Biomedical subjects

Hiroyuki Minakata

Publications and source records attributed to Hiroyuki Minakata.

25 records · Page 2Linked to original sources

PubMed2003

Novel neuropeptides related to frog growth hormone-releasing peptide: isolation, sequence, and functional analysis.

We previously identified in the bullfrog a novel hypothalamic RFamide peptide (SLKPAANLPLRF-NH(2)) that stimulated GH release in vitro and in vivo and therefore was designated frog GH-releasing peptide (fGRP). Molecular cloning of cDNA encoding the deduced fGRP precursor polypeptide further revealed that it encodes fGRP and its related peptides (fGRP-RP-1, -RP-2, and -RP-3). In this study immunoaffinity purification using the antibody against fGRP was therefore conducted to determine whether these three putative fGRP-RPs exist as mature endogenous ligands in the frog brain. The mass peaks of the isolated immunoreactive substances were detected at 535.78, 1034.14, and 1079.71 m/z ([M+2H](2+)), and their sequences, SIPNLPQRF-NH(2), YLSGKTKVQSMANLPQRF-NH(2), and AQYTNHFVHSLDTLPLRF-NH(2), were revealed by the fragmentation, showing mature forms encoded in the cDNA sequences of fGRP-RP-1, -RP-2, and -RP-3, respectively. All of these fGRP-RPs contained a C-terminal -LPXRF-NH(2) (X = L or Q) sequence, such as fGRP. This study further analyzed hypophysiotropic activities of the identified endogenous fGRP-RPs. Only fGRP-RP-2 stimulated, in a dose-related way, the release of PRL from cultured frog pituitary cells; its threshold concentration ranged from less than 10(-7) M. A similar stimulatory action of fGRP-RP-2 on GH release was evident. It was ascertained that fGRP-RP-2 was also effective in elevating the circulating GH and PRL levels when administered systemically. In contrast, fGRP-RPs did not have any appreciable effect on the release of gonadotropins. Thus, fGRP-RP-2 may act as a novel hypothalamic factor on the frog pituitary to stimulate the release of GH and PRL.

Amino Acid Sequence↗

PubMed2003

Cloning of Octopus cephalotocin receptor, a member of the oxytocin/vasopressin superfamily.

We reported that the common octopus, Octopus vulgaris, in common with vertebrates, possesses two members of the oxytocin/vasopressin superfamily: octopressin (OP) and cephalotocin (CT). This was the first observation of its kind in invertebrates. As OP and CT have different biological activities, the presence of specific receptors has been proposed. We cloned the cDNA of an orphan receptor from Octopus brain and found it to encode a polypeptide of 397 amino acids that displays sequences characteristic of G-protein coupled receptors. The orphan receptor showed high homology to receptors of the oxytocin/vasopressin superfamily and seemed to conserve the agonist-binding pocket common to the oxytocin and vasopressin receptors. Xenopus oocytes that express the orphan receptor responded to the application of CT by an induction of membrane Cl(-) currents coupled to the inositol phosphate/Ca(2+) pathway. OP and the other members of the oxytocin/vasopressin superfamily did not activate this receptor. HPLC fractionation of the Octopus brain extract combined with an oocyte assay yielded a single substance that was identical to CT. On the basis of these results, we conclude that the cloned receptor is the CT receptor (CTR). Expression of CTR mRNA in Octopus was detected in the central and the peripheral nervous systems, the pancreas, the oviduct and the ovary. This receptor may mediate physiological functions of CT in Octopus such as neurotransmission, reproduction and metabolism.

Amino Acid Sequence↗

PubMed2003

Insight into tachykinin-related peptides, their receptors, and invertebrate tachykinins: a review.

Tachykinins (TKs) constitute the largest vertebrate neuropeptide family with multifunctions in central and peripheral tissues. In several invertebrate species, two types of structurally related peptides, 'tachykinin-related peptides (TKRPs)' and 'invertebrate tachykinins (inv-TKs)' have been identified. TKRPs, isolated from the nerve and/or gut tissues, contain the common C-terminal sequence -Phe-X-Gly-Y-Arg-NH(2) (X and Y are variable) analogous to the vertebrate TK consensus -Phe-X-Gly-Leu-Met-NH(2), and exhibit vertebrate TK-like contractile activity on invertebrate gut tissues. Inv-TKs have been shown to be present exclusively in the salivary gland of several species, to share vertebrate TK consensus motif, and to possess TK-like potencies on vertebrate, not invertebrate tissues. However, the functional and evolutionary relevance of TKRPs and inv-TKs to vertebrate TKs remains to be understood. Recent studies have revealed that TKRP precursors dramatically differ from vertebrate preprotachykinins in structural organization and that TKRP receptors share structural and functional properties with vertebrate TK receptors. Moreover, the C-terminal arginine in TKRPs has been shown to play an essential role in discriminating their receptors from vertebrate TK receptors. Such recent marked progress is expected to enhance further investigation of biological roles of TKRPs. This review provides an overview of the basic findings obtained previously and a buildup of new knowledge regarding TKRPs and inv-TKs. We also compare TKRPs and inv-TKs to vertebrate TKs with regard to evolutionary relationships in structure and function among these structurally related peptides.

Amino Acid Sequence↗

PubMed2002

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.

Amino Acid Sequence↗

PubMed2002

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.

Amino Acid Sequence↗

PubMed2002

A novel tachykinin-related peptide receptor. Sequence, genomic organization, and functional analysis.

Structurally tachykinin-related peptides have been isolated from various invertebrate species and shown to exhibit their biological activities through a G-protein-coupled receptor (GPCR) for a tachykinin-related peptide. In this paper, we report the identification of a novel tachykinin-related peptide receptor, the urechistachykinin receptor (UTKR) from the echiuroid worm, Urechis unitinctus. The deduced UTKR precursor includes seven transmembrane domains and typical sites for mammalian tachykinin receptors and invertebrate tachykinin-related peptide receptors. A functional analysis of the UTKR expressed in Xenopus oocytes demonstrated that UTKR, like tachykinin receptors and tachykinin-related peptide receptors, activates calcium-dependent signal transduction upon binding to its endogenous ligands, urechistachykinins (Uru-TKs) I-V and VII, which were isolated as Urechis tachykinin-related peptides from the nervous tissue of the Urechis unitinctus in our previous study. UTKR responded to all Uru-TKs equivalently, showing that UTKR possesses no selective affinity with Uru-TKs. In contrast, UTKR was not activated by substance P or an Uru-TK analog containing a C-terminal Met-NH2 instead of Arg-NH2. Furthermore, the genomic analysis revealed that the UTKR gene, like mammalian tachykinin receptor genes, consists of five exons interrupted by four introns, and all the intron-inserted positions are completely compatible with those of mammalian tachykinin receptor genes. These results suggest that mammalian tachykinin receptors and invertebrate tachykinin-related peptide receptors were evolved from a common ancestral GPCR gene. This is the first identification of an invertebrate tachykinin-related peptide receptor from other species than insects and also of the genomic structure of a tachykinin-related peptide receptor gene.

Amino Acid Sequence↗

PubMed2002

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.

Amino Acid Motifs↗