-related research and on the ethical and responsible conduct of research; and, 8. To provide medical students with an introduction to clinical geriatric medicine and career options in geriatrics through structured clinical "shadowing" opportunities.


By utilizing an in vitro cell line established from Drosophila melanogaster, we intend to expolit the genetic, cytogenetic and molecular biological technology of this organism toward the goal of understanding the structure and function of genes. First, we intend to define mRNA from Drosophila cells with respect to their size, poly A content, sequence relatedness, and to localize at least those redundant transcripts as to their genetic sites by means of in situ hybridization. Second, we intend to explore the nature of what appears to be an endosymbiotic double-stranded RNA virus that exists in one of our cell lines.


During the prior year we demonstrated that there are distinct forms of Drd1a-mediated activation of ERK (receptor coupled kinase) signaling in the dorsal striatum and the nucleus accumbens (Gerfen et al., 2008). Using Drd1a- and DARPP32-deficient transgenic mice we show that in the dopamine intact accumbens, psychostimulants activate ERK1/2 via a Drd1a- and DARPP32-dependent mechanism. This mechanism is activated in the dorsal striatum only following dopamine depletion as occurs in Parkinson's Disease. Work done during the past year is a continuation of studies to determine the affects of Drd1a-mediated activation of ERK1/2 in the striatum. To this end we have developed a transgenic mouse model in which ERK1 and ERK2 are deleted in the different subpopulations of striatal neurons. Developing these mice has taken over 4 years of intensive work and we are now at the stage of having the mice available to use them in animal models of Parkinson's Disease with L-DOPA induced dyskinesia. Further work on the cellular mechanisms underlying neurologic and pscyhiatric disorders is limited by the fact that neurons throughout the brain utilize common mechanisms for critical functions such as synaptic plasticity, whereas specific neural systems are affected in different disorders. A large part of the Laboratory is now working with the Gene Expression Nervous System Atlas (GENSAT) project ( funded by NINDS and NIMH ) to produce transgenic mice with Cre-recombinase under the regulation of promoters to drive expression in specific neuron populations. Our part of this work is to characterize the expression of Cre in the brains of such transgenic lines and to provide such lines to the Mutant Mouse Regional Resource Center (MMRRC) for distribution to the research community. In the past year we characterized 50 Cre driver lines and are in the process of characterizing 75 lines for the current year ( Gong et al., 2007)


The overall goal of this project is to test the hypothesis that Specific Language Impairment (SLI) is associated with deficits in acoustic processing. To this end, the mismatch negativity (MMN) of event-related potentials will be used to assess a variety of basic capacities associated with pre-attentive, automatic processing of acoustic events. These capacities include: 1. detection of changes in intensity, frequency and stimulus duration; 2. identifying features of acoustic stimuli that are constant while other features change; 3. determining that the order of two-tones pairs has changed; 4. determining that the serial order of three to five tones has changed; 5. forming an abstract rule as to the direction of change between the frequencies of two-tones pairs across a series of pairs (e.g., that they all rise in frequency from the first to the second tone), while the absolute frequencies of the tone pairs varies; 6. the duration of transient memory upon which the MMN relies. In addition, SLI and control subjects will receive training on the paradigm used in points 1-4 above which yields the poorest MMN to determine whether the MN will become larger in amplitude and/or shorter in latency.


Our studies on novel small molecules inhibitors of HIV integrase have led to the identification of new potent phenanthrene diketoacid (DKA) HIV integrase inhibitors that also inhibit HIV replication in cell culture, with a selectivity index up to 10. In light of the need for more effective and less toxic anti- HIV drugs, these results appear promising and warrant follow-up optimization and preclinical studies. However, since phenanthrenes are polyaromatic hydrocarbons (PAHs) with potential carcinogenicity we propose to investigate these novel phenanthrene diketoacids for carcinogenicity potential. Our hypothesis going forward is that appropriate substitution with electron withdrawing groups can minimize mutagenicity and produce useful anti-HIV agents. This is supported by the example of the new anti-malarial drug halofantrine, a substituted phenanthrene that is effective against multidrug resistant malaria, and is neither mutagenic nor teratogenic. We thus believe that appropriate substitution of our HIV integrase inhibitory phenanthrene DKAs will make them non-mutagenic and non-carcinogenic. Thus the specific aims of this proposal are: 1) to synthesize and test the IN inhibitory activity of new phenanthrene diketoacids with multiple electron withdrawing substituents and 2) to determine the carcinogenicity potential of the phenanthrene DKAs synthesized in Specific Aim 1. We will use the Ames test for in vitro carcinogenicity testing. The success of this project will provide vital information to decide whether or not to go ahead with optimization of the potent phenanthrene DKA HIV integrase inhibitors that we discovered recently and shown to selectively inhibit HIV replication in human peripheral blood mononuclear cells. If this approach to reducing carcinogenicity is successful it will not only provide a means of eliminating carcinogenicity of the phenanthrene DKAs, but may also provide a general method for reducing the carcinogenicity of PAH containing drugs. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: This project is aimed at investigating whether or not a novel class of potent phenanthrene diketo acid HIV integrase inhibitors that have been shown to suppress HIV viral replication in cell culture, are carcinogenic. The results of the research will determine whether to carry on with further work on optimizing this class of new anti-HIV agents towards AIDS therapeutics development. [unreadable] [unreadable] [unreadable] [unreadable]


Erythropoietin is a cytokine required for red blood cell formation. Erythropoietin receptor expression is not restricted to hematopoietic cells and erythropoietin activity extends beyond blood and includes neural protection and cardiac protection demonstrated in select animal models. Using the erythropoietin receptor null mouse that develops a lethal embryonic anemia, we demonstrated that erythropoietin is required for normal endocardium and myocardium development. We also observed erythropoietin receptor expressed on skeletal muscle satellite cells. Erythropoietin stimulates myoblast proliferation and modifies expression of MyoD and myogenin transcription factors to delay cell differentiation and fusion to myotubes. Erythropoietin response is lost with down regulation of its receptor after skeletal muscle differentiation. We test the hypothesis that erythropoietin administration or up regulation of its receptor on myoblasts can contribute to muscle maintenance and/or repair. Specifically we examine erythropoietin protection in myocardial ischemia-reperfusion injury, in myoblast survival in skeletal muscle and in skeletal muscle wound healing. [unreadable] We found that in a mouse model for myocardial ischemia-reperfusion injury, erythropoietin administration significantly reduced infarct size. Erythropoietin receptor is expressed on endothelial and cardiac myocytes and an animal model was created with erythropoietin receptor restricted to hematopoietic and endothelial cells to examine cell specific response. Erythropoietin cardioprotection in this animal model is in progress. [unreadable] Myoblast transplantation for the treatment of myopathies has the potential to retard or stop muscle degeneration. We identified erythropoietin receptor expression on primitive/early myoblasts as well as more mature myoblasts isolated from mouse skeletal muscle. Erythropoietin stimulation of primary myoblasts decreased apoptosis associated with inflammatory cytokine treatment. Forced expression of erythropoietin receptor provides further protection with erythropoietin treatment. Transplantation of myoblasts into mice is being conducted to determine the ability of erythropoietin treatment to increase donor cell survival. [unreadable] In culture, we observed that erythropoietin protected myoblasts from hypoxic stress as well as inflammatory cytokines, suggesting a potential for erythropoietin activity in skeletal muscle repair. The potential of erythropoietin to influence wound healing of skeletal muscle or myoblast survival is being evaluated in a mouse model system of mechanical or toxic injury.[unreadable] These studies address the role of erythropoietin in muscle maintenance and repair. Experiments are designed to elucidate regulatory mechanisms that determine endogenous erythropoietin receptor expression in myogenic cells, to reveal the nature of erythropoietin myoprotection and to illustrate the potential utility of erythropoietin administration in myoblast transplantation. The ability of erythropoietin to stimulate muscle progenitor cells demonstrates its potential to activate proliferation and/or act as a survival factor to prevent apoptosis in multi-organ systems, an activity that is closely linked to the presence and level of receptor expression.


These studies are directed towards definition of the tumor-specific transplantation antigen(s) (TSTA) recognized by Lyt-l+ T effector (Te) and Lyt-2+ T suppressor (Ts) cells responsive to the 51509a (H-2[unreadable]a[unreadable]) methylcholanthrene-induced fibrosarcoma. T cells derived from S1509a-hyperimmune A/J (H-2[unreadable]a[unreadable]) hosts proliferate in vitro in response to soluble tumor-specific protein antigens present in S1509a ascites fluid, predominantly against a 60 to 70 kilodalton molecule with a buoyant density of 1.2-1.32 gm/cc CsCl. Proliferation is specific in that no response is observed with soluble YAC (H-2[unreadable]a[unreadable]) tumor antigens, but appropriate cross-reactivity is exhibited with related SAl (H-2[unreadable]a[unreadable]) soluble antigens. Tumor-specific Ts do not appear to bind the crude antigen preparation, supporting previous observations on the differential specificity of suppressor and effector T cells for S1509a and SAl tumor-associated antigens. A panel of syngeneic monoclonal antibodies (mAbs) reactive with soluble and/or cell-bound 51509a tumor antigens and exhibiting varying degrees of cross-reactivity with other ALJ tumors and with ALJ spleen cells is now being evaluated for recognition of the 51509a TSTA relevant to T-cell immunity. Our second interest is in the administration of anti-class II mAbs as an immunotherapeutic approach to regulating transplantation immunity. Treatment with minor-\or class I-incompatible skin graft prolongs survival 2-\to 3-fold and results in 50% indefinite survival of heterotopic heart allografts. Treatment is associated with delayed development of donor-specific DTH and CTL reactivity concomitant with the appearance of alloantigen-specific T[unreadable]s[unreadable]. Failure to enhance H-2-incompatible graft survival is to the differential recognition of class II versus class I or minor alloantigens by host T[unreadable]s[unreadable]. This therapy may prove beneficial in class II-matched organ transplants, since treatment can be discontinued once suppression has been established, leaving the host response to environmental pathogens intact. (LB)


DESCRIPTION: (Applicant's Abstract) Hormones have been shown to influence the growth characteristics of epithelial ovarian carcinoma, but their mechanism of action is still unclear. In particular, low and high affinity LHRH receptors have been demonstrated on ovarian tumor cells. Also, LHRH agonists have been used successfully in phase II clinical studies of ovarian carcinoma. This project will investigate the antitumor activity of Cetrorelix, the first LHRH antagonist suitable for clinical use. A phase II study of Cetrorelix will be performed on patients with advanced recurrent ovarian cancer. Included in this study is a pharmacological investigation of different dose levels of Cetrorelix to determine the endocrine inhibitory effect of this drug in these ovariectomized patients. In order to understand the hormonal dependency of some ovarian carcinomas, we will evaluate the modulation of hormone and growth factor receptors on the membrane of ovarian tumor cells treated with Cetrorelix. Laboratory results obtained will be compared to clinical responses. The ultimate goal is to predict which tumor is sensitive to this LHRH antagonist by identifying relevant prognostic factors applicable to clinical use.


Actins are ubiquitous, highly conserved proteins that fall into classes: muscle actin (alpha-actin) and nonmuscle or cytoplasmic actins (beta- and gamma -actin). Because of its highly conserved nature, most antibodies to actin has an unusual specificity: in addition to reacting with the immunogen, this antiactin recognizes cytoplasmic vertebrate actin and not skeletal muscle actin. It is therefore an ideal probe for studying the distribution of cytoplasmic actin in skeletal muscle. Our previous immunocytochemical studies of human skeletal muscle showed that cytoplasmic actin is present and associated with membranes. Our more recent immunofluorescence studies of rat diaphragm muscle suggest that this actin may somehow stablize acetylcholine receptors in the postsynaptic membrane at the neuromuscular junction. To understand its function in muscle, an immunological approach will be used to localize cytoplasmic actin in sections of adult skeletal muscle and developing muscle cells at the level of the light and electron microscope. An antibody raised to squid tubulin also will be used to study the organization of microtubules in muscle. Lastly, dystrophic and myasthemic muscle will be examined to identify fine defects in the cytoskeletal structure of these tissues.


For the past three years, the Depression Unit and the Clinical Research Unit have collaborated in a study which surveys a population of individuals maintained on methadone to determine the prevalence of depression. The group studied included about 100 volunteers, and was generally representative of the entire clinic population in terms of such features as age, race and sex. In this research participants were evaluated based on several standardized rating scales measuring depression as characterized by verbal self-report, behavior, and symptoms. On the basis of the survey result, over 30 percent of these individuals were considered to be significantly depressed, including some patients with rather severe degrees of depressive symptoms. In most clinical settings, the degree of depression as shown by members of this group is considered of sufficient intensity to require antidepressant medication. We therefore propose a further study which assesses the efficacy of an antidepressant medication in treating depression in individuals maintained on methadone. The study will include 50 patients who exhibit significant depression at the time of admission to the Methadone Program. The selected individuals will be randomly assigned to receive dosages of the antidepressant Tofranil (imipramine) or a placebo on a daily basis for eight weeks. All medication will be dispensed on a double-blind basis and will be combined with daily methadone dosages. Throughout this drug trial, depression will be measured by self-reports, and by clinical assessment of behavior and symptoms. It is expected that the study will take two years, and will provide a sufficient sample to assess the efficacy of antidepressant medication in treating depression in this population.


Abstract: This cost center is fulfilling an essential service for the EDRN by receiving samples for our specimen reference sets, often aliquoting them to smaller sizes, maintaining these reference sets in freezers, and shipping sets out to investigators who obtain approval to test their biomarkers. These sets are used only for pre-validation or validation studies of biomarkers which is one the chief purposes of the EDRN. Without the maintenance of these reference sets the effectiveness of EDRN to test newly emerging biomarkers would be severely hampered. These reference sets differ than the extensive collections obtained through studies like PLCO or NLST in that the numbers of samples kept are much less being selectively chosen to address a particular clinical situation, include properly matched controls in that clinical context, and are powered statistically to permit adequate analysis of the biomarkers being tested.


Methods of enzymology, chemical and enzymatic kinetics, and synthetic, physical and analytical chemistry are being used to investigate the mechanisms of action of reverse transcriptase (RT) and protease enzymes of HIV-1, with the ultimate goal of developing specific inhibitors for these enzymes. Optical assays for retroviral proteases and P's, developed in this laboratory, are being employed in studies of the effects of reaction conditions and potential inhibitors on the activity of both RT and retroviral proteases. i) A continuous UV spectrophotometric assay described in a previous Report was used to study the elongation, catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I, of a partially self-complementary "hairpin" shaped oligonucleotide consisting of 35 mononucleotide residues. The sensitivity of the method permits the measurement of incorporation of a single base. ii) Concentrations of sodium chloride up to 4.5 M accelerated the hydrolysis of a chromogenic peptide substrate catalyzed by the protease of avian myeloblastosis virus. Similar effects have been observed by us for the cleavage of a peptide substrate catalyzed by HIV-1 protease, and have also been reported by others for cleavage of several peptides by this enzyme. These salt effects appear to be unrelated to the association of subunits that is required for the activity of retroviral aspartyl proteases, since very similar behavior is observed with the monomeric, mammalian enzyme pepsin. iii) We have identified two amino-acid repeat motifs in RT which are conserved in ten HIV-1 and eight HIV-2 isolates, and which occur in a region of the protein that may be involved in association of the p66 and p51 subunits of this enzyme. One motif is a modified "leucine zipper", whereas the other motif involves five tryptophans. Since the activity of HIV-1 RT has been suggested to require subunit association, an understanding of this process may lead to novel approaches to the inhibition of this enzyme.


The in vitro carcinogenesis model derived from organ culture of the whole mammary glands of BALB/c female mice will be used to study the transforming action of the environmental carcinogens, benzo[a]pyrene (B[a]P) and N-diethylnitrosamine (DENA). Appearance of the potentially neoplastic nodule-like alveolar lesions (NLAL) in the mammary glands in vitro after exposure to B[a]P or DENA will be used as a morphological marker for preneoplastic transformation. Carcinogenecity of the mammary epithelial cells transformed in vitro will be confirmed by the ability of cells to produce mammary carcinomas after transplantation into gland-free mammary fat pads of syngeneic virgin mice. The unique organ culture model of mammary cell transformation also will be used to study the mechanics of chemopreventive action of the dietary compounds, a new retinoid, Beta-carotene and selenium, during the neoplastic process induced by DMBA, B[a]P or DENA. Influence of the chemopreventive agents on the molecular events such as cellular accumulation of the electrophilic reactants, DNA alkylation and DNA repair activity initiated by the carcinogenic chemicals will be examined. Attempts will be made to ascertain whether influence of the chemopreventive agents on the molecular events of "initiation" may alter morphological expression of the transformed cells as NLAL in the glands in vitro and/or in the mammary tumors in the mammary fat pads in vivo. Studies also will include determination of the influence of the chemopreventive agents during hormone mediated "promotion" of the initiated (transformed) cells, both in vitro and in vivo. These studies will examine influence of chemopreventive agents at the specific stages of transformation between normal -- preneoplasia and preneoplasia -- neoplasia. The results should provide elucidation o the mechanics of chemopreventive action of these dietary compounds.


The nuclear enzyme DNA topoisomerase I relaxes supercoiled DNA and participates in DNA replication and transcription. Topoisomerase I localizes predominantly to nucleoli, and is the cytotoxic target for drugs of the camptothecin class. Several analogues of this class are currently in Phase I/II clinical trials, and early results of these traials have shown them to have activity against several types of "refractory" solid tumors. Intracellular factors reported to influence the cytotoxicity of the camptothecins include the amount of nuclear enzyme, the drug-induced increase in covalent topoisomerase I-DNA complexes, and the requirement for ongoing DNA synthesis. We suggest that additional, previously unidentified cellular events also significantly affect the cytotoxicity of the camptothecins. Therefore, we propose to test the hypotheses that: 1) Camptothecins limit their own cytotoxicity by altering the subcellular distribution of topoisomerase I and other nuclear proteins and by decreasing the amount of nuclear topoisomerase II, resulting in decreased topoisomerase I-DNA complexes and decreased DNA synthesis; 2) Intermittent, repeated exposures to the camptothecins that allow cells to maintain nuclear enzyme levels and nucleolar integrity, and consequently the ability to form topoisomerase-DNA complexes, will have the greatest cytotoxic effect; and 3) Based on biochemical observations, markers that predict the antitumor effect of topotecan in samples of primary tumors or bone marrow metatases of pediatric patients with neuroblastoma or medulloblastoma cal be identified.


Induced pluripotent stem cells (iPSCs) and their application to tissue engineering and disease modeling have great potential to change current medical practices. Current research is largely focused on devising efficient virus-free protocols to produce large numbers of iPSCs. Direct delivery of proteins obviates the risk of mutagenic insertion and enables more accurate control of the highly sensitive reprogramming process. However, cell-penetrating peptide methods currently provide reprogramming efficiencies that are too low for clinical use. The microfluidic delivery technology proposed has demonstrated its ability to deliver proteins at high efficiencies to human fibroblasts and it eliminates the need fo chemical modification or the use of exogenous compounds. Moreover, preliminary results indicate that the technique can be developed into a universal delivery method capable of delivering a range of macromolecules to different cell types underserved by current technologies. The current prototype is capable of delivering high throughput rates of 10,000-20,000 cells/s and can yield up to 1 million delivered cells per run. This combination of single-cell level control and macro-scale throughput places this device in a unique position relative to existing delivery methods. Aim 1: The mechanism of protein delivery and cell recovery will be investigated to better understand the system and direct its optimization. Preliminary results indicate macromolecular delivery occurs through a pore formation mechanism. To validate this hypothesis, model fluorescent macromolecules and proteins will be used in experiments designed to control against endocytosis and image membrane pores directly. Results will be used to develop a predictive model of the delivery system and conduct optimization studies to improve delivery efficiency, uniformity and cell viability. The design of future device generations will be guided by the gained mechanistic understanding and will aim to incorporate features such as coupling with electroporation. A streamlined version of the system will also be developed for use in collaborating laboratories. Aim 2: The intracellular delivery method will be optimized for protein-based reprogramming of fibroblasts to iPSCs. The robust delivery capabilities of the device will allow studies on the biological aspects of the reprogramming process itself, such as the optimal combination of transcription factors to produce maximum reprogramming efficiency and identification of the role of individual factor in the overall process Moreover, the device will be used to investigate potential improvements by combining other macromolecules, such as microRNA and mRNA, with protein-based reprogramming. In addition to reprogramming applications, such a high throughput microfluidic device platform capable of delivering a range of macromolecules with minimal cell death could enable unprecedented control over cellular function. Hence, in the future, it can be implemented in studies of disease mechanisms, identification of macromolecular therapeutic candidates, stem cell differentiation, and diagnostic applications with reporter cell lines.


This is a biobehavioral, longitudinal investigation of the role of emotion in the development of psychopathology in adolescence. The focus is on (a) the role of multiple components (experience, expression, regulation) of negative emotions (anger, anxiety, sadness) in the evolution of psychopathology, and (b) socialization experiences and biological processes that contribute to emotion dysregulation and disorder. The dysregulated experience and expression of emotion is implicated in both externalizing (antisocial patterns) and internalizing (anxiety, depressed mood) disorders. Adolescence is a critical juncture in the development of these disorders because of both the increased incidence of psychopathology during this time period and the increased differentiation of clinical problems. Four groups of youths are studied (N= 60 in each group): (1) comorbid externalizing and internalizing problems, (2) externalizing problems predominate, (3) internalizing problems predominate, (4) asymptomatic. Half the youth in each group are 11-13 years, the other half 14-16 years in order to conduct age cross-sectional analyses at each time point. Equal numbers of males and females participate, in order to examine sex differences in symptoms and emotion regulation. One theme pertains to the integration of emotions across systems, and how different patterns of emotion relate to psychopathology. A second theme pertains to the developmental changes in how disorders are manifested, e.g. increased differentiation of disorder along gender specific pathways. During the past year a primary focus has been on data collection, and both laboratory sessions and home visits are close to complete. This year will be spent (a) coding, analyzing, and preparing articles for publication based on Time 1 data, and (b) collecting the Time 2 data for longitudinal analyses. - emotions & psychopathology; adolescent clinical problems; co-morbidity; gender - Human Subjects & Human Subjects: Interview, Questionaires, or Surveys Only


The sense of hearing is initialized in the inner ear by signal transduction; i.e., the transformation of sound to electrical signals. Normal cochlear function relies on the integrity and functionality of the hair cells that act as mechano-electric transducers. Cochlear transduction is vulnerable to structural and metabolic alterations in the inner ear that consequently lead to hearing loss; therefore, techniques that can assess cochlear transduction would be a desirable clinical tool to evaluate cochlear function. The long-term goal of the proposed research is to characterize cochlear transduction in cochlear disorders and, in turn, to develop a diagnostic tool that can no invasively distinguish cochlear hearing losses. The transduction processes in the hair cells produce sound or acoustic emissions that are measurable in the ear canal. Evoked by two-tone stimuli, the distortion product otoacoustic emissions (DPOAEs) are generated from the nonlinearity in cochlear transduction. Recently, we have developed a technique to derive a nonlinear transducer function of the inner ear from low-frequency modulation of DPOAEs. The modulation patterns of the DPOAEs provide physiologic indices of cochlear transduction. To continue our research towards the long-term goal, three studies, two in animals and one in the humans, are proposed. In the animal studies, the specific aim is to determine the sensitivity and specificity of the transducer function and the associated indices in detecting cochlear pathologies. The sensitivity of the transduction indices will be tested in animals with moderate temporary hearing loss induced by exposures to pure tones. The specificity will be evaluated by differences in the change of transducer functions obtained from animals with hearing losses produced by different ototoxic drugs. The specific aim of the human study is to obtain normative values, the variability and reliability of the cochlear transduction indices derived from the low-frequency modulation of DPOAEs. Successful completion of the proposed research will provide a solid base for future development of a clinical tool that is helpful in the early identification of cochlear disorders, a critical practice in otology and audiology for treatment and hearing preservation. [unreadable] [unreadable]


The use of alcohol off and on the job by employed adults represents an important social policy issue because it can undermine employee health and productivity, and may further interfere with employers' ability to compete effectively in an increasingly competitive domestic and global economic environment. Therefore, it is imperative that we develop a better understanding of the workplace causes of employee alcohol use both off and on the job. The most frequently explored work-related cause of employee alcohol use is exposure to work stressors. Nonetheless, because past research on work stress and alcohol use has left many issues unresolved or unexplored, it is not surprising that the findings to date have been weak and inconsistent. The proposed study will systematically extend past research on work stress and alcohol use in a number of ways. First, a broad taxonomy of work stressors will be used to determine the general types of work stressors that may be related to employee alcohol use. Second, a broad set of alcohol measures will be used to determine if work stressors are more strongly related to certain dimensions of alcohol involvement. The taxonomy of alcohol use will include several dimensions of both context-free (i.e., overall) alcohol involvement and work-related context-specific (use before work, use during the workday, use after work, and use on off-work days) alcohol involvement. Finally, variables expected to moderate or mediate the relation between work stressors and alcohol use will be examined. Mediating variables include negative affect, inability to relax or unwind after work, physical and psychological fatigue, and the desire to use alcohol (cravings). Moderating variables include person characteristics (affect and performance regulation expectancies, avoidant coping, impulsivity, and stress-related rumination) and environmental characteristics (overall and workplace physical and social availability of alcohol). To explore these issues, a national telephone survey will be conducted using a representative sample of 3,500 employed individuals 18 to 65 years old. The proposed study will help shape future research on work stress and alcohol use by leading to a more comprehensive model of work stress and employee alcohol use. It will contribute to organizational policy and shape future intervention research on workforce and workplace alcohol use by identifying work conditions and vulnerable subgroups that can be the focus of intervention efforts and work redesign. [unreadable] [unreadable] [unreadable]


This project involves the analysis of Missouri vital statistics data to determine if using a cohort method of analysis is biased in understanding the effect of prenatal care on pregnancy outcome. Cohort methods appear to underestimate the effect of prenatal care and suggest that late entry into prenatal care is more beneficial than early entry.


An attenuated monocyte-tropic clone of SIV (SIV/17E-C1) has properties which are unique to a live attenuated SIV vaccine. Infection with SIV/17E-C1 induces an early vigorous type-specific neutralizing antibody response which broadens during the first 7 months postinfection to include activity against SIV/DeltaB670, a heterologous primary isolate. The induction of protective immune responses coincides with the switch from type-specific to group-specific neutralizing antibody, and, at least in part, appears mediated by antibody since sera from protected monkeys passively protected 2 of 4 recipients. A nef-deleted variant of this monocyte-tropic clone, but not the lymphocyte-tropic parent SIVmac239deltanef, also induced detectable class I restricted CTL during the first 6 months postinfection. These unique properties are likely due to a unique gp120 conformation imposed by sequences which enable it to replicate in macrophages, and/or selective presentation to the immune system by the infected macrophage. We propose to further characterize the protective responses induced by SIV/17E-C1 infection, and to utilize this information to design a subunit vaccine that can induce the protective responses observed with the attenuated virus. The protection observed by intravenous challenge with cell-free virus of SIV/e-C1- infected monkeys will be extended to protection against infected cells, delivered both intravenously and at the intestinal mucosal surface. The role of antibody in passive protection will be confirmed by passive transfer of purified immunoglobulin. Antibodies binding to native versus denatured gp120 will be affinity purified, characterized in vitro and similarly analyzed to define the specificity of protective responses. Passive transfer studies will also be performed with monoclonal antibodies derived from SIV/17E-C1-infected monkeys to identify the specific epitope(s) required for protection. The contribution of cellular immunity will be determined by determining the kinetics of class I restricted CTL induction in infected monkeys, and correlating these responses to protection. The efficacy of a purified recombinant gp120 vaccine comprised of SIV/17E-C1 sequences will be evaluated to determine whether these sequences per se uniquely induce protective immunity. The ability of DNA vaccines comprised of SIV/17E sequences, alone or in combination with cytokine expression vectors, to elicit protective responses will be assessed using both direct injection of DNA and the particle delivery system developed by Agracetus. The optimal HIV vaccine strategy is one which is efficacious in both SIV (disease) and HIV (infection) model systems. Thus, the ability of SIV/HIV recombinant viruses to serve as live attenuated vaccines will be determined to extend the observations gained from SIV to HIV glycoproteins. A model for assessing effective SIV subunit vaccines, as well as heterologous (inter- clade) protection, will be established using SHIV constructs.


The Universidad Central del Caribe (UCC), through its Center for Addiction Studies and the Caribbean Basin and Hispanic Addiction Technology Transfer Center (CBHATTC), is requesting support to implement "The Second National Conference on the Hispanic/Latino Family: The Role of the Hispanic/Latino Family as a System of Drug Use Treatment and Prevention," to be held August 18-20, 2004 at the San Juan Marriott in San Juan, Puerto Rico. The Conference is part of the Center for Addiction Studies and CBHATTC's educational commitment to continually pursue the goal of "Blending Research/Science to Practice." The conference is designed both to build a knowledge base among participants regarding up-to-date research findings in each of the themes of the conference to provide an environment in which Hispanic drug treatment providers, others who serve Hispanic populations, and researchers can interact and develop relationships and coalitions that may result in conjoint research endeavors. The conference program is structured around the subtheme of "From molecules/genes to community to culture," and is comprised of pre-conference workshops, plenary sessions, workshops, and poster sessions. Major topics to be covered include the neuroscience of addiction; family-centered approaches to prevention and treatment of substance use; community and cultural factors which may facilitate or impede the family's ability to address substance use among its members; and issues of trauma and violence, sexual identity, and co-occurring disorders as they relate to substance abuse and its treatment and prevention. A Conference Proceedings document is to be developed in Spanish and English, which will be available to all states through the SAMHSA/CSAT ATTC Network and the NIDA National Hispanic Science Network. The conference approach is guided by elements of Hispanic culture, and the theories of Diffusion of Innovation and Successful Technology Implementation.


Acquired immune deficiency syndrome (AIDS) occurs at a very high rate in Zaire and attacks females at approximately the same rate as males. It is clear that the epidemiology of the disease in Africa differs from that seen in the United States and studies of the disease in Zaire could lend valuable information in understanding and combatting the disease in this country. For this reason, a Collaborative AIDS Project in Zaire (CAPZ) has been established by the Centers for Disease Control and the NIAID to study epidemiologic and immunologic aspects of this disease in Zaire. The study is being set up, laboratory facilities prepared and equipment being shipped. Dr. Francis will be responsible for the laboratory aspects of the project and for some epidemiologic studies of special interest to the NIAID and Dr. Mann will be responsible for most epidemiologic study design and implementation. It is planned that a clinician will be added to the project in the near future. The project is estimated to take two years to complete.


PROJECT SUMMARY This application entitled ?Circadian Rhythms of Skin Barrier, Pruritus and Inflammation in Atopic Dermatitis,? is being submitted by Dr. Anna Fishbein at Northwestern University Feinberg School of Medicine/ Ann & Robert H. Lurie Children's Hospital of Chicago for a Mentored Patient-Oriented Research Career Development Award. Dr. Fishbein is a pediatrician, allergist and immunologist with a background in translational research. Eczema (atopic dermatitis) is the most common chronic skin disease of childhood, affecting 10% of US children. Nocturnal eczema is the itchy nighttime exacerbation of atopic dermatitis (AD) that afflicts more than 50% of children with AD, resulting in devastating scratch and sleep disturbance. Circadian rhythms are 24 hour biological cycles that appear to underlie the nocturnal worsening of atopic dermatitis. The objective of this project is to elucidate how circadian rhythm dysregulation is related to underlying AD pathophysiology. Dr. Fishbein's novel approach will apply state of the art tools to test her overarching hypothesis that the circadian system drives skin barrier disruption, pruritus and inflammation in nocturnal atopic dermatitis flare. She will test the specific hypotheses that 1) blunting of the normal skin barrier function rhythm in AD is associated with increased scratch and sleep disturbance and 2) melatonin increases release of TH2/TH22 cytokines that are known to disrupt skin barrier function and increase itch. Modified constant routine protocol (28 hours in a controlled-laboratory setting with dim light) will isolate the contribution of the circadian system to atopic dermatitis. The specific aims to test the hypotheses are: 1) To determine how changes in the skin barrier function rhythm exacerbate nocturnal atopic dermatitis flare; and 2) To determine the role of melatonin in atopic inflammation in AD. Through the proposed research, Dr. Fishbein will work towards her long-term goal of becoming an independent translational physician investigator by developing chronotherapy (timed based treatment) for atopic dermatitis. Her short-term goal is to apply circadian biology to study skin barrier, pruritus and inflammation to determine the mechanism of nocturnal AD flare. Her training goals for this award are to: 1) build a foundation in circadian research techniques and molecular assessment of rhythms to study inflammatory skin disease, 2) enhance her knowledge base in immunology with a focus on circadian rhythms in T-cell biology, 3) advance personalized medicine for children with AD and 4) become an independently funded investigator. Dr. Fishbein's clinical training and prior experience in patient-oriented research make her an ideal candidate to accomplish this multidisciplinary project. Ann & Robert H. Lurie Children's Hospital of Chicago/Northwestern University provides an optimal training environment with top-notch expertise. Primary mentor Dr. Phyllis Zee is an expert in circadian biology and has successfully trained many academicians. Co-mentors Dr. Amy Paller and Dr. Robert Schleimer are world-renowned researchers in atopic dermatitis and immunology. .


DESCRIPTION: We intend to refine the affinity-reagent discovery process so that it is not only cheaper and faster than currently possible, but also so that we produce reagents that are more versatile and useful than current monoclonal antibodies. We propose to use in vitro combinatorial recombination of pre-defined synthetic complementarity determining regions (CDRs) of single chain variable fragment (scFv) antibodies to synthesize a novel type of highly diverse synthetic display library. We have termed this new library-type as a 'pre-defined CDR' (PDC) library. This novel library system will have properties that will also enable it to greatly facilitate both the downstream affinity-reagent selection and the maturation processes. Methods for testing this library using microfluidics and emulsion screening are discussed.


This proposal is to develop a revolutionary new type of angioscope. The new technology will achieve the following improvements compared to existing angioscopes: 1)a factor of two brighter image, 2)a 67% improvement in resolution, 3)a factor of five greater flexibility, and 4)the ability to make quantitative measurements such as the degree of vascular stenosis. The broad objectives of this project are to fabricate angioscopes of several sizes with this new technology. They will be designed to be plug-in compatible with the already installed base of electronic cameras, illumination systems, and monitors. The specific aims of Phase I are : 1)to fabricate image guides of various resolution, 2)characterize the optical and mechanical properties of these guides, and 3)design an advanced angioscope which incorporates the new technology and has the ability to perform quantification, and spectroscopy. Heart and vascular disease is the single largest killer in the USA. This new approach to angioscopy will provide access to 100% of the vascular tree with unprecedented image quality, and quantification. The proposed technology has never previously been used in endoscopy. The successful demonstration of the feasibility of this technology will have wide ranging implications for innovation in all aspects of medical and industrial imaging. PROPOSED COMMERCIAL APPLICATION: The world wide market for angioscopes is about $20M. The new technology being proposed will make a substantially better product at a lower cost and permit penetration of this market. Success in the small diameter endoscope market will be expanded into the rest of medical endoscopy and industrial imaging, which is a several hundred million dollar market.


The purpose of this project is twofold: 1) gain additional training in conducting research and primary data collection/analyses in developing countries, with a focus on issues related to early intervention for children with disabilities; and 2) to obtain more knowledge about the influence of psychosocial, resource and cultural factors on the needs of children with disabilities and their families in developing countries. The candidate wishes to pursue a career in international rehabilitation, with the ultimate goal of contributing to improved conditions for children with disabilities and their families in developing countries. Her immediate goal is to combine her backgrounds in cross-cultural and disability studies to address the global issue of the factors that influence the conditions of children with disabilities and families. The candidate's long-term goals include embarking on a career of independent research and teaching in international rehabilitation for children with disabilities and their families and conducting field research. This award will allow the candidate to benefit from a period of mentored research in international rehabilitation for children and gain necessary experience in conducting field research and primary collection in a developing country. The candidate plans to spend about five months per year for this five-year project primarily in Hanoi, Vietnam, and the remainder of the year at the New York State Institute for Basic Research in Developmental Disabilities in the U.S. under the guidance of Drs. Peter Vietze, Tran Trong Hai, Judith Ladinsky and Nguyen Viet Nhan. During this time, she will attend relevant seminars and courses for additional training in the field, including courses in Vietnamese language, advanced statistics, and longitudinal research methods at the City University of New York and New York University. The early intervention study will involve a comprehensive assessment of children with disabilities at the time the parent enters the treatment study and again at 3, 6, 12, 18, and 24 months. The primary aims of the study are: 1) to examine the level of child's and family variables over the course of the intervention program; 2) to explore potential factors that cause these changes including stressors and family patterns; and 3) to test whether changes in child adaptation, the hypothesized variables, and the relation between parent and child symptoms differ as a function of the type of services they received. It is expected that the study will represent a unique opportunity to further our understanding of the familial context for child disabilities that can serve as a guide for the development of intervention programs for young children with disabilities in developing countries.


Despite great scientific advances over the past decade, the translation of these discoveries to the benefit of patients has been hampered by a number of traditional barriers. The overall goal of the iWioyinit Siimai ImstitiLiiites of ClBrnical &Tiramslatioinial Sciences is to establish a new research paradigm at Mount Sinai, which will create an integrated multidisciplinary environment that will enable translation of cutting-edge research more quickly and easily from "bench-to-bedside-to-implementation" in the community;train and create an "academic home" for the clinical and translational investigators of tomorrow;and supply the governance, facilities, resources and opportunities needed to elevate the clinical research enterprise so that clinical investigators can take full advantage of Mount Sinai's Translational Research Institutes and potential collaborators in Mount Sinai Hospital and its affiliates, the School of Medicine and partnering institutions, and other CTSA programs around the country. This transformation will be achieved by fulfilling the following Specific Aims: (1) redesign the institutional research enterprise infrastructure and governance by integrating research functions across administrative, departmental and intellectual boundaries, enhancing and promoting interactions between basic scientists and clinical investigators, and streamlining administrative procedures to facilitate the start-up of clinical trials and the dissemination of results;(2) establish a Translational Discoveries Program to facilitate and expedite clinical research by providing consultation, oversight and facilities for clinical and translational research, engaging the surrounding community and our affiliates to rapidly translate health benefits to the general population, and developing new methodologies to improve clinical trial design and reduce participant burden;(3) train and support the translational investigators of tomorrow by overseeing a multi-disciplinary, doctoral degree-granting program in clinical and translational research for all health care professionals, developing a program to recruit, enhance career development and retain clinical and translational researchers, providing an academic home where new collaborations can be formulated;(4) establish an Experimental Therapeutics&Technologies Program to stimulate the development of new methodologies in clinical and translational research by developing an institution-wide mentored program to identify and develop novel clinical and translational research projects; and (5) establish an institution-wide biomedical information infrastructure that can connect basic researchers, clinical researchers, caregivers and laboratories with an integrated network of information. RELEVANCE (See instructions): The establishment of the ilfloynt Siinao Institutes of Clinical &Tiranslational Sciemces would create an effective, efficient and centralized research administrative structure, foster and reward interdisciplinary collaborations, educate and retain the clinical and translational investigators of the future, enable translation of basic scientific discoveries into clinical practice, and deliver to our diverse community new therapies and a standard of care that would have been unimaginable even five years ago.


Accumulating evidence suggests that cerebral amyloid beta (Abeta) deposition, which begins rapidly following traumatic brain injury (TBI), occurs in response to axonal and oxidative damage. Our long-term goal is to validate the use of positron emission tomography (PET) imaging with a novel molecular probe, [18F]FDDNP, for visualizing Abeta plaques, in vivo, in the setting of acute TBI. We hypothesize that the amount of [18F]FDDNP binding will correlate with both the concentration of diffusible Abeta in the cerebrospinal fluid and patient outcome. The Specific Aims are to 1) confirm the specificity of [18F]FDDNP for cerebral Abeta deposition [18F]FDDNP uptake following TBI using autoradiography and immunohistochemistry of brain specimens and, 2) determine the relationship between cerebral Abeta plaque deposition, assessed by [18F]FDDNP-PET imaging, and CSF Abeta concentration acutely following TBI. [18F]FDDNP-PET has been used successfully in imaging amyloid pathology in Alzheimer's disease patients, allowing early diagnosis and improved understanding of the disease. We believe that the ability to visualize amyloid deposition in vivo with PET, in a broad range of TBI injury severity, will allow us for the first time to assess the incidence, time course, and regional distribution. More importantly, it may also offer a noninvasive tool to monitor the efficacy of treatments aimed at decreasing amyloid deposition (including neuro-inflammation modulators). In brief, the research design entails studying a range of mild to severely head-injured patients acutely after injury (3-10 days) with FDDNP-PET. The results may have important public health implications in that improved understanding of the pathological consequences of cerebral amyloid deposition may lead to medical treatments that will improve outcome following TBI. [unreadable] [unreadable] [unreadable]


This interdisciplinary study is primarily designed to assess the effect of a prototype intervention program designed to improve cardiovascular health of rural, American Indian, Hispanic, and Anglo fifth graders. The basic program will be compared with a control condition and a condition in which the program is supplemented by peer modeling. The program will involve instruction about the heart and cardiovascular system, obesity, nutrition, exercise, smoking and self-control techniques for changing behaviors related to cardiovascular health. A variety of instructional methods including parental and community involvement, lessons in food preparation and storage, and an emphasis on cultural tradition as well as traditional audiovisual and written materials will be used. A secondary purpose of the study will be to collect some descriptive data on health-related knowledge and attitudes in a population which has not previously been studied. Measures of short-term and long-term effects will include a) physical measures: height, weight, blood pressure, heart rate, and skinfolds, b) physical fitness: the one mile run/walk, c) knowledge measures about the heart and cardiovascular system, nutrition, obesity, smoking, exercise and behavior change, d) self-report measures of eating, exercise and smoking behaviors, e) measures of attitudes toward obesity and self-perception of their own weight and fitness, f) a posttest assessment of their feelings about the program, for those not in the control groups. It is hypothesized that subjects in both experimental conditions will show changes on all measures at the end of the semester in which the major part of the material is presented. Maintenance of the changes is expected to be superior for those in the peer modeling condition, who will have the opportunity to serve as peer models in subsequent years. The focus on incorporating behavioral changes into the life styles of the participants is hoped to lead to permanent reductions in risk factors related to cardiovascular disease.


PROJECT SUMMARY/ABSTRACT The Cancer Genetics and Epigenetics (CGE) Program of Winship Cancer Institute of Emory University is a laboratory-based basic science program that seeks to better understand how altered genetic and epigenetic components of the genome contribute to the initiation and progression of human cancers. Under the leadership of Paula Vertino, PhD (leader) and Jin-Tang Dong, PhD (co-leader) the CGE Program includes 27 core members drawn from 14 departments and three schools across Emory University, including the School of Medicine, Emory College, and Rollins School of Public Health. The goals of the CGE program are to better understand the mechanisms that govern the maintenance of genome stability and proper gene regulatory networks, how these become corrupted in cancer cells, and how their disruption contributes to the initiation and progression of cancer. The CGE Program seeks to promote the expanded application of genomic technologies in the molecular classification of cancer phenotypes and outcomes. The scientific focus of the program is organized around three inter-related and complimentary themes: (1) DNA Damage, Repair, and Cellular Responses to Stress, (2) Epigenetics and Gene Regulation, and (3) Cancer Genetics and Genomics. Over the last competitive cycle CGE Program members have published 256 cancer-relevant scientific articles. Of these, 55 (21%) were intra- and 105 (41%) were inter-programmatic collaborations, and 123 (48%) represented a collaboration with another cancer center or other academic organization. As of March 31, 2016, CGE held $9.1 million in annual total cancer-relevant research funding, of which approximately $3 million (33%) was awarded from the NCI. Strong scientific synergism has led to important discoveries. Key insights into the mechanism of DNA double strand break repair, how replication stress is resolved, and radiation mutagenesis are leading to important predictors of chemotherapy and radiation response. Landmark investigations into the structure and function of the TET dioxygenase enzymes have revealed oxidized methylcytosine residues to be more than a transient intermediate in the turnover of 5mC, but a distinct component of the epigenetic `code' governing gene expression programs. Significant strides have been taken towards the successful translation of the program's genetics/genomics efforts, including new insight into the contribution of key `driver' gene mutations and the unique cellular vulnerabilities that such alterations unmask, an RNA-based biomarker panel for the early detection of aggressive prostate cancer, a combined RNA-DNA test for risk stratification in oropharyngeal carcinomas, and the implementation of a clinical genomics workflow to guide decision making in several cancer types. Overall, the CGE Program promotes center-wide goals for improvements in cancer outcomes across the state of Georgia by identifying of key points of cancer cell vulnerability that can be exploited towards new therapeutic opportunities and the development of genomic and epigenomic signatures as predictors of clinical outcomes and population risk.


Project Summary Overwhelming evidence suggest that mitochondria plays a causative role in diabetic endothelial dysfunction. How mitochondria become dysfunctional remains enigmatic. Our exciting preliminary data indicate that diabetes instigates aberrant mitochondrial fission by suppressing autophagy-dependent degradation of the dynamin-related protein 1 (DRP1), a molecule essential for mitochondrial fission. The central hypothesis of this application that selective impairment autophagy-mediated DRP1 degradation in diabetes instigates vascular endothelial dysfunction and accelerates atherosclerosis. This hypothesis will be tested using gain-/loss-of-function strategies in both animal models and cultured cells. Aim 1 is to test the hypothesis that defective vascular autophagy increases mitochondrial fission in the development of endothelial dysfunction and atherosclerosis by characterizing the spatial and temporal dynamics of vascular autophagy, mitochondrial fission, and endothelial dysfunction in Akita mice (type 1 diabetes) and ob/ob mice (type 2 diabetes), determining whether inhibition of autophagy aggravates mitochondrial fission, endothelial dysfunction, and atherosclerosis in diabetic LDLr-/-/beclin1 heterozygous (beclin1+/-, with reduced autophagy) mice and their littermates, and examining whether enhanced endothelial autophagy lessens oxidative stress and improves endothelial function in diabetic Atg7 endothelium-specific transgenic mice (with enhanced autophagy). Aim 2 is to delineate the mechanisms by which diabetes-inhibited autophagy increases mitochondrial fission. This aim will test the hypothesis that diabetes induces mitochondrial fission by inhibiting autophagic degradation of DRP1. Proposed studies are significant as the completion of this proposal will provide the rationales for developing new therapeutics to prevent/treat cardiovascular complications in diabetes.


Rapid eye movement (REM) sleep is a behavioral state characterized by activation of the cortical and hippocampal EEG, rapid eye movements and muscle atonia. While some progress has been made in recent years in the effort to delineate 1) the locus of the pontine switching circuitry for REM sleep, 2) the neurotransmitters regulating REM phenomenon, i.e., muscle atonia, activation of the cortical and hippocampal EEG, and 3) how dysfunction of this circuitry may form the neuropathologic basis of REM sleep behavior disorder, major gaps remain in our knowledge. Recent work by our laboratory has revealed the presence of mutually inhibitory REM-off and REM-on areas in the mesopontine tegmentum that may form the neuroanatomical basis of the switching circuitry for REM sleep. These findings, which form the basis of the present research plan, posit a REM switching circuitry model that is analogous to an electronic 'flip-flop'switch. In this flip-flop switch arrangement, GABAergic REM-on neurons (located in the sublateraldorsal tegmental nucleus (SLD)) inhibit GABAergic REM-off neurons (located in the ventrolateral periaqueductal gray matter (vlPAG) and lateral pontine tegmentum (LPT)) and vice versa. Inside this pontine brainstem "switch" the REM-on area contains two populations of glutamatergic neurons, the first of which projects to the basal forebrain and regulates EEG components of REM sleep and the second which projects to the ventromedial medulla and spinal cord and regulates atonia during REM sleep. To demonstrate the critical role of glutamatergic SLD neurons in producing REM without atonia, we will selectively eliminate glutamatergic neurotransmission in the SLD by stereotaxically injecting an adeno-associated virus containing the gene for Cre recombinase (AAV-Cre) into the SLD of conditional knock-out mice with lox-P modified alleles of the vesicular glutamate transporter 2 (VGLUT2) genes. We will similarly eliminate GABAergic neurotransmission in the SLD and LPT by stereotaxically injecting AAV-Cre into mice with lox-P modified alleles of the vesicular GABA transporter (VGAT). Finally, we will examine the role of the ventromedial medulla in REM atonia by combining injections of orexin-saporin into rats and AAV-Cre injections into VGAT and VGLUT2 mice. Findings from the present proposal will provide a context for understanding the pathophysiologic mechanisms and etiological bases for a variety of sleep disorders, including REM sleep behavior disorder.


This research focuses on the central unsolved problem of psychiatric epidemiology, the probem of how to measure psychiatric disorders independently of treatment status in the general population. Its major aim is to test the sensitivity and specificity of the newly developed Psychiatric Epidemiology Research Interview (PERI) as a first-stage instrument to screen functional psychiatric disorders among adults in a demographically complex urban population. To this end, we will investigate PERI's relationship to other potential screening instruments on a sample of 600 adults from the general population and calibrate PERI on a sample of 500 psychiatric patients with DSM-III diagnoses of schizophrenia and the schizophreniform psychoses, affective disorders, various types of disorder under the heading of neurosis, anti-social personality, and other selected personality disorders. The research setting is Washington Heights, a section of Manhattan in New York City that surrounds Columbia Presbyterian Medical Center. It is a demographically complex urban area containing large percentages of blacks and Purerto Ricans as well as members of more advantaged ethnic groups. The results should provide a major step toward the development and testing of a two-stage procedure for case identification and classification. The findings should also be important for needs assessment and for the planning and evaluation of mental health services in Washington Heights.


Protein-protein interactions underlying molecular recognition are studied, utilizing monoclonal antibodies (Mabs) specific for the protein hen egg white lysozyme (HEL), a protein which has long served as a prototype for investigating the specificity of immune recognition. The X-ray structures of HyHEL-5 Fab complexed with 2 single site-directed mutants of HEL have been solved and refined: R68K which reduces the affinity of the complex by a factor of over 10 to the 3rd power, and R45K, which reduces affinity by only 10-fold. These results represent the first time structures have been obtained for 3 antigens, differing at only a single critical residue, complexed to the same antibody, and will provide valuable insight about the role of Arginine side chains in protein-protein interactions. We are beginning to define fundamental principles that will allow prediction of function from structure, principles that are critical to such applications as antibody design and vaccine development. We are also approaching the problem of vaccine development by investigating immunogenicity and protective epitopes in Shigella flexnerii. In addition, experiments with specific-pathogen- free and conventional BALB/c mice have established that plasmacytoma- refractory SPF BALB/cAnPt mice have naive T cell responses as well as associated restricted B cell responses. These results suggest a significant influence of antigenic exposure, particularly viral antigens, on development of the specificity repertoire and plasmacytomagenesis.


The feasibility of a device to enable precise and repeatable calibration of single-breath lung diffusion (DLCO) instruments will be investigated. The calibrator will enable the accuracy of DLCO instruments to be verified between pulmonary laboratories, allowing greater confidence in a diagnosis based on the single breath DLCO test. This study will verify the performance of a unique gas collection and mixing system which will allow generation of precise carbon monoxide (CO) and Helium (HE) gas fractions without using gas analyzers. This will allow production of a DLCO calibrator which will not require any calibration of its own.


Continued study of human fetal hemoglobin in normal and abnormal hematological conditions will be made to increase insight into the genetics and control of the linked beta, gamma, and delta structural genes. In particular, examination of cord bloods will be made to gain more information about a newly detected but poorly understood anomaly in the regulation of the gamma genes. These studies will be made in collaboration with Prof. T.H.J. Huisman of the Medical College of Georgia. It is hoped to complete the sequence studies on the catalases. If possible, studies of sequence in lactoperoxidase will be carried further.


We are requesting beam time to investigate how the presence of methane affects the structure of intermediate Q in M. tricosporium methane monooxygenase. We would like to determine when and how methane interacts with the diiron active site.


The major goal of this research project is to understand the molecular mechanisms by which steroid and peptide hormones regulate the expression of specific genes in differentiated cells. We will focus our attention on the chick transferrin and ovalbumin genes because these two egg white genes represent well-characterized examples of tissue-specific expression and hormonal regulation. The ovalbumin gene is expressed exclusively in the oviduct in response to estrogen and progesterone; however, tissue culture experiments have recently demonstrated that this steroid response is completely dependent on the additional presence of an insulin-related growth factor (IGF). The transferrin gene is expressed and regulated in at least three differentiated tissues: the oviduct, liver, and testis. Our specific objectives are to (1) study the IGF-mediated events which modulate the response of the ovalbumin gene to estrogen (2) identify the DNA sequences that interact with steroid receptors and regulate transcription of the transferrin and ovalbumin genes (3) explore the mechanisms which lead to gene commitment and tissue-specific expression of the transferrin gene. Our approach will utilize hormone-responsive oviduct cells in culture as an in vivo assay system for regulatory proteins and DNA sequences that can be introduced into the living cells by microinjection. The tissue-specific expression of the transferrin gene will be examined by introducing the chick transferrin gene into the mouse genome via oocyte microinjection. The expression and regulation of the chick gene in differentiated mouse tissues will then be examined. We expect these studies to further elucidate the basic mechanisms by which reproductive hormones control the developmental program of growth, differentiation, and specific gene expression in target tissues.