Michael Stern - US grants

We have recently completed an epidemiologic survey of cardiovascular risk factors and diabetes in Mexican Americans and Anglos living in San Antonio, TX. Our current results indicate that Mexican Americans have excess diabetes and related cardiovascular risk factors over and above that which can be explained on the basis of their excess obesity alone. In the present project we propose to carry-out a further epidemiologic survey aimed at elucidating the additional factors beyond obesity which contribute to diabetes and cardiovascular risk in this ethnic group. We plan to test two new hypotheses relating to insulin resistance which flow directly from our current results. We will study 3,858 Mexican American and Anglo men and women (ages 25-64) randomly sampled from the San Antonio population. Sampling will be carried out in such a way that we will be able to match Mexican Americans and Anglos on obesity, thereby enabling us to study ethnic differences in diabetes and cardiovascular risk factors independent of the confounding effects of obesity. To further our long-range objective of elucidating the lifestyle vs. the genetic contribution to diabetes and cardiovascular risk in Mexican Americans we will continue to study acculturation and genetic markers (including gene polymorphism and HLA phenotyping) in the new project. By pooling the new project with the original data base we will have a sufficiently large sample for an eventual prospective epidemiologic study of diabetes and cardiovascular risk factors in Mexican Americans which is one of our long-range objectives. We also plan to study the relative prevalence of diabetic complications in 400 Mexican American and Anglo diabetics (195 from our original survey and 205 projected in the new survey). The assessment of complications will include the following: eye, visual acuity, tonometry, fundus photography; renal, serum creatinine, urinary protein, urine cultures; neurological, nerve conduction studies and vibratory sensory threshold; and peripheral vascular, resting and post-ischemic ankle blood pressures by Doppler ultrasound.

Dynamic laser light scattering is a powerful method for studying microscopic fluctuations of molecular and particulate systems, which has been widely applied in biophysics. Its applicability to complex systems such as intact cells is limited by non-selective scattering from structures which are not under study. Based on our extensive theoretical studies, we propose to develop a novel non-linear optical fluctuation technique, quasi-coherent double resonance fluorescence photon correlation (QDORF) which will permit selective measurement of the translational, rotational and chemical fluctuations of specific labeling chromophores within cells, and apply the technique to studies of cardiac excitation-contraction coupling. We will construct apparatus which will simultaneously excite the labeling molecules at wavelengths resonant with two coupled spectral transitions. Fluorescence emitted at the sum of the exciting frequencies contains a quasi-coherent component whose sidebands are directly related to fluctuations in the position, orientation and chemical environment of the labeling molecules. The spectrum of this component will be analyzed by heterodyne photon correlation. To validate the theoretical principles, we will measure QDORF spectra from merocyanine dyes and other large conjugated molecules, in free solution of various viscosities, and partially immobilized on colloids. We will make QDORF measurements from isolated single cardiac myocytes, using injected labels and native chromophores, to determine the sensitivity and sources of interference in the cellular environment. In the latter part of the study we will collaborate with synthetic dye chemists to develop QDORF dyes capable of chelating calcium. The chemical kinetic QDORF fluctuation spectrum from such a dye is predicted to yield a linear estimate of intracellular calcium concentration in several compartments simultaneously. Numerous other biophysical applications of QDORF are foreseen.

DESCRIPTION: (Adapted from Investigator's Abstract) The "Hispanic paradox" refers to the concept that all-cause and cardiovascular (CV) mortality are widely thought to be lower in Hispanics, including Mexican Americans, than in non-Hispanic whites in the U.S. The data supporting this paradox are derived almost exclusively from vital statistics which are subject to various biases including ethnic misclassification and incomplete ascertainment of deaths, both of which tend to underestimate Hispanic mortality. Therefore, the possibility exists that the Hispanic paradox is an artifact. Cohort data with complete ascertainment of vital status have the potential of eliminating or at least markedly ameliorating most of these biases. The preliminary mortality data, based on 50,148 person-years of follow-up, suggest that all-cause mortality is actually higher in Mexican Americans than in non-Hispanic whites in San Antonio (odds ratio of 1.54). The plan is to perform a 15- to 24-year mortality follow-up with 92,164 person-years of follow-up and 907 deaths projected by the year 2003, to form a more precise estimate of the ethnic odds ratio. The additional number of deaths will also permit exploration of the potential time dependencies in the ethnic mortality ratio, and will allow examination of the effects of potentially explanatory covariates. These will include, in addition to the biochemical parameters, hemodynamic and anthropometric variables such as socioeconomic status, health care access and utilization and migrant status. Of the 5,158 individuals (approximately 65% Mexican American) in the original cohort, 3,666 were examined twice, 7-8 years apart. Therefore, it will be possible to examine the effects on mortality of changes in risk factors, e.g., whether weight change (voluntary or involuntary) influences mortality and whether the excess mortality in patients with impaired glucose tolerance is confined to those who convert to diabetes. There are also plans to examine whether Mexican-American diabetics have a higher mortality than non-Hispanic diabetics. Finally, there are plans to validate a sample of 200 Mexican-American and 200 non-Hispanic white death certificates by medical record review. The purpose of this project is to determine whether Mexican Americans, deemed to have died of cardiovascular causes based on medical record review, are more or less likely to have cardiovascular codes (ICDA 390-459) listed on their death certificates than non-Hispanic whites deemed to have died of these causes. The investigators note that this will aid in the interpretation of data relating to possible ethnic differences in cause-specific mortality based on death certificate coding.

heart contraction; myocardial ischemia /hypoxia; oxygen transport; caffeine; heart cell; single cell analysis; calcium channel blockers; cellular pathology; ryanodine; adenine nucleotides; calcium transporting ATPase; sarcoplasmic reticulum; laboratory rat; guinea pigs; laboratory rabbit;

This project will focus on the role of hyaluronidase and hyaluronidase inhibitors in fetal wound healing. The fetal wound has the unique ability to heal without inflammation and scar formation. Hyaluronic acid (HA) is prominent in the extracellular matrix whenever rapid tissue proliferation, regeneration and repair occur. This is the situation in the early stages of postnatal wound repair, prior to scar formation. In the postnatal wound, elevated levels of HA occur early but are transient, due to the appearance of wound hyaluronidase. In the fetal wound, however, the elevated HA levels are prolonged. Since HA inhibits cell differentiation, its prolonged presence in the healing fetal wound may explain the lack of scar formation by the fetus. This study will examine differences between repairing adult and fetal wounds in the rate of degradation of HA. We hypothesize that this rate of degradation is modulated by wound and serum hyaluronidase, possibly mediated through the presence of a hyaluronidase inhibitor. This study will develop sensitive and convenient assays to determine hyaluronidase and hyaluronidase inhibitor levels in adult and fetal sheep wounds as a function of time after wounding and gestational age. In addition, we will purify and characterize hyaluronidase inhibitor from fetal serum. The understanding and application of the mechanisms involved in this lack of scar formation would be of obvious use to all surgical specialties.

DESCRIPTION: (Adapted from Applicant's Abstract) The proposed research is a genetic epidemiologic study of non-insulin dependent diabetes mellitus (NIDDM) in families of randomly selected diabetic Mexican Americans, a high risk population for diabetes. A strong genetic component in NIDDM has long been recognized, although no clear pattern has yet been identified. Recent studies have suggested that insulin resistance may also be an inherited trait. The investigators propose to conduct pedigree studies to examine the relationship between NIDDM and four candidate genes affecting glucose metabolism (two glucose transporter genes (GLUT1 and GLUT4), the insulin receptor gene (INSR), and Amylin, a new candidate gene) and two marker phenotypes found previously to be associated with NIDDM in population studies (Rh blood group and haptoglobin). Approximately 720 individuals from 60 families will be studied over 5 years. Probands will be randomly selected from among all diabetic Mexican Americans identified in the San Antonio Heart Study, a population-based study of cardiovascular disease and diabetes. Subjects will receive a clinical examination during which fasting and two-hour levels of glucose, insulin, and C-peptides will be determined and anthropometric measurements will be obtained. Diabetes will be diagnosed according to World Health Organization criteria. Blood samples will also be drawn for analysis of DNA polymorphisms at the candidate gene loci. Lymphocytes from all participants will be transformed with Epstein-Barr virus and used to create a DNA bank as a future resource. Complex segregation analysis will be used to determine whether major genes influence the inheritance of NIDDM or serum glucose, insulin, and C-peptide levels. Quantitative trait linkage analysis will be used to determine whether any major genes identified in the segregation analyses are linked with polymorphisms of the candidate genes.

One process that is important for nervous system function is synaptic transmission, the process by which neurons communicate with each other and with target muscle cells. Neuronal ion channels play key roles in controlling this process. A more complete understanding of the mechanisms by which synaptic transmission can be regulated requires identification of the ion channel structural and regulatory components. However many of these components have as yet resisted molecular characterization. The long-term objective of this work is to use genetic methodology in Drosophila to identify and characterize these components. With genetic methodology, the genes that regulate synaptic transmission are identified by mutation. Because any gene can be mutated, any protein can be identified by mutation regardless of abundance, homology to previously characterized proteins or even prior knowledge of existence. Thus this approach provides a unique way identifying novel classes of functionally important molecules not accessible by other means. Once identified, the roles of these genes in controlling synaptic transmission are determined with electrophysiological assays, and finally the genes are cloned and sequenced which enables the encoded products to be studied at the molecular level. I previously identified mutations in three new genes that interact behaviorally with Shaker, the structural gene for the A type potassium channel. Electrophysiological analysis of these new mutants has shown that each exhibits aberrant synaptic transmission at the larval neuromuscular junction as a result of aberrant excitability of the motor neuron. In the present application, further functional and molecular characterization of these three genes is proposed. The phenotypes of flies lacking each gene, as well as overexpressing each gene, will be determined. Possible synergistic interactions among the genes will be tested by construction and analysis of double mutants. Effects of each gene on nerve terminal structure and electrophysiological properties will be determined. To facilitate cloning of these genes, mutagenesis with P-elements and X-rays will be performed. Isolation and sequence analysis of cDNAs from these genes will provide clues as to the function of the gene products and provide material for further studies. These genes might encode ion channel subunits or regulatory molecules such as protein kinases, G-proteins, or calcium binding proteins. Because such genes are well conserved in evolution, human homologues of these genes will likely exist and might be involved in hereditable disorders of the nervous or neuromuscular system. In addition, because potassium and calcium channel functions are required for non-neural processes such as the control of blood pressure, insulin release and the activation of T-lymphocytes, these human homologues might be defective in hereditable disorders of these processes as well. Therefore I expect that the study of ion channel structure and regulation in Drosophila will have general medical significance. In the future, this genetic approach will be used further to identify and analyze additional components that control the important process of synaptic transmission.

We are currently carrying out an epidemiologic survey of 35-64 year old Mexican residents of several low-income colonias in Mexico City in which we expect to enroll a total of 296 subjects by Feb 28, 1993. Preliminary results indicate that, compared to age-matched, low-income Mexican Americans in San Antonio, Mexicans in Mexico City may have "carbohydrate-induced lipemia" (i.e., high carbohydrate diet, hypertriglyceridemia and low HDL). Moreover, Mexican diabetics are more hyperglycemic than Mexican American diabetics. These findings raise questions about the relative rates of cardiovascular disease and microvascular complications of diabetes in Mexicans vs. Mexican Americans. To address these questions we propose to carry out a 3.5 year prospective followup of our Mexico City cohort to determine the incidence of type II diabetes. We anticipate examining 1952 of the original 2296 subjects. All participants will receive a medical history interview and a health habits interview, the latter covering diet, physical activity, cigarette smoking, and alcohol use. Participants will also undergo a clinic examination which will include a glucose tolerance test with insulins, lipids and lipoproteins including measurement of "small, dense LDL", blood pressure,, anthropometrics, and an EKG. Baseline and follow-up EKGs will be Minnesota-coded to assess both prevalence and incidence of EKG abnormalities. Special examinations to further assess cardiovascular disease and microvascular complications of diabetes will include: ankle-arm blood pressure ratios to assess peripheral vascular disease to be performed on all subjects by Doppler ultrasound; ultrasound examination to assess carotid disease in 400 randomly selected subjects; and fundus photography to assess diabetic retinopathy in the anticipated 298 diabetics and 100 non-diabetic controls. Fundus photographs will be read by the University of Wisconsin Reading Center. Diabetic nephropathy will be assessed by clinical proteinuria and microalbuminuria in the same subset of patients. All prevalence and incidence rates will be compared to the corresponding rates for Mexican Americans and non-Hispanic whites in San Antonio in whom all of the above described measurements are currently being made using comparable methodology.

The purpose of this project is to carry out a systematic search of the genome for susceptibility genes for non-insulin dependent diabetes (NIDDM). The study will be carried out in Mexican American families currently enrolled in the San Antonio Family Diabetes Study (SAFADS), a project currently funded by NIDDKD (R01 DK42273, N. Stern, PI). To date, 432 individuals from 29 different families have been enrolled into the SAFADS; our recruitment target is 720 total individuals, and we anticipate achieving this target by June 1994. SAFADS families are ascertained on a low-income Mexican American NIDDM proband. Blood lymphocytes are currently being EBV transformed for SAFADS participants and cell lines have been/are being established to provide a renewable source of DNA. The current project will use SAFADS families as a resource for conducting a gene search. Our strategy will be first to seek preliminary evidence for linkage by typing markers on an initial set of 177 individuals from 23 families already enrolled and secondly, to verify suspected linkages by typing markers on additional family members from the initial pedigrees and by typing other SAFADS families. We will use approximately 250 highly polymorphic PCR-based microsatellite markers that span the entire genome at regular intervals and then use linkage analysis to search for linkage between these markers and NIDDM or its precursor traits (i.e., glucose, insulin, or C-peptide levels). Our simulations indicate that if NIDDM were inherited as a Mendelian dominant trait, we would have excellent power of detecting evidence for linkage on the initial set of pedigrees. As new markers are typed and added to the database, we will perform two- point linkage analyses to screen for linkage. Those markers with suggestive evidence for linkage will be used in combined segregation and linkage analyses and in multipoint analysis. If evidence for linkage persists, additional markers will be typed in that region to "zero in" on the NIDDM susceptibility gene(s). The phenotypes to be considered in these analyses are NIDDM, the quantitative traits, glucose, insulin, and C-peptide levels, and a risk score derived from a predictive model for NIDDM.

This project will focus on the role of hyaluronidase and hyaluronidase inhibitors in fetal wound healing. The fetal wound has the unique ability to heal without inflammation and scar formation. Hyaluronic acid (HA) is prominent in the extracellular matrix whenever rapid tissue proliferation, regeneration and repair occur. This is the situation in the early stages of postnatal wound repair, prior to scar formation. In the postnatal wound, elevated levels of HA occur early but are transient, due to the appearance of wound hyaluronidase. In the fetal wound, however, the elevated HA levels are prolonged. Since HA inhibits cell differentiation, its prolonged presence in the healing fetal wound may explain the lack of scar formation by the fetus. This study will examine differences between repairing adult and fetal wounds in the rate of degradation of HA. We hypothesize that this rate of degradation is modulated by wound and serum hyaluronidase, possibly mediated through the presence of a hyaluronidase inhibitor. This study will develop sensitive and convenient assays to determine hyaluronidase and hyaluronidase inhibitor levels in adult and fetal sheep wounds as a function of time after wounding and gestational age. In addition, we will purify and characterize hyaluronidase inhibitor from fetal serum. The understanding and application of the mechanisms involved in this lack of scar formation would be of obvious use to all surgical specialties.

One process that is important for nervous system function is synaptic transmission, the process by which neurons communicate with each other and with target muscle cells. Neuronal ion channels play key roles in controlling this process. A more complete understanding of the mechanisms by which synaptic transmission can be regulated requires identification of the ion channel structural and regulatory components. However many of these components have as yet resisted molecular characterization. The long-term objective of this work is to use genetic methodology in Drosophila to identify and characterize these components. With genetic methodology, the genes that regulate synaptic transmission are identified by mutation. Because any gene can be mutated, any protein can be identified by mutation regardless of abundance, homology to previously characterized proteins or even prior knowledge of existence. Thus this approach provides a unique way identifying novel classes of functionally important molecules not accessible by other means. Once identified, the roles of these genes in controlling synaptic transmission are determined with electrophysiological assays, and finally the genes are cloned and sequenced which enables the encoded products to be studied at the molecular level. I previously identified mutations in three new genes that interact behaviorally with Shaker, the structural gene for the A type potassium channel. Electrophysiological analysis of these new mutants has shown that each exhibits aberrant synaptic transmission at the larval neuromuscular junction as a result of aberrant excitability of the motor neuron. In the present application, further functional and molecular characterization of these three genes is proposed. The phenotypes of flies lacking each gene, as well as overexpressing each gene, will be determined. Possible synergistic interactions among the genes will be tested by construction and analysis of double mutants. Effects of each gene on nerve terminal structure and electrophysiological properties will be determined. To facilitate cloning of these genes, mutagenesis with P-elements and X-rays will be performed. Isolation and sequence analysis of cDNAs from these genes will provide clues as to the function of the gene products and provide material for further studies. These genes might encode ion channel subunits or regulatory molecules such as protein kinases, G-proteins, or calcium binding proteins. Because such genes are well conserved in evolution, human homologues of these genes will likely exist and might be involved in hereditable disorders of the nervous or neuromuscular system. In addition, because potassium and calcium channel functions are required for non-neural processes such as the control of blood pressure, insulin release and the activation of T-lymphocytes, these human homologues might be defective in hereditable disorders of these processes as well. Therefore I expect that the study of ion channel structure and regulation in Drosophila will have general medical significance. In the future, this genetic approach will be used further to identify and analyze additional components that control the important process of synaptic transmission.

DESCRIPTION: (Adapted from Applicant's Abstract) The proposed research is a genetic epidemiologic study of non-insulin dependent diabetes mellitus (NIDDM) in families of randomly selected diabetic Mexican Americans, a high risk population for diabetes. A strong genetic component in NIDDM has long been recognized, although no clear pattern has yet been identified. Recent studies have suggested that insulin resistance may also be an inherited trait. The investigators propose to conduct pedigree studies to examine the relationship between NIDDM and four candidate genes affecting glucose metabolism (two glucose transporter genes (GLUT1 and GLUT4), the insulin receptor gene (INSR), and Amylin, a new candidate gene) and two marker phenotypes found previously to be associated with NIDDM in population studies (Rh blood group and haptoglobin). Approximately 720 individuals from 60 families will be studied over 5 years. Probands will be randomly selected from among all diabetic Mexican Americans identified in the San Antonio Heart Study, a population-based study of cardiovascular disease and diabetes. Subjects will receive a clinical examination during which fasting and two-hour levels of glucose, insulin, and C-peptides will be determined and anthropometric measurements will be obtained. Diabetes will be diagnosed according to World Health Organization criteria. Blood samples will also be drawn for analysis of DNA polymorphisms at the candidate gene loci. Lymphocytes from all participants will be transformed with Epstein-Barr virus and used to create a DNA bank as a future resource. Complex segregation analysis will be used to determine whether major genes influence the inheritance of NIDDM or serum glucose, insulin, and C-peptide levels. Quantitative trait linkage analysis will be used to determine whether any major genes identified in the segregation analyses are linked with polymorphisms of the candidate genes.

One process that is important for nervous system function is synaptic transmission, the process by which neurons communicate with each other and with target muscle cells. Neuronal ion channels play key roles in controlling this process. A more complete understanding of the mechanisms by which synaptic transmission can be regulated requires identification of the ion channel structural and regulatory components. However many of these components have as yet resisted molecular characterization. The long-term objective of this work is to use genetic methodology in Drosophila to identify and characterize these components. With genetic methodology, the genes that regulate synaptic transmission are identified by mutation. Because any gene can be mutated, any protein can be identified by mutation regardless of abundance, homology to previously characterized proteins or even prior knowledge of existence. Thus this approach provides a unique way identifying novel classes of functionally important molecules not accessible by other means. Once identified, the roles of these genes in controlling synaptic transmission are determined with electrophysiological assays, and finally the genes are cloned and sequenced which enables the encoded products to be studied at the molecular level. I previously identified mutations in three new genes that interact behaviorally with Shaker, the structural gene for the A type potassium channel. Electrophysiological analysis of these new mutants has shown that each exhibits aberrant synaptic transmission at the larval neuromuscular junction as a result of aberrant excitability of the motor neuron. In the present application, further functional and molecular characterization of these three genes is proposed. The phenotypes of flies lacking each gene, as well as overexpressing each gene, will be determined. Possible synergistic interactions among the genes will be tested by construction and analysis of double mutants. Effects of each gene on nerve terminal structure and electrophysiological properties will be determined. To facilitate cloning of these genes, mutagenesis with P-elements and X-rays will be performed. Isolation and sequence analysis of cDNAs from these genes will provide clues as to the function of the gene products and provide material for further studies. These genes might encode ion channel subunits or regulatory molecules such as protein kinases, G-proteins, or calcium binding proteins. Because such genes are well conserved in evolution, human homologues of these genes will likely exist and might be involved in hereditable disorders of the nervous or neuromuscular system. In addition, because potassium and calcium channel functions are required for non-neural processes such as the control of blood pressure, insulin release and the activation of T-lymphocytes, these human homologues might be defective in hereditable disorders of these processes as well. Therefore I expect that the study of ion channel structure and regulation in Drosophila will have general medical significance. In the future, this genetic approach will be used further to identify and analyze additional components that control the important process of synaptic transmission.

This study is part of the San Antonio Family Heart Study, the first comprehensive genetic epidemiological study of atherosclerosis and its correlates in Mexican Americans. Its goal is to detect and map new polymorphic genes that influence variation in susceptibility to cardiovascular disease in Mexican Americans. Because non insulin dependent diabetes mellitus and obesity are risk factors for cardiovascular disease and are common in this population, the pleiotropic effects of diabetes and obesity-related genes on quantitative correlates of cardiovascular disease will also be studied. More than 40 extended families will be studied and a complete risk factor profile will be developed.

The high-affinity sulfonyurea receptor (SUR1) expressed on the beta cells of the pancreas is a key component in glucose-stimulated insulin secretion. The investigators have previously reported linkage of 2-hour plasma glucose levels to a region near the SUR1 gene in Mexican Americans and observed a silent variant in exon 31 of the gene which was associated with high insulin concentrations in non-diabetic members of Mexican American type 2 diabetic families. It is not known whether this is due to primary oversecretion of insulin or compensatory insulin response to insulin resistance. This study will compare insulin secretion and insulin resistance in 15 non-diabetic Mexican Americans homozygous for this variant of the SUR1 gene and 15 subjects who are homozygous for the wildtype allele and who were previously genotyped participants of the San Antonio Family Diabetes Study.

Cell migration events play crucial roles in normal development, and aberrant migratory behavior, such as the migrations of metastatic cancer cells, can cause life-threatening diseases. Thus, a detailed understanding of the mechanisms that control the movement of cells is important both for our general understand of development as well as for providing targets for therapeutic reagents to combat metastatic cancer progression. Cell migration requires the interplay of the migration cell with the guidance causes and substrates which influence its movement. The long- range objective of this proposal is to understand the molecular basis for how migration cells read the extracellular cues that influence their migrations and integrate this information to result in directed cell movement. This proposal seeks to obtain a comprehensive understanding for the mechanisms guiding the migrations of the sex myoblasts (SMs) in the nematode Caenorhabditis elegans to attain this goal. The SMs in C. elegans hermaphrodites migrate anteriorly to final positions that flank the center of the developing gonad. The migrations of the SMs are known to be guided by a gonad-dependent attractive cue that appears to be a fibroblast growth factor (EGL-17) and a gonad-independent mechanism that requires the function of three genes, unc-53, unc-71, and unc-73. In the absence of the gonad-dependent attraction, the SMs are kept posterior by a gonad-dependent repulsion. In males, a similar set of SMs migrate posteriorly, although the components that directly regulate the sexually- dimorphic migrations of the SMs are not known. To accomplish our long-term objective, this proposal has the following specific aims: (1) Analyze the extend of involvement of the EGL-15 FGF receptor in the various SM migration mechanisms and determine whether it acts within the SMs by mosaic analysis. (2) Investigate whether EGL-17(FGF) emanates from the gonad to attract the SMs to their final positions. (3) Develop tissue-specific promoters to help probe the roles and sites of action of SM migration components. (4) Analyze the molecular basis of the gonad-independent mechanism by molecularly characterizing unc-71 and studying mutants that affect the gonad-independent sexually dimorphic direction of SM migration. (5) Identify components of the repelling to probe its role in normal SM migration guidance. Using these approaches, we hope to gain a better understanding of how the multiple mechanisms that influence the SMs cooperate to ensure reproducible precisely targeted migrations.

DESCRIPTION: (Adapted from the Investigator's Abstract) The investigators have now completed a genome scan on the genotyping set of the San Antonio Family Diabetes Study (SAFADS) population which consists of 444 individuals from 27 extended pedigrees ascertained on a low income, Mexican American proband with type 2 diabetes. Multipoint variance components linkage analysis revealed strong evidence for linkage between a region on chromosome 10q25.3-26.1 and the trait type 2 diabetes (LOD = 2.88, p = 0.00014) and age of diabetes onset (LOD = 3.75, p = 1.6x10-6). The investigators now propose to perform genetic disequilibrium mapping of the 10 cM region centered on the marker D10S587 which is the point of maximum signal. A number of publicly available SNPs have been mapped, but preliminary analysis of five of these suggested that they were uninformative in the investigators' Mexican American population. However, single strand conformational polymorphism (SSCP) analysis of these five and an additional 13 efficiently revealed either known variants or identified new polymorphisms. They therefore propose to develop up to 150 new SNPs from known SNP and EST clones mapped to the 10q25.3-26.1 region for their linkage disequilibrium mapping project. All 444 SAFADS participants in the genotyping set will then be typed for each SNP. Once a region is determined to harbor a diabetes susceptibility gene, a BAC contig spanning the region of highest genetic linkage disequilibrium will be constructed and DNA sequenced to identify open reading frames. Approximately 50 additional SNPs will be generated from the diabetes candidate genes which are identified, and these will be tested in SAFADS families. Disequilibrium mapping will be performed by joint linkage/association analyses using 1) the multipoint variance components approach and 2) the gamete competition model. They will also employ a novel statistical functional genomics approach to localizing functional variants and polymorphisms exhibiting the highest disequilibria with the putative susceptibility loci.

DESCRIPTION: (adapted from applicant's abstract) Proper function of the nervous system requires specific interactions between neurons and glia, and yet the precise mechanisms by which these interactions occur remain incompletely understood. The Drosophila segmental nerve, which comprises a layer of motor and sensory axons surrounded by an inner (peripheral) and outer (perineural) glial layer, provides a genetic system for the analysis of this intercellular signaling. Mutations in push, a gene identified and cloned in the PI's lab, and Nf1, the Drosophila homolog of the gene responsible for neurofibromatosis in humans, each increase the thickness of the perineural glial layer. In addition, the effect of push and Nf1 mutations are strongly potentiated by mutations in ine which encodes a neurotransmitter transporter. The effect of push mutations in perineural glial growth is also enhanced by mutations in eag which encodes a potassium channel subunit. Both push and Nf1 encode intermediates in the receipt of intercellular signaling pathways mediated by the PACAP neuropeptide, or by the amn-encoded protein, which is PACAP-related. Thus their results are consistent with a model in which perineural growth in Drosophila is controlled by two interacting neurotransmitter-mediated signaling pathways, one controlled by Amn and acting through push and Nf1, and the second controlled by substrate neurotransmitter of ine and acting through eag. The PI proposes a further dissection of these intercellular signaling pathways. Three specific questions are asked. First, is the Amn signal released from peripheral glia and received by perineural glia or is the signal released from neurons, received by peripheral glia and then signaled by a relay mechanism to perineural glia? Second, does the Eag potassium channel act in the Amn signaling cell, or in the Amn receiving cell, to modulate the effect of Amn signaling on perineural growth? Third, in other PACAP or Amn signaling pathways described, additional intermediates have been implicated, including PKA, Ras and Raf. Which of these intermediates participate in Amn signaling in the Drosophila segmental nerve? These experiments will provide new molecular insights into the nature of neuron/glia signaling. In addition, it appears that defects in signaling with this pathway models, at least in part, the tumor formation that occurs in neurofibromatosis. Thus the studies proposed here are anticipated to enable the development of new models for the role of Nf1 in tumor formation, and perhaps enable the development of new pharmacological strategies.

The long-term objective of this proposal is to understand the molecular basis of signaling specificity by fibroblast growth factor receptors (FGFRs). Fibroblast growth factors (FGFs) initiate many different types of biological events, including cell proliferation, angiogenesis, differentiation, cell migration, and cell survival. While much work has been done to elucidate one key FGFR signaling pathway, we have only a rudimentary understanding of the mechanisms that confer specific outcomes to FGF triggered events. FGFs play important roles in human health and disease. FGFs are known to be involved in many important developmental and homeostatic events, and aberrant FGF signaling is responsible for a number of skeletal and craniofacial syndromes. Studies in model organisms have helped elucidate the function of FGFRs and the signaling pathways they utilize. This proposal seeks to extend this understanding by analyzing FGFR signaling specificity in the nematode Caenorhabditis elegans, a model system with tremendously reduced cellular and molecular complexity. C. elegans possesses a single FGFR (EGL-15) and two known FGFs (LET-756 and EGL-17) that mediate several distinct functions: (1) an essential function; (2) guidance of the migrating sex myoblasts (SMs); and (3) the inhibition of sex muscle differentiation. This proposal will focus on how the ligands, specificity determinants on the receptor, and different signal transduction components combine to generate specific biological outcomes of FGFR signaling. We will analyze the specificity determinants on EGL-15 by structure/function studies. We will identify other components required for specific EGL-15 functions by using genetic screens for modifiers of EGL-15-induced phenotypes and by two-hybrid screens for proteins that interact with the specificity determinants on EGL-15. In this way, we will gain a clearer understanding of how specific responses are elicited by the activation of FGF receptors.

In many organisms, growth occurs primarily during early juvenile phases. When a specific body size is reached, the organism stops growing and transitions to a sexually mature adult (at puberty in mammals and at metamorphosis in insects). Nevertheless, it is unclear how organisms sense when they are large enough for this transition. In the fruitfly, Drosophila, metamorphosis is triggered by the steroid hormone ecdysone, synthesized in an endocrine tissue called the prothoracic gland (PG). One key activator of ecdysone synthesis is the protein hormone, PTTH. Elimination of PTTH delays ecdysone synthesis and causes large and developmentally delayed adults to form. The cellular "machinery" within the PG by which PTTH triggers ecdysone synthesis has not been identified, and yet this identification is critical to understanding how the timing of metamorphosis, and hence final body size, is regulated. This research project will test the hypothesis that PTTH activates ecdysone synthesis via a regulatory pathway (MAP kinase pathway) in the PG. Transgenes inhibiting or activating the MAP kinase pathway will be expressed specifically in the PG to determine if these transgenes either block the ability of PTTH to induce ecdysone synthesis, or activate ecdysone synthesis in the absence of PTTH. In addition, transgenes will be used to increase or decrease PTTH synthesis, and the effects of changes in PTTH levels on MAP kinase activity will be determined. Successful completion of these experiments will provide essential information on the machinery used by organisms to control the timing of developmental transitions and hence final body size. In addition, this project will support the development of the next generation of scientists by training undergraduate and graduate researchers in genetic and molecular techniques.

Project Summary Degeneration of neurons or muscle are observed in several human pathologies, including Alzheimer's, Parkinson's and the hereditary spastic paraplegias (HSP) for neuronal degeneration, and disuse atrophy, cancer cachexia, and sepsis for muscle degeneration. Despite many recent advances, the molecular mechanism(s) underlying these degenerative processes remain incompletely understood. To generate such mechanistic insights, the PIs have recently established a Drosophila model for the HSPs. The specific focus is in atlastin (atl, Spastic Paraplegia Gene 3A), which encodes an ER fusion protein. Based on the previous observation that atl knockdown in neurons causes progressive, age-dependent locomotor deficits, the we asked if this knockdown also caused progressive cellular degeneration. Investigations into the adult thoracic musculature revealed that atl loss from either neuron or muscle caused progressive degeneration associated with a number of other pathologies including accumulation of aggregates containing ubiquitin, increased reactive oxygen species (ROS), and activation of the JNK/Foxo stress response pathway. Administering the drug rapamycin, which inhibits the Tor kinase, or decreasing Tor gene dosage reversed many of these pathologies at least partially, indicating that atl loss might activate muscle Tor. Muscle Tor and Foxo activation have also been observed in denervation-induced muscle atrophy. In this application, experiments are proposed to elucidate the mechanisms by which atl loss causes progressive muscle pathologies. Aim #1 will test the hypothesis that muscle Tor is activated by atl loss, determine if Tor activity is sufficient as well as necessary for atl loss phenotypes, and test the prediction that Tor activity promotes muscle degeneration by inhibiting autophagy. Aim #2 will examine the causal relationship between activated Tor and increased ROS, and between ROS and the JNK/Foxo stress pathway. In particular, we will test two non-mutually exclusive hypotheses explaining Foxo activation; first, that activated Tor increases ROS, which in turn is responsible for JNK activation, and finally Foxo activation, and second, that activated Tor activates its target S6K, which in turn down-regulates insulin signaling, thus decreasing activity of the Foxo inhibitor Akt. Aim #3 will test the hypothesis that neuronal atl loss activates muscle Tor by attenuating glutamatergic neuromuscular transmission. In particular, it will be determined if deletion of one glutamate receptor, previously shown to be sufficient to activate muscle Tor, will cause similar muscle pathologies as is observed by neuronal atl knockdown. In addition, it will be determined if neuronal atl loss confers neuronal phenotypes similar to those conferred by glutamate receptor deletion. Successful completion of these experiments will provide novel and critical mechanistic insights linking defective synaptic input conferred by atl loss to muscle degeneration. The PIs anticipate that these experiments will also provide mechanistic insights applicable to neuronal degeneration as well, which will give these experiments a broad medical relevance.

Genetic or pharmacological interventions that decrease synaptic transmission often induce a signal produced in the postsynaptic cell that acts in a retrograde manner to increase transmitter release from the presynaptic cell. At some synapses, generation of this signal requires an increased concentration of the phospholipid phosphatidic acid (PA) in the postsynaptic cell, which then activates the ?Target of rapamycin? (Tor) kinase. Tor activation, in turn, increases translation of mRNAs encoding the retrograde signal. Although increased protein synthesis is the most prominent Tor output, Tor also inhibits autophagy by phosphorylating and inhibiting several Atg proteins; this autophagy impairment might explain the association of activated Tor with a number of neurodegenerative and muscle degenerative disorders. The Hereditary Spastic Paraplegias (HSPs) represent one family of neurodegenerative disorders caused by mutations in any of over 70 different genes. We recently developed a Drosophila model for the HSP caused by mutations in atlastin (atl, SPG3A), which encodes an ER fusion GTPase. Using this model, we found that neuronal atl loss both decreased evoked transmitter release at the larval neuromuscular junction and caused progressive muscle degeneration. Because this degeneration, as well as associated locomotor and muscle pathologies, was partially suppressed by either decreasing Tor gene dosage or by administering the Tor inhibitor rapamycin, we hypothesize that neuronal atl loss activates muscle Tor. In this proposal, we will determine the role of [PA] in mediating the muscle degeneration and related pathologies caused by atl loss. In aim #1 we use mass spectrometry and an in vivo fluorescent PA reporter to test the hypothesis that neuronal atl loss increases muscle [PA]. In aim #2, we will determine the functional role of altered muscle [PA] in muscle degeneration. In particular, we will adjust muscle [PA] levels by overexpressing or introducing mutations in genes encoding PA-metabolizing enzymes including PLD, DAG Kinase and PA phosphatase. We hypothesize that increasing muscle [PA] will be sufficient to cause muscle degeneration whereas decreasing muscle [PA] will prevent muscle degeneration caused by neuronal atl loss. If we find, as expected that, increasing muscle [PA] is sufficient to cause muscle degeneration, we will then determine if this degeneration is dependent on Tor. In aim #3 we will investigate the Tor dependent retrograde signaling pathway that occurs at the Drosophila larval neuromuscular junction. This pathway is triggered by deletion of gluRIIA, one of the two alternative subunits of post-synaptic muscle glutamate receptors. We will determine if muscle PA is both necessary and sufficient to generate this retrograde signal. If successful, these studies will establish a link between impaired synaptic transmission and postsynaptic cell degeneration and elucidate the role of postsynaptic PA in this process. We anticipate that these studies will provide critical mechanistic insights into the HSPs as well as in the process of degeneration in diseases such as Alzheimer?s with great clinical importance.