Peter C. Agre - US grants

The long term goal of this proposal is to identify the primary molecular defects in erythrocyte membrane skeletons causing certain hereditary hemolytic anemias and employ these mutations to probe the intricacies of normal membrane skeleton architecture. Recently developed biochemical methods will be systematically used to measure specific protein concentrations, binding interactions, and possible structural differences in spectrin and other RBC membrane skeleton proteins. I. Is partial spectrin deficiency the principal structural defect in hereditary spherocytosis (HS), and why is the spectrin deficient? Concentrations of spectrin, ankyrin, band 3 and other proteins will be determined by analysis of SDS-PAGE slabs and by RIAs. Effects probably secondary to spectrin deficiency will be measured including lipid deficiencies, increased osmotic fragility, certain biophysical parameters, and clinical severity. The possibility of progression of spectrin deficiency will be approached with density gradient fractionation and in vitro aging. HS spectrin will be examined for intrinsic defects by 2D peptide fingerprinting, electron microscopy, and specific binding interactions between spectrin chains and associated proteins. Possible roles for newly identified calmodulin binding proteins, tropomyosin, and myosin will be sought by comparative analyses of HS and normal RBCs. II. What are the molecular defects in 6 recessive mouse mutations resulting in spectrin-deficient spherocytosis resembling non-dominant human HS? RBC membranes and proteins will be biochemically analyzed as described for human HS. Investigation of the biosynthesis, assembly and turnover of these proteins will be conducted utilizing immunoprecipitations of in vitro 35S-methionine labeled spectrin, ankyrin and band 3. III. Significance of membrane skeleton linkage with the membrane and possible pathological consequences thereof will be studied in RBC membranes from individuals with defective ankyrin binding sites, individuals with reduced 4.1, and Ca2+ ionophore induced spheroechinocytes with predominantly 2.3 anykyrin. Possible roles for newly identified calmodulin-binding proteins, tropomyosin, and myosin will be investigated in these cells. Band 3 oligomerization and 43K fragment analysis will be conducted on RBCs with reduced ankyrin sites. 4.1 binding will be measured in 4.1 deficient RBCs. The importance of ankyrin 2.3 will be sought.

Alcoholism is known to produce clinically significant hematologic disorders including macrocytosis and sideroblastic anemia, but the molecular basis of these problems remains unclear. The major objective of this proposal is to employ recently developed membrane protein biochemical techniques in order to better understand the molecular pathology of red cell membrane damage in alcoholism. Potential long term clinical benefits from this study may include the development of laboratory tests to determine who among drinkers is developing specific forms of membrane pathology, the quantitation of which may guage the longitudinal severity an individual's alcohol consumption. Red cells from normal humans or lab rats will be compared to red cells from humans or lab rats suffering from excessive alcohol intake of a) acute or b) chronic duration or c) during recovery from a or b. Three basic lines of investigation will be followed: I. Evaluation of fatty acid acylations of the RH polypeptide and other major red cell acyl-proteins. Thioesterification and turnover of palmitic acid and other radiolabeled fatty acids onto free -SH groups will be assessed in an attempt to identify and quantitate specific functionally important red cell membrane protein sulfhydryl damage in alcoholism. The synthesis and stability of the Rh polypeptide itself will be assessed by immunological and physical analyses. II. A newly recognized Mr 28kDa red cell membrane channel protein will be evaluated in the different states of alcoholism. Synthesis and stability of 28kDa as well as possible alterations in the N-glycosylated subpopulation of 28kDa will be evaluated by immunological, chemical, physical and northern analyses. III. Potential defects in other red cell membrane proteins including spectrin, ankyrin, protein 4.1, and adducin will be assessed by high resolution gel electrophoresis, radioimmunoassays, protein binding assays, and membrane biophysical methods.

membrane channels; erythrocyte membrane; molecular pathology; protein structure function; protein biosynthesis; polysaccharides; posttranslational modifications; phosphorylation; messenger RNA; kidney disorder; dyserythropoietic anemia; congenital hemolytic anemia; cell differentiation; chemotherapy; lens; gene expression; RNA splicing; renal tubule; membrane reconstitution /synthesis; complementary DNA; Xenopus oocyte; laboratory rabbit; laboratory mouse; embryo /fetus cell /tissue; molecular cloning; human subject; urine; chimeric proteins; electron microscopy; cryopreservation;

membrane channels; erythrocyte membrane; molecular pathology; protein structure function; posttranslational modifications; phosphorylation; messenger RNA; polysaccharides; protein biosynthesis; complementary DNA; dyserythropoietic anemia; congenital hemolytic anemia; cell differentiation; chemotherapy; lens; gene expression; RNA splicing; renal tubule; kidney disorder; membrane reconstitution /synthesis; Xenopus oocyte; embryo /fetus cell /tissue; laboratory rabbit; laboratory mouse; molecular cloning; urine; human subject; protein sequence; electron microscopy; chimeric proteins; cryopreservation;

DESCRIPTION: The Red Cell Gordon Conference has provided an informal atmosphere for in depth discussions relating to the cellular and molecular biology of the erythrocyte as well as clinically pertinent issues. The meeting has traditionally drawn leading scientists and clinicians from around the world. The 1995 meeting should be especially noteworthy, because of major recent advances in several areas. Eight sessions are planned, each featuring an introductory talk, three full lectures and a selection of short talks in the following areas: 1. Structure and associations of membrane skeleton proteins. 2. Structures, functions, and physical behavior of bilayer-spanning proteins. 3. Hematopoiesis I, Early development and stem cells. 4. Hematopoiesis II, Erythropoietin and its receptor. 5. Control of red cell gene expression, Trans regulation. 6. Control of red cell gene expression, Cis regulation. 7. Disease models and therapies I, Control of gene expression. 8. Disease models and therapies II, Membranes-site of pathology and treatment. Two afternoon poster sessions will feature a mixture of presentations from all disciplines. Ten individuals with the most exciting posters will be asked to give 15 min. presentations on the final evening, rather than having a single keynote speaker. The diversity of interests and richness of discussions have led to numerous scientific collaborations stemming from past Red Cell Gordon Conferences, and the same benefits should arise from the conference planned for August 1995.

water channel; protein structure function; RNA splicing; erythrocyte membrane; protein isoforms; protein degradation; salivary glands; lung; renal tubule; brain; molecular pathology; gene expression; membrane reconstitution /synthesis; Xenopus oocyte; immunoelectron microscopy; molecular cloning; laboratory mouse; laboratory rabbit; genetically modified animals; urinalysis; yeasts; immunocytochemistry; human subject; complementary DNA; in situ hybridization;

DESCRIPTION: The human red cell membrane is the model upon which our general understanding of plasma membranes is based. Several membrane transport proteins have first been identified in red cells, and during previous years of this project, a 28kDa protein was discovered, purified, cloned, expressed and functionally defined in Dr. Agre's laboratory. Now designated AQP1, this protein is the first recognized membrane water transport molecule. While multiple homologous aquaporins are now becoming recognized in other tissues, physical studies of human red cell AQP1 are revealing its structure at subnanometer resolution and are providing advanced insight into the biophysical transport function of the molecule. Here Dr. Agre proposes studies to establish the molecular structure of AQP1 at near-atomic resolution and to define the behavior of AQP1 in membranes. Aim I. Structure and function of purified AQP1 protein. High resolution cryoelectron microscopic analysis of membrane crystals containing red cell AQP1 will be undertaken to elucidate the 3D structure of AQP1 at better than 3 resolution. Yeast and other heterologous systems will be developed to express mutagenized AQP1 molecules with specific epitopes for affinity-purifications, metal binding, definition of the aqueous pore, identification of the sites of ion repulsion and assembly of individual subunits into tetramers. Functional analysis of mutagenized forms of AQP1 will be determined by direct measurement of the water permeability of AQP1 proteins in yeast microsomal vesicles and in reconstituted proteoliposomes. Aim II. AQP1 protein in cell membranes. The Colton blood group antigens result from a polymorphism in the first extracellular loop of the AQP1 protein. Using fluorescently labeled-anti-Co, Dr. Agre plans to characterize the surface equilibrium distributions of AQP1 on normal red cells by immunofluorescence, immunoprecipitations, and flow sorting. Kinetic distributions of AQP1 in red cell membranes will be undertaken with anti-Co by measurement of fluorescence recovery after photobleaching. Similar studies will be performed on enzymatically modified red cells and red cells from patients with sickle cell anemia and other congenital hemolytic states. These abnormal red cells and AQP1 deficient red cells will also be examined for membrane water permeabilities. To fully define the molecular determinants of the Co antigen, nonerythroid cells will be studied for Co expression by transfection with mutagenized forms of AQP1.

DESCRIPTION: (provided by applicant): Numerous physiological and pathophysiological processes involve the transport of water across cell membranes. The molecular identity of water transporters became known with discovery of the aquaporin family of membrane water channels. Four of the ten known mammalian aquaporins are expressed in anterior segment of eye. This application addresses the molecular mechanisms regulating function of human AQPO, AQP1, AQP3, and AQP5 and dysfunction of these proteins in clinical disorders of eye. Aim I. Analysis of aquaporin proteins in normal human eye. New reagents will be prepared including plasmids encoding wild-type, site-directed mutant, and epitope-tagged human AQPO, AQP1, AQP3, and AQP5. Antibodies specific for the N- and C-termini of the human proteins will be raised in rabbits and affinity-purified. The biophysical functions of human aquaporins will be expressed in Xenopus laevis oocytes and analyzed at baseline and after activation. Human aquaporins will be expressed in yeast and purified for reconstitution into proteoliposomes for permeation studies, into planar bilayers for analysis of electrophysiological properties, and into membrane crystals for structural studies. The distribution of these aquaporins will be defined in normal human eye. Aim II. Analysis of aquaporin proteins in clinical disorders of eye. The distribution of human aquaporins will be defined in tissues from patients with cataract or Sjogren's syndrome. Basic mechanisms by which AQPO and AQP5 may contribute to these disorders will be sought including defects in water and solute permeation, membrane trafficking, subunit oligomerization, and internalization, Physiological deficits will also be evaluated in rodent models of AQPO degradation and AQP5 deficiency.

[unreadable] DESCRIPTION (provided by applicant): Regulation of water and acid-base metabolism is one of the most important homeostatic functions in mammals. We have recently identified and characterized a new homolog of aquaporin family, aquaporin-6 (AQP6) from rat kidney. AQP6 is quite different from the other members of the aquaporin family. AQP6 protein resides exclusively in the membranes of intracellular vesicles of acid secreting intercalated cells in renal collecting duct where it colocalizes with H+-ATPase. AQP6 functions as a pH-gated anion channel rather than a water channel. AQP6-null mice are used in studies of acid-base regulation, urinary concentrating and diluting capacity, and to investigate the significance of AQP6-mediated ion transport. The structure-function relationships of AQP6 are addressed to elucidate the unique mechanisms of gating and ion permeation by AQP6. AQP6 proteins are expressed and purified for structural studies. These multiple approaches in transgenic animal physiology, molecular biology, electrophysiology, and biophysics may facilitate definition of the biological and physiological relevance of AQP6 at a molecular level in hopes of providing new insight into problems in water and acid-base homeostasis. [unreadable] [unreadable] Aim I. Phenotypic analysis of AQP6-null mice. A gene targeting approach has been applied to create a mouse lacking AQP6. Renal clearance studies will be performed in metabolic cages under different conditions including water loading or deprivation and chronic acidosis or alkalosis. Isolated perfused renal tubule studies will be performed to examine the role of AQP6 in water, proton, and bicarbonate transport. [unreadable] [unreadable] Aim II. Structure-function relationship of AQP6. Site-directed mutations will be introduced to identify the key residues for ion permeation and gating of AQP6. Functions of mutants will be assessed by osmotic water permeability and ion conductance in Xenopus laevis oocytes. Patch-clamp analysis will be performed to characterize single-channel properties of AQP6 and to examine the effects of nitric oxide on AQP6 in mammalian cells. Heterologous expression system will be used for structural studies. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This is a renewal application for years 28-32 of GM07171 which supports the Medical Scientist Training Program (MSTP) at Duke University. One of the first MSTP's, this Program has been continually funded by the National Institutes of General Medical Sciences, the first ten years as GM01678 (1966-1975) and the subsequent 26 years as GM07171 (1975-2001). The Program currently has an award of 36 funded positions and is requesting the same number of slots in this renewal. However, the Program has grown significantly since the previous application in 1996 when there were 42 students. Currently, there are 57 students and the Program will reach a new steady state of 70-84 students. This number represents 10 to 12% of the Medical School class. The additional funding is provided by the Medical and Graduate Schools, the Duke Medical Alumni Association, and the Duke Endowment. In addition, an $800,000 reserve fund has been created to cover unexpected financial needs by the Program. As of this writing, 14 of 16 applicants accepted by the Program have indicated their intention to matriculate here. The current Program student body is quite diverse with 17.6% underrepresented minority enrollment and a gender distribution that is 44% female. A large pool of applicants (200-300 per year) has allowed us to recruit an outstanding group of students. The structure of the Program is unchanged since the prior application. It consists of a basic science core year, a clinical core year, graduate school, and return to a final clinical year. The average program length is 7.2 years. Students may spend their graduate years in the traditional Medical School basic sciences, Chemistry, Zoology, and Biomedical Engineering. Greater flexibility is now available to our students who may also opt a new Biostatistics and Bioinformatics department, Economics, or Cognitive Neuroscience/Psychology.

DESCRIPTION (provided by applicant): Aquaglyceroporins form a subfamily of the aquaporin water channel family-transporting water, glycerol and urea. Human red blood cells [RBCs] contain the aquaglyceroporin AQP3;mouse RBCs contain AQP9. Plasmodia causing malaria contain aquaglyceroporins -- P. falciparum causes human malaria and expresses PfAQP;P. berghei causes mouse malaria and expresses PbAQP. Glycerol is used by the intracellular malaria parasite [merozoite] for production of glycerolipids. Import of glycerol requires transport of the solute across three membranes - the RBC plasma membrane [PM], the parasitophorous vacuolar membrane [PVM], and the plasmodia plasma membrane [PPM]. We hypothesize that aquaglyceroporins are involved in malarial infection and may provide a new pathway of potential therapeutic importance. Aim I. To understand the contribution of aquaglyceroporins to glycerol transport by RBC membranes, the copy numbers will be determined for AQP3 in human and AQP9 in mouse RBCs. We will also compare the water, glycerol, and urea permeability of aquaglyceroporin null RBCs to wildtype cells. Aim II. The biophysical functions of aquaglyceroporin PbAQP from P. berghei will be characterized. In addition the localization and expression of PbAQP will be defined during the life cycle of the organism. Aim III. Aquaglyceroporin null cells will allow us to understand the role of these channels in malarial infection. The pathogenicity of PbAQP null P. berghei will be compared to that of wildtype parasites. The role of AQP9 will be determined by comparing the parasitemia of wildtype mice to that of AQP9 null mice. The mouse model of malaria will also be evaluated in the insect stages of Plasmodium by studying the expression of PbAQP in the insect and the effect of PbAQP disruption on the proliferation of P. berghei in the mosquito and the transmission of P. berghei from the mosquito to the mouse. Aim IV. Investigation of the human malaria parasite, P. falciparum, will be crucial for understanding roles for aquaglyceroporins in human malarial infection. Human RBCs will be infected with PfAQP null or wildtype P. falciparum, and the parasitemia compared. To determine the role of the human RBC aquaglyceroporins in malaria, parasitemia of wildtype RBCs will be compared to AQP3 null RBCs. Malaria is a major cause of disease and death of children in many underdeveloped countries. The parasites causing malaria invade red blood cells and multiply, causing massive cellular destruction. The goal of this application is to define the pathway for uptake of the nutrient glycerol by red blood cells and malaria parasites, with hope that this may reveal new avenues for prevention or treatment of malaria.

DESCRIPTION (provided by applicant): The proposed Center of Excellence for Malaria Research will focus on the epidemiology, vector biology and genetic diversity of malaria parasites under conditions typical of much of southern Africa. The research will be conducted in three endemic areas with different levels of malaria transmission and stages of control. The main research site will be located at the Malaria Institute at Macha (MIAM) in the Choma District of the Southern Province of Zambia. This is a semi-arid region that has a history of intense seasonal malaria transmission. While chemotherapy to reduce deaths from malaria has been in effect for over 20 years, no vector control interventions were implemented until 2007. The second field site will be at the Tropical Disease Research Centre (TDRC) in the Nchelenge District in the Luapula Province in northern Zambia, in the environs of Lake Mweru. This is a high transmission area with hyper- to holo-endemic malaria. The third field site will be at the Biomedical Research Training Institute (BRTI) in Mutasa District in Manicaland Province, Zimbabwe. This region was under an effective malaria control program for about 50 years but the program was terminated in the late 1990's. Consequently, there has been a resurgence of malaria. The ecological, geographical and demographic differences between these research areas provide a range of epidemiological conditions to investigate malaria transmission and control in southern Africa, specifically spatio-temporal patterns of transmission, vector biology and parasite diversity. Studies of malaria epidemiology will focus on hospital, clinic and community-based prospective studies to track spatial and temporal changes in transmission. Population genetic studies of the vector and parasite will allow characterization of the genetic heterogeneity associated with natural and imposed bottle necks and the changing frequency of insecticide resistance and antimalarial resistance mutations. The project Cores, consisting of Environmental Surveillance, Genomics and Data Management/Biostatistics provide links between the three research areas. An integrated relational data base will link information across space and time. The Training/Career Development Program will be coordinated at the University of Zambia and the Johns Hopkins School of Public Health and will take advantage of local capacity to strengthen their training programs to ensure the Center of Excellence for Malaria Research is sustained after the completion of this program.

The Administrative Core based at the Johns Hopkins Malaria Research Institute (JHMRI) at the Bloomberg School of Public Health (JHSPH) will be responsible for the overall administration, coordination and management of the Center of Excellence for Malaria Research in southern Africa. Dr. Peter Agre, a Nobel Prize winning physician-scientist and Director ofthe JHMRI, will be the Principal Investigator and the Core Leader ofthe Administrative Core. Dr. Agre, in conjunction with the Research Area Project Leaders and the Core Leaders (Data Management/Biostatistics, Environmental Surveillance, and Genomics) will have overall responsibility for managing, coordinating and supervising Center activities, and ensuring that project milestones are met within the proposed timelines. Dr. William Moss, Research Area A Project Leader, will assist Dr. Agre in these activities as Project Co-investigator. In addition to Dr. Agre, the Administrative Core at JHMRI will consist of a Research Administrator, Genevieve Nixon, and a Senior Financial Analyst, Meredith Piplani. Dr. Clive Shiff will assist with the Training and Career Development Program as part ofthe Administrative Core. Utilizing existing recruitment structures and candidate selection process, the University of Zambia (UNZA) will recruit at least two post-doctoral candidates, junior faculty or established investigators to participate in a JHSPH- and UNZA-led training program designed to prepare scientists for careers in malaria research. Project activities will also be administered and managed at the Centers of Excellence established in southern Zambia, specifically the Malaria Institute at Macha (MIAM), Tropical Diseases Research Centre (TDRC) and University of Zambia (UNZA) in Zambia and the Biomedical Research and Training Institute (BRTI) in Zimbabwe. Each of these sites will have an overall project supervisor, administrative assistant and financial analyst. Communication between JHMRI and the sites in southern Africa will be facilitated through a project website that will allow for the rapid transmission of information and documents between all project scientists. Over the course ofthe project, and as a consequence of training, career development and capacity building, project administration and coordination will increasingly be based at the sites in Zambia and Zimbabwe. The goal is to build the capacity of endemic country scientists, to conduct high quality malaria research, to establish a sustainable Center of Excellence for Malaria Research, and to further malaria control. The Administrative Core is prepared to host all other Centers for the first Annual Workshop in Victoria Falls, Zambia.

DESCRIPTION (provided by applicant): Continued training in The Molecular and Cellular Bases of Infectious Diseases (MCBID) is proposed for 8 PhD students and 3 postdoctoral fellows selected from large pools of highly qualified applicants. The training program is uniquely situated in the Molecular Microbiology and Immunology Department (MMI) within the Johns Hopkins Bloomberg School of Public Health. The 29 training faculty have a wide range of experience and expertise in viruses, bacteria and parasites causing human disease and in the vectors and environmental factors associated with emergence and transmission of these pathogens. The training program has been funded since 1994 and has produced scientists working in many areas of academia and government on problems related to infectious diseases, vaccine development and the public's health. The goal of the MCBID training program is to provide students with both a firm foundation in the basic disciplines necessary for the study of infectious diseases and a perspective that will enable them to apply their knowledge creatively to public health problems. Each student is expected to complete 1) a series of required courses in the basic disciplines of cell and molecular biology, biochemistry, and immunology, 2) courses in virology, bacteriology, parasitology, and disease ecology, 3) courses in research ethics and public health perspectives, and 4) elective courses relevant to thesis topic and long-term career goals. Elective courses are chosen from among courses available in MMI, other departments in the School of Public Health, or in other Divisions of the University. Students will also complete 3 11-week laboratory rotations during the first year. Student progress is monitored by a Thesis Advisory Committee and the Graduate Program Committee. The goals of the postdoctoral training program are 1) to provide focused training in those areas of the molecular and cellular basis of infectious diseases in which program faculty have special expertise;2) to provide an opportunity for doctoral degree holders trained in more traditional environments to broaden their exposure to problems of public health importance and to evaluate their career goals in terms of public health issues;and 3) to prepare the PDF for an independent career in the biological sciences. RELEVANCE : This program is highly relevant to national interests in the areas of emerging infectious diseases, as it trains students and postdoctoral fellows broadly not only in both the molecular aspects of pathogen biology and disease pathogenesis, but also in the ecology of disease emergence and the role of vectors in pathogen transmission.

DESCRIPTION (provided by applicant): Continued training in The Molecular and Cellular Bases of Infectious Diseases (MCBID) is proposed for 8 PhD students and 3 postdoctoral fellows selected from large pools of highly qualified applicants. The training program is uniquely situated in the Molecular Microbiology and Immunology Department (MMI) within the Johns Hopkins Bloomberg School of Public Health. The 29 training faculty have a wide range of experience and expertise in viruses, bacteria and parasites causing human disease and in the vectors and environmental factors associated with emergence and transmission of these pathogens. The training program has been funded since 1994 and has produced scientists working in many areas of academia and government on problems related to infectious diseases, vaccine development and the public's health. The goal of the MCBID training program is to provide students with both a firm foundation in the basic disciplines necessary for the study of infectious diseases and a perspective that will enable them to apply their knowledge creatively to public health problems. Each student is expected to complete 1) a series of required courses in the basic disciplines of cell and molecular biology, biochemistry, and immunology, 2) courses in virology, bacteriology, parasitology, and disease ecology, 3) courses in research ethics and public health perspectives, and 4) elective courses relevant to thesis topic and long-term career goals. Elective courses are chosen from among courses available in MMI, other departments in the School of Public Health, or in other Divisions of the University. Students will also complete 3 11-week laboratory rotations during the first year. Student progress is monitored by a Thesis Advisory Committee and the Graduate Program Committee. The goals of the postdoctoral training program are 1) to provide focused training in those areas of the molecular and cellular basis of infectious diseases in which program faculty have special expertise; 2) to provide an opportunity for doctoral degree holders trained in more traditional environments to broaden their exposure to problems of public health importance and to evaluate their career goals in terms of public health issues; and 3) to prepare the PDF for an independent career in the biological sciences. RELEVANCE : This program is highly relevant to national interests in the areas of emerging infectious diseases, as it trains students and postdoctoral fellows broadly not only in both the molecular aspects of pathogen biology and disease pathogenesis, but also in the ecology of disease emergence and the role of vectors in pathogen transmission.