William Harvey - US grants

The long term objective is to analyse the structure, function regulation of the plasma membrane H+ V-ATPase in Manduca sexta, to isolate the k+/2H+ antiporter and to understand how activity of the ATPase-antiporter couple alkalinizes the larval midgut lumen. A concomitant objective is to provide a new basis for vector mosquito control by using cDNA probes and antibodies from the transport model, M. sexta to study midgut alkalinization in a disease vector model, Aedes aegypti. The hypothesis is that the high pH of both caterpillar and mosquito midgut is generated by an H+ translocating, plasma membrane V-ATPase. The voltage component of the protonmotive force energized the apical membrane and drives electrophoretic K+/H+ exchange by a novel K+/2H+ antiporter. A structure/function analysis of the ATPase- antiporter couple will be worthwhile in itself and will provide molecular details of the alkalinization mechanism. Selective inhibition of the antiporter should prevent mosquito midgut alkalinization, eventually killing larvae with little environmental impact. Aim 1 is to analyse the structure and function of the V1 complex; ATP binding and hydrolysis as well as subunit assembly will be studied using recombinant subunits produced by in vitro transcription/translation or over-expression; eventually the catalytic site will be systematically modified by site directed mutagenesis. Aim 2 is to analyse the regulation of subunit gene expression using an existing genomic DNA library; promoter elements will be isolated and their activity will be monitored in transfected cells using reporter genes. Aim 3 is to isolate the K+/2H+ antiporter by protein biochemistry as well as to clone and sequence its encoding cDNA by hybridization cloning with cDNA probes from vertebrate Na+/H+ antiporter, by complementation cloning in Na+/H+ antiport-deficient yeast or by expression cloning in Xenopus oocytes. Aim 4 is to analyze the alkalinization mechanism by comparing ATPase-antiporter structure/function in the structurally complex caterpillar midgut with that in the structurally simple mosquito midgut using in situ hybridization and immunocytochemistry and to evaluate alkalinization blocking with microencapsulated amiloride derivatives. The proposal has SCIENTIFIC MERIT because energization of animal cell plasma membranes by a protonmotive force and reversal of acidification direction by secondary active cation/proton antiport are both novel concepts. It has HEALTH RELEVANCE because the new basic science is immediately applied to a disease vector model.

This project's long term goal is to determine the mechanism of action of electrically driven amino acid, potassium ion cotransport (symport) of insects, to assess its role in K+ homeostasis, and to identify symport inhibitors. The working hypothesis is that the twenty naturally occurring L-amino acids, AAs, move from highly alkaline midgut contents to neutral across brush border membranes. Identification, isolation, and determination of the primary structure of the symporter proteins are required to reach the goal. Brush border membrane vesicles, BBMV, isolated from feeding, fifth instar larvae of the tobacco hornworm, Manduca sexta, will be used to study AA uptake by rapid filtration and fluorescence quenching techniques. Three aims lead naturally toward the goal. In Aim 1 symporter substrate sub-groups are identified through cation-gradient and counter-transport studies of AA-K+ uptake using labeled AA, rapid filtration techniques. These techniques have been used successfully to study AA uptake kinetics in eel BBMV but are novel in the study of insects. In Aim 3 high affinity amino acid analogues are identified and used to label symport proteins. The project will pave the way for isolating symporter proteins, for cloning symporter cDNA, and for determining the primary structure of the proteins. AA analogues which interfere with AA-K+ symporter proteins in Lepidoptera may be developed as environmentally safe agents for insect control. The extremely high lumen pH (approaching 11.5) and positive PD (approaching 240 mV) of Lepidopteran midgut may be reflected in unusual AA-cation symporters, unlike those of mammals and birds. Since larval mosquito midguts also have high lumen pH, such AA symport-inhibiting analogues may be selectively toxic to mosquito larvae and be useful in disease vector control.

The overall goal is to characterize lepidopteran midgut K(+) homeostasis; according to the midgut model, K(+) enters midgut cells via channels in the basal membranes and is propelled across goblet cell apical membrane, GCAM, to goblet cavity via an electrogenic H(+) pump in parallel with an nH(+)/K(+) antiporter; the apical membrane is energized to a PD >180 mV. The PD supports a lumen pH >10 compared to the cell pH of 7.0; it drives amino acid/K(+) symport from lumen to columnar cells, resulting in amino acid absorption and completing the K(+) homeostatic cycle. This renewal application deals with GCAM. The hypothesis is that a proton pumping electrogenic V-ATPase and an electrogenic nH(+)/K(+) antiporter inserted into a cation impermeable lipid bilayer are key components of the homeostatic pathway. Aim 1 is to complete sequencing cDNAs encoding GCAM V-ATPase subunits of 67 and 56 kDa and to clone and sequence cDNAs for 43, 28, and 16 kDa subunits. Aim 2 is to solubilize and purify the antiporter protein(s). Aim 3 is to clone and sequence the cDNA encoding the antiporter(s). Aim 4 is to localize the V-ATPase and nH(+) /K(+) antiporter in K(+) transporting epithelia and describe the ontogeny of GCAM from vacuolar membranes. GCAM will be isolated by sonication and gradient centrifugation. Antiporter will be solubilized with nonionic detergents and purified by density gradient centrifugation and/or FPLC using reconstitution into liposomes as an assay. Midgut larval cDNA library screening will use either antibodies to midgut proteins or oligonucleotide probes to highly homologous components from other sources. ATPase and antiporter localization will use fluorescent and gold labelled antibodies to appropriate subunit proteins in both light and EM immunocytochemistry. The midgut model may provide insight into V-ATPase energization of plasma membranes of other insects and into energization of vertebrate urinary and bone membranes. Because the midgut model deals with an unusual cation impermeable apical membrane protecting cells from a highly alkaline lumen, it has provided insight into the action of the Bacillus thuringiensis endotoxin; it may lead to other environmentally safe caterpillar and mosquito larval controls.

DESCRIPTION (provided by the applicant): Taking advantage of the complete genome as well as the morphological simplicity and cellular accessibility of larval midgut in An. gambiae, we will create a comprehensive physiological model that integrates molecular, cellular, and electrochemical mechanisms of epithelial transport. In particular, we wish to understand the integrative physiology of lumen alkalinization, which is critical for larval digestion and nutrient uptake. We postulate that alkalinization and nutrient uptake depend upon transepithelial and longitudinal ion gradients, which are generated by specific spatial distribution and electrochemical interaction between primary H + V-ATPases and secondary acid/base transporters in the larval midgut. We have identified eight genes in the An. gambiae genome, which encode the putative acid/base transporters and designed tissue-specific cDNA library from An. gambiae larvae, which dramatically increased the cloning efficiency. We have also developed unique nanoscale analytical approaches, which allow us to analyze epithelial tissue with subcellular resolution. To explore the integrative physiology and functional genomics of the mosquito midgut, we will complete four specific aims. Aim 1 uses preliminary bioinformatics data to clone acid/base transporters from An. gambiae larvae; the gene products will be evaluated by electrochemical analysis of transcript expression in Xenopus oocytes. Aim 2 localizes the transporters along the midgut in whole-mounts of An. gambiae larvae using in situ hybridization with transcript-specific dioxygenin-RNA probes. The apical/basal (polar) integration of the transporters and H v V-ATPases will be determined by confocal microscopy of immuno-labeling preparations. Aim 3 seeks to determine electrochemical motive forces across basal/apical membranes and phosphorylation potentials in specified midgut cells using capillary electrophoresis and ion selective microelectrodes. Trans-membrane voltages will be measured with microelectrodes in midgut of intact and semi-intact An. gambiae larvae. Aim 4 examines the mechanism, efficiency, and role of electrochemical coupling between H + V-ATPase and identified transporters in midgut alkalinization using non-invasive self-referencing ion-selective microelectrodes (SERIS LIX) and time-lapse photography of a lumen alkalinization profile in vivo. With this proposed study, integration of the molecular and electrochemical mechanism of a specific physiological process will be defined for the first time, which is crucial not only for understanding midgut alkalinization in disease vector mosquitoes but other types of transporting epithelia as well. Since the work is to be done on a malaria vector, it has health relevance because the larval midgut is the target for Bti, TMOF, and other larvicides and is an apt model for the plasmodium interaction with the adult-female midgut.

The Virginia-North Carolina Alliance for Minority Participation (VA-NC AMP), a Phase I Louis Stokes Alliance for Minority Participation (LSAMP), is well poised to participate in the National Science Foundation's initiative to broaden participation of underrepresented populations in the science, technology, engineering, and mathematics (STEM) disciplines and workforce. Eight colleges and universities in Virginia and North Carolina comprise the proposed VA-NC AMP: Four historically black colleges and universities (HBCU), including two Master's Colleges and Universities; and four Doctorate-Granting/Research-Extensive Universities, forging a strong public-private institutional partnership dedicated to student success across the Alliance.

VA-NC AMP's goal is to double the number of STEM baccalaureate degrees awarded to students from underrepresented populations in STEM fields from an Alliance yearly average base, measured over academic years 2001 through 2005, of 524 to 1,053 by the end of 2012.

DESCRIPTION (provided by applicant): The Hampton University (HU) Biomedical Research Center (BRC) will be a genuine national resource: a state-of-the-art, interdisciplinary, research facility situated on campus at an Historically Black University. The BRC will become a flagship for integrated research and training on a small university campus. Unfortunately, HU does not currently have any dedicated modern research buildings. There also exists a critical national need for diversity in the educational pipeline and a need for growth in quality graduate programs. The BRC will develop minority scientists and create skilled professional and a diverse workforce. HU is uniquely poised to capitalize on this challenge. This proposed facility will create sufficient research space capacity, critically needed to attract competitive investigators, while supporting the expert training of young scientists. The BRC will consolidate and greatly improve productivity of research activities currently dispersed across campus and located off campus at hospital facilities, federal laboratories, and other institutions in our market. The clustering of these research activities into a single research facility will support interconnected scientific growth in an interdisciplinary environment for faculty, staff and students. This will also leverage the existing research strengths supporting related research areas, which can be explored within the foundation of an existing solid scientific base. Compelling scientific questions will be assailed from multiple fronts, in the BRC. This will create a dynamic environment where researchers will be encouraged to look both at and beyond their focused areas to incorporate into their studies strategic information from other sciences as well.

DESCRIPTION (provided by applicant): The Hampton National Research Mentoring Network (NRMN) Consortium planning grant is designed to provide an innovative approach to networking and mentorship experiences for individuals from backgrounds underrepresented in biomedical/behavioral research from the undergraduate to junior-faculty level. It will bring together successful, but currently disparate mentoring and networking programs from geographically and racially/ethnically diverse entities, representing different missions and organizational goals. The mission of the proposed Consortium will be to recruit individuals into the biomedical, behavioral and clinical research enterprise to facilitate translation of fundamental scientific discoveries and clinical observations within NIH's scientific mission bi-directionally between bench and bedside. The vision of the consortium is to transform good outcomes to great outcomes by implementing the following aims. Aim 1: Implement a six-month planning initiative leading td Hampton University's (HU) establishment of partnerships among institutions of higher learning, professional associations as well as mentoring organizations to create a national mentoring network. Aim 2: Assess existing mentoring strategies that can be effectively coordinated and delivered on a national basis, and develop new programs as needed. Aim 3; Delineate innovative mentoring methods, including social media mechanisms, that will be integrated into the five-year NRMN award proposal. To achieve the aims herein, the following methods will be implemented: (1) a representative democratic governance structure will be adapted with strong emphasis on consensus building for decision-making; (2) assessment of mentoring needs and evidenced-based best practices for the target communities of mentees and mentors; (3) determination of leading edge mentoring innovations; and (4) the design of a strong social media background to facilitate and provide access to mentee-to mentee and mentee-to-mentor networks. The outcomes of implementing these planning grant activities will provide the data needed to prepare a competitive NRMN mentoring grant application. In sum, completing the vision of the Hampton NRMN Consortium will lead to an increase in.the pool of independent biomedical researchers, which will significantly contribute to the diversity of the future research workforce.