John Law - US grants

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An accurate, efficient modern instrument for amino acid analysis is required by a diverse group of investigators. This would be utilized for determining amino acid composition of proteins and peptides, as well as for free amino acids in physiological fluids. Additional equipment is needed to upgrade the protein structure facility by providing automated operation and data processing, as well as a reliable source of pure water for the sensitive chromatographic steps.

Dr. John Law and his colleagues at the University of Arizona will establish a Biological Research Center for Insect Science (CIS). The CIS will aim research at 1) discovering new aspects of insect biology that will aid in the design of biorational methods for controlling insects; 2) development of model systems using insects that will yield fundamental knowledge useful in the study of mammalian systems; and 3) searching for new useful products and processes from insects. Programs will include research, training, education and outreach, with a strong emphasis on molecularly-oriented interdisciplinary research. The CIS will sponsor meetings and symposia in insect science, and have strong international linkages. An industrial associates program will facilitate technology transfer. Individual projects will include research on insect: cuticle structure and function, digestion and nutrition, proteins and hormones, endocrine systems, reproductive systems and central nervous systems. Other studies will focus on molecular, sensory, behavioral and ecological factors in insect-plant interactions; insect communication and behavior; insecticide action; caste differentiation in social insects; and insect population biology, systematics and evolution. Through the results of its research and training programs, the CIS should have a worldwide impact on fundamental and applied biology. Insects share co-dominance of the biosphere with humans. The relationship ranges from competitive to cooperative, as they compete for resources, carry disease, pollinate plants, and process organic wastes. The importance of understanding insect biology was brought out as some chemical pesticides were abandoned for social and environmental concerns, and as the effectiveness of others decreased when resistant strains developed. In fact, much of our knowledge of insects derives from studies of one organism, the common fruit fly Drosophila, which has been exploited for decades in the study of genetics and animal development. The remarkable new experimental tools provided by recent advances in biochemistry and molecular biology now allow new approaches to the study of fundamental aspects of the biology of many other insects. Besides contributing to the development of new agents and strategies for pest control, such studies should provide a wealth of information on genetics, social behavior, animal development, and nervous system function. To this end, the CIS will provide multidisciplinary research and education programs on insect science. This center will be built on a strong base of existing faculty and will coordinate with an existing Biotechnology Center to expand collaborative efforts in basic research, to train present and future scientists, and, in collaboration with industry, to probe the potential uses of insects in applied research.

A group of investigators pursuing diverse research projects require sensitive instrumentation for protein and peptide sequencing, amino acid analysis and DNA sequencing. Among the projects to be pursued with these instruments are structure of polypeptides, cDNA and genes important in lipid transport, egg production, oxidative reactions, neurotransmission, heat shock and endocrinology.

The long-term objectives of this study are to define lipid mobilization, transport and metabolism in insects and to characterize the biochemical processes involved in formation of the insect egg. The relationship of these studies to human health is two fold. First, the processes of mobilization, transport and metabolism of lipids in insects has direct counterpoints in mammals, and study in the invertebrate animal model can provide new insight into the mammalian processes. Second, detailed studies of egg production and lipid metabolism in insects can suggest new general methods of insect control that will be applicable to insect vectors of disease. Using the tobacco hornworm, Manduca sexta, we will study lipid mobilization in an in vitro fat body system. Factors involved in mobilization (lipoproteins, apoproteins, lipid transfer factor, adipokinetic hormone) will be defined by varying their structure and lipid composition. Specific delivery of lipids to different peripheral tissue, especially flight muscle and prothoracic glands (site of steroid hormone synthesis) will also be studied in vitro. Factors involved in specific lipid delivery (e.g. diacylglycerol to flight muscle, cholesterol to prothoracic glands) will be defined and characterized. Endocytotic receptors in the ovary that are responsible for selecting specific hemolymph proteins and internalizing them in the oocyte will be isolated, characterized, cloned and sequenced. This should provide information on evolution and diversity of membrane receptors. Specific inhibitors (antibodies, peptides) of endocytosis will be obtained and used to produce eggs lacking specific proteins in order to determine the function of these materials in developing embryos.

This proposal requests funds to purchase multi-user instrumentation for biomolecular processing, purification and analysis. An Automated Laboratory Workstation would assist processing DNA sequencing reactions. A High Performance Gel Electrophoresis Unit, a Synthetic Peptide Preparative Purification System, and a Biomolecule Preparative Purification System would accomplish a wide range of macromolecule purifications. A Solution Analytical Capillary Electrophoresis Unit would permit advanced detection of biomolecules for a wide range of research needs. The main users would be researchers in the Colleges of Agriculture, Arts and Sciences, and Medicine, and the Arizona Research Laboratories at the University of Arizona. The instruments will be housed in the Macromolecular Structure Facility established by the Division of Biotechnology. Skilled prsonnel will operate, maintain and train other users. The specific research that would be enhanced by these instruments includes: the molecular biological basis for cell differentiation and morphology; plant gene expression; plant responses to environmental stresses (salt, low water,,heat); RNA processing, protein transport and gene expression in chlorplasts; cyanelle molecular biology; expression of insect proteases inhibitors; insect endocrinology; structure/function studies of insect proteins; insect protein and lipid biochemistry; total immune system evolution; peptide/protein conformational constraints and receptor recognition; biopolymer/Ligand interactions; and chromatography support design.

This proposal requests funds to purchase two multi-user instruments for oligonucleotide and peptide synthesis. An Automated Oligonucleotide Synthesizer would allow uninterrupted core DNA service by replacing an old, undependable and inefficient instrument. A Multiple Peptide Synthesizer would allow improvements in basic research projects currently hampered by the expense and time required for synthesis of a large number of peptide analogs. The major users would be NIH/NSF funded researchers in the Colleges of Agriculture, Arts and Sciences, Medicine, and the Arizona Research Laboratories (an interdisciplinary division). The instruments would be housed in the Macromolecular Structures Facility established by the Division of Biotechnology. Skilled personnel will operate, maintain and train other users. THe specific research that would be enhanced by these instruments includes: the molecular biological basis for cell differentiation and morphology; plant gene expression and responses to biological stresses (salt, low water, heat); heat shock proteins; RNA processing, protein transport and gene expression in chloroplasts; yeast mRNA stability; gene transcription regulation and self-processing; cytokeratin immunology and biochemistry; expression of insect protease inhibitors; insect endocrinology; insect protein, lipid, and structure function studies; total immune system evolution; electron transport protein biochemistry and studies on peptide/protein conformational constraints, hormone receptor recognition and structure/function studies.

Recent reports have established that insects, like vertebrates, have iron-binding proteins, transferrin and ferritin. This suggests that iron metabolism in insects may be very similar to that in vertebrates. Therefore, the insect could be used as a model for studying iron metabolism, and the results could be applicable in vertebrate systems. Furthermore, a detailed study of the insect system will give new insights into strategies for insect control and for disrupting the interaction between parasites and insect disease vectors. We propose to: 1) characterize ferritin from Manduca sexta and develop molecular probes for it; 2) determine the presence and characteristics of transferrin receptors from tissue of this insect; 3) determine if the translational regulatory expression of ferritin and the transferrin receptor is similar to that of vertebrates; 4) search for ferritin receptors and characterize them; and 5) develop an in vitro insect tissue model for iron absorption. The molecular probes developed in this project will be used to explore iron metabolism in insects that are less amenable for biochemical studies, particularly mosquitoes.

The long-term objectives of this study are to define lipid mobilization, transport and metabolism in insects and to characterize the biochemical processes involved in formation of the insect egg. The relationship of these studies to human health is two fold. First, the processes of mobilization, transport and metabolism of lipids in insects has direct counterpoints in mammals, and study in the invertebrate animal model can provide new insight into the mammalian processes. Second, detailed studies of egg production and lipid metabolism in insects can suggest new general methods of insect control that will be applicable to insect vectors of disease. Using the tobacco hornworm, Manduca sexta, we will study lipid mobilization in an in vitro fat body system. Factors involved in mobilization (lipoproteins, apoproteins, lipid transfer factor, adipokinetic hormone) will be defined by varying their structure and lipid composition. Specific delivery of lipids to different peripheral tissue, especially flight muscle and prothoracic glands (site of steroid hormone synthesis) will also be studied in vitro. Factors involved in specific lipid delivery (e.g. diacylglycerol to flight muscle, cholesterol to prothoracic glands) will be defined and characterized. Endocytotic receptors in the ovary that are responsible for selecting specific hemolymph proteins and internalizing them in the oocyte will be isolated, characterized, cloned and sequenced. This should provide information on evolution and diversity of membrane receptors. Specific inhibitors (antibodies, peptides) of endocytosis will be obtained and used to produce eggs lacking specific proteins in order to determine the function of these materials in developing embryos.

Immobilized metal ion affinity chromatography (IMAC) has proved useful in isolating proteins present in trace amounts from complex mixtures. The objective of this award is to characterize and map several IMA-adsorbents and to use the results of these studies to formulate and test protocols for the isolation of proteins from crude mixtures using cascade mode multi-affinity chromatography (CASMAC). The first goal is to characterize new gels with regard to chelating agents, metal ions and chromatography conditions. Among other methods, adsorption isotherms and NMR will be used. Secondly, these gels will be mapped using retention studies of amino acids, and model peptides and proteins. Thirdly, the investigators plan to develop and test protocols that allow fractionation of complex protein mixtures using CASMAC. It is anticipated that this work will result in making adsorbents available to the scientific community for both IMAC and CASMAC that offer incremental affinities for proteins and peptides, and that are specifically targeted for interaction with different amino acid side groups. Further the investigators plan to develop a rational approach using a series of affinity techniques to separate complex protein mixtures effectively.

All insects provide essential nutrients to their eggs. Failure to provide these nutrients will result in failure of the embryo to develop. Determination of the molecular events of transport and sequestration of nutrients in the egg should provide new targets for specific control of insect pests, especially vectors of human and animal diseases. We will investigate the transport of two essential nutrients--iron and lipids--to the oocytes of the mosquito Aedes aegypti. Since the adult female must feed on vertebrate blood in order to produce eggs, we will study the utilization of both inorganic and heme iron from the blood meal for egg production. Focus will be on the carrier proteins for transport through the hemolymph and the uptake and storage in the oocyte. This will involve iron-binding proteins, transferrin and ferritin, and receptors that allow iron to enter cells. Lipid transport from the fat body to the oocyte involves release, probably facilitated by a lipid transport particle, transport by lipoproteins, unloading at the ovary facilitated by lipoprotein lipase and a receptor for binding the lipoprotein to the cell membrane. Attempts will be made to assess the effectiveness of interfering with the function of the various macromolecular components. The ultimate goal is to provide systems that will allow development of specific materials that inhibit insect reproduction.

DESCRIPTION (Adapted from the Applicant's Abstract): Iron is an essential nutrient for nearly all life forms. It is also a dangerous toxin that must be controlled in biological systems. Proeins that have a high affinity for binding ionic iron or are capable of sequestering it so that it cannot exhibit its harmful effects are produced by virtually all organisms. These same proteins sometimes are used as a defense against parasites and pathogens by depriving them of a nutrient source of iron. The use of the iron-binding proteins transferrin and ferritin as a protection against iron poisoning or defense against pathogens and parasites will be investigated in flies and mosquitoes. It is known the transferrin synthesis is increased by bacterial infection of these insects. This is a part of the insect innate immune system.

The biotechnology and life sciences industry has become one of the primary economic forces in Pittsburgh, projecting 20% growth in the need for qualified technicians in the next ten years. If the frontiers of knowledge in Pittsburgh's biotechnology research and development community are to be advanced and scientific breakthroughs are to occur, trained biotechnicians must be available to meet the demand. The aim of the Biotechnology Workforce Collaborative (BWC) is to widen the spectrum of available, skilled workers in the region by enhancing ongoing partnerships with local stakeholders to a burgeoning program for the training of biotechnology/life sciences laboratory technicians.

The objectives and methods include:
(a) Academic training at the college resulting in the associate degree.
(b) A laboratory internship in the laboratories of Allegheny General Hospital, Allegheny Singer Research Institute (ASRI), the Pittsburgh Tissue Engineering Initiative (PTEI); and the University of Pittsburgh Medical Center McGowan Institute for Regenerative Medicine.
(c) Career development support for individual students starting at the early stages of participant preparation for college work and continuing in assistance for those students upon employment at various research laboratories.
(d) Workgroup meetings to further develop and refine the training approach for laboratory technicians.

Intellectual Merit: Recruitment efforts focus on developing three staggered cohorts of students. Both academic and employment criteria used in the field are being applied to the selection of promising candidates. Economically and educationally disadvantaged students are being supported by the project that integrates the associate degree for biotechnology technician with the workforce needs of research laboratories that specialize in molecular genetics, tissue engineering and regenerative medicine, and innovation in the application of research results for health care. The collaborative effort includes an internship and long term support for career development of biotechnology/life sciences laboratory technicians.

Broader Impacts: Based on gender, ethnicity, and geography, the cohorts broaden the participation of underrepresented groups in the biotechnology field, predominantly serving the biological sciences. As the BWC continues to become a major player in the Pittsburgh region, this project has an important role in advancing discovery and understanding while promoting teaching, training, and learning between the academic and industry partners. It enhances the infrastructure for research and education as a feeder to meet employment needs and as a critical link to industry.