Novel fish hypothalamic neuropeptide.

Recently, we identified novel avian and amphibian hypothalamic neuropeptides that inhibited gonadotropin release and stimulated growth hormone release. They were characterized by a similar structure including the C-terminal LPLRF-NH2 motif. To clarify that the expression of these novel hypothalamic neuropeptides is a conserved property in vertebrates, we characterized a cDNA encoding a similar novel peptide, having LPLRF-NH2 from the goldfish brain, by a combination of 3' and 5' rapid amplification of cDNA ends (RACE). The deduced peptide precursor consisted of 197 amino acid residues, encoding three putative peptide sequences that included -LPXRF (where X is L or Q) at their C-termini. Mass spectrometric analyses revealed that a tridecapeptide (SGTGLSATLPQRF-NH2) was derived from the precursor in the brain as an endogenous ligand. Southern blotting analysis of reverse-transcriptase-mediated PCR products demonstrated a specific expression of the goldfish peptide gene in the diencephalon. In situ hybridization revealed the cellular localization of goldfish peptide mRNA in the nucleus posterioris periventricularis in the hypothalamus. Immunoreactive cell bodies were also restricted to the the nucleus posterioris periventricularis and the nervus terminalis and immunoreactive fibers were distributed in several brain regions including the nucleus lateralis tuberis pars posterioris and pituitary. Thus, the goldfish hypothalamus expresses a novel neuropeptide containing the C-terminal -LPQRF-NH2 sequence, which may possess multiple regulatory functions and act, at least partly, on the pituitary to regulate pituitary hormone release.

Isolation and characterization of a GnRH-like peptide from Octopus vulgaris.

Gonadotropin-releasing hormone (GnRH) is the key peptide in the hypothalamo-hypophysial-gonadal axis, the core of regulation of reproduction in vertebrates. In this study, an octopus peptide with structural features similar to vertebrate GnRHs was isolated from brains of Octopus vulgaris. This peptide showed luteinizing hormone-releasing activity in quail anterior pituitary cells. A cDNA encoding the precursor protein was cloned. The RT-PCR transcripts were expressed in the supraesophageal and subesophageal brains, peduncle complex, and optic gland. The presence of the peptide in the different brain region was confirmed with enzyme-linked immunosorbent assay and time-of-flight mass spectrometric analysis. Immunoreactive neuronal cell bodies and fibers were observed in the subpedunculate lobe that controls the optic-gland activity. Optic gland nerves and glandular cells in the optic gland were immunostained. The isolated peptide may be octopus GnRH that contributes to octopus reproduction not only as a neurohormone but also as an endocrine hormone.

A novel rat hypothalamic RFamide-related peptide identified by immunoaffinity chromatography and mass spectrometry.

Recently, cDNAs encoding novel RFamide-related peptides (RFRPs) have been reported in the mammalian brains by a gene database search and the deduced RFRPs have been suggested to participate in neuroendocrine and pain mechanisms in the rat. Two peptides have been predicted to be encoded in the cDNA of rodent RFRPs. To assess precise functions of rodent RFRPs in the brain, in the present study we identified a naturally occurring RFRP in the rat hypothalamus by immunoaffinity purification combined with mass spectrometry (MS). The affinity chromatography showed that the rat hypothalamus contained RFRP-like immunoreactivity. The immunoreactive material was analyzed by a nanoflow electrospray ionization time-of-flight MS followed by tandem MS analysis. The mass peak corresponding to octadecapeptide was detected at 1010.54 m/z ([M+2H](2+)) and its sequence, ANMEAGTMSHFPSLPQRF-NH(2), was revealed by the fragmentation, showing a mature form encoded in the cDNA sequence of RFRPs. The identified endogenous RFRP will aid not only in defining its physiological roles but also facilitate the development of its agonists and antagonists in the rodent brain.