Studies will be conducted on the endocrine control of insect molting and development, tracing at the cellular, biochemical and molecular levels the means by which a brain neuropeptide, prothoracicotropic hormone (PTTH), stimulates the prothoracic glands to synthesize and secrete a steroidal prohormone, 3-dehydroecdysone (3dE) which is ultimately converted peripherally to the major molting hormone, 20-hydroxyecdysone (20-HE). Analyses will continue on the role of PKA and S6 kinase, phosphatase and protein synthesis (general and specific) in the transductory pathway linking peptide hormone receptor and enhanced ecdysteroidogenesis. The role of protein synthesis in terms of PTTH enhanced beta-tubulin synthesis by using molecular probes and immunocytochemistry. In the second specific aim, ecdysteroidogenesis will be studied by dissecting the biosynthetic pathway between cholesterol and 3dE using classical biochemical and molecular paradigms. The biochemical paradigms will utilize suicide inhibition probes recently synthesized in the principal investigator s laboratory. Characterization of what appears to be a unique prothoracic gland cytochrome P450 involved in 3dF biosynthesis (hydroxylation) will proceed utilizing high affinity probes and monoclonal antibodies. The third portion of this project deals with ecdysteroid action using Drosophila oogenesis as a model system to study ecdysteroid - juvenile hormone interactions as well as those between the genes for the ecdysteroid receptor (EcR) and ultraspiracle which form heteromers.
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