The means by which hormones interact and control growth and development will be examined using insert metamorphosis as a model system. Precisely timed tobacco hornworm larvae raised on an artificial diet will be the primary experimental organisms. We will attempt to purify and characterize the two forms of the brain neurohormone (PTTH) that initiate cellular reporgramming and the molting cycle, using chromatographic and electrophoretic methods and our newly devised specific and sensitive in vitro assay. Once purified, will be generated and an RIA will be developed so as to titer the hormone and immunocytochemically trace its transport from the site of synthesis (prothoracicotropes) to the neurohemal organ. The role of PTTH in stimulating the prothoracic glands to synthesize and secrete ecdysone, via cAMP, will be studied using organ culture techniques to quantify protein kinase activity (protein phosphorylation). ACtive and inactive prothoracic glands will be investigated as well as glands stimulated by crude extracts and purified PTTH. The role of PTTH in embryogenesis, pupal diapause, and diapause-break will also be explored. Studies will continue on the regulation of the mitochondrial cytochrome P450 ecdysone monooxygenase that converts the steroid prohormone, ecdysone, to the molting hormone, 20-hydroxyecdysone, with emphasison control by juvenile hormone and neural-factors, using our sensitive radioenzymological assay. We will investigate the relationship between the very specific juvenile hormone binding protein of insect hemolymph and target cell cytosol receptors utilizing competitive binding protein techniques developed in this laboratory. Using very high specific activity tritiated ecdysteroid and nominally high specific activity juvenile hormone, receptors for both hormone classes will be localized in endocrine gland so as to examine feedback relationships between the glands and the growth hormones. These studies should provide evidence for the means by which animals regulate and maintain hormone titers and how environment cues are transduced into endocrine mediated events. These phenomena could shed light on endocrinologically based diseases and may lead to new concepts for insect control, insects being vectors of numerous human diseases and the indirect cause of malnutrition.
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