Evaluation of allatostatin pseudopeptide analogs and quantification of allatostatin expression in the cockroach Diploptera punctata.

摘要

YXFGLa-allatostatins (ASTs) were originally identified on the basis of their ability to inhibit juvenile hormone biosynthesis in vitro by corpora allata from the cockroach, Diploptera punctata. In the invertebrates, they are now known to be a ubiquitous, multifunctional family of peptides.; Although ASTs have been shown to affect important physiological processes in vitro, they are susceptible to inactivation by peptidases in the haemolymph, gut and other internal tissue membranes. We designed and synthesized a series of D. punctata-AST derived pseudopeptides, in which sterically bulky components were incorporated into the active core region to block peptidase attack. Peptidase cleavage and bioactivity were investigated in these analogs and the results used to design and synthesize the first pseudopeptide analog of an insect neuropeptide resistant to degradation by both haemolymph and membrane-bound peptidases. Injection of this analog into mated female D. punctata significantly inhibited the endogenous rates of JH biosynthesis and growth of basal oocytes. It is likely that the biological activity of AST(b)&phis;2 is attributable, at least in part, to its resistance to degradation. Disruption of critical reproductive and/or developmental processes by AST-pseudopeptide analogs could provide novel and selective strategies for future insect pest management.; The cloning of the Dippu-AST preprohormone provided the necessary information to study Dippu-AST expression. We have developed (and optimized) a sensitive and specific quantitative competitive reverse-transcriptase polymerase chain reaction (QC-RT-PCR) method to quantify Dippu-AST expression. Using this technique, we have quantified levels of Dippu-AST mRNA in the brain of females (mated and virgin) and males, and in the midgut of mated females during early adult life. Levels of Dippu-AST expression changed significantly in the brains of mated and virgin females and in the midgut. We have also used this technique to show, for the first time, Dippu-AST expression in both the common and lateral oviducts, in addition to the ovary, in adult female D. punctata. The quantification of Dippu-AST mRNA has yielded insights into the function of ASTs. Furthermore, the quantification of Dippu-AST expression under normal conditions, will provide a basis for comparison of Dippu-AST expression following various physiological manipulations.