Sarjeet Gill - US grants

The long-term objective is to provide the tools which will allow the assessment of risks to man upon exposure to epoxides. To do so we must understand the relative contribution of various epoxide metabolizing enzymes (EME's) in the degradation/activation of epoxides, some of which are cytotoxic, mutagenic and/or carcinogenic, in vivo. Thus cell free systems will initially be used, both to develop methodology and to better understand the various EME's, in particular the cytosolic and mitochondrial epoxide hydrolases (EH's) in the cell. Knowledge gained from these studies will then be used for hepatocyte culture and isolated liver systems, and finally to in vivo systems. The specific aims of this proposal are: I. Evaluate the relative role of EME's in cell free systems. This aim will develop assay methodology, monitor metabolism of endogenous epoxides, localize the mitochondrial EH, monitor the relative distribution of EME's in different species, and evaluate various compounds as inhibitors of EME's. The cytosolic EH will be purified, antibodies developed, and immunoassays (ELISA) developed. II. Evaluate EME's in hepatocytes. Levels of EME's will be determined and epoxide metabolism monitored. The effect of various inducers and inhibitors on epoxide metabolism in hepatocytes will be assessed. The cytotoxicity and genotoxicity of selected epoxides will be evaluated. III. Determine epoxide metabolism in isolated liver, and finally, IV. Perform initial experiments to determine epoxide metabolism in vivo. Various methodologies will be used. Epoxide hydration and/or conjugation will be monitored by partition, tlc, glc, and/or hplc assays. Cytosolic EH purification will be by ion exchange, hydrophobic, affinity and gel filtration chromatography, while characterization will include pI, substrate selectivity, kinetics, and active site analysis. Hepatocytes will be isolated following procedures by Seglen and monolayer cultures used. Pharmacokinetics and epoxide metabolism in isolated liver and in vivo will be determined by monitoring and identifying metabolites in perfusion media, blood, urine, feces and tissues. The practicality of using selective epoxide substrates, inducers, inhibitors and different species to unravel the complexity and relative role of the various EME's in the vivo metabolism of epoxides will be assessed.

The long-term objective is to understand the mechanism(s) by which the endotoxins of Bacillus thuringiensis (B.t.) exert their toxic effect. The endotoxin, although bacterial in origin is a mechanicl insecticide. Its use in insect control programs is increasing annually, however, inspite of this we do not have reliable information on its mode of action and the basis for its selective toxicity both between insects and mammals, and between insect species. The specific aims of this proposal are: I. Purify and characterize the protoxin and endotoxin of B.t., and develop antibodies and selective immunossays to endotoxin. II. Determine the toxicity of B.t. toxins. The toxicity of crystal, protoxin and endotoxin will be compared in both cell culture (insect, mammalian) and mouse. In addition, the effect of proteases (insect, mammalian, bacterial) on endotoxin structure, and the effect of chemical modification of endotoxin on its toxicity will be evaluated. III. Determine the mode of action of the endotoxin. The effect of protoxin and endotoxin on cell membranes, and on adenylate cyclase and protein synthesis will be determined. In addition, cellular uptake of the endotoxin will be monitored and initial experiments on characterizing cell membrane receptors for the endotoxin will be performed. Purification of the protoxin and endotoxin will be performed by a variety of biochemical techniques including ion exchange, hydrophobic and gel filtration chromatography, and polyacrylamide gel electrophoresis (PAGE) and preparative isoelectricfocusing (IEF). Antibodies will be raised in rabbits and enzymelinked immunosorbent assays developed. The endotoxin will be characterized by a number of biochemical and chemical methods. Assays will be developed using cell culture and radiolabeling techniques for evaluating endotoxin toxicity before and after chemical modification. Effect of proteases on endotoxin will be monitored by cytotoxicity, PAGE and IEF assays. Effect of endotoxin on cell membranes will be evaluated by electro-physiological techniques and ion channel probes. Cellular uptake and receptor binding will be monitored by radiolabeled ligands, while endotoxin effects on protein synthesis and adenylate cyclase will be determined by amino acid incorporation and enzyme assays, respectively. The research proposed will provide a better understanding of B.t. endotoxin structure, mode of action and basis of selective toxicity. Such an understanding will allow us to evaluate whether B.t. endotoxins can be used as selective membrane probes which may be useful in targeting immunotoxins in the treatment of disease.

The overall objective of the research proposed is to evaluate the effects of epoxidized toxicants, both naturally occurring and man- made, on the murine immune system. A large number of these epoxidized compounds are toxic, and as humans are exposed to these compounds either from industrial or environmental sources, epoxides pose significant hazards to human health. The specific objectives of the proposal are: 1) Evaluate the immunosuppressive (or immunopotentiative) effects of selected epoxides; 2) Characterize the mechanism(s) of immunosuppression; and 3) Attempt to modify these immunosuppressive effects. These epoxidized compounds will be examined for their effects on the immune system at sublethal doses. Three experimental protocols will be used. The first will involve a totally in vivo system with epoxide treatment, immunization and analysis of immune function performed in vivo; the second a combination of in vivo treatment with epoxides and in vitro evaluation of immune function, while the third protocol will be totally in vitro. In addition to pathology, both cell and humoral-mediated immunity will be monitored. Among the parameters of immune function determined will be the effects on host resistance, delayed hypersensitivity, mitogen and cytotoxic T-lymphocyte response, Natural Cell Mediated Cytotoxicity (both NK and NC), antibody response to antigen challenge, macrophage function and lymphokine production. Standard immunologic assays will be utilized to asses these functions. The effects of epoxides on cell populations will also be assessed with the aid of specific antibodies and a fluorescence activated cell sorter. We will also determine the effect of epoxide treatment on lipid peroxidation and glutathione levels, and evaluate how this affects immune response. In addition, the capability of lymphocytes to metabolize epoxides will also be determined. Finally we will attempt to characterize the mechanisms by which epoxides exert their immunosuppressive effects. In particular, the ability of epoxides to interfere with target recognition and binding and its subsequent killing of target cell will be determined by single cell binding assays, for both cytotoxic T-lymphocyte response and natural killer activity. Macrophage function will be determined by IL1 production, phagocytosis and antigen presentation.

This proposal's long-term objective is to characterize the proteins involved in organic molecule transport across insect cell membranes. Membrane protein mediated neurotransmitter, amino acid, and sugar transport systems are essential for insect homeostasis. In contrast to glucose and amino acid transport in bacteria, in yeast and in mammals, our knowledge of these transport processes in insects is rudimentary. More recently plasma membrane neurotransmitter transporters have been identified and cloned in vertebrates. We have preliminary information that similar neurotransmitter transporters, some of which appear to be unique, occur in insects. The specific objectives of this current proposal are to characterize plasma membrane and vesicular neurotransmitter transporters in the lepidopteran, Manduca sexta. M. sexta is being used as a model insect species because its nervous system is much better defined than that of mosquitoes. Once the M. sexta neurotransmitter systems are characterized these probes could be used to identify mosquito neurotransmitter transport. Significant amino acid homology exists between various vertebrate plasma membrane neurotransmitter transporters. Utilizing this fact oligonucleotide primers will be made using the conserved domains as templates for primer design. The primers will then be used for the polymerase chain reaction (PCR) amplification of the putative M. sexta plasma membrane neurotransmitter transporter. These PCR products or alternatively rat plasma membrane and vesicular neurotransmitter transporters will be used for screening of M. sexta cDNA libraries.. However, if this strategy fails, alternative protocols, ie. expression cloning of the transporters will be attempted. Once the cDNA are isolated, they will be expressed and their function and pharmacology determined using established methods. Characterization of these transporter proteins will facilitate further research on transmitter recycling in the nervous system in general, and in the insect brain in particular. Further, the proposed studies will aid more precise determination of transmitter distribution in the insect central nervous system. Such knowledge may enable us to target these transporters as a site for manipulating insect populations. The control of insects, in particular mosquitoes, is critical for the disruption of many mosquito borne diseases of man.

DESCRIPTION (Adapted from the Applicant's Abstract): This research's long-term objective is to understand ion regulation in insects, particularly in mosquitoes. Our present grant has determined the structure of many key proteins involved in ion regulation in Aedes aegypti Malpighian tubules. Further we have preliminary data that show enhanced V-ATPase transcript levels following a blood meal. In the present proposal we plan to build on these findings. Our objective in this proposal in to molecularly characterize the proteins that are involved in Na+ and C1- transport in mosquitoes. In the first objective we plan to functionally characterize a Na+/K+/2C1- cotransporter that was isolated from A. aegypti Malpighian tubules. This cotransporter is significantly divergent from known mammalian and insect cotransporters. Further while the V-ATPase plays a crucial role in ion regulation in mosquitoes, other proteins facilitate actual cation transport. Hence in the second objective we will also characterize the cation/antiporter CDNA clone isolated from the midgut and/or Malpighian tubule of A. aegypti. In the third objective we will attempt to evaluate changes in Malpighian tubules function following a blood meal. Therefore we will follow up our preliminary studies on V-ATPase regulation. We will determine shether V1 and V0 dissociation- reassociation occurs in Malpighian tubules after blood feeding ans also whether these genes are transcriptionally regulated. Data from these experiments will help elucidate mechanisms of change in ion transport in mosquito Malpighian tubules that occurs following a blood meal. In addition we will evaluate whether the A. aegypti Na+/K+/2C1- cotransporter is regulated. In summary, this proposal is directed towards elucidating the molecular processes involved in ion secretion following a blood meal in mosquitoes. Characterization of these processes may aid future work that could focus on the disruption of these regulatory processes in an important vector of human disease.

9977158

Abstract

This award will be used to acquire an advanced light microscope laboratory that will enable new research projects to be undertaken in the biological sciences at the University of California, Riverside (UCR). This laboratory will consist of an upright confocal scanning laser microscope, an inverted microscope for micromanipulation/injection, and a computer workstation for advanced image data analysis in a mult-user, state-of-the-art facility. These light microscopes will be housed in the currently existing centralized electron microscopy laboratory, thereby creating a Centralized Laboratory for Advanced Microscopy. The light microscopes will service over 30 well established research programs in 12 departments in the College of Natural and Agricultural Sciences. The users in this proposal specifically need this instrumentation for studies of fertilization, oocyte transport, angiogenesis, cell receptors, cell adhesion and dye transfer, cell junctions in neurosecretory cells, molecular motors, ion transport, plant pollination, trafficking of viral proteins in plants, and transcytosis. The equipment will allow Principle Investigators at UCR to extend their current research programsm and will enable initiation of many new projects not currently possible on the UCR campus. The light microscope laboratory has been carefully integrated and designed to be flexible, thereby meeting the broad range of needs of a core facility. The centralized laboratory where the light microscope will be housed is currently staffed by a full-time manager, and the university will provide an additional 50% Staff Research Associate for daily operation and maintainance of the light microscopes and workstation. Training on the light microscopes will be provided in an undergraduate course in Cell Biology, a graduate level class in microscopy, and by one-on-one interaction with the Staff Research Associate. UCR has the highest percentage of minority students of any UC campus and one of the highest nationally. Many students receiving training will be minority students.

DESCRIPTION (Adapted From The Applicant's Abstract): This grant is targeted towards the PI's long-term focus to characterize the structure and function of the midgut of mosquitoes. This grant focuses upon elucidating the molecular processes involved in nutrient uptake in Aedes aegypti. Through a molecular approach to identify transport processes at the midgut, the PI has isolated cDNA's for 3 amino-acid transporters, a potential protein needed to form a functional heterodimer with the amino acid transporters, and a putative sucrose transporter from the midgut of Aedes aegypti. In objective 1, the PI proposes to continue in the isolation of full-length cDNA clones for two of the proteins for which he has partial cDNAs. These cDNAs will be expressed in a heterologous expression system (Xenopus oocytes) and this system used to characterize the substrate selectivity of each of the amino acid transporters and the ion selectivity of the transport process. In the second objective, similar experiments will be performed for the sucrose transporter. As the final objective, the in vivo regulation of these transporters will be analyzed. The expression of the genes will be analyzed in vivo to determine if there is developmental regulation and modulation following either sugar or blood feeding.

A capillary DNA sequencer and associated instruments are requested to establish a core DNA sequencing facility at the University of California, Riverside. The principal investigator and seven other major users, all with NIH funding will need increased sequencing capability to identify DNA isolated in their research programs. The campus presently does not have any DNA sequencing facility where these investigators have ready access with reasonable costs. These investigators are involved in health- related research that include elucidation of biochemical processes in human disease bearing mosquitoes and their transformation, liver specific gene expression radiation induced mutagenesis and genomic instability, regulation of p53-mediated transcription, role of receptor tyrosine kinases and cell cycle genes in the shear stress-induced activation. These users wish to identify development of EST database for mosquitoes, DNA microarray development, mutation analysis, polymorphisms, determination of cDNAs and genes, and confirmation of expression constructs. All of the NIH funded grants would benefit from a core DNA facility on campus. The facility would also be of help for other investigators with NSL and USDA funding to elucidate sex- determination genes, identifying genes involved in sporulation and infection cycles in fungi, genes involved in plant defense mechanisms, and genetics of abiotic stress tolerance.

DESCRIPTION (provided by applicant): Bacillus thuringiensis subsp. israelensis has been used in the field for over twenty years without resistance development in any target insect. In contrast, mosquito resistance to Bacillus sphaericus has been observed in the field in many countries. This remarkable difference in the propensity to develop resistance to two mosquitocidal Bacillus strains is likely due to the presence of multiple toxins in B. thuringiensis subsp. israelensis. However, mosquitoes are able to rapidly develop resistance to individual toxins from this strain. A major reason for the apparent inability of mosquitoes to develop resistance to B. thuringiensis subsp. israelensis is the presence of cytolytic (Cyt) toxins in this and other mosquitocidal strains. The precise mechanism by which Cyt toxins interact with the insecticidal crystalline (Cry) toxins is not known, and therefore forms the basis for this proposal. We hypothesize that Cyt and Cry toxins interact in the membrane facilitating the formation of pores by either protein. In this proposal, our emphasis is on Cry11Aa and Cyt1Aa toxins of B. thuringiensis subsp. israelensis, which interact synergistically to enhance mosquitocidal activity of each toxin. The lack of resistance development is also due to two factors: the different molecular targets involved in the action of Cry and Cyt toxins, and secondly, the synergism between the Cyt and Cry toxins. Hence the objectives of the proposal are to first characterize the mechanism by which the Cry11Aa exerts its toxic effects, and the second is to understand the molecular basis of synergism between the Cry and Cyt toxins. In the first objective we will identify the domains involved in the receptor and toxin, identify the receptor(s), and finally isolate the receptor involved. Once the receptor is isolated we will use dsRNA-mediated gene silencing to determine its functional role as a Cry11A receptor. In the second objective our focus is on membrane binding of the toxins and the subsequent pore forming processes of both toxins. These two objectives are critical to our understanding of why no mosquito species have developed resistance to Bacillus thuringiensis subsp. israelensis. This project is also a joint proposal between three different investigators to best use the expertise of each laboratory.

[unreadable] DESCRIPTION (provided by applicant): Formulations of bacterial strains with insecticidal activity have been used for the control of insect pests in public health for more than two decade. For example, Bacillus thuringiensis subsp. israelensis has been used with remarkable success for the control of Simulium vectors in West Africa, resulting in the reduction of onchocerciasis, a disease of critical importance in that region. Similarly, this bacterium together with B. sphaericus has been used successfully against both Aedes and Culex in many developed and developing countries, thereby attenuating diseases, such as dengue and West Nile. However, the use of both these bacteria against Anopheles vectors of malaria has been much more limited, in part due to their lower activity against mosquitoes of this genus. In contrast Clostridium bifermentans subsp malaysia, another Gram positive spore forming strain isolated from Malaysia, has the highest toxicity to a number of Anopheles spp. Preliminary evidence supports our hypotheses, which are: a) that the mosquitocidal activity of this strain is due to toxins produced by the bacteria, and b) that toxins from this strain are novel. The focus of this R21proposal is therefore to identify the genes and isolate the toxic proteins using mosquito bioassays to drive both processes, and to characterize their activity against Anopheles, Aedes and Culex mosquitoes. This objective lays the ground work for our long-term objective of characterizing the molecular basis of selective activity of these toxins to different mosquito species. To achieve the objective proposed we will use both genomic and proteomic approaches. In the former approach, we have identified cosmid clones, prepared from genomic DNA, that are toxic to An. Stephensi larvae, and in this proposal we will characterize the critical gene(s) involved in toxicity using transposon mutagenesis and cosmid sequencing. As an additional approach we will purify the toxins involved using classical purification techniques, in conjunction with assays using larval mosquitoes, which will allow for monitoring purification of toxic proteins. Finally the toxins will be expressed to confirm their role in mosquitocidal toxicity. The proposed research will lead to the identification of new and novel mosquitocidal proteins. These protein toxins will provide additional tools that can be used to delay resistance development in mosquito vectors of human diseases and also, provide tools for optimization of mosquitocidal toxins that could be generated by modern biotechnological tools. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Bacillus thuringiensis subsp. israelensis (Bti) has been used in the field for over twenty years without resistance development in any target insect. In contrast, mosquito resistance to B. sphaericus has been observed in the field in many countries. This remarkable difference in the propensity to develop resistance to two mosquitocidal Bacillus strains is likely due to the presence of multiple toxins in Bti. However, mosquitoes are able to rapidly develop resistance to individual toxins from this strain. A major reason for the apparent inability of mosquitoes to develop resistance to Bti is the presence of cytolytic (Cyt) toxins in this and other mosquitocidal strains. During the last grant period we showed that key to the lack of mosquito resistance to Bti was how two toxins in this strain, namely Cry11A and Cyt1A toxins interact. We showed Cyt1A acts as a surrogate receptor for Cry11Aa. Importantly Cyt1Aa binds through Cry11A loop domains that are also involved in binding endogenous Aedes aegypti receptor proteins. Our research showed Aedes receptor proteins include cadherin, alkaline phosphatases (ALP), and aminopeptidases (APNs). All three classes bind mosquitocidal toxins with relatively high affinity, unlike in lepidopterans where only cadherin binds with high affinity. This implies both APN and ALP of mosquitoes could act as primary rather than as secondary receptors as in moths, suggesting a possible difference in the mode of action of mosquitocidal and lepidopteran Cry toxins. We believe however, there is conservation in the mode of action of Bt Cry toxins. Therefore we hypothesize a similar mode of action of action occurs in mosquitoes as in lepidopterans. In this proposal we plan to test this hypothesis. Consequently, we hypothesize that: (i) cadherin is a key protein which mediates initial binding to mosquitocidal Cry toxins and is essential for larval toxicity;ii) ALPs (and APNs) act as secondary receptors that allow toxin targeting to the cell membrane. Also in the previous proposal we showed Cyt1A plays a critical role in acting as a surrogate receptor for Cry11Aa. We therefore hypothesize that (iii) Cyt1A also is a receptor for other Cry toxins in Bti, and synergizes the toxicity of mosquitocidal Cry toxins. We will test this hypothesis and also elucidate the mechanism by which Cyt toxins insert into the membrane to act as surrogate receptors. This project is also a proposal to continue a successful collaboration between three different investigators to best use the expertise of each laboratory. PUBLIC HEALTH RELEVANCE: B. thuringiensis subsp. israelensis are used for control of human disease vectors, such as species of Simulium, Aedes, Culex, and Anopheles. Elucidating mechanisms of Cry toxin action in aids our understanding of how mosquito and black fly control is achieved and also enables us to determine mechanisms by which resistance can occur. This mode of Cyt1A toxin action investigation also illuminates mechanism of synergism between mosquitocidal Cry and Cyt toxins. Finally, elucidation of mechanisms by which the Cyt1Aa toxin acts also will help us understand how other cytolytic toxins of human health significance cause toxicity.

? DESCRIPTION (provided by applicant): Programs that control disease-transmitting vectors often use insecticides, both chemical and biological. Thus Bacillus and Lysinibacillus sp. have been used worldwide for over four decades for successful control of mosquitoes and blackflies. But a novel strain, Clostridium bifermentans (Cb) malaysia, the most toxic bioinsecticide to Anopheles has not been used, even though being an anaerobe; it is much easier to culture with minimal equipment. The lack of knowledge of what the toxins are in Cb malaysia prevents its utilization. Thus our long-term goal is to understand Cb malaysia mosquitocidal action, facilitating its use for the control of anopheles larval mosquitoes, thereby attenuating adult populations and malarial transmission. The overall objective of the current proposal is to identify the anopheline active toxins and elucidate their mechanism of action. To identify the mosquitocidal toxins involved, we generated a loss of function Cb malaysia mutant. Genomes of this mutant, Cbm-77, together with that of wild-type Cb malaysia and of the non-mosquitocidal type strain Cb were sequenced. The data obtained demonstrated that a loss of a megaplasmid in the Cbm-77 mutant, or its absence in Cb, results in total loss of mosquitocidal activity. Hence, we hypothesize the mosquitocidal activity is encoded by the plasmid, and specific genes in this plasmid are responsible for high Cb malaysia toxicity to anophelines. Mass spectrometry data confirmed that the anopheline-toxic proteins were encoded by the megaplasmid, and these proteins form a complex. The plasmid has two toxin encoding loci, cry and ctox; the former consists of a single operon, while the second locus has a cmp (clostridal mosquitocidal protein) operon and two additional genes, p47 and ha41. Therefore in the first aim we will validate our preliminary results by: a) identifying the critical anopheline active toxins in the ctox locus. Sine this locus encodes proteins that are similar to those produced by other Clostridium strains we hypothesize that the HA41 protein is involved in midgut membrane binding, while the CMP, which has an endopeptidase motif, is the active toxin that could cleave SNARE complexes required for vesicular release. Preliminary evidence supports both hypotheses. We will test these hypotheses in the next two aims: b). Characterize the binding of the Cb malaysia toxins to midgut membranes; and c). Define the intracellular target site for the Cb malaysia toxins. Completion of this proposal will show the utility of this novel bacterium because of its ease of use. Its novel mode of toxin action also complements that of toxins from B.t. israelensis and L. sphaericus, facilitating its use as a biocontrol agent for anopheline control. Its novel mechanism of toxicity would broaden the spectrum of available anopheline-active bioinsecticides, and will also help attenuate resistance development.