新しい脳ペプチド分離の新戦略

2001 Fiscal Year Annual Research Report

• Project/Area Number: 11354010
• Research Institution: Natiomal Institute of Genetics
• Principal Investigator: 藤澤 敏孝 国立遺伝学研究所, 個体遺伝研究系, 助教授 (60000262)
• Co-Investigator(Kenkyū-buntansha): 筒井 和義 広島大学, 総合科学部, 教授 (20163842) ; 小泉 修 福岡女子大学, 人間環境学部, 教授 (50094777) ; 岡戸 信男 筑波大学, 基礎医学系, 教授 (50060140) ; 松島 治 広島大学, 理学部, 教授 (80127711) ; 藤本 正昭 島根大学, 生物資源科学部, 教授 (90081728)
• Keywords: 脳ペプチド / ラット / トリ / 両生類 / 無脊椎動物

Research Abstract

1.ヒドラペプチドの組織的単離・同定:360のペプチドを単離し、内122種についてアミノ酸配列を決定し、38種を化学合成し生物活性検定に供した。最終的には新規神経ペプチド6種、上皮ペプチド5種を同定し、これらをコードする遺伝子全てを同定した。また、抗ペプチド抗体の作成、生物活性検定を順調にこなし、非常に興味あるペプチド分子を多く得ることが出来た。
2.抗PKVamide(Hym-176)抗体を用いたラット脳の免疫組織染色:我々は既に抗GLWamide抗体を用いてラット脳の免疫組織染色を行い、抗GLWamide抗体陽性細胞が腹側被蓋野に存在することを観察している。同様に、抗PKVamide抗体陽性細胞が視床下部弓状核腹側の外足部に観察された。これらのことからヒドラの神経ペプチドの類似ペプチドが脊椎動物の脳にも存在すると予想された。
3.抗ペプチド抗体3種(抗RFamide、抗GLWamide、抗PKVamide)を利用した脳ペプチドの分離:ヒドラを含む腔腸動物の神経ペプチド抗体を利用し、ELISA法を用い脊椎動物の脳から抗体と反応するペプチドの単離を行った。抗RFamide抗体を用いて、鳥類脳の視床下部からRFamideファミリーに属する新規のゴナドトロピン放出抑制ペプチドを同定した。両生類からもC-末端がRFamideの成長ホルモン放出ペプチドを同定した。また、ラット脳から抗GLWamide抗体と反応するペプチドを既に同定したが、抗PKVamide抗体と反応するペプチドも単離した。
4.その他の脳ペプチド:マダコ脳からマダコ循環系に作用するペプチド2種、生殖系に作用するペプチド3種を得た。またフナ脳から同定したC-RFa(mide)は視床下部で産生され、下垂体でプロラクチン(PRL)放出を促進し、PRLは血流で標的細胞(塩細胞と考えられる)に達し、浸透圧調整を行うと予想された。

Research Products

• [Publications] Iwao, K.: "A cnidarian neuropeptide of the GLWamide family induces metamorphosis of reef-building corals in the genus Acropora"Coral Reef. (In press).
• [Publications] Mochizuki, K.: "Universal occurrence of the vasa-related genes among metazoans and their germline expression in Hydra"Development, Genes and Evolution. 211. 299-308 (2001)
• [Publications] Bosch, T.C.G.: "Polyps, peptides and patterning"Bioessays. 23. 420-427 (2001)
• [Publications] Harafuji, N.: "Enhancement of foot formation in Hydra by a novel epitheliopeptide, Hym-323"Development. 128. 437-446 (2001)
• [Publications] Mochizuki, K.: "Expression and evolutionary conservation of nanos-related genes in Hydra"Development, Genes and Evolution. 210. 591-602 (2000)
• [Publications] Cardenas, M.: "Selective protein kinase inhibitors block head specific differentiation in Hydra"Cellular Signaling. 12・9-10. 649-658 (2000)
• [Publications] Takahashi, T.: "A novel neuropeptide, Hym-355, positively regulates neuron differentiation in Hydra"Development. 127. 997-1005 (2000)
• [Publications] Kumpfmueller, G.: "Identification of an astacin matrix metalloprotease as target gene for Hydra foot activator peptides"Development, Genes and Evolution. 209. 601-607 (1999)
• [Publications] Grens, A.: "Pedibin/Hym-346 lowers positional value thereby enhancing foot formation in hydra"Development. 126. 517-524 (1999)
• [Publications] Okado, N.: "A serotonin malfunction hypothesis by finding clear mutual relationships between several risk factors and symptoms associated with sudden infant death syndrome"Med. Hypoth.. (In press).
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• [Publications] Naka, F.: "Enriched environments increase noradrenaline contents in the mouse brain"Brain Res.. 924. 124-126 (2002)
• [Publications] Okado, N.: "Biogenic amine-synapse mechanism for mental retardation and developmental disabilities"Brain Devel. 23. S11-S15 (2001)
• [Publications] Abe, S.: "Effects of single and repeated phencyclidine administration on the expression of metabotropic glutamate receptor subtype mRNAs in rat brain"Neuropsychopharmacology. 25・2. 173-184 (2001)
• [Publications] Sakata-Haga, H.: "Differential localization and colocalization of two neuron-types of sodium-dependent inorganic phosphate cotransporters in rat forebrain"Brain Res.. 902・2. 143-155 (2001)
• [Publications] Narita, N.: "Serotonin transporter gene variation is a risk factor for sudden infant death syndrome in the Japanese population"Pediatrics. 107・4. 690-692 (2001)
• [Publications] Maxishima, M.: "Serotonin 2A receptor-like immunoreactivityis detected inastrocytes but not in oligodendrocytes of rat spinal cord"Brain Res.. 881・1-2. 270-273 (2001)
• [Publications] Shutoh, F.: "Long term depletion of serotonin leads to selective changes in glutamate receptor subunits"Neurosci. Res.. 38・4. 365-371 (2000)
• [Publications] Yamaguchi, M.: "Time-course effects of a single administration of cocaine on receptor binding and subunit mRNAs of GABA(A) receptors"Mol. Brain Res.. 81・1-2. 155-163 (2000)
• [Publications] Masuda, T.: "The involvement of axonin-1/SC2 in mediating notochord-derived chemorepulsive activities for dorsal root ganglion neurites"Dev. Biol.. 224・2. 112-121 (2000)
• [Publications] Ikemoto, K.: "Human midbrain dopamine neurons express serotonin 2A receptor : an immunohistochemical demonstration"Brain Res.. 853・2. 377-380 (2000)
• [Publications] Maeshima, T.: "The central distribution pattern of primary afferent fibers innervating the thigh muscle posterior iliotibialis in the chicken"J. Brain Res.. 3. 389-390 (1999)
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• [Publications] Asai, D.: "Preparation of the antibody which discriminates the conformations of ligand-bound and ligand-free estrogen receptors : Applications to the quantitative analysis of ligand bindings"Peptide Science. 2000. 411-412 (2001)
• [Publications] Takahashi, T.: "A novel neuropeptide, Hym355, positively regulates neuron differentiation in hydra"Development. 127. 997-1005 (2001)
• [Publications] Hatta, I.: "Identification of a homolog of actin-binding protein, ABP-280, localized at epithelial cell-cell boundaries in hydra"Zoological Science. 16. 439-443 (1999)
• [Publications] Inoue, M.: "Localization of the neuropeptide NGIWYamide in theholothurian nervous system and its effects on muscular contraction"Proc. R. Soc. Lond. B.. 266. 993-1000 (1999)
• [Publications] Tsutsui, K.: "Biosynthesis and biological actions of neurosteroids in the avian brain"Avian Poultry Biol. Reviews. (In press).
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• [Publications] Sakamoto, H.: "Dendritic spine formation in response to progesterone synthesized de novo in the developing Purkinje cell in rats"Neurosci. Lett.. (In press).
• [Publications] Ukena, K.: "A novel rat hypothalamic RFamide-related peptide identified by immunoaffinity chromatography and mass spectrometry"FEBS Lett.. 512. 255-258 (2002)
• [Publications] Tsutsui, K.: "The Zoological Society Prize : Biosynthesis and biological actions of neurosteroids in the brain"Zool. Sci.. 18. 1-2 (2002)
• [Publications] Koda, A.: "A novel amphibian hypothalamic neuropeptide : Isolation, localization and biological activity"Endocrinology. 143. 411-419 (2002)
• [Publications] Sakamoto, H.: "Effects of progesterone synthesized de novo in the developing Purkinje cell on its dendritic growth and synaptogenesis"J. Neurosci.. 21. 6221-6232 (2001)
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• [Publications] Ubuka, T.: "Developmental changes in galanin in lumbosacral sympathetic ganglionic neurons innervating the avian uterine oviduct and galanin induction by sex steroids"J. Endocrinol.. 170. 357-368 (2001)
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• [Publications] Matsunaga, M.: "Expression and localization of cytochrome P45017 α-hydroxylase/c17, 201yase in the avian brain"Brain Res.. 899. 112-122 (2001)
• [Publications] Ukena, K.: "Distribution of novel RFamide-related peptide-like immunoreactivity in the mouse central nervous system"Neurosci. Lett.. 300. 153-156 (2001)
• [Publications] Satake, H.: "Characterization of a cDNA encoding a novel avian hypothalamic neuropeptide exterting an inhibitory effect on gonadotropin release"Biochemical J.. 354. 379-385 (2001)
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• [Publications] Sakamoto, H.: "Existence of galanin in lumbosacral sympathetic ganglionic neurons that project to the quail uterine oviduct"Endocrinology. 141. 4402-4412 (2000)
• [Publications] Tsutsui, K.: "A novel avian hypothalamic peptide inhibiting gonadotropin release"Biochem. Biophys. Res. Commun.. 275. 661-667 (2000)
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• [Publications] Kohchi, C.: "Avian galanin : Cloning of complementary DNAs and characterization of transcripts in different tissues"J. Exp. Zool.. 287. 183-190 (2000)
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• [Publications] 筒井和義: "総説:小脳プルキンエ細胞におけるニューロステロイドの合成と作用"自律神経(自律神経学会編). 37. 235-243 (2000)
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• [Publications] Takase, M.: "Pregnenolone, pregnenolone sulfate and cytochrome P450 side-chain cleavage(P45Oscc)in the amphibian brain and their seasonal changes"Endocrinology. 140. 1936-1944 (1999)
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• [Publications] Ukena, K.: "Expression and activity of 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase in the rat Purkinje neuron during neonatal life"Endocrinology. 140. 805-813 (1999)
• [Publications] Tsutsui, K.: "Neurosteroids in vertebrate brains"Comp. Biochem. Physiol. C.. 124. 121-129 (1999)
• [Publications] Tsutsui, K.: "Production and function of neurosteroids in the cerebellar Purkinje neuron"J. Mol. Med.. 4. 49-56 (1999)
• [Publications] 筒井和義: "総説:脳がステロイドを合成する"ファルマシア(日本薬学会編). 35. 112-117 (1999)
• [Publications] 筒井和義: "総説:鳥類の微小脳の繁殖戦略-産卵誘起ペプチドとその作用機構"比較生理生化学(比較生理生化学会編). 16. 191-199 (1999)
• [Publications] Seale, A. P.: "Isolation and characterization of a homologue of mammalian Prolactin-releasing peptide from the tilapia brain and its effect on prolactin release from the tilapia pituitary"General and Comparative Endocrinology. (In press).
• [Publications] Wang, X.: "Immunohistochemical localization of C-RFamide, a FMRF-related peptide, in the brain of the goldfish, Carassius auratus"Zoological Science. 17. 1067-1074 (2000)
• [Publications] Wang, X.: "Carassius RFamide, a novel FMRFa-related peptide, is produced within the retina and involved in retinal information processing in cyprinid fish"Neuroscience Letters. 289. 115-118 (2000)
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• [Publications] Nomura, T.: "Purification and some properties of alanine racemase from a bivalve mollusc Corbicula japonica"J. Exp. Zool.. 288. 1-9 (2001)
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• [Publications] Matsushima, O.: "Annelid bioactive peptides : A comparative, view"Trends in Comp. Biochem. Physiol.. 7. 131-141 (2000)
• [Publications] Muneoka, Y.: "Comparative aspects of invertebrate neuropeptides"Acta Biol. Hungarica. 51. 111-132 (2000)
• [Publications] Csoknya, M.: "Distribution of Eisenia tetradecapeptide immunoreactive neurons in the nervous system of earthworms"Acta Biol. Hungarica. 51. 395-408 (2000)
• [Publications] Fujisawa, Y: "The Aplysia Mytilus inhibitory peptide-related peptides : Identification, cloning, processing, distribution, and action"J. Neurosci.. 19・21. 9618-9634 (1999)
• [Publications] Satake, H.: "Characterization of a cDNA encoding a precursor polypeptide of a D-amino acid-containing peptide, achatin-I and localized expression of the achatin-I and fulicin genes"Eur. J. Biochem.. 261. 130-136 (1999)
• [Publications] Fujino, Y.: "Possible functions of oxytocin/vasopressin-superfamily peptides in annelids with special reference to reproduction and osmoregulation"J. Exp. Zool.. 284. 401-406 (1999)
• [Publications] Nagahama, T.: "Localization of the leech excitatory peptide, a member of the GGNG peptides, in the central nervous system of a leech (Whitmania pigra) by immunohistochemistry and in situ hybridization"Cell Tissue Res.. 297. 155-162 (1999)
• [Publications] Minakata, H.: "Comparison of precursor structures of the GGNG peptides derived from the earthworm Eisenia foetida and the leech Hirudo nipponia"Biosci. Biotechnol. Biochem.. 63. 443-445 (1999)
• [Publications] Satake, H.: "Evidence for conservation of the vasopressin/oxytocin superfamily in annelida"J. Biol. Chem.. 274. 5605-5611 (1999)
• [Publications] Iwakoshi, E.: "Isolation and characterization of a GnRH-like peptide from Octopus vulgaris"Biochem. Biophys. Res. Comm.. (In press).
• [Publications] In, Y: "Comformational comparison of μ-selective endomorphin-2 with its C-terminal free acid in DMSO solution, by 'H NMR spectroscopy and molecular modeling calculation"J. Peptide Res.. 58・5. 399-412 (2001)
• [Publications] Satake, H.: "Characterization of a cDNA encoding a novel avian hypothalamic neuropeptide exerting an inhibitory effect on gonadotropin release"Biochemical J.. 354・2. 379-385 (2001)
• [Publications] In, Y.: "Functional analysis of C-terminal amide in bioactivity of endomorphin-2 : Conformational compariso with its unamidated peptide in DMSO solution"Peptide Science. 2000. 313-316 (2001)
• [Publications] Kawada, I.: "Identification of multiple urechistachykinin peptides, gene expression, pharmacological activity, and detection using mass spectrometric analyses"Peptides. 21・12. 1777-1783 (2000)
• [Publications] Fujisawa, Y.: "Fulicin regurates the female reproductive organs of the snail, Achatina fulica"Peptides. 21・8. 1203-1208 (2000)
• [Publications] Iwakoshi, E.: "Cardioactive peptides isolated from the brain of a Japanese octopus, Octopus minor"Peptides. 21・5. 623-630 (2000)
• [Publications] Minakata, H.: "Structure and function of molluscan neuropeptides -Cardioactive peptides isolated from the cephalopod, Octopus minor"Peptide Science. 1999. 171-174 (2000)
• [Publications] Ikeda, T.: "The importance of C-terminal residues of vertebrate and invertebrate tachykinins for their contractile activities in gut tissues"FEBS Letters. 461・3. 201-204 (1999)
• [Publications] Kawada, T.: "Characterization of a nevel cDNA sequence encoding invertebrate tachykinin-related peptides isolated from the echiuroid worm, Urechis unicinctus"Biochem. Biophys. Res. Commun.. 263・3. 848-852 (1999)
• [Publications] Fujisawa, Y.: "The Aplysia Mytilus inhibitory peptide-related peptides : identification, cloning, processing, distribution, and action"J Neurosci.. 19・21. 9618-9634 (1999)
• [Publications] Satake, H.: "Characterization of cDNA and expression of mRNA encoding an Achatina cardioexcitatory RFamide peptide"Peptides. 20・11. 1295-1302 (1999)
• [Publications] Tsutsui, K.: "A novel avian hypothalamic peptide. In : Recent Advances in Comparative Endocrinology, John Yuh-Lin Yu(ed)"Institute of Zoology, Academia Sinica, Taipei, Taiwan. 8 (2001)
• [Publications] Tsutsui, K.: "Steroidogenesis in the avian brain. In : Avian Endocrinology, A. Dawson and C. M. Chaturvedi (eds)"Journal of Endocrinology Ltd. 18 (2001)
• [Publications] Tsutsui, K.: "Novel cerebellar function : Neurosteroidsin the Purkinjeneuron and their genomic and non-gemonicactions. In Neural Plasticity, Developmentand Steroid Hormone Action, R. J. Handa et al. (eds.)"CRC Press, Boca Raton, USA. 19 (2001)
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• [Publications] Sakamoto, H.: "Sympathetic ganglionic galanin neurons evoke avian oviposition by innervating the uterine oviduct : A novel neuronal mechanism of avian oviposition. In : Perspective in Comparative Endocrinology, H. J. Th. Goos et al. (eds. )"Monduzzi Editore, Sorrento, Italy. 7 (2001)
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• [Publications] 筒井和義: "小脳における神経ステロイドの合成と作用「ステロイドホルモンと脳科学:性・ストレス・脳をめぐって」"第14回「大学と科学」公開シンポジウム組織委員会編. 8 (2000)
• [Publications] 藤本正昭: "比較生理生化学"魚類脳から単離されたペプチドC-RFaとその機能(印刷中).
• [Publications] Wang, X.: "A candidate for fish prolactin-releasing peptide isolated from Japanese crucian carp. In Recent Advances in Comparative Endocrinology. Ed. by J. Y. L. Yu,"Proceedings of 4^<th> Congress of the Asia and Oceania Society for comparative Endocrinology, Taipei. 10 (2001)
• [Publications] Iwakoshi, E.: "Isolation and characterization of a GnRH-like peptide from Octopus vulgaris. In : Perspective in Comparative Endocrinology, H. J. Th. Goos et al. (eds.)"Monduzzi Editore, Sorrento, Italy. 6 (2001)