We have prepared highly purified tetanus toxin by both the cell extract and culture filtrate procedures. The amino acid composition of toxin prepared by both procedures was completed and agrees with that reported by other laboratories. The ordered structure of toxin prepared by both methods was estimated from their ultraviolet circular dichroic spectra and are similar. We are now preparing several large peptides from the toxin molecule. Digestion of toxin, cell extract or culture filtrate preparations with papain followed by gel filtration allows the separation of at least one large atoxic peptide and a fraction containing small peptides. We have separated filtrate toxin, by gel electrophoresis in the presence of appropriate reducing agents, into two large peptides referred to as heavy and light chains. Cell extract toxin must first be "nicked" by trypsin before it can be completely separated into similar peptides. These large peptides will now be treated with cyanogen bromide which should produce about 8 peptides and 5 peptides from the heavy and light chains respectively. Fragmentation with trypsin should yield an appreciably larger number of peptides. Peptides from each fragmentation procedure will be separated and sequenced. If a peptide is too large for overlap other enzymes will be used. The amino acid composition of peptides, prepared by cyanogen bromide and trypsin cleavage from both cell extract and culture filtrate toxin will be compared. This should detect any difference in the two toxins. There is no apparent reason to sequence toxin prepared by both methods. These experiments should allow us to compare the amino acid sequence of this unusual protein with its secondary structure and that of other nontoxic soluble proteins. It should also enhance our understanding of the molecular mechanism of the pathology of tetanus and ultimately aid in the prevention and care of this deadly disease. BIBLIOGRAPHIC REFERENCES: Robinson, John P., Pieklesimer, John B. and Puett, David. Tetanus Toxin: The Effect of Chemical Modifications on Toxicity, Immunogenicity and Conformation. JBC 250, 7435-7442, 1975. Wahba, Nagi, Felch, James W., Shih, J. Wai-Kuo and Hash, John H. The N,O-Diacetylmuramidase of Chalaropsis Species IV. Tryptic Peptides. J. Biol. Chem. 250, 3709-3712, 1975.


To further define potential abnormalities in parathyroid (PTH) secretion that may occur with aging.


These studies aim to identify new methods of gene transfer into hematopoietic stem cells using the genetic disease canine leukocyte adhesion deficiency (CLAD) as a model. We are using the canine model to identify new vectors for gene transfer and conditioning regimens to enable sufficient numbers of gene modified hematopoietic stem cells to engraft and reverse the disease phenotype. The canine form of this disease is an optimal model for these studies since: 1) the defect involves a membrane receptor on the surface of leukocytes, flow cytometry allows fascile detection and analysis of the number of gene corrected cells;2) low levels of gene-corrected cells result in reversal of the disease phenotype;and 3) studies in the canine model have been predictive of success in humans in the field of hematopoietic stem cell biology. The presence of a human counterpart to the canine disease allows the results from the animal model to be directly extrapolated to humans. The long-term objective of these studies is to develop strategies that will allow levels of expression of CD18 in hematopoietic cells of children with leukocyte adhesion deficiency (LAD) that are sufficient to reverse the clinical phenotype. We have utilized this model to test retroviral and foamy viral gene transfer into the bone marrrow cells of dogs. The foamy viral vector demonstrated greater efficacy and a more favorable integration profile than the conventional retroviral vectors. The foamy viral vector-treated dogs are being followed for the durability of the correction, and for any possible genotoxicity from the vector. To date, there has been no genotoxicity. These results represent the first successful use of a foamy virus (FV) vector to treat a genetic disease, and they suggest that foamy virus vectors will be effective in treating human hematopoietic diseases. We are currently testing third-generation foamy viral vectors and lentiviral vectors with cellular promoters and lentiviral vectors with cellular rather than viral promoters in our canine model.


Variation in bitter taste perception may play a key role in diet selection, which has an uncontestable effect on human health. Although bitter compounds are often associated with toxic substances a large number of nutritionally significant food sources contain bitter phytochemicals (e.g., broccoli [1], spinach [2]) many of which can have beneficial value [1, 3] Understanding how bitter taste is perceived and detected is of great importance to understanding diet selection [4] and increasing the acceptability of pediatric medicines [19]. Under normal feeding and drinking conditions taste compounds must inescapably mix with saliva before reaching their receptor targets but very little work has been done examining how salivary proteins modulate taste stimuli and alter bitter taste perception. Seventy percent of all salivary proteins make up a class of proteins referred to as proline-rich proteins (PRPs). PRP production is induced in rats by dietary exposure to tannic acid, a class of plant compounds that animals, including humans, regularly consume. It has been hypothesized that PRPs alter the acceptability of tannic acid by binding to tannins and thereby reduce the perceived intensity of the tannic acid solution [10, 11]. There is evidence to suggest that these proteins may have a similar relationship with other bitters. They are produced in close proximity to bitter taste receptors [13]. Genes for PRPs and bitter taste receptors are interspersed on the same chromosome [5] and lastly, gene linkage studies have implicated a role for PRPs bitter acceptance in humans and mice [15-18]. Specific Aim 1 will establish if long term exposure to bitter compounds can increase the expression of PRPs and establish whether oral exposure is necessary and sufficient to cause induction. Saliva will be analyzed for PRP content before and after exposure to diets adulterated with bitter compounds, including quinine and tannic acid. To isolate the site of exposure necessary for PRP induction rats will also be given tannic acid via either oral or gastric exposure. The ability or inability to induce PRP production is not sufficient to predict interactios between the proteins and the bitter tastants. Therefore, Specific Aim 2 will establish if PRP induction can alter the detection threshold for and superthreshold responsiveness to multiple bitter stimuli. Rats will be used to derive psychophysical detection curves of several bitter tastants in the presence and absence of PRPs. Likewise, responsiveness in the suprathreshold range will be assessed in the presence and absence of PRPs with a series of brief-access tests. Collectively, these experiments will test the hypothesis that PRPs alter taste sensitivity for bittr taste stimuli and ultimately blunt unconditioned taste avoidance responses to these compounds. Together these aims could inform methods for increasing the palatability of healthy phytochemicals and pediatric medicines.


The Florida Pediatric Community Clinical Oncology Program is a consortium of four Florida hospitals (All Children's Hospital, Jacksonville Wolfson Children's Hospital, Orlando Regional Medical Center, and Sacred Heart Children's Hospital) which seeks to affiliate with the Pediatric Oncology Group as its research base. These four hospitals complement the three major university teaching hospitals which are also Pediatric Oncology Group members and, in total, comprise the Florida Association of Pediatric Tumor Programs. The goal of the Florida Pediatric CCOP is to make available the latest advances in cancer care to patients in Florida through participation in clinical trials. The CCOP participants will utilize an existing Statewide Patient Information Reporting System as a log of all patients seen in the state. Patients will be registered on POG protocols through the POG Statistical Office which is co-located with the Central Office of the Florida Pediatric CCOP. This unique arrangement affords the opportunity to ensure that the majority of eligible patients are registered on appropriate POG protocols. An excess of 45 patient registrations are anticipated per year through the four hospitals which comprise the Florida Pediatric CCOP. POG quality control procedures will be extended to include the Florida CCOP, ensuring compliance with protocols and external review of pathology, radiotherapy, surgery, and chemotherapy. The Florida Pediatric CCOP extends the latest treatment advances through the opportunity to participate in clinical trials to a significant segment of the Florida population.


DESCRIPTION (appended verbatim from investigator's abstract): The guidance of axons to their targets during embryonic development is an intricate process requiring remarkable accuracy. The Eph family of receptor tyrosine kinases, and their ligands, the ephrins, proscribe many of these early axon guidance events. It is our hypothesis that oligomerization of these molecules controls both their signaling and their posttranslational modification, and in doing so confers upon them the accuracy and specificity of function necessary to shape the nervous system. Aim 1 will determine the mechanism of ephrin B2 oligomerization, and its importance for receptor activation. Analytical ultracentrifugation will be used to determine whether ephrin B2 undergoes homophilic binding reactions. Further, an ephrin B2 binding protein, ABP, will be assayed for its ability to oligomerize ephrin B2. We will also use our newly developed system, fusing ephrin B2 to the lac repressor, to produce dimeric and tetrameric forms of the ephrin, which will be directly compared for their capacity to activate receptors. Aim 2 and aim 3 will determine whether oligomerization of ephrin B2 drives its phosphorylation and degradation, respectively. The phosphorylation state of ephrin B2 will be measured after being clustered by mechanisms independent of the receptors. Ephrin B2 undergoes proteolysis in chick embryo fibroblasts. The proteolysis appears to be dependent upon clustering. We will determine whether dimers or tetramers undergo proteolysis. We will also use mutagenesis of ephrin B2 to determine which sequences are required for proteolysis. The fourth aim will determine the importance of the biochemical interactions elucidated in the first three aims to the regulation of axon guidance. Derivatives of ephrin B2, designed to be resistant to posttranslation modifications, will be compared for their capacity to repel motor axons. In vitro studies will use the stripe assay, which tests the preference of an axon to extend over alternating stripes, containing, or lacking the appropriate chemorepellant. In vivo studies, will use the assay we have established to observe any deviations from normal of the motor nerve pathways, following ectopic expression of the ephrin B2 derivative from a retroviral vector in chick embryos. Through these comparative studies of ephrin function we will determine the post translation mechanisms which regulate their axon guidance activity.


The University of Kentucky, being a land grant and comprehensive institution, has a tradition of excellence in interdisciplinary research. Such an academic environment will allow study for the overall theme of our SBRP research, which focuses on the toxicology of Superfund chemicals and how health effects of exposure can be modulated by both intrinsic and extrinsic factors, namely genetics and nutrition, respectively. The investigators will integrate five biomedical and two non-biomedical projects to concentrate on chlorinated organics (e.g., polychlorinated biphenyls, trichloroethylene) as model toxins. Chlorinated organics are prevalent in most Superfund sites, including those found in Kentucky. The preliminary findings suggest that nutrition and dietary habits can markedly influence mechanisms of toxicity of the above-mentioned Superfund chemicals. Thus, a major objective of our SBRP is to explore the paradigm that nutrition can modify Superfund chemical toxicity. The investigators hypothesize that highly refined diets, i.e., diets high in fats or calories and low in fruits and vegetables (antioxidants), are associated with an observed national epidemic in chronic diseases, and that populations associated with such dietary habits are more prone to Superfund chemical insult. Biomedical projects will focus on chronic diseases such as cardiovascular disease, cancer, obesity, hypertension, and diabetes. In addition, non-biomedical projects will explore novel techniques for both remediation (detoxification) and biosensors associated with PCBs and other chlorinated organics. Results from the interdisciplinary research will be utilized for informative/educational, technology transfer, training, policy and translational purposes as part of the objectives of the Research Translation, Community Outreach, and Training Cores. Nutrition may be the most sensible means to develop primary prevention strategies of diseases associated with many environmental toxic insults. Thus, the research may lead to novel dietary recommendations at the national level for populations at risk, i.e., people residing near Superfund sites.


Support through the K23 Award will provide the Candidate the necessary time, resources, and mentorship to integrate his clinical interests in substance use disorders with his experience in patient-oriented emergency medicine research. His long-term career plan involves using the knowledge gained from the tobacco smoking and nicotine dependence studies outlined in this proposal as a model for future studies of other substances that are commonly abused by emergency department patients, such as alcohol, cocaine, opiates, and "club" drugs. The emergency department (ED) represents a significant portion of all healthcare delivered in the United States, with 100 million ED visits annually. Approximately 50% of the patients seen in the ED have no source of regular outpatient care, and the ED acts as a healthcare "safety net" for people with inadequate healthcare insurance. Healthy People 2010 and other government publications have encouraged screening and interventions for tobacco smoking to be conducted during all healthcare encounters, regardless of the setting. However, the extant literature, as well as the Candidate's own pilot studies, indicate that such practices are rare in ED settings. This is true despite the fact that many of the populations shown to be most recalcitrant in their smoking, such as the economically disadvantaged and minorities, are more likely to use the ED for their acute and primary healthcare. If smoking is not addressed with these patients in the ED, it may not get addressed at all. Numerous studies have shown that brief, ED-based interventions for problem behaviors, like alcohol and substance abuse, are both feasible and effective. The noted lack of ED-based screening and intervention for tobacco smoking and nicotine dependence appears due mainly to a lack of relevant research and advocacy rather than a lack of need or barriers prohibiting such practices. The Candidate's career development plan will incorporate needed course work, field experience, and mentorship in data management, study design, statistics, and tobacco research methodology. The research plan concentrates on ED-based tobacco research, beginning with a surveillance study and culminating in a Stage 1A/B behavioral therapy development study of a Behavioral Change Counseling intervention based on principles of Motivational Interviewing. The research plan includes preparing and submitting an R01 in Year 2.


Two new and unusual products of V(D)J recombination in lymphoid cells have been identified. Normal recombination joins the two signal sequences that identify the rearrangement site and, in the complementary reaction, joins the fragments of coding sequences that flank the signals. In extrachromosomal plasmid substrates, these products are found, but there is also a class of "hybrid joints" that link one signal to the coding flank of its partner signal. A second class of "open and shut" events rejoins each signal to its own flank, and is identifiable because of nucleotides lost and/or added at the joint. Both these products supply evidence that DNA strand alignment is not precisely specified in V(D)J recombination. A specific defect of V(D)J recombination has been identified in lymphoid cell lines derived from mice with the severe combined immune deficiency (scid) mutation. In extrachromosomal substrates, these cells make signal joints (though with greater imprecision than normal) but are unable to make coding joints. This failure appears sufficient to explain the absence of functional B and T cells in scid mice.


An initial objective of the proposed research is the elucidation of the complete genomic sequences of the retinoid-binding proteins (RBPs), cellular retinol-binding protein (cRBP), cellular retinol-binding protein (cRBP), cellular retinoic acid-binding protein (cRABP) and membrane-associated retinoic acid-binding protein (mRABP). RBPs will be purified to homogeneity and their amino-terminal ends sequenced. The amino acid sequences of these proteins and a codon frequency derived from a known, highly homologous protein will be the basis for the synthesis of several oligonucleotides. These oligonucleotides will be used as primary probes in screening complete mouse cDNA libraries; the derived cDNAs will be used to identify the complete genomic clone of cRBP, cRABP, and mRABP. Retroviruses will be constructed that contain either a normal or inverted (anti-sense) genomic sequence (from or near the amino terminal end extending 3 prime to include any primary intron) of the resolved RBP genes. These retroviruses will be infected into cell lines that have been selected for (i) their expression of RBPs cRABP+, cRBP or cRABP+/cRBP+ and (ii) their ability to respond to relatively low concentrations of retinoids retinoic acid+, or retinol+. Changes in cell characteristics (e.g., proliferation, transformation, terminal differentiation) in the presence and absence of (i) an additionally expressed RBP genomic sequence or anti-sense message; (ii) retinoids, and (iii) the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, will also be evaluated. These experiments will (i) provide the complete amono acid as well as genomic sequence for cRBP, cRABP and MRABP; (ii) establish the requirement for RBPs in the control of cytodifferentiation, (iii) allow characterization of RBP secondary structures; and (iv) provide data for the prospective development of anti-retinoid and anti-RBP compounds that may be of significant value in chemoprevention.


Multiple system atrophy (MSA) is a progressive, degenerative neurological disorder characterized by parkinsonism, ataxia & dysautonomia. The cardinal pathological feature of MSA is the presence of glial cytoplasmic inclusions composed of alpha-synuclein (SYN) in oligodendrocytes. Recent studies suggest that abnormal SYN accumulation in neurons & glia leads to cellular dysfunction & neurodegeneration. During the previous funding period we developed in vitro & in vivo models of MSA showing that mitochondrial damage & hyperphosphorylated SYN aggregate generation may contribute to the pathogensis of MSA. However, mechanisms by which these pathways promote oligodendrogial dysfunction & neurodegeneration are unclear. In this renewal we will investigate the role of mitochondrial dysfunction in SYN phosphorylation & toxicity. Our central hypothesis is that oxidative stress due to mitochondrial dysfuntion may promote G-protein coupled receptor kinase (GRK) activation & toxic SYN phosphorylation. The main objective is to investigate neurodegeneration in MSA-like SYN transgenic (tg) models to determine if reducing SYN accumulation represents a therapeutic strategy for MSA. Aim 1. In order to determine the role of hyperphosphorylated SYN accumulation in oligodendrocytes in the mechanisms of neurotoxicity, we will analyze SYN accumulation & neurodegeneration in myelin basic protein (MBP)-SYN tg mice expressing wild-type (wt) human SYN or a nonphosphorylatable SYN mutant (S129A). MBP-SYNwt tg mice will be crossed with GRK2- or GRKS-deficient mice & MBP-SYNwt tg mice & MBP-SYN(S129A) tg mice will receive intra-cerebral infections with lentivirus expressing GRK2 or GRK5 under a oligodendroglial specific promoter (MBP). Aim 2. In order to determine the role of mitochondrial dysfunction & oxidative stress on GRK activation & SYN phosphorylation, MBP-SYN (wt and S129A) mice & oligodendroglial cells will be challenged with 3- nitropropionic acid. Aim 3. In order to determine if neuronal impairments in MSA models can be ameliorated by reducing SYN aggregation or inhibiting GRKs, MBP-SYN wt tg mice will be treated with rifampicin or GRK blockers. Behavioral performance, neurodegeneration, SYN oligomerization & phosphorylation & GRK activity will be assessed.


Apoptosis now is recognized to be an important mechanism of cell death during development and after injury. In neurons, it is well known that growth factor withdrawal can produce apoptotic cell death, both in vivo, and in vitro. Recently, our lab has shown that ionizing radiation (IR) induces apoptosis in post-mitotic dorsal root ganglion (DRG) neurons in vitro. The ability of IR to induce apoptosis dramatically declined over the first 3 weeks in culture to the point where 21 day old DRG neurons did not undergo radiation-induced apoptosis. Previous work in non-neuronal cells has shown that apoptosis occurring as a result of IR-produced DNA damage requires induction of p53. Increased expression of p53 results in activation of a number of downstream genes, including p21, GADD45, and mdm-2, but recent evidence suggests that it is the interaction between p53 and members of the bc1-2 gene family (including bc1-2, bc1-x(L), and bax) that determines whether apoptotic cell death will occur. The experiments described in this proposal will specifically address the individual roles of, and interactions between, p53, bax, bc1-2, and bc1- x(L) in regulating the apoptosis program in post-mitotic DRG neurons exposed to ionizing radiation. We will also examine the roles that these proteins play in producing the temporal change in sensitivity of DRG neurons to IR. By identifying the interactions critical for determining whether these neurons survive or die following exposure to IR, we may be able to design rational strategies for protecting neurons from other potentially lethal insults.


We propose to investigate the interface between EcoRI endonuclease and its cognate DNA recognition sequence by solution biochemical methods. The research is divided into two complementary efforts which will investigate the enzyme-DNA interface from both sides, i.e., we propose to: 1) Localize the points on the DNA which interact with the enzyme by determining the effects of base substitutions within and immediately surrounding the cannonical hexanucleotide, GAATTC. We will measure the effects of sequence changes on the enzyme kinetics, dissociation constants and the ability of the endonuclease to protect bases from Maxam-Gilbert reagents. 2) Localize points on the endonuclease which interact with the DNA by determining which amino acid residues can be: (A) Crosslinked to synthetic oligonucleotides containing the photo-activatable base analog 5-bromodeoxyuridine at unique points in the recognition sequence. (B) Protected by the DNA from reacting with reagents which modify exposed amino acid residues. We will also seek to determine whether or not the polypeptide chain is comprised of domains, one of which retains most of the recognition specificity. These experiments make extensive use of techniques developed in this laboratory for specifically binding DNA containing the recognition site to the enzyme under conditions which abolish catalysis. The ultimate goal of this research is to elucidate molecular mechanisms of sequence specific DNA-protein interactions.


Project Summary/Abstract Despite intensive national efforts to address childhood obesity, the epidemic persists. Recognizing the challenges, the National Institutes of Health (NIH) and the Institute of Medicine (IOM) have called for a systems-oriented approach to address childhood obesity. We hypothesize that among low-income, ethnically diverse overweight and obese children, aged 2-12 years, a systems approach to child obesity incorporating secondary prevention programs embedded within primary prevention will reduce body mass index (BMI) z-scores compared to primary prevention alone. Intervention strategies will be adapted from the portfolio of evidence-based programs developed by study investigators. An assets-based assessment will be conducted with research and community partners in disadvantaged neighborhood catchment areas in Austin and Houston; data will inform integration of intervention strategies and provide a structure for program sustainability. Our specific study aims are: Aim 1: To implement and evaluate a primary prevention obesity program in low-income, ethnically diverse catchment areas in Austin and Houston. Baseline and 2-year follow-up data on the prevalence of child overweight, risk factors, and the availability of healthcare services and community programs will be collected in 2 demonstration (n=1614) and 2 control catchment areas (n=1614). Parent, child and community data will provide matched comparison data to assess the efficacy of the primary prevention intervention. Aim 2: To implement and evaluate the efficacy of a systems approach to child obesity on reducing obesity by embedding a 12-month family-based secondary prevention program within the community primary prevention program. The secondary prevention weight management program will be conducted with overweight/obese children and their families in the primary prevention catchment areas. Families of overweight/obese children (total n=576), aged 2-12 years, will be randomly assigned to either the 12-month secondary prevention program (experimental) or prevention program alone (control), stratified by age (2-5, 6-8, and 9-12 y). Outcomes include child BMI z-score, quality of life measures, and program use indicators. Aim 3: To quantify the incremental cost-effectiveness of the 12-month family-based secondary prevention program relative to primary prevention alone for child obesity. Activity Based Costing methods will be used to quantify the incremental cost of delivering the secondary prevention program relative to primary prevention. Public Health Relevance: If this research trial is proven efficacious and cost-effective, the demonstration project could be disseminated widely to address obesity among underserved, at risk children. Program sustainability will be ensured through active involvement of community partners within healthcare, childcare, schools and community sectors.


The viral Gag proteins control many aspects of the HIV-1 replication cycle. The Gag precursor drives the assembly of virus particles in the infected cell, and, through putative interactions with the transmembrane envelope (Env) glycoprotein gp41, directs Env incorporation into virus particles. Following infection, the Gag proteins play a central role in uncoating and assist in the reverse transcription process. The assembly of type C retroviruses and lentiviruses, including HIV-1, takes place at the plasma membrane (PM). It is well established that this process is promoted by Gag proteins; however, the mechanisms by which assembly is specifically targeted to the PM remain to be determined. Recent studies have suggested that the PM contains microdomains with distinct protein and lipid compositions. One type of microdomain, the lipid raft, is enriched in sphingolipids and cholesterol and can be isolated as detergent-resistant membrane (DRM). Rafts have been shown to play essential roles in a variety of biological processes, often by acting as a target site for proteins involved in a common pathway. Last year, we reported that a large portion of membrane-bound Gag was recovered in DRM (Ono and Freed, PNAS, 2001). To address the specificity of Gag-raft association, we examined the kinetics of this association. We found that DRM association of Gag occurred more slowly than binding of Gag to membranes, suggesting that HIV-1 Gag is specifically targeted to PM rafts after it binds membrane. We observed that the determinant for raft association maps to the N-terminus of Gag, but that Gag-Gag interactions facilitate or stabilize raft association. To address the physiological relevance of Gag-raft association, we analyzed the impact of cholesterol depletion, which disrupts raft structure, in HIV-1-producing cells. We found that cholesterol depletion markedly and specifically reduces virus release. Moreover, virus released from cholesterol-depleted cells displays reduced infectivity. Interestingly, cells expressing a mutant Gag with substitutions in the p6 late domain motif show no reduction in virus release. These results identify the association of Gag with PM rafts as an important step in HIV-1 production, and suggest possible links between rafts and late steps in virus assembly. Recent work has been aimed at defining the mechanism by which raft disruption impairs virus production. These results also suggest that cholesterol depletion prevents the efficient binding of Gag to membrane. The p6 domain of HIV-1 is located at the C-terminus of the Gag precursor protein Pr55Gag. Previous studies indicated that p6 plays a critical role in HIV-1 particle budding from virus-expressing HeLa cells. We performed a detailed mutational analysis of the N-terminus of p6 to define sequences required for efficient virus release. By examining the effects of p6 mutation in biological and biochemical analyses and by electron microscopy, we determined the role of p6 in particle release in a panel of cell lines as well as in peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM). The results (Demirov et al., J. Virol. 2002) indicate that: i) the highly conserved P-T/S-A-P motif located near the N-terminus of p6 is remarkably sensitive to change; even conservative mutations in this sequence induce profound virus-release defects in HeLa cells. ii) Single and double amino acid substitutions outside the P-T/S-A-P motif have no significant effect on particle release. iii) The introduction of stop codons one or two residues beyond the P-T-/S-A-P motif blocks virion release, whereas truncation four residues beyond P-T/S-A-P has no effect on particle production in HeLa cells. iv) Though the effects of p6 mutation on virus replication in T-cell lines are cell-type dependent, even in T-cell lines in which p6 mutations block virus replication, these changes have little or no effect on particle release. v) p6-mutant particles produced in T-cell lines exhibit a defect in virion-virion detachment, resulting in the production of tethered chains of virions. vi) Transient heterokaryons produced between HeLa cells and a T-cell line display a T cell-like phenotype with respect to the requirement for p6 in particle release, indicating that the activity that suppresses the release defect phenotype is dominant in this cell system. vii) Finally, in primary MDM, mutation of p6 results in marked defects in both particle release and virus replication. These results reveal a strong cell-type dependent requirement for p6 in virus particle release. A variety of lines of evidence suggest a connection between retroviral L domains function and the host ubiquitination machinery. Recently, it was demonstrated that the product of tumor susceptibility gene 101 (TSG101), which contains at its N-terminus a domain highly related to ubiquitin conjugating (E2) enzymes, binds HIV-1 Gag in a p6-dependent fashion. We examined the impact of overexpressing the N-terminal region of TSG101 on HIV-1 particle production. Intriguingly, we observe that this domain (referred to as TSG-5') potently inhibits virus production (Demirov et al., PNAS 2002). Examination of cells coexpressing HIV-1 Gag and TSG-5' by electron microscopy reveals a defect in virus budding very reminiscent of that observed with p6 L domain mutants. In addition, the effect of TSG-5' is dependent upon an intact p6 L domain; the assembly and release of virus-like particles produced by Gag mutants lacking a functional p6 PTAP motif is not significantly affected by TSG-5'. Furthermore, assembly and release of murine leukemia virus and Mason-Pfizer monkey virus are insensitive to TSG-5'. TSG-5' is incorporated into virions, confirming the Gag/TSG101 interaction in virus-producing cells. Mutations that inactivate the p6 L domain block TSG-5' incorporation. These data demonstrate a link between the E2-like domain of TSG101 and HIV-1 L domain function, and raise the possibility that TSG101 derivatives could serve as potent and specific inhibitors of HIV-1 replication by blocking virus budding. To define the mechanism by which TSG-5' inhibits virus release, we investigated the importance of TSG-5'/Gag interaction in the virus release inhibition. We observed that a mutation in TSG-5' that prevents Gag from interacting with this truncated protein eliminates its ability to disrupt particle release. These results suggest that direct binding to Gag, rather than disruption of cellular sorting machinery, is responsible for the ability of TSG-5' to block budding. We also investigated the effect of overexpressing longer forms of TSG101, including the full-length protein, on virus budding. We observe that overexpressing other truncated mutants as well as the full-length protein, disrupt budding. Interestingly, the ability of full-legth TSG101 to inhibit virus release when overexpressed does not require binding to Gag. Rather, as indicated by confocal microscopy experiments, overexpressing the full-length protein intereferes with the cellular endosomal sorting machinery. To complement our studies on HIV-1, we are investigating the role that TSG101 and other host factors play in the budding of a diverse array of retroviruses, including murine leukemia virus, human T cell leukemia virus, bovine leukosis virus, Mason-Pfizer monkey virus, and equine infectious anemia virus.


We have documented age-related and degeneration-related changes in the metabolism of collagens,[unreadable] proteoglycans, and matrix-degrading enzymes in the human intervertebral disc. Our studies (i) confirmed the[unreadable] pivotal roles that cytokines and growth factors play in intervertebral disc degeneration and repair, (ii)[unreadable] demonstrated that certain growth factors inhibit the autocrine production of cytokines and that cytokine[unreadable] inhibitors can change basal levels of synthesis of matrix molecules and metalloproteinases, and (iii) found[unreadable] that biglycan was expressed in greater amounts than other small proteoglycans during late stages of disc[unreadable] degeneration and in the discs of old donors and plays an important regulatory role in matrix homeostasis.[unreadable] Our data suggest that an intricate balance of growth factors, cytokines and regulatory molecules is important[unreadable] for the maintenance of tissue homeostasis. Over the next five years, we propose to investigate the[unreadable] mechanisms of action and the interplay between growth factors, cytokines and various regulatory molecules[unreadable] in order to determine their respective roles in tissue homeostasis and their potential use in promoting tissue[unreadable] repair. Hypothesis 1: The effects of growth factors on the intervertebral disc are regulated by the production[unreadable] of cytokines by intervertebral disc cells and by the presence of inhibitory factors; growth factors have a[unreadable] negative feed back on the production of these cytokines. Hypothesis 2: The regulatory mechanisms of[unreadable] matrix homeostasis by cytokines and growth factors vary at different stages of disc degeneration because of[unreadable] changes in the cellular microenvironment in situ within the disc. The response to cytokines and growth[unreadable] factors and the production and activation of matrix-degrading enzymes will be different at advanced stages[unreadable] of disc degeneration. Hypothesis 3: The inhibition of cytokine action by cytokine blockers and the inhibition[unreadable] of cytokine production by growth factors can delay or reverse the degenerative status of intervertebral disc[unreadable] cells in the cellular microenvironment that can be observed in the early and middle stages of disc[unreadable] degeneration. Low back pain is responsible for enormous human suffering, high health care costs and[unreadable] significant socioeconomic losses. Although the etiology of back pain is often unknown, the intervertebral[unreadable] disc is a significant source of back problems. The results from this study will advance the field of biological[unreadable] treatment and tissue engineering for intervertebral disc degeneration.


Project Summary/Abstract The overall goal of this proposal is to generate, maintain, and distribute a new collection of 1,500 custom engineered Drosophila melanogaster stocks that will allow versatile manipulation of genes that are highly conserved in humans but currently lack an available mutant allele. Utilizing cutting edge CRISPR technology, this collection or 'kit' will represent a powerful new set of tools that will benefit virtually all Drosophila geneticists; as such it is likely that there will be a high demand for this resource for many years to come. Many of the most important advances in our understanding of human development have come from studies using the fruit fly as an animal model system. Since many parallels exist between Drosophila and mammals in terms of the underlying molecular mechanisms controlling biological processes, knowledge gained from research in Drosophila can be either directly applied or readily adapted to understanding human biology and disease. During Phase I of this project, we optimized the conditions for CRISPR/Cas9 mediated insertion of a custom CRIMIC cassette and correctly targeted 55 out of 63 genes attempted. This high rate of success (87%), indicates that we are perfectly poised to scale up our efforts to a larger set of genes. In this Phase II proposal, we aim to generate CRIMIC targeted alleles for an additional 1,500 conserved genes that currently lack an available mutant allele. The primary benefit of such a collection is that the CRIMIC insertion not only generates a strong loss-of-function or null allele of the targeted gene, but can also serve as a docking site for further engineering, such as transcript or protein tagging or creating point mutations that can be used to model human disease pathology. In coordination with other existing collections and the ongoing Gene Disruption Project (GDP), our Specific Aims are to: Aim 1. Select 1,800 gene targets and construct 1,800 gRNA/donor pairs Aim 2. Generate and validate a collection of 1,500 targeted gene stocks Aim 3. Create and maintain an online searchable database for targeted genes Harnessing the benefits of new technologies, this collection will fully complement existing collections and the ongoing GDP effort. Once generated, these combined resources will cover the vast majority genes in the Drosophila genome that are directly relevant to human disease.


1) Evolutionary classification of P-loop NTPases As a part of my ongoing research on the P-loop NTPases, which constitute the largest set of monophyletic protein domains in most proteomes, we completed the evolutionary classification of the AAA+ ATPases and the P-loop kinases. These studies helped us to identify the early diversification events in the history of these proteins. We provide evidence that the P-loop NTPases first differentiated into two classes, namely the KG class (Kinase, GTPase class) and the ASCE class (Additional strand conserved glutamate) that includes the AAA+, RecA-like, PilT/VirD4-like and ABC ATPases. These classes had several representatives that could be traced back to the last universal common ancestor of all life forms suggesting that they had undergone a vast radiation even before that stage. Hence they provide insights into some of the earliest aspects of protein evolution. 2) Analysis of the novel protease families A multi-pronged strategy including extensive sequence searches, structural modeling, and analysis of contextual information extracted from domain architectures, genetic screens, and large-scale protein-protein interaction analyses was employed to predict previously undetected components of the eukaryotic ubiquitin (Ub) signaling system. Two novel groups of proteins that are likely to function as de-ubiquitinating and de-SUMOylating peptidases (DUBs) were identified. The first group of putative DUBs, designated PPPDE superfamily (after Permuted Papain fold Peptidases of DsRNA viruses and Eukaryotes), consists of predicted thiol peptidases with a circularly permuted papain-like fold. In addition to eukaryotic proteins, the PPPDE superfamily includes predicted proteases from several groups of double-stranded RNA viruses and one single-stranded DNA virus. The apparent recruitment of DUBs for viral polyprotein processing seems to represent a common theme in evolution of viruses. The second group of putative DUBs identified in this study is the WLM (Wss1p-like metalloproteases) family of the Zincin-like superfamily of Zn-dependent peptidases, which are linked to the Ub-system by virtue of fusions with the UB-binding PUG (PUB), Ub-like, and Little Finger domains. More specifically, genetic evidence implicates the WLM family in de-SUMOylation. If validated experimentally, the WLM family proteins will represent the first case of a Zincin-like metalloprotease involvement in Ub-signaling. 3) Evolution of the nuclear membrane and nuclear pore complex The presence of a distinct nucleus, the compartment for confining the genome, transcription and RNA maturation, is a central (and eponymous) feature that distinguishes eukaryotes from prokaryotes. Structural integrity of the nucleus is maintained by the nuclear envelope (NE). A crucial element of this structure is the nuclear pore complex (NPC), a macromolecular machine with over 90 protein components, which mediates nucleo-cytoplasmic communication. Given the indispensability of these structures for nuclear function, the natural history of the nucleus can only be understood in terms of the origin and subsequent evolution of NE and NPC components. We investigated the provenance of the conserved domains found in these perinuclear proteins and reconstructed a parsimonious scenario for NE and NPC evolution by means of comparative-genomic analysis of their components from the available sequences of 28 sequenced eukaryotic genomes. We show that the NE and NPC proteins were tinkered together from diverse domains, which evolved from prokaryotic precursors at different points in eukaryotic evolution, divergence from pre-existing eukaryotic paralogs performing other functions, and de novo. It is shown that several central components of the NPC, in particular, the RanGDP import factor NTF2, the HEH domain of Src1p-Man1, and, probably, also the key domains of karyopherins and nucleoporins, the HEAT/ARM and WD40 repeats, have a bacterial, most likely, endosymbiotic origin. The specialized immunoglobulin (Ig) domain in the globular tail of the animal lamins, and the Ig domains in the nuclear membrane protein GP210 are shown to be related to distinct prokaryotic families of Ig domains. This suggests that independent, late horizontal gene transfer events from bacterial sources might have contributed to the evolution of perinuclear proteins in some of the major eukaryotic lineages. Snurportin 1, one of the highly conserved karyopherins, contains a cap-binding domain which is shown to be an inactive paralog of the guanylyl transferase domain of the mRNA-capping enzyme, exemplifying recruitment of paralogs of pre-exsiting proteins for perinuclear functions. We infer an autogenous scenario of nuclear evolution in which the nucleus emerged in the primitive eukaryotic ancestor (the ?prekaryote?) as part of cell compartmentalization triggered by archaeo-bacterial symbiosis. A pivotal event in this process was the radiation of Ras-superfamily GTPases yielding Ran, the key regulator of nuclear transport. A primitive NPC with approximately 20 proteins and a Src1p-Man1-like membrane protein with a DNA-tethering HEH domain are inferred to have been integral perinuclear components in the las common ancestor of modern eukaryotes.


Male and female 10-12 year old children of men with Alcohol Use Disorder (AUD) (high risk) and control fathers (low risk) will be studied at baseline and on two follow-ups (ages 12-14 and 16) to developmentally clarify liability for an Alcohol Use Disorder. In addition to obtaining behavioral measures, the project will measure cognitive, electrophysiological and eye movement processes using a multitrait multimethod (MTMM) paradigm. High risk subjects (N=400) are hypothesized to perform deficiently compared to low risk subjects (N=400) on measures that differentially reflect prefrontal cortex functioning and to be comparable on non-prefrontal measures. Within the developmental framework encompassing transition from late childhood to mid-adolescence, it is hypothesized that the manifest behavioral dysregulation (due to prefrontal cortex impairment) is associated with disruptive parent-child interactions and affiliation with deviant peers. The resulting maladjustment, namely, an externalizing disposition, predisposes to precocious alcohol exposure, early age intoxication and, by mid-adolescence, heavier alcohol involvement. In effect, individual liability (dysregulation explicable by prefrontal cortex dysfunction), via interaction with the family and peer environments, biases the developmental trajectory toward an externalizing adjustment style (proximal outcome) and problem alcohol consumption (distal outcome). Confirmation of the individual basis of AUD liability would point to innovative approaches to prevention by identifying the specific components of risk which are subsumed within well-known functional neuroanatomical systems. Furthermore, this project will afford the opportunity to elucidate AUD etiology as the culmination of person- environment interactions.


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. We have proposed a role for anxiety in the initiation and maintenance of SIB. To test this hypothesis directly, we investigated hormonal and behavioral responses of adult male rhesus macaques to various doses of the benzodiazepine (BDZ) receptor inverse agonist FG 7142.


It is proposed to develop a sensitive and specific method of quantitative radioimmunoassay to measure gut-related autoimmune phenomena in Lewis-strain rats induced by highly purified syngeneic and xenogeneic intestinal mucins. Tritiated ovine salivary mucin (OSM) will be studied first in its reaction with antibodies raised in Lewis-strain rats to Lewis rat intestinal sialylmucins. After parameters of the reaction have been delineated, the technique will be used to study the time-course of antibody development in Lewis rats immunized with xenogeneic and syngeneic sialylmucins. Differential radioimmunoassays will be used to determine the various classes and subclasses involved in the immune reactions. Adsorption techniques coupled with radioimmunoassays will be used to determine the various end groups of mucins represented in the syngeneic rat antiserum reactions. Dose-response characteristics of the antibody responses will be studied by dosage manipulation one at a time, or antigen, of adjuvant (Mycobacterium butyricum), and of ancillary adjuvant (Bordetella pertussis) at fixed doses of the other two components. The requirements of T-cell dependency will be assessed in Lewis rats that have been thymectomized as adults, irradiated with 900 rads whole-body, reconstituted with 10 to the 7th power bone-marrow cells from normal Lewis rat donors, and challenged with sialylmucin preparation. Finally, it will be determined, by the method of passive transfer into normal recipients of donor lymphocytes, sensitized to sialylmucin, whether an experimental inflammatory bowel disease can be induced and whether coproantibodies and/or humoral antibodies to sialylmucins can be detected in the recipients by the proposed method of radioimmunoassay.


We will study the metabolism and action of methyl-n-amylnitrosamine (MNAN) in the esophagus as follows: (1) We will examine the production of hydroxy and keto metabolites of MNAN in freshly removed esophagus, forestomach and other tissues of rodents. We will complete studies of changes with age of this metabolism in key tissues of suckling rats and hamsters, determine whether these changes are due to intrinsic or extrinsic factors, examine effects of agents that induce individual P-450s, and determine configurations of the hydroxy-MNAN metabolites. (2) We will develop systems for culturing adult rat esophagus, while still maintaining MNAN metabolism. We will study MNAN metabolism in cultures of newborn and adult rat forestomach, hamster forestomach and hamster esophagus, and by freshly incubated adult and fetal human esophagus, and study effects of inducers and other agents on these cultures. (3) We will examine MNAN metabolism by rat liver and esophageal microsomes and reconstituted cytochrome P-450 ("P-450") systems to determine which P-450s metabolize MNAN. Gel electrophoresis and specific antibodies will identify the individual P-450s in adult and developed esophagus and forestomach. (4) We will examine MNAN alkylation of DNA to give 06- and N7 alkylguanine and 04-alkylthymine, using 3H-MNAN or radioimmunoassay. In vivo persistence of alkylated bases in esophageal DNA will be measured, and DNA alkylation by 2- and 4-oxo-MNAN. (5) We will determine whether MNAN is taken up more extensively than dimethylnitrosamine by rat esophagus. (6) We will compare carcinogenicity of MNAN injected into newborn, 3-day-old and adult rats and hamsters, test for carcinogenicity of 2- and 4-oxo-MNAN in adult rats, and determine whether inducers can affect MNAN carcinogenicity. These studies are designed to investigate (a) why MNAN is a specific carcinogen for the rat esophagus, (b) what intrinsic or extrinsic factors control the striking changes in MNAN metabolism from newborn to adult rats and hamsters, and (c) whether human esophagus could be susceptible to nitrosamine carcinogenesis.


We continue to study the streptococcal infections associated with acute glomerulonephritis (AGN) and acute rheumatic fever (ARF) in Trinidad by identifying the streptococcal strains isolated and/or the type specific antibodies which develop. We also assess patient-response to these infections by titrating antistreptolysin O and antihyaluronidase in serum samples and compare these responses in patients with ARF or AGN and in normal children. We also shall study the response to streptococcal DNAse B in representative patients and school-children. We are examining local immunological responses in patients with ARF by assaying: 1) saliva, for immunoglobulins, ASO, AH and anti group A carbohydrate and 2) joint fluid, for immunoglobulins and complement components. We have completed observations on the course of serum B1C globulin and fibrin degradation products in patients with AGN. These were made in view of the possibility that activation of the coagulation system with subsequent activation of plasmin may be responsible for the decreased C3 found in these patients. We found no evidence for this occurrence. We also are studying the effects of both "nephritogenic" and "rheumatogenic" streptococcal strains in peritoneal chambers on rats, according to the studies of Lindberg and Vosti.


Studies on DNA-DNA homology between bacteria of food and human clinical origin will be performed to determine the human pathogenic potential of refrigerated food spoilage bacteria in the genera Pseudo- monas and Achromobacter. The bacteriostatic properties of the fluorescent pigment produced by certain refrigerated food spoilage psuedomonads will be studied with respect to inhibition of Achromobacters on fish and meat. The fluorescent pigment from Pseudomonas septica will be purified, and its mechanism of bacterial inhibition (DNA, RNA, or protein synthesis) determined. The use of gas chromatography and mass spectroscopy will be applied to the genus Pseudomonas in an attempt to further characterize the identify refrigerated food spoilage bacteria of this genus. The mechanism of temperature restriction of an obligately psychrophilic phage-host system involving an intense fish spoilage strain of Pseudomonas putrefaciens is to be studied.


This facility, operating since 1990, provides cost-effective and accurate chemical analyses for molecules relevant to cancer research of Cancer Center (CC) investigators. It is also a base for consultation related to the quantitation of analytes and strategies in analytical chemistry. Services and expertise in advanced methods are available including state-of-the-art techniques, particularly liquid chromatography mass spectrometry (LCMS) and multiplexed immunoassays. In addition, routine analyses and determinations of clinical markers are offered to investigators. A host of new assays have been implemented in the current cycle due to needs of CC members. No other laboratories are available to provide many of these services in the State of Hawai'i, particularly regarding type, speed, cost, and combinations of assays, many of which are custom designed by this core to fulfill the specific requirements of CC projects. The high quality of services is continually monitored by taking part in internal and external quality assurance programs including the participation in round robin tests organized by the National Institute of Standards and Technology. The availability of this core provides state-of-the-art services, improves feedback to the investigators, stimulates inter-disciplinary collaborations, and provides cost effective service to CC investigators. The current use at full capacity is anticipated to continue for the foreseeable future. Newly funded projects requiring services of the resource will outnumber projects terminating in the coming years. Equipment obtained through the CCSG support grant in the current cycle has greatly enhanced the potential of this resource. This facility has shown remarkable growth: its productivity increased by 84% since the last cycle and has grown accordingly in personnel and revenue. The great value of this core is underscored by a total of 79 cancer related peer reviewed publications during FY06-11 that involved services by the ABSR (58% inter-programmatic, 13% intra-programmatic).


Hip fracture and resultant surgical repair entail a significant loss of muscle mass and function. Both injury and surgery, in and of themselves, initiate a cascade of events which lead to the loss of muscle mass. The general premise of this project is that this initial loss of muscle protein during surgery and the accompanying convalescence predispose a generally compromised patient to poor functional or morbidity outcomes. Further, we propose that muscle protein is lost due to a significant alteration in the muscle protein metabolism. The general goal of this project is to investigate interventions designed to ameliorate the negative muscle balance between protein synthesis and protein breakdown. We hypothesize the enhancement of muscle anabolism is required for these patients to recover muscle strength and function. We first propose to restore muscle protein synthesis throughout hospitalization with the administration of an essential am/no acid formula. We further propose that the amelioration of hypercortisolemia after surgery will result in a decreased muscle protein breakdown. The amelioration of hypercortisolemia after surgery will be accomplished with a common antifungal agent, ketoconazole, in order to investigate the resultant affects on muscle protein breakdown. We will measure lean body mass and muscle volume, as well as functional outcomes before surgery and discharge from acute hospitalization to determine the efficancy of these interventions. We will utilize stable isotope methodology to quantify muscle protein metabolism after acute hospitalization. We will also amplify these results by investigating cellular markers of protein breakdown to corroborate our metabolic endpoints. [unreadable] [unreadable] [unreadable]


Our long-term goal is to determine how cells establish and then maintain progressive motility in response to nondirectional external signals during wound repair. In wound repair, fibroblasts and endothelial cells repopulate the immature matrix to form both the supporting matrix and vasculature required to regenerate the tissue structures. The initial migration is driven by signals that arise from within the wound bed. However, once within the wound bed, the cells must distribute often m the absence of a gradient of signals. A central question is how cells establish the asymmetry required forprogressive motility. Soluble growth factors induce the migration of both the fibroblasts and endothelial cells. This motility requires asymmetry of biophysical cell processes within the cell. At the front, the cells must extend lamellipodla and form new adhesions, while rear de-adhesion and retraction is required. Between these two cell regions, contractility occurs to bring the cell body forward. Key molecular switches have been identified which regulate each biophysicalprocess. What remains unknown is how these signaling pathways initiate the biophysical processes in their correct spatial orientation. While an external signaling gradient would be an attractive explanation, the literature suggests that for eukaryotic cells, the key regulators are intracellular. This would be particularlz true for chemokinetic agents, such as EGFR and VEGFR ligands, which can induce progressive cell motility even in the absence of an external gradient. We hypothesize that cellular asymmetry needed for productive motility is accomplished by subeytoplasmie restriction of the activation of key biochemical switches. We propose to test the following molecular mechanisms: I. That PLCgamma activity is limited to the leading lamellipod by cdc42. We will use imaging and molecular perturbations to determine where and how PLCgamma/-1, required for front-direct actin reorganization, interacts with cdc42, the small GTPase that orchestrates the motility vector. These studies are based on preliminary data. II. That m-calpain drives de-adhesion in the cell body and trailing edge secondary to restricted availability of phospho-inositides. We will determine how m-calpain activity is restricted to the cell body and tail region, based on preliminary findings of phospho-inositides being required for m-calpain activation by growth factors. III. That contractile forces are asymmetric within the cell during EGF-induced motility. Biophysical deconstruction will identify quantitative and qualitative differences in contractility across a motile cell. These investigations wil! define molecular bases of the spatial restriction of signals and responses. This will enable the design of'smart scaffolds for cell and tissue engineering directing the synthesis of both the matrix and the vascular bed that is reauired to sunnort tissue function.


This research study is looking at the interactions of the combination of ritonavir and saquinavir and efavirenz.


Epithelial Na channels (ENaC) are expressed in the distal nephron where they serve as the final sites of Na reabsorption, thereby playing a key role in the regulation of extracellular fluid volume and blood pressure (BP). ENaC gain-of-function mutations are associated with hypertension, whereas loss-of-function mutations are associated with hypotension. ENaCs are comprised of three structurally related subunits termed ?, ? and ?. These subunits are assembled into a trimer within the endoplasmic reticulum (ER) and, following ER exit, the subunits undergo post-translational processing. In particular, cleavage at defined sites of the extracellular domain of the ? subunit by the proteases furin and prostasin has a key role in activating ENaC in heterologous systems, presumably by releasing an inhibitory domain within the subunit's ectodomain. Experiments are proposed to first define the functional role of ? ENaC in vivo by generating mice with distal nephron-specific ? ENaC disruption. These will be followed by studies assessing the functional role of ? ENaC subunit proteolysis in vivo by generating mice that express ? ENaC subunits in the distal nephron that lack key defined proteolytic cleavage sites for furin or prostasin. The specific aims are: 1. Generation and characterization of mice lacking ? ENaC selectively in ENaC-expressing cells in the kidney. The proposed studies are directed at generating a mouse model that has disrupted ? ENaC expression in the nephron. To accomplish this, two mice will be used: 1) a mouse expressing Cre recombinase either in principal cells of the collecting duct or in the entire nephron;and 2) a mouse containing loxP-flanked exons critical to ? ENaC function. Breeding of these animals will result in distal nephron-knockout of ? ENaC. These ? ENaC knockout animals will allow us to define the role of ? ENaC in the regulation of blood pressure, renal electrolyte excretion, and collecting duct ion transport. 2. Generation and characterization of mice with renal-specific reconstitution of wild-type or mutant ? ENaC. These studies are designed to define the role of ? ENaC proteolysis in the in vivo regulation of ENaC activity. Prostasin-dependent cleavage of the ? subunit, in concert with furin cleavage, fully activates the channel. The proposed studies are directed at generating a mouse model that expresses a ? subunit with mutations in the furin or prostasin cleavage sites selectively within the distal nephron. To accomplish this, native renal ? ENaC will be disrupted and ? ENaC expression reconstituted using;1) a ? subunit with a mutation in the furin cleavage site;2) a ? subunit with a mutation within the prostasin cleavage site;or 3) a wild type ? subunit (as a control). The introduced ? subunits will bear epitope tags to facilitate detection and proteolytic processing. These animals will allow us to (i) define the extent of proteolytic processing of the ? subunit at the prostasin or furin cleavage sites under basal conditions and in the setting of volume depletion;and (ii) determine the role of ? subunit proteolytic processing at the prostasin or furin cleavage sites in the expression of functional channels. PUBLIC HEALTH RELEVANCE: The proposed studies are designed to determine the physiologic role of cleavage of the epithelial sodium channel in the kidney. The studies will define the importance of two different cleavage sites within the sodium channel in regulation of kidney sodium and potassium excretion and maintenance of normal blood pressure. This work has the potential to uncover mechanisms involved in the development and/or maintenance of hypertension and disorders of potassium excretion.


Mechanical ventilation (MV) is a clinical tool to sustain pulmonary gas exchange in patients that are incapable of maintaining adequate alveolar ventilation. Although MV is often a life-saving intervention, prolonged MV promotes problems in weaning patients from the ventilator. While several factors can contribute to difficult weaning, weak inspiratory muscles are a major factor. In this regard, MV results in diaphragmatic inactivity that promotes the rapid onset of inspiratory muscle weakness due to diaphragmatic atrophy and contractile dysfunction (known as ventilator-induced diaphragm dysfunction (VIDD)). Although MV-induced diaphragmatic atrophy occurs due to both increased protein breakdown and decreased protein synthesis, the mechanism(s) that regulate these processes are poorly understood and thus, no clinical therapy exists. Hence, our long-term goal is to identify biological targets that will assist in the development of a therapeutic strategy to preven VIDD and therefore, protect against weaning difficulties. Although numerous signaling events can contribute to VIDD, we predict that the forkhead boxO (FoxO) family of transcription factors are important because they regulate the expression of genes involved in both the ubiquitin-proteasome and the autophagy pathway of proteolysis. Moreover, gene targets of FoxO may also contribute to the depression of protein synthesis. HYPOTHESIS: We will test the hypothesis that activation of FoxO-dependent transcription is required for the decreased protein synthesis, increased expression of proteasome/autophagy genes, and fiber atrophy that occurs in the diaphragm during MV. APPROACH: Our hypothesis will be tested in a well-established rat model of MV. Cause and effect will be determined by using an adeno-associated virus vector for gene delivery to express a dominant negative FoxO to prevent activation of FoxO target genes in the diaphragm during prolonged MV. SPECIFIC AIMS: Aim 1 will establish if FoxO-dependent transcription is essential for the rapid decrease in diaphragmatic protein synthesis that is observed during prolonged MV. Aim 2 will determine if FoxO-dependent transcription is required for the MV-induced increase in the expression of proteasome and autophagy genes along with diaphragmatic atrophy. SIGNIFICANCE: Collectively, this work can provide the foundation for new therapeutic strategies in the prevention of VIDD, a major contributor to the inability to wean patients from the ventilator.


Individuals with apolipoprotein (Apo) E deficiency develop hypercholesterolemia and atherosclerosis. Similarly, ApoE-deficient (ApoE-/-) mice elevate plasma ApoB48-carrying lipoproteins and develop atherosclerosis in a manner that resembles the human disease. The primary cause of atherosclerosis in ApoE-deficient patients and mouse models is the deposition of ApoE-deficient, ApoB48-carrying (E-/B48) lipoproteins in the arterial wall. The deposited lipoproteins recruit monocytes into the arterial intima and transform them into macrophages, which participate in the pathogenesis of atherosclerosis mainly through two mechanisms: 1) releasing inflammatory mediators, and 2) forming foam cells. An uncontrolled macrophage uptake of E-/B48 lipoproteins could be a mechanism underlying foam cell formation in ApoE-/- mice. Our laboratory recently demonstrated that E-/B48 lipoproteins reduce cellular cholesterol efflux from macrophages and down-regulates lysosomal hydrolase expression. In addition, the degradation of E-/B48 lipoproteins by macrophages declined over time. These novel findings indicate that both reduced cholesterol efflux and decreased degradation of E-/B48 lipoproteins could contribute to foam cell formation. Our preliminary studies also revealed that incubation of macrophages with E-/B48 lipoproteins enhanced eukaryotic translation initiationfactor 21 (eIF-2a) phosphorylation, which is linked to one of the unfolded protein response (UPR) signaling pathways. Thus, interaction of E-/B48 lipoproteins with macrophages may activate UPR signaling pathways, which in turn regulate gene expression and induce atherogenic events, such as triggering foam cell formation. In the proposed studies, we will test the hypothesis that activation of UPR signaling pathways is a mechanism by which E-/B48 lipoproteins regulate gene expression, induce foam cell formation and promote atherosclerosis. This project includes four specific aims: 1) to determine whether E-/B48 lipoprotein-induced changes in gene expression result from altered transcription or translation or both in mouse macrophages;2) to determine whether activation of UPR signaling pathways is a mechanism underlying E-/B48 lipoprotein-induced gene expression changes;3) to determine whether activation of UPR pathways is a mechanism underlying E-/B48 lipoprotein-induced foam cell formation;and 4) to determine the effect of inhibiting eIF-2a phosphorylation on atherosclerosis in ApoE-/- mice. If our hypothesis is correct, inactivation of UPR signaling pathways will attenuate E-/B48 lipoprotein-induced gene expression, and suppress foam cell formation and atherosclerosis development. PUBLIC HEALTH RELEVANCE: Myocardial infarction and stroke are the leading caused of death in the United Sates. Atherosclerosis is the primary cause of myocardial infarction and stroke. Formation of lipid-laden foam cells in the vessel wall is the early stage of atherosclerosis. The goal of this proposal is to determine the involvement of endoplasmic reticulum stress in foam cell formation. Endoplasmic reticulum stress is a cellular event that reduces the level of some proteins but increases the level of other proteins in cells. Our preliminary studies indicate that lipoproteins obtained from mice deficient in apolipoprotein E can cause endoplasmic reticulum stress and induce foam cell formation. The experiments designed in this proposal will study whether endoplasmic reticulum stress is the cause of foam cell formation. Finding from this work should contribute to understanding of the molecular mechanism of foam cell formation, and provide strategies for treatment or prevention of atherosclerosis by inhibition of endoplasmic reticulum stress.


This project undertakes an analysis of the mechanisms of transformation and gene regulation by human adenoviruses. Deletion mutants of adenovirus type 2 (Ad 2), isolated from laboratory stocks, or constructed in vitro, will be used to identify regions of the viral genome involved in transformation or the regulation of gene expression. The phenotypes of mutants with apparent defects in transformation or regulation will be carefully studied to determine how the deleted regions of the genome are involved in these processes.


This application requests continuation of funding for a very successful and well-established post-doctoral training program (dating back to 1991) in Major Psychoses and Translational Clinical Neuroscience. It seeks to integrate training in translational clinical neuroscience with an emphasis on the study of schizophrenia and other major psychoses using a lifespan trajectory perspective. That is, trainees may study illnesses during their characteristic age of onset, but they may also examine them from the perspectives of neurodevelopmental and/or aging processes. This program in integrative translational clinical neuroscience will place strong emphasis on all three aspects: integrative, translational/clinical, and neuroscience. The ultimate goal is to train a group of young investigators who will combine a high level of sophistication about the complexities of clinical phenomena with a firm grounding in some specific area of neuroscience, such as neuroimaging, cognitive neuroscience, and genetics/genomics/molecular biology. The program supports five trainee slots/year. Trainees remain in the program for a minimum of two years, and many elect to continue their training for a total of three years because of the complexities inherent in doing sophisticated integrative translational clinical neuroscience. The minimum experience required for entry into the program is a doctoral degree (M.D., Ph.D.), but many of our trainees have also completed residency training and have M.D./Ph.D.s. Relevance An integration of basic and clinical science will, we hope, help identify the mechanisms and causes of the major psychoses and to suggest improved methods for treating or preventing them. The disorders studied through this program (schizophrenia, depression, and bipolar illness) are three of the top ten contributors to the global burden of disease, according to a recent study by the World Health Organization. Thus it is crucial that we train a young generation of investigators who will advance knowledge about these diseases over the coming decades. PUBLIC HEALTH RELEVANCE: The disorders studied through this program (schizophrenia, depression, and bipolar illness) are three of the top ten contributors to the global burden of disease, according to a recent study by the World Health Organization. Thus it is crucial that we train a young generation of investigators who will advance knowledge about these diseases over the coming decades. 20


The main objectives of this project are: 1) to measure the effect of polycyclic hydrocarbons on the microsomal enzymes of macrophages and lymphocytes; 2) to develop a dependable, convenient in vitro assay for aryl-hydrocarbon hydroxylase (AHH) in human peripheral blood monocytes; and 3) to find whether interaction of polycyclic hydrocarbons with macrophages facilitates the induction and/or expression of contact hypersensitivity to these compounds.


The absence of a suitably HLA-matched donor precludes hematopoietic cell transplantation (HCT) in many patients, as the risks of graft-versus-host disease (GVHD) and rejection are formidable when extensive HLA barriers are transgressed. HLA-mismatched HCT without rejection or severe GVHD would make this treatment available to patients lacking a matched donor, and could maximize graft-versus-tumor (GVT) effects. The toxicity of allogeneic HCT can be diminished using non-myeloablative conditioning. The major goal of this Program Project is to develop non-myeloablative approaches to HLA-mismatched HCT, while minimizing GVHD and exploiting the potent GVT effect of anti-MHC GVH alloreactivity. Two major strategies will be explored in animal models, and a third will be characterized mechanistically. Nonmyeloablative allogeneic HCT that includes T cell depletion of the recipient and donor inoculum can lead to mixed hematopoietic chimerism across extensive MHC barriers without GVHD. When donor lymphocyte infusions (DLI) are administered to established murine mixed chimeras, the GVH alloresponse associated with subsequent DLI leads to powerful GVT effects without GVHD. Recipient-derived professional antigenpresenting cells (APC) in mixed chimeras maximize GVH alloresponses and thereby maximize GVT, while GVHD, a disease of epithelial target tissues, is avoided. We have taken these observations from mice to a unique large animal (pig) model and into clinical trials for the treatment of leukemias and lymphomas. Two of the Projects (Project 1 in mice, Project 3 in large animals) focus on the administration of delayed DLI to mixed allogeneic chimeras prepared with non-myeloablative conditioning that includes T cell-depleting mAbs, in order to achieve maximal GVL effects without GVHD. The goals of expanding donor hematopoietic stem cells (HSC) ex vivo and in vivo and improving immune recovery in Project 4 will ultimately be applicable to the clinical application of this approach, since large numbers of purified (T cell-depleted) donor HSC may be required to achieve engraftment across extensive HLA barriers. Project 4 includes an initial clinical evaluation of the ability of PTH to expand the numbers of mobilized HSC in patients failing conventional mobilization treatment. Large animal studies to evaluate the use of PTH to enhance allogeneic HSC engraftment (Project 3) will provide a critical bridge between the pre-clinical and clinical applications of these strategies (Project 4). Like Project 1, Project 2 involves confinement of the GVH alloresponse to the lymphohematpoietic system, via a novel strategy involving IFN-gamma. A mechanistic understanding of this phenomenon will lead to the development of novel strategies for separating GVHD and GVL clinically. Core A will provide multicolor (up to 11-color) flow cytometry and high speed cell sorting services to support all of these projects. Core B (Administrative) will be responsible for administering the grant, and Core C will provide statistical and database support for all of the Projects.


This proposal seeks continued support for the development and continuation of an interdisciplinary Center for Research in Oral Biology. The central goal or purpose of the Center is to assist in the national effort to reduce the toll of oral disease and to promote the general level of oral health. The Center is achieving this goal by developing and supporting meritorious and relevant research projects, by creating an intellectual and physical environment conducive to optimal research productivity, by providing opportunities for significant interaction between investigators with diverse backgrounds, by producing both basic and applied knowledge related to the oral region, and by encouraging the incorporation of relevant research findings into dental education, dental practice, and the surrounding community. The types of projects supported have been the subject of intensive discussion within the Center and by the Scientific Review Committee, and within the committee appointed by the Vice President for Health Affairs to study the future pattern of the Center. The emphasis in the past has been on high quality basic research programs. This emphasis continues, but we are increasing our percent effort directed toward application of basic research findings to problems of patient diagnosis and care and to measures which will lead to improvement of the oral and dental status of the population.


The broad objective of the Opportunities Fund Management Core (OFMC) will be to facilitate early stage support for innovative, exploratory, and R&D projects, particularly those which have the potential to lead to practical products in the fields of radiation biodosimetry and prediction and prevention of acute and delayed injuries. The OFMC will support 1) Pilot projects (basic and translational research) and 2) Advanced Development projects that lead to Investigational New Drug (IND)-enabling studies and Medical Countermeasure (MCM) development. The OFMC will generate an SOP (Standard Operating Procedure) for the operation of the Core, which after Steering Committee approval, will be the template for its operation. The OFMC will coordinate the process of soliciting proposals, as well as coordinating the review of the proposals by an external panel of experts convened via teleconference for the purpose. The OFMC will tally the scores given by the review panel at the teleconference, and present the rank ordered list to the Steering Committee for funding decisions. Innovative and translational research will be strongly emphasized in the review process. The OFMC will also be responsible for coordinating all necessary documentation of financial, IRB, and IACUC requirements with sub-recipient institutions, as well as financial oversight and reporting. The OFMC will also coordinate annual progress reports, and collect progress reports with renewal requests to be considered by the Steering Committee. The OFMC will also help to facilitate Presentation opportunities for the pilot project recipients. For instance, pilot awardees may be given the opportunity to prepare a poster for the annual CMCRC meeting, and will be encouraged to present at national meetings, such as the Radiation Research Society Annual Meeting. !


Abstract Bioluminescence imaging (BLI) systems are installed in thousands of facilities and labs, and with their straightforward and low cost workflow for longitudinal studies, are the most commonly used preclinical modality for assessing tumor models in rodents. There currently is no high throughput and low cost system enabling BLI images to be combined with anatomical images of soft tissue to confirm tumor volume, context, or vascularity. In our Phase I work, we demonstrated the feasibility of mapping data from our whole body ultrasound (US) system to 2D BLI images. The additional US data dramatically reduced inter-user quantification variability of the BLI signal (>80%). Furthermore, we showed that our clinically translatable microvessel imaging technology, acoustic angiography, can be mapped to the BLI data. Those tumor microvessels were analyzed using patented vessel analysis algorithms, yielding quantifiable vascular morphology metrics, previously shown to be reliable predictors of tumor malignancy and response to therapy in humans. Thus our commercialized hybrid modality US+BLI device, the Alpheidae Platform, will allow angiogenic tumors and anti-angiogenic therapies to be studied in ways current in vivo imaging tools do not allow. In Phase II, we propose to bring the Alpheidae Platform to market, leveraging whole body tissue and vascular US imaging to improve cancer research with BLI. The team includes experts in optical imaging system design, photoacoustic system design, and US imaging system design. Specifically, in Phase II, we will address the following aims: (Aim 1) Hardware R&D for multi-animal tri-modality imaging. Six animals will be scanned sequentially by our robotic system in each of the three modes. Throughput for six animals will be <15 min. (Aim 2) Software and algorithm R&D to enable automated targeted US and PA acquisitions based on BLI images, and leveraging algorithms for improved spatial resolution in both US and PA datasets. (Aim 3) Validation studies within in vivo tumor drug response study. Device performance will be assessed by comparison to standard BLI in a murine breast cancer model. Its capacity to reliably predict eventual drug response within one week of starting therapy will be the criteria for success. Once Phase II is completed we will have created a novel high throughput and portable tool enabling tumor tissue and microvessel images to be mapped to BLI data. Importantly from a commercial perspective, the Alpheidae Platform can be sold for a fraction of what competitive systems cost. From a translational research perspective, the device includes a clinically translatable US microvessel imaging approach for tumor assessment, and thus forms a direct link between preclinical findings in mice and actionable clinical cancer assessment protocols.


Receptors for transplantation antigens may be visualized directly on unsensitized cells with the use of an anti-idiotypic antisera. It should be possible to eliminate these cells specifically by treatment of the unsensitized population with sera directed at the specific binding site. Sera have been raised to cells cytotoxic for transplantation alloantigen in strain combinations selected so that only the variable portion of the antigen specific receptor could be recognized. Bona fide anti-idiotypic sera have not been raised. Refinements in the approach under way include 1) use of cytotoxic populations as immunogen with a demonstrably high proportion of receptor-bearing cells, 2) use of parent strains differing only by a point mutation to restrict the heterogeneity of the immune response of one against the other, and 3) use of continuously cultured cytotoxic cell lines to increase the homogeneity of the receptors used as immunogen.


Occupational exposure to arylamines used in the manufacturing of industrial dyes was the first known cause of human bladder cancer. These carcinogenic arylamines, specifically, 4-aminobiphenyl (4-ABP), beta-naphthylamine, and benzidine, are also contained in tobacco smoke, and are the most likely causative agents responsible for the raised rates of bladder cancer in smokers. Oxidation of arylamines is recognized as a critical first step in turning these chemical species into their carcinogenic metabolites capable of causing DNA damage to urothelial cells. Hair dyes represent another substantial source of arylamines in humans, and we recently showed that sustained use of permanent hair dyes in women is a risk factor for bladder cancer, especially among those deficient in arylamine detoxifying enzymes. Subsequently, 4-ABP was detected in bottles of commercial hair dyes bought in a US store. Then, using hemoglobin adducts of 4-ABP as a biomarker of exposure, we showed that other, presumably diffuse, sources of 4-ABP exposure may be related to bladder cancer in nonsmokers. Thus, the latest evidence indicates arylamine exposure as the primary cause of bladder cancer in the United States. Ten years ago, we reported that a reason for the 3-fold increased risk of bladder cancer in US white versus Chinese men despite their comparable smoking habits may be the higher prevalence in the former population of individuals with deficient arylamine-detoxifying enzymes, which are under genetic control. We capitalized on this finding and launched a population-based case-control study involving both a Los Angeles and a Shanghai, China component to explore genetic factors that play a major role in determining bladder cancer risk in arylamine-exposed individuals. This already completed database consists of roughly 1300 cases of incident bladder cancer (750 cases in Los Angeles, 550 cases in Shanghai) and about an equal number of control subjects. Our initial goals were to investigate the roles of selected polymorphic, arylarnine-metabolizing genotypes/phenotypes in bladder cancer, and in the Los Angeles component, to use hemoglobin adducts as biomarkers of exposure to arylamines in examining nonsmoking-related bladder cancer. In this application, we aim for a more comprehensive understanding of the arylamine-bladder cancer etiologic link through the following extensions on our Los Angeles/Shanghai database: (1) Completion of arylamine hemoglobin adduct measurements on Shanghai subjects in order to compare the respective effects of these adducts on risk between Los Angles and Shanghai study subjects;(2) Addition of genotypes involved in arylamine metabolism, in cellular response to oxidative stress and in DNA repair;and (3) Development of a Bayesian hierarchical statistical model to allow for an efficient, pathway-driven examination of multiple gene-arylamine interactions in bladder cancer. The ultimate goal of this research is to assess individual bladder cancer risk for the purpose of preventive interventions.


Recent literature in mental health services has identified a serious gap between what is known about mental disorders and their treatment from university-based clinical research and what services are actually provided to consumers in typical community practice settings. This IP-RISP "Biosocial Factors in Rehabilitation for Schizophrenia" will build a partnership between university-based clinical services researchers and practitioners and consumers from a psychosocial rehabilitation service agency. The goal of this IP-RISP is to implement a research and practice agenda that will translate recent findings on psychobiological deficits in schizophrenia and their remediation into community based rehabilitation practice. We will address questions relevant to: (i) improving the effectiveness of community-based psychosocial rehabilitation interventions for functional disability in schizophrenia, and (ii) adapting and infusing new knowledge and new interventions into psychosocial rehabilitation practice settings in the community. This IP-RISP will address four practice-research aims and two infrastructure aims. The four practice-research aims are: 1) To introduce knowledge on functional outcomes and psychobiological factors to practitioners and consumers of psychosocial rehabilitation services at Portals;2) To partner with agency clinicians and consumers in adapting and infusing (transporting) psychosocial intervention strategies for the remediation of psychobiological deficits into existing rehabilitation services;3) To partner with agency clinicians and consumers in implementing controlled pilot research on whether targeted psychobiological service elements increase the effectiveness of psychosocial rehabilitation services in the community by improving consumer outcomes;4). To facilitate the sustainability of the new service configurations by incorporating consumer, practitioner, and administrator perspectives. The two infrastructure aims are: 5). To develop a shared university/agency infrastructure consisting of a database and shared clinical-research personnel specifically: a). To increase the clinical utility of an existing database used to measure psychosocial outcomes in a community psychosocial rehabilitation setting, and b). To train and support clinical-research personnel who will participate in both clinical and research agendas at the community service setting. 6). To build a platform for ongoing psychobiological data collection consisting of an agency-based laboratory for assessing and measuring relevant psychobiological variables such as neurocognition, social cognition, and psychophysiological responsivity. The aims will be met during four activity phases consisting of: a) partnering among agency staff, consumers, and researchers;b) introducing and adapting new service elements into rehabilitation practice;c) implementing pilot research on the effectiveness of the new intervention elements;and d) assessing the implementation experience.


PRODUCTION OF TWO THOUSAND CAPSULES OF 4 MG AZA-TDCPOP: 03/04/19 THROUGH 03/03/20. The batch of product was produced by the contractor and sent to clinical repository. The shelf-life was also evaluated. The drug product will be used in the NCI clinical trials.


Program Summary. Amyotrophic lateral sclerosis (ALS) affects an estimated 30,000 individuals in the United States and 450,000 worldwide. The disease is characterized by loss of motor neurons leading to loss of muscle control and muscle wasting. Patients typically progress from diagnosis to death in 3-5 years. While there are two approved therapies to treat ALS, their effects are limited. The lack of available treatments for ALS patients remains a significant unmet medical need. Unlike numerous other rare diseases, ALS is not monogenic, thus it is unlikely that a single therapy will be able to address the entire patient population. Despite recent advances in sequencing technology, 90% of ALS cases are sporadic making determination of a genetic cause more difficult. Nonetheless, many of the genes originally found in familial cases also appear in the sporadic population including C9orf72, and Optineurin. One of the genes that has recently been shown to be associated with ALS in a dominant fashion is tank binding kinase 1 (TBK1). Using patient derived cells, it has been shown that the mutations lead to a haploinsufficiency and loss of function. Furthermore, studies on TBK1 have shown that it is a key regulator of autophagy and mitophagy and interacts with Optineurin, p62, and Ubiquilin, all of which have also been shown to be associated with ALS. To develop therapies for TBK1 patients, we propose to develop a cellular system with TBK1 haploinsufficiency that can be used to develop an assay to assess the efficacy of potential drug candidates to increase TBK1 function from the remaining allele. We propose to use CRISPR-Cas9 to disrupt a single allele of the TBK1 gene in human induced pluripotent stem cells (hiPSCs) as a phenotypic model of TBK1 patients. Once we have generated a single allele knockout of TBK1, we will assess the phenotype in hiPSC-derived motor neurons in comparison to parental cells. Disease relevant phenotypes will be examined including levels of activated TBK1, autophagy, and mitophagy. Our drug discovery efforts are focused on increasing the levels of activated TBK1 and thus rescuing any deficiency found in the downstream pathways of autophagy and mitophagy. We have identified a molecular target that modulates TBK1 activity and have preliminary data showing that pharmacological modulation of this target leads to an increase in mitophagy in human microglial cells. We are continuing to pursue this target and anticipate that we will have a significant number of compounds to assay once the TBK1 knockout cells are available. The demonstration of activity in a cellular model of ALS is crucial to support our ultimate goal of developing a therapeutic for this patient population. In addition, many ALS associated genes, including TBK1, have also been shown to be associated with Frontotemporal Temporal Dementia (FTD), and autophagy and mitophagy defects are associated with several neurodegenerative diseases, thus this work may have implications for drug discovery beyond ALS.


Description (from applicant): One of the long standing unanswered questions in one of the most frequently studied models of carcinogenesis, chemical hepatocarcinogenesis, is what is the cell of origin of hepatocellular carcinomas (HCC)? The classic cellular pathway, championed by Emmanuel Farber, is that HCC develop from altered hepatocytes progressively through foci and preneoplastic nodules. However, studies on oval cells, which appear early during hepatocarcinogenesis, indicate that HCC may arise from "bipolar" liver progenitor cells located within terminal ducts or from pluripotent progenitor cells located adjacent to the ducts. In addition, recent studies on female mice and rats receiving bone marrow transplants from male donors indicated two possible origins of liver stem cells: from bipolar progenitor cells in the terminal ducts or from periductal stem cells. It is further postulated that these periductal progenitor cells may arise from circulating bone marrow stem cells either by differentiation into liver cells or that bone marrow cells may fuse with liver cells. In Specific Aim 1, the applicant proposes to develop a transgenic Fischer rat line expressing EGFP which can be used as a donor for bone marrow transplantation studies. In Specific Aim 2, three models of carcinogenesis: a. Continuous DEN, which features foci of altered hepatocytes preceding HCC with little or no oval cells will be used to determine if HCC arise from hepatocytes; b. Solt-Farber model, which features prominent proliferation of ductal oval cells to determine if HCC arise from bipolar ductal precursor cells; c. Choline-deficiency-ethionine, a model which features periductular proliferation of oval cells will be used to determine if preneoplastic lesions or HCC arise from periportal oval cells. In each of these protocols, DDPIV- (canalicular enzyme) female rats will receive a bone marrow transplant from DDPIV+, EGFP+, male donors. The origin of the populations of cells responding by proliferation will be identified by the presence of donor or recipient markers, and the types of cells identified through labeling with both liver lineage and littoral cell markers, when appropriate. In Specific Aim 3, the possibility of fusion of donor bone marrow stem cells with recipient liver cells will be tested and the possibility of fusion in etiology, progression or metastasis of HCC determined. Knowledge of the cellular origin of cancer is critical for understanding how cancer evolves and for developing prevention strategies.


ABSTRACT Pattern recognition receptors sense pathogen-associated molecular patterns and mediate the earliest host innate immune response to infection. The cytosolic nucleotide oligomerization domain (NOD) like receptors (NLRs) are a highly conserved group of cytosolic proteins that play a central role in the immune response to diverse microorganisms, environmental insults and cellular danger signals. We have recently demonstrated a role for Nlrp6 in the control of enteric virus infection. Nlrp6 controls enteric virus infection in the intestine by interacting with a RNA sensor, Dhx15. Here we propose to define the mechanisms of NLRP6-mediated anti-viral pathways, to identify whether other helicases may also interact with NLRP6 (Aim 1), whether other relevant RNA viruses are recognized through this pathway, and whether mutations may underlie certain human cases of vaccine failures (Aim 2). A fuller understanding of the triggers and physiologic function and signaling pathways for NLRs will provide key insights to immune mechanisms involved in host defense and immune-mediated diseases.


The acetylcholine receptor of Torpedo californica is an oligomeric glycoprotein which is embedded in the membrane of the multinucleated electroplaque cells of the electric organ. The arrangement of the subunits of the receptor may be an important indication of their function. I propose to study this arrangement by chemical crosslinking in order to map the nearest neighbor proteins of each subunit and by surface labeling, enzymatic proteolysis, and chemical fragmentation in order to determine the exposure of each subunit at the cytoplasmic and external surfaces of the membrane. A primary culture of the electroplaque precursor cells will be isolated for the purpose of studying the biogenesis of the acetylcholine receptor. Both receptor synthesis and degradation will be investigated. The synthesis of the receptor may be regulated at the transcriptional, posttranscriptional or translational levels. The control mechanisms will be studied by isolating mRNA and using this mRNA as a template for in vitro translation.