John H. 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.

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.

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.

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.