Expression of the allatostatin gene in endocrine cells of the cockroach midgut.

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RESUMO

Cockroach allatostatins are neuropeptides that have been isolated from the brain of Diploptera punctata and shown to inhibit juvenile hormone production by the corpora allata. Enzyme-linked immunoassay and immunocytochemistry with antisera to two allatostatins, ASB2 (AYSYVSEYKRLPVYNFGL-NH2) and ASAL (APSGAQRLYGFGL-NH2), revealed that allatostatins were located not only in the insect brain but also in several peripheral tissues including the cockroach midgut and hindgut. Allatostatin-like immunoreactivity was found in nerve fibers of the stomatogastric nervous system as well as in intrinsic endocrine cells of the midgut. Midgut extracts were shown to be biologically active in an allatostatin bioassay and to contain several allatostatin-like peptides, including the octadecapeptide ASB2, which was identified by mass spectrometry following HPLC purification. Reverse transcription of brain mRNA followed by PCR with degenerate oligonucleotides for ASB2 and ASAL yielded a 338-bp fragment of the allatostatin gene that encoded six allatostatins. In situ hybridization with this probe confirmed that an allatostatin gene is expressed in intrinsic endocrine cells of the midgut. Reverse transcription of midgut mRNA followed by PCR and sequencing of the product revealed that the same gene is expressed in the midgut and in the brain. Allatostatins are thus an example of insect "brain-gut peptides" and we suggest that their function may not be restricted to the regulation of juvenile hormone production.

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