MF研究者総覧

研究者データベース

詳細
酒井　隆一(サカイ　リユウイチ)
水産科学研究院　海洋応用生命科学部門　生物資源化学分野
教授
researchmap個人ページ
Last Updated :2024/05/09

基本情報

所属

  • 水産科学研究院　海洋応用生命科学部門　生物資源化学分野

職名

  • 教授

科研費研究者番号

  • 20265721

J-Global ID

研究キーワード

  • メタボロミクス   トロンボポエチン受容体   グルタミン酸受容体   興奮性アミノ酸   海綿   ダイシハーベイン   神経毒   免疫組織化学   アミノ酸   興奮性神経毒   ミクロネシア連邦   ポリアミン   ペプチド   海洋天然物   抗菌性   ホヤ   ガレクチン   イソグアニン   天然物化学   カイニン酸   細胞毒性   組織免疫染色   Dysidea herbacea   生理活性物質   海綿(スポンジ)   バイオミネラリゼーション   GABA受容体   局在性   ベタイン   抗しゅよう性   中枢神経   海洋天然物化学   Marine Natural Products chemistry   

研究分野

  • ライフサイエンス / 神経科学一般
  • ライフサイエンス / 水圏生命科学

担当教育組織

職歴

  • 2010年 - 2012年 北海道大学 水産科学研究科(研究院) 教授
  • 1992年 - 1994年 イリノイ大学 Urbana-Champaign 博士研究員
  • 1987年 - 1991年 イリノイ大学 Urbana-Champaign 博士課程
  • 1985年 - 1987年 ハーバーブランチ海洋学研究所（米国）
  • 1984年 - 1985年 琉球大学理学研究科修士課程

学歴

  •         - 1992年   イリノイ大学大学院   化学研究科   有機化学
  •         - 1992年   University of Illinois at Urban
  •         - 1985年   琉球大学   理学研究科   海洋学
  •         - 1983年   琉球大学   理学部   海洋
  •         - 1983年   琉球大学

所属学協会

  • 日本分子生物学会   日本農芸化学会   日本水産学会   アメリカ化学会(American chemical Society)   日本化学会   

研究活動情報

論文

  • Ryuichi Sakai, Ken Matsumura, Hajime Uchimasu, Kei Miyako, Tohru Taniguchi, V Raghavendra Rao Kovvuri, Anjana Delpe Acharige, Kenneth G Hull, Daniel Romo, Lakkana Thaveepornkul, Sarin Chimnaronk, Hiroko Miyamoto, Ayato Takada, Hiromi Watari, Masaki J Fujita, Jiro Sakaue
    The Journal of organic chemistry 2024年04月01日 
    Mellpaladines A-C (1-3) and dopargimine (4) are dopamine-derived guanidine alkaloids isolated from a specimen of Palauan Didemnidae tunicate as possible modulators of neuronal receptors. In this study, we isolated the dopargimine derivative 1-carboxydopargimine (5), three additional mellpaladines D-F (6-8), and serotodopalgimine (9), along with a dimer of serotonin, 5,5'-dihydroxy-4,4'-bistryptamine (10). The structures of these compounds were determined based on spectrometric and spectroscopic analyses. Compound 4 and its congeners dopargine (11), nordopargimine (15), and 2-(6,7-dimethoxy-3,4-dihydroisoquinolin-1-yl)ethan-1-amine (16) were synthetically prepared for biological evaluations. The biological activities of all isolated compounds were evaluated in comparison with those of 1-4 using a mouse behavioral assay upon intracerebroventricular injection, revealing key functional groups in the dopargimines and mellpaladines for in vivo behavioral toxicity. Interestingly, these alkaloids also emerged during a screen of our marine natural product library aimed at identifying antiviral activities against dengue virus, SARS-CoV-2, and vesicular stomatitis Indiana virus (VSV) pseudotyped with Ebola virus glycoprotein (VSV-ZGP).
  • Kenji Morokuma, Hiromi Watari, Maika Mori, Ryuichi Sakai, Raku Irie, Masato Oikawa
    Tetrahedron 133623 - 133623 2023年08月
  • Ryuichi Sakai, Naoko Goto-Inoue, Hiroshi Yamashita, Naoya Aimoto, Yuto Kitai, Tadashi Maruyama
    iScience 26 7 107250 - 107250 2023年07月
  • Hiromi Watari, Reimi Kishi, Satoko Matsunaga, Takayuki Nishikawa, Yuji Sawada, Akito Honda, Masaki J Fujita, Ryuichi Sakai
    Chemistry Letters 52 3 185 - 189 2023年03月05日
  • Koya Yoshizawa, Akira Matsura, Masaya Shimada, Sumire Ishida‐Ishihara, Fuyu Sato, Takahiro Yamamoto, Kan Yaguchi, Eiji Kawamoto, Taruho Kuroda, Kazuya Matsuo, Nobuyuki Tamaoki, Ryuichi Sakai, Yasuhito Shimada, Mithilesh Mishra, Ryota Uehara
    Molecular Oncology 17 6 1148 - 1166 2023年02月11日 
    Tetraploidy is a hallmark of cancer cells, and tetraploidy‐selective cell growth suppression is a potential strategy for targeted cancer therapy. However, how tetraploid cells differ from normal diploids in their sensitivity to anti‐proliferative treatments remains largely unknown. In this study, we found that tetraploid cells are significantly more susceptible to inhibitors of a mitotic kinesin (CENP‐E) than are diploids. Treatment with a CENP‐E inhibitor preferentially diminished the tetraploid cell population in a diploid–tetraploid co‐culture at optimum conditions. Live imaging revealed that a tetraploidy‐linked increase in unsolvable chromosome misalignment caused substantially longer mitotic delay in tetraploids than in diploids upon moderate CENP‐E inhibition. This time gap of mitotic arrest resulted in cohesion fatigue and subsequent cell death, specifically in tetraploids, leading to tetraploidy‐selective cell growth suppression. In contrast, the microtubule‐stabilizing compound paclitaxel caused tetraploidy‐selective suppression through the aggravation of spindle multipolarization. We also found that treatment with a CENP‐E inhibitor had superior generality to paclitaxel in its tetraploidy selectivity across a broader spectrum of cell lines. Our results highlight the unique properties of CENP‐E inhibitors in tetraploidy‐selective suppression and their potential use in the development of tetraploidy‐targeting interventions in cancer.
  • Hiromi Watari, Hiromu Kageyama, Nami Masubuchi, Hiroya Nakajima, Kako Onodera, Pamela J Focia, Takumi Oshiro, Takashi Matsui, Yoshio Kodera, Tomohisa Ogawa, Takeshi Yokoyama, Makoto Hirayama, Kanji Hori, Douglas M Freymann, Misa Imai, Norio Komatsu, Marito Araki, Yoshikazu Tanaka, Ryuichi Sakai
    Nature communications 13 1 7262 - 7262 2022年11月25日 
    N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that traffics to the cell surface and constitutively activates the receptor. However, the molecular basis for this mechanism is elusive because oncogenic activation occurs only in the cell-intrinsic complex and is thus cannot be replicated with external agonists. Here, we describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC), a homodimerized lectin with calcium-dependent fucose-binding properties. In-depth characterization of lectin-induced activation showed that, similar to oncogenic activation, sugar chain-mediated activation persists due to limited receptor internalization. The strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. Overall, the existence of sugar-mediated MPL activation, in which the mode of activation is different from the original ligand, suggests that receptor activation is unpredictably diverse in living organisms.
  • Kazunori Otsuka, Masayoshi Miyahara, Sara Takaki, Ryoya Wakabayashi, Kei Miyako, Raku Irie, Satoshi Takamizawa, Ryuichi Sakai, Masato Oikawa
    European Journal of Organic Chemistry 2022 36 2022年09月27日
  • Hiromi Watari, Hiromu Kageyama, Nami Masubuchi, Hiroya Nakajima, Kako Onodera, Pamela Focia, Takumi Oshiro, Takashi Matsui, Yoshio Kodera, Tomohisa Ogawa, Takeshi Yokoyama, Makoto Hirayama, Kanji Hori, Douglas Freymann, Norio Komatsu, Marito Araki, Yoshikazu Tanaka, Ryuichi Sakai
    2022年04月01日
  • Shuntaro Tsukamoto, Oriel Hlokoane, Kei Miyako, Raku Irie, Ryuichi Sakai, Masato Oikawa
    RSC Advances 12 34 22175 - 22179 2022年 
    By interchanging the order of reactions, two types of skeletons were created and a neuroactive artificial glutamate analog was developed.
  • Mohamed Farghali, Yuhendra AP, Israa M.A. Mohamed, Masahiro Iwasaki, Suchon Tangtaweewipat, Ikko Ihara, Ryuichi Sakai, Kazutaka Umetsu
    Journal of Environmental Chemical Engineering 9 6 106405 - 106405 2021年12月
  • A. P. Yuhendra, Mohamed Farghali, Israa M. A. Mohamed, Masahiro Iwasaki, Suchon Tangtaweewipat, Ikko Ihara, Ryuichi Sakai, Kazutaka Umetsu
    BIOCHEMICAL ENGINEERING JOURNAL 175 2021年11月 
    Utilization of macroalgal biomass through the anaerobic digestion (AD) system can overcome algal pollution while providing alternative and renewable energy as fuel scarcity increases. To achieve this objective, Sargassum fulvellum biomass was used as the feedstock for the batch AD process. Original-sized algal biomass (So) of 106 mu m-4.75 mm particle size, reduced-sized biomass of 75-850 mu m (mechanically pretreated, Sr), chemically pretreated reduced-sized wet biomass with 40 mL/L (Sac1), 20 mL/L (Sac2) of 2 M HCl and with 10 mL/L (Sal1) and 5 mL/L (Sal2) of 6 N NaOH, and biologically pretreated original particle size biomass (Se) with 1 mL/L of cellulase enzyme were employed as feedstocks for digestion. Mechanical pretreatment of Sargassum fulvellum (Sr) without chemical addition resulted in 142.91 +/- 0.004 mL CH4/gVS, which is higher than chemically pretreated reduced-sized macroalgae in So, Sac1, Sac2, Sal1, and Sal2 by 52.34%, 9.83%, 15.89%, 12.73%, and 18.26%, respectively. Biological treatment reduced methane yield in Se by 9.49% than the original-sized algal biomass (So). In addition, the rate of hydrolysis and maximum biomethane production potential improved after mechanical pretreatment by a maximum of 45.60% and 48.71%, respectively. This study indicates that the utilization of marine biomass as an alternative resource for biomethane production can be achieved, with an optimum methane production from mechanically pretreated macroalgae without chemical addition.
  • Hiromi Watari, Hiromu Kageyama, Nami Masubuchi, Hiroya Nakajima, Kako Onodera, Pamela J. Focia, Takumi Oshiro, Takashi Matsui, Yoshio Kodera, Tomohisa Ogawa, Takeshi Yokoyama, Makoto Hirayama, Kanji Hori, Douglas M. Freymann, Norio Komatsu, Marito Araki, Yoshikazu Tanaka, Ryuichi Sakai
    2021年10月29日 
    ABSTRACT N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that constitutively activates the receptor. However, the molecular basis for this mechanism has not been studied because no external agonists existed. We describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC), a homodimerized lectin with calcium-dependent fucose-binding properties. ThC-induced activation persists due to limited receptor internalization. The strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. MPL is subject to sugar-mediated activation, where the kinetics differ from those of cytokines. This result suggests the presence of diverse receptor activation pathways in human thrombopoiesis. One-sentence summary A marine sponge lectin catalyzes thrombopoietin receptor dimerization and activation, exhibiting strong synergy with thrombopoietin, and modulates internalization of the receptor.
  • Raku Irie, Kei Miyako, Satoko Matsunaga, Ryuichi Sakai, Masato Oikawa
    Journal of natural products 84 4 1203 - 1209 2021年04月23日 
    The structure of protoaculeine B, the N-terminal residue of the marine peptide toxin aculeine B, is revised to the cis-1,3-disubstituted tetrahydro-β-carboline framework. We prepared two truncated model compounds that lack a long-chain polyamine using the one-step Pictet-Spengler reaction of tryptophan and compared their NMR, mass spectra, and chemical reactivity with those of the natural protoaculeine B. The synthetic models reproduced the profiles of the natural product well, which confirmed the appropriateness of the structure revision.
  • Kenji Morokuma, Shuntaro Tsukamoto, Kyosuke Mori, Kei Miyako, Ryuichi Sakai, Raku Irie, Masato Oikawa
    Beilstein Journal of Organic Chemistry 17 540 - 550 2021年02月24日 
    Herein, we report the enantiospecific synthesis of two artificial glutamate analogs designed based on IKM-159, an antagonist selective to the AMPA-type ionotropic glutamate receptor. The synthesis features the chiral resolution of the carboxylic acid intermediate by the esterification with ʟ-menthol, followed by a configurational analysis by NMR, conformational calculation, and X-ray crystallography. A mice in vivo assay showed that (2R)-MC-27, with a six-membered oxacycle, is neuroactive, whereas the (2S)-counterpart is inactive. It was also found that TKM-38, with an eight-membered azacycle, is neuronally inactive, showing that the activity is controlled by the ring C.
  • Kei Miyako, Yoko Yasuno, Tetsuro Shinada, Masaki J. Fujita, Ryuichi Sakai
    JOURNAL OF NATURAL PRODUCTS 83 10 3156 - 3165 2020年10月 
    Fourteen aromatic metabolites (6-19) were isolated from an aqueous extract of the solitary tunicate Cnemidocarpa irene collected in Hokkaido, Japan. The structures of the metabolites were determined based on the spectroscopic interpretations, including one- and two-dimensional NMR, mass spectra, UV, and circular dichroism data. The biopterin analogue 10 modulated the behavior of mice after intracerebroventricular injection and showed a weak affinity to ionotropic glutamate receptor subtypes. Analyses of fluorescent coelomic fluid of the tunicate revealed that pterin 12 was responsible for the fluorescence of the blood cells, while beta-carbolines 1 and 3 were fluorescent compounds in the serum. The metabolic profiles in adults, juveniles, larvae, and eggs of the animal differed substantially, suggesting that the metabolism of the animal, especially biosynthesis of aromatic secondary metabolites, changes over different life stages.
  • Raku Irie, Masayoshi Miyahara, Shota Nakamura, Akito Honda, Ryuichi Sakai, Masato Oikawa
    Journal of natural products 83 9 2769 - 2775 2020年09月25日 [査読有り][通常論文]
     
    By establishing the procedures for sequential deprotections, reaction monitoring, purification, and handling, for the first time, we achieved the total synthesis of the proposed structure for protoaculeine B (2), which is a highly hydrophilic and polycationic amino acid. The NMR and mass spectra and chemical reactivity of the synthetic sample differed from those of natural protoaculeine B, which indicates the necessity for revision of the originally reported structure.
  • Satoko Matsunaga, Hiroki Ikeda, Ryuichi Sakai
    Molecules (Basel, Switzerland) 25 13 2020年07月03日 [査読有り][通常論文]
     
    The scallop Mizuhopecten yessoensis accumulates carotenoids in the ovary during the maturation stage. Its conspicuous pink color implies the presence of carotenoprotein. However, the carotenoprotein from the scallop ovary has never been isolated and characterized, probably due to its instability and complexity. Here, we developed an extraction and isolation procedure for the carotenoprotein by employing a basic buffer containing potassium bromide to facilitate its efficient extraction from the ovary, and we succeeded in obtaining the carotenoprotein, termed pectenovarin. The carotenoid composition of the pectenovarin was similar to that of the ovary. The N-terminal and internal amino acid sequences of pectenovarin showed a high similarity to those of vitellogenin, the precursor of egg yolk protein under analysis.
  • Naoko Goto-Inoue, Tomohiko Sato, Mizuki Morisasa, Hiroshi Yamashita, Tadashi Maruyama, Hiroki Ikeda, Ryuichi Sakai
    Scientific reports 10 1 656 - 656 2020年01月20日 [査読有り][通常論文]
     
    Giant clams have evolved to maximize sunlight utilization by their photosymbiotic partners, while affording them protection from harmful ultraviolet (UV) light. The presence of UV absorbing substances in the mantle is thought to be critical for light protection; however, the exact localization of such compounds remains unknown. Here, we applied a combination of UV liquid chromatography (LC), LC-mass spectrometry (MS), MS imaging, and UV micrography to localize UV absorbing substances in the giant clam Tridacna crocea. LC-MS analysis revealed that the animal contained three classes of mycosporines: progenitor, primary, and secondary mycosporines. MS imaging revealed that primary and secondary mycosporines were localized in the outermost layer of the mantle; whereas progenitor mycosporines were distributed throughout the mantle tissue. These findings were consistent with the results of UV micrography, which revealed that the surface layer of the mantle absorbed UV light at 320 ± 10 nm. This is the first report indicating that progenitor and primary mycosporines are metabolized to secondary mycosporines by the giant clam and that they are differentially localized in the surface layer of the mantle to protect the animal from UV light.
  • Tsukamoto Shuntaro, Itagaki Hiyori, Morokuma Kenji, Miyako Kei, Ishikawa Yuichi, Sakai Ryuichi, Oikawa Masato
    Heterocycles 101 1 91 - 98 2020年 [査読有り][通常論文]
     
    Herein we report improved enantiospecific synthesis and some structure-activity relationships of our heterotricyclic artificial glutamate analogs bearing seven-membered ring for the C-ring. Starting from readily available oxanorbornene rac-3, optically pure (2R)-TKM-107, (2R)-IKM-154, and the antipodes were synthesized in total nine steps for each. Mice in vivo assay indicated that only the (2R)-enantiomer was active in both cases. Behaviors phenotypes observed in the mice assay suggested that these compounds are similar in mode of action to that of IKM-159 but with discrete potency.
  • Saki Umetsu, Mamoru Kanda, Ichiro Imai, Ryuichi Sakai, Masaki J Fujita
    Molecules (Basel, Switzerland) 24 24 2019年12月10日 [査読有り][通常論文]
     
    Questiomycin A (1) along with three new compounds, questiomycins C-E (2-4), were isolated from culture of Alteromonas sp. D, an algicidal marine bacterium, guided by algal lethality assay using the raphidophyte, Chattonella antiqua, one of the causative organisms of harmful algal bloom. The structures of 1-4 were assigned on the basis of their spectrometric and spectroscopic data. Compounds 1 to 4 exhibited algicidal activity against C. antiqua with LC50 values ranging from 0.18 to 6.37 M. Co-cultivation experiment revealed that 1 was produced only when the microalgae and the bacterium are in close contact, suggesting that some interactions between them trigger the biosynthesis of questiomycins. These results suggested that the algicidal bacteria such as Alteromonas sp. D can control microalgae chemically in marine ecosystem.
  • Hiromi Watari, Hiroya Nakajima, Wataru Atsuumi, Takanori Nakamura, Takeshi Nanya, Yuji Ise, Ryuichi Sakai
    Comparative biochemistry and physiology. Toxicology & pharmacology : CBP 221 82 - 88 2019年07月 [査読有り][通常論文]
     
    We screened 868 marine extracts in search of hematopoietic molecules resulted in findings of several extracts that proliferated Ba/F3-HuMpl cells but not the cells expressed with other hematopoietic cytokine receptors, EPO and G-CSF. Separation of the most potent extract of a Micronesian sponge Corticium sp., guided by the cell proliferation assay using Ba/F3-HuMpl cells resulted in an isolation of thrombocorticin (ThC), a novel 14 kDa protein as an active principal. ThC displayed concentration-dependent proliferation of Ba/F3-HuMpl cells, and had a stronger activity than that of eltrombopag, a small molecule drug used to treat thrombocytopenia. ThC induced phosphorylation of STAT5, suggesting that it activates Jak/STAT pathway as in the case of TPO. These results together indicated that ThC is a specific agonist for c-Mpl, although the size and shape differs largely from TPO. Here we present isolation, characterization and biological activity of ThC.
  • Ryuichi Sakai, Kota Tanano, Takumi Ono, Masaya Kitano, Yusuke Iida, Koji Nakano, Mitsuru Jimbo
    Marine drugs 17 4 2019年04月08日 [査読有り][通常論文]
     
    A novel protein, soritesidine (SOR) with potent toxicity was isolated from the marine sponge Spongosorites sp. SOR exhibited wide range of toxicities over various organisms and cells including brine shrimp (Artemia salina) larvae, sea hare (Aplysia kurodai) eggs, mice, and cultured mammalian cells. Toxicities of SOR were extraordinary potent. It killed mice at 5 ng/mouse after intracerebroventricular (i.c.v.) injection, and brine shrimp and at 0.34 µg/mL. Cytotoxicity for cultured mammalian cancer cell lines against HeLa and L1210 cells were determined to be 0.062 and 12.11 ng/mL, respectively. The SOR-containing fraction cleaved plasmid DNA in a metal ion dependent manner showing genotoxicity of SOR. Purified SOR exhibited molecular weight of 108.7 kDa in MALDI-TOF MS data and isoelectric point of approximately 4.5. N-terminal amino acid sequence up to the 25th residue was determined by Edman degradation. Internal amino acid sequences for fifteen peptides isolated from the enzyme digest of SOR were also determined. None of those amino acid sequences showed similarity to existing proteins, suggesting that SOR is a new proteinous toxin.
  • Masaki J Fujita, Yusuke Goto, Ryuichi Sakai
    Marine drugs 16 9 2018年09月19日 [査読有り][通常論文]
     
    The biosynthetic gene cluster for bisucaberin B (1, bsb gene cluster), an N-hydroxy-N-succinyl diamine (HSD)-based siderophore, was cloned from the marine bacterium Tenacibaculum mesophilum, originated from a marine sponge. The bsb gene cluster consists of six open reading frames (ORFs), in contrast to the four ORFs typically seen in biosynthetic gene clusters of the related molecules. Heterologous expression of the key enzyme, BsbD2, which is responsible for the final biosynthetic step of 1 resulted in production of bisucaberin B (1), but not bisucaberin (2) a macrocyclic counterpart of 1. To date, numbers of related enzymes producing macrocyclic analogues have been reported, but this work represents the first example of the HSD-based siderophore biosynthetic enzyme which exclusively produces a linear molecule rather than macrocyclic counterparts.
  • Hiroki Shiozaki, Masayoshi Miyahara, Kazunori Otsuka, Kei Miyako, Akito Honda, Yuichi Takasaki, Satoshi Takamizawa, Hideyuki Tukada, Yuichi Ishikawa, Ryuichi Sakai, Masato Oikawa
    Organic letters 20 11 3403 - 3407 2018年06月01日 [査読有り][通常論文]
     
    A synthetic strategy for accessing protoaculeine B (1), the N-terminal amino acid of the highly modified peptide toxin aculeine, was developed via the synthesis of the fully protected natural homologue of 1 with a 12-mer poly(propanediamine). The synthesis of mono(propanediamine) analog 2, as well as core amino acid 3, was demonstrated by this strategy. New amino acid 3 induced convulsions in mice; however, compound 2 showed no such activity.
  • Ken Matsumura, Tohru Taniguchi, James D. Reimer, Shuntaro Noguchi, Masaki J. Fujita, Ryuichi Sakai
    Organic Letters 20 10 3039 - 3043 2018年05月18日 [査読有り][通常論文]
     
    A new tris-guanidine alkaloid, KB343 (1), was isolated from the aqueous extract of a Palauan zoantharian, Epizoanthus illoricatus. The structure of 1 was determined on the basis of spectral analyses of 1 and its derivatives. The absolute configuration for 1 was determined upon comparison of the CD spectrum of 1 to those obtained from density functional theory calculations. The structure of 1 is highly unusual, as three guanidine groups are present in one ring system.
  • Yuta Oda, Quang Zhang, Satoko Matsunaga, Masaki J. Fujita, Ryuichi Sakai
    CHEMISTRY LETTERS 46 8 1272 - 1274 2017年08月 [査読有り][通常論文]
     
    New mycosporine-like amino acids (MAA) LC-343 (1) and mycosporine-ethanolamine (2) along with known MAAs asterina-330 (3) and shinorine (4) were isolated from the Micronesian marine sponge Lendenfeldia chondrodes. The structures of 1 and 2 were determined on the basis of their spectroscopic and spectrometric data as well as chemical degradation. LC-343 (1) is the first MAA possessing Nmethylated iminium substructure. Absorption maximum (lambda(max)) for each 2, 3, 4, and 1 was 310, 330, 332, and 343, respectively.
  • Akiko Kita, Mitsuru Jimbo, Ryuichi Sakai, Yukio Morimoto, Ryota Takeuchi, Hiroshi Tanaka, Takashi Takahashi, Kunio Miki
    GLYCOBIOLOGY 27 8 696 - 700 2017年08月 [査読有り][通常論文]
     
    A symbiosis-related lectin, SLL-2, from the octocoral Sinularia lochmodes, distributes densely on the cell surface of microalgae, Symbiodinium sp., an endosymbiotic dinoflagellate of the coral, and is also shown to be a chemical cue that transforms dinoflagellates into a nonmotile (coccoid) symbiotic state. SLL-2 binds to the sugar chain of the molecule similar to Forssman antigen pentasaccharide (GalNAc alpha 1-3GalNAc beta 1-3Gala1-4Gal alpha 1-4Glc) on the surface of microalgae with high affinity. Here we report the crystal structure of the complex between SLL-2 and Forssman antigen tetrasaccharide (GalNAc alpha 1-3GalNAc beta 1-3Gala1-4Gal beta) at 3.4 angstrom resolution. In an asymmetric unit of the crystal, there are two hexameric molecules with totally 12 sugar recognition sites. At 9 in 12 sites, the first and second saccharides of the Forssman antigen tetrasaccharide bind directly to galactopyranoside binding site of SLL-2, whereas the third and fourth saccharides have no interaction with the SLL-2 hexameric molecule that binds the first saccharide. The sugar chain bends at alpha-1,4-glycosidic linkage between the third and fourth saccharides toward the position that we defined as a pyranoside binding site in the crystal structure of the complex between SLL-2 and GalNAc. The structure allowed us to suggest a possible binding mode of the Forssman antigen pentasaccharide to SLL-2. These observations support our hypothesis that the binding of SLL-2 to the cell surface sugars of zooxanthella in a unique manner might trigger some physiological changes of the cell to adapt symbiosis with the host coral.
  • Shoko Motohashi, Mitsuru Jimbo, Tomohiro Naito, Takefumi Suzuki, Ryuichi Sakai, Hisao Kamiya
    MARINE DRUGS 15 6 2017年06月 [査読有り][通常論文]
     
    Egg lectins occur in a variety of animals ranging from mollusks to vertebrates. A few examples of molluscan egg lectins have been reported, including that of the sea hare Aplysia kurodai; however, their biological functions in the egg remain unclarified. We report the isolation, determination of primary structure, and possible functions of A.kurodai lectin (AKL) from the egg mass of A. kurodai. We obtained AKL as an inseparable mixture of isoproteins with a relative molecular mass of approximately 32 kDa by affinity purification. The hemagglutinating activity of AKL against rabbit erythrocytes was inhibited most potently by galacturonic acid and moderately by xylose. Nucleotide sequencing of corresponding cDNA obtained by rapid amplification of cDNA ends (RACE) allowed us to deduce complete amino acid sequences. The mature polypeptides consisted of 218- or 219-amino acids with three repeated domains. The amino acid sequence had similarities to hypothetical proteins of Aplysia spp., or domain DUF3011 of uncharacterized bacterial proteins. AKL is the first member of the DUF3011 family whose function, carbohydrate recognition, was revealed. Treatment of the egg with galacturonic acid, an AKL sugar inhibitor, resulted in deformation of the veliger larvae, suggesting that AKL is involved in organogenesis in the developmental stage of A. kurodai.
  • Tadokoro Y, Nishikawa T, Ichimori T, Matsunaga S, Fujita MJ, Sakai R
    ACS omega 2 3 1074 - 1080 2017年03月31日 [査読有り][通常論文]
     
    New brominated β-carbolines irenecarbolines A (1) and B (4) along with known β-carbolines 2 and 3 and a new 8-oxoisoguanine derivative, 5, were isolated from a solitary ascidian, Cnemidocarpa irene. The structures of these compounds were determined on the basis of their spectral data. All, except for 3, inhibited the action of acetylcholinesterase (AchE). The activities of 1 and 5 were comparable to those of galantamine, a clinically used AchE inhibitor. Compounds 1 and 2 were found to be present in high concentrations in blood, and fluorescence was observed in certain types of cells found in the blood of the tunicate.
  • Hlokoane Oriel, Itagaki Hiyori, Chiba Manami, Noda Taiki, Takasaki Yuichi, Miyako Kei, Sakai Ryuichi, Ishikawa Yuichi, Oikawa Masato
    Heterocycles 96 3 453 - 460 2017年 [査読有り][通常論文]
  • Hajime Uchimasu, Ken Matsumura, Masashi Tsuda, Keiko Kumagai, Mai Akakabe, Masaki J. Fujita, Ryuichi Sakai
    TETRAHEDRON 72 45 7185 - 7193 2016年11月 [査読無し][通常論文]
     
    Novel guanidine alkaloids dopargimine (1) and mellpaladines A-C (2-4) were isolated from a Palauan Didemnidae tunicate. The structures of 1-4 were elucidated on the basis of spectral data along with chemical reactions. A putative biosynthetic building block of 1-4, 4-guanidinobutyric acid (5), and dimeric polysulfur dopamine, as well as lissoclibadins 11 (6a) and 12 (6b) were also isolated. Compounds 1-3 bound to synaptic receptors, and modulated behavioral profiles of mice after intracerebroventricular injection. (C) 2016 Elsevier Ltd. All rights reserved.
  • Koichi Fukushima, Yuichi Ishikawa, Ryuichi Sakai, Masato Oikawa
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 26 21 5164 - 5167 2016年11月 [査読有り][通常論文]
     
    Monocyclic analog of neuroexcitatory neodysiherbaine has been designed and stereoselectively synthesized in 0.40% yield over total 24 steps starting from D-ribose, by employing domino aldol-Cannizzaro reaction and stereoselective aldol reaction for construction of two quaternary carbon stereogenic centers at C4 and C6 positions, respectively. The hyperactivity of neodysiherbaine in mice was found to deteriorate in the novel analog, upon intracerebroventricular injection. (C) 2016 Elsevier Ltd. All rights reserved.
  • Manami Chiba, Yuichi Ishikawa, Ryuichi Sakai, Masato Oikawa
    ACS COMBINATORIAL SCIENCE 18 7 399 - 404 2016年07月 [査読有り][通常論文]
     
    Here, we report an unprecedented, highly diastereoselective Prins-Ritter reaction of aldehydes, homoallylic alcohols, and nitriles in a three-component coupling reaction for the synthesis of tetra-cis-substituted 4-amidotetrahydropyrans. In this study, the reaction was not only applied for carbohydrate-based heterobicycles but also for more complex heterotricycles, showing acceptable levels of conversion yield (42-97% BRSM) and exclusive diastereoselectivity. Furthermore, the latter heterotricycles were converted to nine analogues of our neuronal receptor ligands IKM-159 and MC-27. An in vivo assay by intracerebroventricular injection in mice suggested that the substituent at C9 of the novel analogues interferes with the molecular interactions with the AMPA receptor, which was originally observed in the complex of IKM-159 and the GluA2 ligand binding domain. Our research has thus shown the power of a multicomponent coupling reaction for the preparation of a structurally diverse compound collection to study structure activity relationships of biologically active small molecules.
  • Akiko Kita, Mitsuru Jimbo, Ryuichi Sakai, Yukio Morimoto, Kunio Miki
    GLYCOBIOLOGY 25 9 1016 - 1023 2015年09月 [査読有り][通常論文]
     
    D-Galactose-binding lectin from the octocoral, Sinularia lochmodes (SLL-2), distributes densely on the cell surface of microalgae, Symbiodinium sp., an endosymbiotic dinoflagellate of the coral, and is also shown to be a chemical cue that transforms dinoflagellate into a non-motile (coccoid) symbiotic state. SLL-2 binds with high affinity to the Forssman antigen (N-acetylgalactosamine(GalNAc)alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-ceramide), and the presence of Forssman antigen-like sugar on the surface of Symbiodinium CS-156 cells was previously confirmed. Here we report the crystal structures of SLL-2 and its GalNAc complex as the first crystal structures of a lectin involved in the symbiosis between coral and dinoflagellate. N-Linked sugar chains and a galactose derivative binding site common to H-type lectins were observed in each monomer of the hexameric SLL-2 crystal structure. In addition, unique sugar-binding site-like regions were identified at the top and bottom of the hexameric SLL-2 structure. These structural features suggest a possible binding mode between SLL-2 and Forssman antigen-like pentasaccharide.
  • Manami Chiba, Chikako Fujimoto, Ryuichi Sakai, Masato Oikawa
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 25 9 1869 - 1871 2015年05月 [査読有り][通常論文]
     
    Ligands for neuronal receptors are important for understanding the biological functions as well as for treatment of neuronal diseases associated with. Here, we report diverted synthesis and biological evaluation of four C-ring analogs of IKM-159, a subtype-selective inhibitor for (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid (AMPA)-type ionotropic glutamate receptor. Starting from iodinated 7-oxanorbornene 7, those analogs 3-6 were successfully synthesized in 7.0-33% yields over 8-11 steps via a common intermediate 13. Intracerebroventricular injection of those analogs on mice showed that introduction of oxo group on the C-ring (analogs 4, 5) or cleavage of the C-ring (analog 6) caused significant loss of the activity, while the ether analog 3 still retain the suppressed motor activity, indicating the importance of the C-ring in the neuronal activity of IKM-159. (C) 2015 Elsevier Ltd. All rights reserved.
  • Masaki J. Fujita, Ryuichi Sakai
    MARINE DRUGS 12 9 4799 - 4809 2014年09月 [査読有り][通常論文]
     
    The siderophore avaroferrin (1), an inhibitor of Vibrio swarming that was recently identified in Shewanella algae B516, was produced by heterologous expression of the biosynthetic gene cluster cloned from a deep-sea sediment metagenomic DNA, together with two analogues, bisucaberin (2) and putrebactin (3). Avaroferrin (1) is a macrocyclic heterodimer of N-hydroxy-N-succinyl cadaverine (4) and N-hydroxy-N-succinyl-putrescine (5), whereas analogues 2 and 3 are homodimers of 4 and 5, respectively. Heterologous expression of two other related genes from culturable marine bacteria resulted in production of compounds 1-3, but in quite different proportions compared with production through expression of the metagenomic DNA.
  • Satoko Matsunaga, Reimi Kishi, Kazunori Otsuka, Masaki J. Fujita, Masato Oikawa, Ryuichi Sakai
    ORGANIC LETTERS 16 11 3090 - 3093 2014年06月 [査読有り][通常論文]
     
    A new polyamine-modified indole derivative protoaculeine B (1) was isolated from Okinawan marine sponge Axinyssa aculeata. The structure of 1 was assigned on the basis of spectral data along with chemical transformations. Because the structure of 1 greatly inferred the N-terminal amino acid for highly modified peptide toxin aculeines, the probable structure for aculeine B was proposed on the basis of high-resolution mass spectral analysis.
  • Bryan A. Copits, Claire G. Vernon, Ryuichi Sakai, Geoffrey T. Swanson
    JOURNAL OF PHYSIOLOGY-LONDON 592 10 2079 - 2096 2014年05月 [査読有り][通常論文]
     
    Key points Lectins are a family of small evolutionarily conserved sugar-binding proteins that regulate a diverse array of physiological processes ranging from immune system activation to cancer cell metastasis. Ionotropic glutamate receptor function can be modulated by plant-derived lectins, but the physiological relevance of this activity is unclear as no analogous function has been identified in animal lectins. We found that a variety of vertebrate lectins, including human brain-expressed galectin-1, modulated glutamate receptor kinetics in a subunit and lectin-dependent manner, which critically depended on complex oligosaccharide processing. Galectin application slowed neuronal kainate receptor currents from nociceptive dorsal root ganglion neurons. We propose that brain-expressed galectins are potential endogenous modulators of neuronal glutamate receptors, which may play important roles in diseases of altered cellular excitability, such as epilepsy or chronic pain. AMPA and kainate receptors are glutamate-gated ion channels whose function is known to be altered by a variety of plant oligosaccharide-binding proteins, or lectins, but the physiological relevance of this activity has been uncertain because no lectins with analogous allosteric modulatory effects have been identified in animals. We report here that members of the prototype galectin family, which are beta-galactoside-binding lectins, exhibit subunit-specific allosteric modulation of desensitization of recombinant homomeric and heteromeric AMPA and kainate receptors. Galectin modulation of GluK2 kainate receptors was dependent upon complex oligosaccharide processing of N-glycosylation sites in the amino-terminal domain and downstream linker region. The sensitivity of GluA4 AMPA receptors to human galectin-1 could be enhanced by supplementation of culture media with uridine and N-acetylglucosamine (GlcNAc), precursors for the hexosamine pathway that supplies UDP-GlcNAc for synthesis of complex oligosaccharides. Neuronal kainate receptors in dorsal root ganglia were sensitive to galectin modulation, whereas AMPA receptors in cultured hippocampal neurons were insensitive, which could be a reflection of differential N-glycan processing or receptor subunit selectivity. Because glycan content of integral proteins can be modified dynamically, we postulate that physiological or pathological conditions in the CNS could arise in which galectins alter excitatory neurotransmission or neuronal excitability through their actions on AMPA or kainate receptors.
  • Shingo Sasaki, Hiroto Suzuki, Hitoshi Ouchi, Tomohiro Asakawa, Makoto Inai, Ryuichi Sakai, Keiko Shimamoto, Yoshitaka Hamashima, Toshiyuki Kan
    ORGANIC LETTERS 16 2 564 - 567 2014年01月 [査読有り][通常論文]
     
    A practical total synthesis of kainoid MFPA (5) was achieved in only six steps, via a novel Ni-catalyst-mediated asymmetric conjugate addition reaction. Furthermore, a fluorescein-based fluorescent ionotropic glutamate receptor probe 28 was efficiently synthesized from a precursor derived from a synthetic intermediate of 5.
  • Ryuichi Sakai, Geoffrey T. Swanson
    NATURAL PRODUCT REPORTS 31 2 273 - 309 2014年 [査読有り][通常論文]
     
    This review covers the isolation, chemical structure, biological activity, structure activity relationships including synthesis of chemical probes, and pharmacological characterization of neuroactive marine natural products; 302 references are cited.
  • Masaki J. Fujita, Koji Nakano, Ryuichi Sakai
    Molecules 18 4 3917 - 3926 2013年04月 [査読有り][通常論文]
     
    A siderophore, named bisucaberin B, was isolated from Tenacibaculum mesophilum bacteria separated from a marine sponge collected in the Republic of Palau. Using spectroscopic and chemical methods, the structure of bisucaberin B (1) was clearly determined to be a linear dimeric hydroxamate class siderophore. Although compound 1 is an open form of the known macrocyclic dimer bisucaberin (2), and was previously described as a bacterial degradation product of desferrioxamine B (4), the present report is the first description of the de novo biosynthesis of 1. To the best of our knowledge, compound 1 is the first chemically characterized siderophore isolated from a bacterium belonging to the phylum Bacteroidetes. © 2013 by the authors.
  • Takuya Ueda, Yuka Nakamura, Caleb M. Smith, Bryan A. Copits, Akira Inoue, Takao Ojima, Satoko Matsunaga, Geoffrey T. Swanson, Ryuichi Sakai
    GLYCOBIOLOGY 23 4 412 - 425 2013年04月 [査読有り][通常論文]
     
    Here we report the bioactivity-guided isolation of novel galectins from the marine sponge Cinachyrella sp., collected from Iriomote Island, Japan. The lectin proteins, which we refer to as the Cinachyrella galectins (CchGs), were identified as the active principles in an aqueous sponge extract that modulated the function of mammalian ionotropic glutamate receptors. Aggregation of rabbit erythrocytes by CchGs was competed most effectively by galactosides but not mannose, a profile characteristic of members of the galectin family of oligosaccharide-binding proteins. The lectin activity was remarkably stable, with only a modest loss in hemagglutination after exposure of the protein to 100 degrees C for 1 h, and showed little sensitivity to calcium concentration. CchG-1 and -2 appeared as 16 and 18 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, whereas matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry indicated broad ion clusters centered at 16,216 and 16,423, respectively. The amino acid sequences of the CchGs were deduced using a combination of Edman degradation and cDNA cloning and revealed that the proteins were distant orthologs of animal prototype galectins and that multiple isolectins comprised the CchGs. One of the isolectins was expressed as a recombinant protein and exhibited physico-chemical and biological properties comparable with those of the natural lectins. The biochemical properties of the CchGs as well as their unexpected activity on mammalian excitatory amino acid receptors suggest that further analysis of these new members of the galectin family will yield further glycobiological and neurophysiological insights.
  • Lina Juknaite, Yutaro Sugamata, Kazuya Tokiwa, Yuichi Ishikawa, Satoshi Takamizawa, Andrew Eng, Ryuichi Sakai, Darryl S. Pickering, Karla Frydenvang, Geoffrey T. Swanson, Jette S. Kastrup, Masato Oikawa
    JOURNAL OF MEDICINAL CHEMISTRY 56 6 2283 - 2293 2013年03月 [査読有り][通常論文]
     
    IKM-159 was developed and identified as a member of a new class of heterotricyclic glutamate analogues that act as AMPA receptor-selective antagonists. However, it was not known which enantiomer of IKM-159 was responsible for its pharmacological activities. Here, we report in vivo and in vitro neuronal activities of both enantiomers of IKM-159 prepared by enantioselective asymmetric synthesis. By employment of (R)-2-amino-2-(4-methoxyphenyl)ethanol as a chiral auxiliary, (2R)-IKM-159 and the (2S)-counterpart were successfully synthesized in 0.70% and 1.5% yields, respectively, over a total of 18 steps. Both behavioral and electrophysiological assays showed that the biological activity observed for the racemic mixture was reproduced only with (2R)-IKM-159, whereas the (2S)-counterpart was inactive in both assays. Racemic IKM-159 was crystallized with the ligand-binding domain of GluA2, and the structure revealed a complex containing (2R)-IKM-159 at the glutamate binding site. (2R)-IKM-159 locks the GluA2 in an open form, consistent with a pharmacological action as competitive antagonist of AMPA receptors.
  • Masaki J. Fujita, Ryuichi Sakai
    Bioscience, Biotechnology and Biochemistry 77 12 2467 - 2472 2013年 [査読有り][通常論文]
     
    Desferrioxamines E (1), D2 (2), X1 (3), and X2 (4), four macrocyclic N-hydroxy-N-succinyl diamine-based siderophores, were produced efficiently by heterologous expression of a fusion biosynthetic gene cluster. This expression system consisted of three genes (mbsA-C) from marine metagenomic DNA and one gene (dfoCC) from the terrestrial bacterium Erwinia amylovora. The first three genes are functional in the production of the common monomers N-hydroxy-N-succinyl cadaverine (5, HSC) and N-hydroxy-N-succinyl putrescine (6, HSP), whereas dfoCC catalyzes the oligomerization and the macrocyclization reactions of compounds 5 and 6 to form compounds 1-4. This fusion gene cluster system provides a convenient expression platform for various biosynthetic genes of HSC-HSP based siderophores by simply switching the fourth gene by the cassette process.
  • Douglas M. Freymann, Yuka Nakamura, Pamela J. Focia, Ryuichi Sakai, Geoffrey T. Swanson
    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 68 9 1163 - 1174 2012年09月 [査読有り][通常論文]
     
    The galectins are a family of proteins that bind with highest affinity to N-acetyllactosamine disaccharides, which are common constituents of asparagine-linked complex glycans. They play important and diverse physiological roles, particularly in the immune system, and are thought to be critical metastatic agents for many types of cancer cells, including gliomas. A recent bioactivity-based screen of marine sponge (Cinachyrella sp.) extract identified an ancestral member of the galectin family based on its unexpected ability to positively modulate mammalian ionotropic glutamate receptor function. To gain insight into the mechanistic basis of this activity, the 2.1 angstrom resolution X-ray structure of one member of the family, galectin CchG-1, is reported. While the protomer exhibited structural similarity to mammalian prototype galectin, CchG-1 adopts a novel tetrameric arrangement in which a rigid toroidal-shaped 'donut' is stabilized in part by the packing of pairs of vicinal disulfide bonds. Twofold symmetry between binding-site pairs provides a basis for a model for interaction with ionotropic glutamate receptors.
  • Ryuichi Sakai, Satoko Matsunaga, Mitsuru Jimbo, Martin B. Gill, L. Leanne Lash-Van Wyhe, Geoffrey T. Swanson
    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 244 2012年08月 [査読有り][通常論文]
  • Takuya Ueda, Yuka Nakamura, Pamela J. Focia, Caleb Smith, Akira Inoue, Takao Ojima, Satoko Matsunaga, Douglas M. Freymann, Ryuichi Sakai, Geoffrey T. Swanson
    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 244 2012年08月 [査読有り][通常論文]
  • Masaki Unno, Masanobu Shinohara, Koichiro Takayama, Hideharu Tanaka, Kenta Teruya, Katsumi Doh-ura, Ryuichi Sakai, Makoto Sasaki, Masao Ikeda-Saito
    JOURNAL OF MOLECULAR BIOLOGY 413 3 667 - 683 2011年10月 [査読有り][通常論文]
     
    Dysiherbaine (DH) and neodysiherbaine A (NDH) selectively bind and activate two kainate-type ionotropic glutamate receptors, GluK1 and GluK2. The ligand-binding domains of human GluK1 and GluK2 were crystallized as bound forms with a series of DH analogues including DH, NDH, 8-deoxy-NDH, 9-deoxy-NDH and 8,9-dideoxy-NDH (MSVIII-19), isolated from natural sources or prepared by total synthesis. Since the DH analogues exhibit a wide range of binding affinities and agonist efficacies, it follows that the detailed analysis of crystal structure would provide us with a significant opportunity to elucidate structural factors responsible for selective binding and some aspects of gating efficacy. We found that differences in three amino acids (Thr503, Ser706 and Ser726 in GluK1 and Ala487, Asn690 and Thr710 in GluK2) in the ligand-binding pocket generate differences in the binding modes of NDH to GluK1 and GluK2. Furthermore, deletion of the C-9 hydroxy group in NDH alters the ligand conformation such that it is no longer suited for binding to the GluK1 ligand-binding pocket. In GluK2, NDH pushes and rotates the side chain of Asn690 (substituted for Ser706 in GluK1) and disrupts an interdomain hydrogen bond with Glu409. The present data support the idea that receptor selectivities of DH analogues resulted from the differences in the binding modes of the ligands in GluK1/GluK2 and the steric repulsion of Asn690 in GluK2. All ligands, regardless of agonist efficacy, induced full domain closure. Consequently, ligand efficacy and domain closure did not directly coincide with DH analogues and the kainate receptors. (C) 2011 Elsevier Ltd. All rights reserved.
  • Satoko Matsunaga, Mitsuru Jimbo, Martin B. Gill, L. Leanne Lash-Van Wyhe, Michio Murata, Ken'ichi Nonomura, Geoffrey T. Swanson, Ryuichi Sakai
    CHEMBIOCHEM 12 14 2191 - 2200 2011年09月 [査読有り][通常論文]
     
    A novel family of functionalized peptide toxins, aculeines (ACUs), was isolated from the marine sponge Axinyssa aculeate. ACUs are polypeptides with N-terminal residues that are modified by the addition of long-chain polyamines (LCPA). Aculeines were present in the sponge extract as a complex mixture with differing polyamine chain lengths and peptide structures. ACU-A and B, which were purified in this study, share a common polypeptide chain but differ in their N-terminal residue modifications. The amino acid sequence of the polypeptide portion of ACU-A and B was deduced from 3' and 5' RACE, and supported by Edman degradation and mass spectral analysis of peptide fragments. ACU induced convulsions upon intracerebroventricular (i.c.v.) injection in mice, and disrupted neuronal membrane integrity in electrophysiological assays. ACU also lysed erythrocytes with a potency that differed between animal species. Here we describe the isolation, amino acid sequence, and biological activity of this new group of cytotoxic sponge peptides.
  • Chang-Shen Qiu, Leanne Lash-Van Wyhe, Makoto Sasaki, Ryuichi Sakai, Geoffrey T. Swanson, Robert W. Gereau
    PAIN 152 5 1052 - 1060 2011年05月 [査読有り][通常論文]
     
    The ionotropic glutamate receptor subunit, GluK1 (GluR5), is expressed in many regions of the nervous system related to sensory transmission. Recently, a selective ligand for the GluK1 receptor, MSVIII-19 (8,9-dideoxy-neodysiherbaine), was synthesized as a derivative of dysiherbaine, a toxin isolated from the marine sponge Lendenfeldia chondrodes. MSVIII-19 potently desensitizes GluK1 receptors without channel activation, rendering it useful as a functional antagonist. Given the high selectivity for GluK1 and the proposed role for this glutamate receptor in nociception, we sought to test the analgesic potential of MSVIII-19 in a series of models of inflammatory, neuropathic, and visceral pain in mice. MSVIII-19 delivered intrathecally dose-dependently reduced formalin-induced spontaneous behaviors and reduced thermal hypersensitivity 3 hours after formalin injection and 24 hours after complete Freund's adjuvant-induced inflammation, but had no effect on mechanical sensitivity in the same models. Intrathecal MSVIII-19 significantly reduced both thermal hyperalgesia and mechanical hypersensitivity in the chronic constriction injury model of neuropathic pain, but had no effect in the acetic acid model of visceral pain. Peripheral administration of MSVIII-19 had no analgesic efficacy in any of these models. Finally, intrathecal MSVIII-19 did not alter responses in Tail-flick tests or performance on the accelerating RotaRod. These data suggest that spinal administration of MSVIII-19 reverses hypersensitivity in several models of pain in mice, supporting the clinical potential of GluK1 antagonists for the management of pain. (C) 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
  • Tsuyoshi Sakurada, Martin B. Gill, Shanti Frausto, Bryan Copits, Keiichi Noguchi, Keiko Shimamoto, Geoffrey T. Swanson, Ryuichi Sakai
    JOURNAL OF MEDICINAL CHEMISTRY 53 16 6089 - 6099 2010年08月 [査読有り][通常論文]
     
    Marine organisms have yielded a variety of metabolites with neuropharmacological applications. Here we describe the isolation and pharmacological characterization of four novel, neurologically active purines 1-4, isolated from Haplosclerida sponges collected in the Republic of Palau. The structures were determined by analyses of spectral and X-ray data. Compound 1 induced convulsions upon intracerebroventricular injection into mice, with a CD50 value of 2.4 nmol/mouse. Purines 2-4 were active in mouse bioassays at higher doses. The seizurogenic activity of 1 was correlated with inhibition of neuronal GABAergic transmission, with only a modest impact on excitatory signaling, in electrophysiological recordings from hippocampal neurons. Despite having a purine template structure, the inhibitory activity of 1 was not prevented by a nonselective adenosine receptor antagonist. Thus, 1 represents a novel substituted purine that elicits convulsions through its actions on inhibitory neurotransmission. These 8-oxoisoguanine analogs comprise a new family of compounds closely related in structure to endogenous neurosignaling molecules and commonly used CNS stimulants.
  • Masato Oikawa, Minoru Ikoma, Makoto Sasaki, Martin B. Gill, Geoffrey T. Swanson, Keiko Shimamoto, Ryuichi Sakai
    BIOORGANIC & MEDICINAL CHEMISTRY 18 11 3795 - 3804 2010年06月 [査読有り][通常論文]
     
    Here, we report our second-generation synthesis of 12 artificial glutamate analogs, starting from heterotricycle intermediates 3a-3d, readily prepared in three steps including tandem Ugi/Diels-Alder reactions. The new synthesis employs imidate intermediates for the deoxygenation of pyrrolidones (10a-10d to 6a-6d), and each advanced intermediate 6a-6d was diversified into three glutamate analogs (1a-1d, 5a-5d, 7a-7d) in 1-2 steps. In vitro electrophysiological assays revealed that the new piperidine-type analog 7c alters neuronal function with lower potency than 1a. Conversely, intracranial injection of 7c into mice produced a greater degree of hypoactivity than 1a. Our recent investigation has revealed that this series of compounds antagonizes AMPA-type glutamate receptor-mediated currents in a subtype selective manner. The more efficient syntheses of this novel set of neuroactive molecules will facilitate their pharmacological characterization. (C) 2010 Elsevier Ltd. All rights reserved.
  • L. Leanne Lash-Van Wyhe, Pekka A. Postila, Koichi Tsubone, Makoto Sasaki, Olli T. Pentikainen, Ryuichi Sakai, Geoffrey T. Swanson
    NEUROPHARMACOLOGY 58 3 640 - 649 2010年03月 [査読有り][通常論文]
     
    Kainate receptor antagonists have potential as therapeutic agents in a number of neuropathologies. Synthetic modification of the convulsant marine toxin neodysiherbaine A (NDH) previously yielded molecules with a diverse set of pharmacological actions on kainate receptors. Here we characterize three new synthetic analogs of NDH that contain substituents at the C10 position in the pyran ring of the marine toxin. The analogs exhibited high-affinity binding to the GluK1 (GluR5) subunit and lower affinity binding to GluK2 (GluR6) and GluK3 (GluR7) subunits in radioligand displacement assays with recombinant kainate and AMPA receptors. As well, the natural toxin NDH exhibited similar to 100-fold selectivity for GluK2 over GluK3 subunits, which was attributable to the C8 hydroxyl group in NDH. We used molecular dynamic simulations to determine the specific interactions between NDH and residues within the ligand-binding domains of these two kainate receptor subunits that contribute to the divergent apparent affinities for the compound. These data demonstrate that interactions with the GluKI subunit are preserved in analogs with substitutions at C10 in NDH and further reveal the determinants of selectivity and pharmacological activity of molecules acting on kainate receptor subunits. which could aid in design of additional compounds that target these receptors. (C) 2009 Elsevier Ltd. All rights reserved.
  • Masato Oikawa, Minoru Ikoma, Makoto Sasaki, Martin B. Gill, Geoffrey T. Swanson, Keiko Shimamoto, Ryuichi Sakai
    EUROPEAN JOURNAL OF ORGANIC CHEMISTRY 2009 32 5531 - 5548 2009年11月 [査読有り][通常論文]
     
    In this article a regioselective domino metathesis reaction of unsymmetrical 7-oxanorbornenes, readily available by a tandem Ugi/Diels-Alder reaction as a key step, promoted by the Hoveyda-Grubbs second-generation catalyst in the presence of electron-rich vinyl acetate as a cross metathesis (CM) substrate is reported. The mechanism for the unusually high regioselectivity observed in the CM reaction was investigated, and a reaction course where a Fischer-type carbene ["Ru"= CH(OAc)] generates a steric interaction is proposed. The metathesis products were further converted to four artificial glutamate analogues whose structures were inspired by naturally derived excitatory glutamate analogues, dysiherbaine and neodysiherbaine. Interestingly, one of the synthetic analogues (28a) induced a cataleptic state in mice. Further electrophysiological studies suggest that 28a might inhibit excitatory synaptic transmission by a yet unknown indirect pathway. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)
  • Karla Frydenvang, L. Leanne Lash, Peter Naur, Pekka A. Postila, Darryl S. Pickering, Caleb M. Smith, Michael Gajhede, Makoto Sasaki, Ryuichi Sakai, Olli T. Pentikainen, Geoffrey T. Swanson, Jette S. Kastrup
    JOURNAL OF BIOLOGICAL CHEMISTRY 284 21 14219 - 14229 2009年05月 [査読有り][通常論文]
     
    The prevailing structural model for ligand activation of ionotropic glutamate receptors posits that agonist efficacy arises from the stability and magnitude of induced domain closure in the ligand-binding core structure. Here we describe an exception to the correlation between ligand efficacy and domain closure. A weakly efficacious partial agonist of very low potency for homomeric iGluR5 kainate receptors, 8,9-dideoxyneodysiherbaine (MSVIII-19), induced a fully closed iGluR5 ligand-binding core. The degree of relative domain closure, similar to 30 degrees, was similar to that we resolved with the structurally related high affinity agonist dysiherbaine and to that of L-glutamate. The pharmacological activity of MSVIII-19 was confirmed in patch clamp recordings from transfected HEK293 cells, where MSVIII-19 predominantly inhibits iGluR5-2a, with little activation apparent at a high concentration (1 mM) of MSVIII-19 (<1% of mean glutamate-evoked currents). To determine the efficacy of the ligand quantitatively, we constructed concentration-response relationships for MSVIII-19 following potentiation of steady-state currents with concanavalin A (EC(50) = 3.6 mu M) and on the nondesensitizing receptor mutant iGluR5-2b(Y506C/L768C) (EC(50) = 8.1 mu M). MSVIII-19 exhibited a maximum of 16% of full agonist efficacy, as measured in parallel recordings with glutamate. Molecular dynamics simulations and electrophysiological recordings confirm that the specificity of MSVIII-19 for iGluR5 is partly attributable to interdomain hydrogen bond residues Glu(441) and Ser(721) in the iGluR5-S1S2 structure. The weaker interactions of MSVIII-19 with iGluR5 compared with dysiherbaine, together with altered stability of the interdomain interaction, may be responsible for the apparent uncoupling of domain closure and channel opening in this kainate receptor subunit.
  • Swanson GT, Sakai R
    Progress in molecular and subcellular biology 46 123 - 157 2009年 [査読有り][通常論文]
  • Minoru Ikoma, Masato Oikawa, Martin B. Gill, Geoffrey T. Swanson, Ryuichi Sakai, Keiko Shimamoto, Makoto Sasaki
    EUROPEAN JOURNAL OF ORGANIC CHEMISTRY 2008 31 5215 - 5220 2008年11月 [査読有り][通常論文]
     
    A highly regioselective domino metathesis reaction of 7-oxanorbornene was developed that employed an intramolecular association of an amide carbonyl group to a ruthenium metal centre. By using this reaction, twelve glutamate analogues inspired by dysiherbaine were efficiently synthesized over 12-14 steps; one of the analogues exhibited bioactivity consistent with central nervous system depression. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)
  • Ryuichi Sakai, Kazufumi Yoshida, Atsuko Kimura, Kanae Koike, Mitsuru Jimbo, Kazuhiko Koike, Atsushi Kobiyama, Hisao Kamiya
    CHEMBIOCHEM 9 4 543 - 551 2008年03月 [査読有り][通常論文]
     
    The cellular origin of dysiherbaine, a morine-sponge toxin, was investigated immunohistochemically by using an anti-dysiherbaine antibody. Dysiherboine-like immunoreactivity was found to be localized in spherical cells harbored in the sponge mesohyl. A combination of ribosomal RNA gene (rDNA) analysis and cell-morphology analysis revealed that the spherical cells were Synechocystis cyanobacteria. However the sponge, identified as Lendenfeldia chondrodes on the basis of its rDNA sequence, appeared to contain two different chemotypes-dysiherboine-producing (DH+) and nondysiherbaine-producing (DH-)-both of which inhabited the some region. Synechocystis cells in the DH- sponge were not labeled with antibody, although the 16S rDNA gene profile of the cyonobacteria in the DH- sponge was indistinguishable from that of the cyanobacterio in the DH+ sponge. On the basis of these results, we hypothesize that dysiherbaine is a metabolite of certain varieties of endosymbiotic Synechocystis sp.
  • L. Leanne Lash, James M. Sanders, Nobuyuki Akiyama, Muneo Shoji, Pekka Postila, Olli T. Pentikainen, Makoto Sasaki, Ryuichi Sakai, Geoffrey T. Swanson
    JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 324 2 484 - 496 2008年02月 [査読有り][通常論文]
     
    Antagonists for kainate receptors (KARs), a family of glutamate-gated ion channels, are efficacious in a number of animal models of neuropathologies, including epilepsy, migraine pain, and anxiety. To produce molecules with novel selectivities for kainate receptors, we generated three sets of analogs related to the natural marine convulsant neodysiherbaine (neoDH), and we characterized their pharmacological profiles. Radioligand displacement assays with recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and KARs demonstrated that functional groups at two positions on the neoDH molecule are critical pharmacological determinants; only binding to the glutamate receptor (GluR)5-2a subunit was relatively insensitive to structural modifications of the critical functional groups. NeoDH analogs in which the L-glutamate congener was disrupted by epimerization retained low affinity for GluR5-2a and GluR6a KAR subunits. Most of the analogs showed agonist activity in electrophysiological recordings from human embryonic kidney-T/17 cells expressing GluR5-2a KARs, similar to the natural convulsant neoDH. In contrast, 2,4-epi-neoDH inhibited glutamate currents evoked from both GluR5-2a and GluR6a receptor-expressing cells. Therefore, this compound represents the first compound to exhibit functional antagonist activity on GluR5-2a and GluR6a KAR subunits without concurrent activity on AMPA receptor subunits. Finally, binding affinity of the synthetic ligands for the GluR5-2a subunit closely correlated with their seizurogenic potency, strongly supporting a role for receptors containing this subunit in the convulsant reaction to KAR agonists. The analogs described here offer further insight into structural determinants of ligand selectivity for KARs and potentially represent useful pharmacological tools for studying the role of KARs in synaptic physiology and pathology.
  • Makoto Sasaki, Koichi Tsubone, Kunimori Aoki, Nobuyuki Akiyama, Muneo Shoji, Masato Oikawa, Ryuichi Sakai, Keiko Shimamoto
    JOURNAL OF ORGANIC CHEMISTRY 73 1 264 - 273 2008年01月 [査読有り][通常論文]
     
    [GRAPHICS] A rapid and efficient total synthesis of dysiherbaine (1), a potent and subtype-selective agonist for ionotropic glutamate receptors, has been accomplished. A key intermediate 15 was synthesized by two approaches. The first synthetic route utilized compound 9, an advanced intermediate in our previous total synthesis of neodysiherbaine A, as the starting point, and the cis-oriented amino alcohol functionality on the tetrahydropyran ring was installed by using an intramolecular S(N)2 cyclization of N-Boc-protected amino alcohol 20. An alternative and even more efficient synthetic approach to 15 featured stereoselective introduction of an amino group at C8 by iodoaminocyclization prior to constructing the bicyclic ether skeleton. The amino acid appendage was efficiently constructed by a catalytic asymmetric hydrogenation of enamide ester 36. The synthetic route developed here provided access to several dysiherbaine analogues, including 9-epi-dysiherbaine (38), 9-deoxydysiherbaine (39), 9-methoxydysiherbaine (40), and N-ethyldysiherbaine (41). The preliminary structure-activity relationship studies revealed that the presence and stereochemistry of the C9 hydroxy group in dysiherbaine is important for high-affinity and selective binding to glutamate subtype receptors.
  • Satoko Matsunaga, Ryuichi Sakai, Mitsuru Jimbo, Hisao Kamiya
    CHEMBIOCHEM 8 14 1729 - 1735 2007年09月 [査読有り][通常論文]
     
    Two distinct marine organisms, diatoms and sponges, deposit dissolved silicates to construct highly architectural and species-specific body supports. Several factors such as proteins, long-chain polyamines (LCPAs), or polypeptides modified with LCPAs are known to be involved in this process. The LCPAs contained in the silica walls of diatoms are thought to play pivotal roles in the silica deposition. In sponges, however, a protein called silicatein and several other proteins hove been reported to be the factors involved in the silica deposition. However, no other factors involved in this process have been reported. We have identified the LCPAs from the marine sponge Axinyssa aculeata and present here some evidence that sponge-derived LCPAs can deposit silica and that the LCPA derivatives are associated with spicules. The results indicate a common chemistry between sponges and diatoms, the two major players in the biological circulation of silicon in the marine environment. A wide variety of organisms are known to utilize silica in their biological processes. Polyamines or other functional molecules might be involved, in combination with proteins, in their biosilicification process.
  • Matsubara H, Nakamura-Tsuruta S, Hirabayashi J, Jimbo M, Kamiya H, Ogawa T, Muramoto K, Cellular Origin of Dysiherbaine, a Marine, Sponge-derived Excitatory Amino Acid, 共著 in, Press Chembiochem, Sakai R, Yoshida K, Kimura A, Koike K, Jimbo M, Koike K, Kobiyama A, Kamiya H, Modulating effect of acorn, barnacle C-Type, lectins on the crystallization of calcium carbonate, 共著 in, Press Fish Sci, Matsubara H, Hayashi T, Ogawa T, Muramoto K, Jimbo M, Kamiya H
    Biosci Biotechnol Biochem 71 71 2 1729 - 1735 Japan Society for Bioscience, Biotechnology, and Agrochemistry 2007年08月01日 [査読無し][通常論文]
     
    The sugar-binding specificities of C-type lectins isolated from marine invertebrates were investigated by frontal affinity chromatography (FAC) using 100 oligosaccharides. The lectins included BRA-2 and BRA-3, multiple lectins from the hemolymph of the acorn barnacle, Megabalanus rosa, and BRL from the acorn barnacle, Balanus rostatus. The diverse sugar-binding specificities of the C-type lectins were determined by FAC analysis. BRA-2 recognized α2-6 sialylation but not α2-3 sialylation on glycans. On the other hand, BRA-3 showed high affinity for oligosaccharides with α-linked non-reducing terminal galactose, but not for sialylated forms, and BRL showed enhanced recognition activity towards Lewisx and Lewisa epitopes.
  • Mitsuru Jimbo, Rika Usui, Ryuichi Sakai, Koji Muramoto, Hisao Kamiya
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 147 2 164 - 171 2007年06月 [査読有り][通常論文]
     
    Three L-rhamnose-binding egg lectins, TBL1, TBL2 and TBL3, were isolated from the eggs of the Far East dace Tribolodon brandti by a combination of affinity chromatography on L-rhamnose-Sepharose 6B gel and reversed-phase HPLC. L-rhamnose is a common inhibitor of the purified lectins and strongly inhibited the hemagglutinating activity of TBL2 and TBL3, but less weakly that of TBL1. L-arabinose, which has the same hydroxyl group orientation at C2 and C4 as L-rhamnose, and D-galactose showed no inhibitory activity against TBL1 but showed weak inhibitory activity against TBL2 and TBL3. The open reading frames of the cDNAs of TBL1, TBL2 and TBL3 encoded for mature proteins of 207, 189, and 293 amino acid residues, respectively. A BLAST homology search showed that the TBLs have about 40% homology to the carbohydrate recognition domains of rhamnose-binding lectins in salmonid eggs. The tandem repeated domains present in TBL1, TBL2 and TBL3 were two, two and three, respectively. TBL2 was exclusively expressed in ovary, while TBL1 and TBL3 were expressed mainly in ovary and weakly in various tissues including gill, heart, kidney, liver, spleen and testis. (C) 2007 Elsevier Inc. All rights reserved.
  • Jie-Fei Cheng, Jong-Soo Lee, Ryuichi Sakai, Elizabeth A. Jares-Erijman, Maria V. Silva, Kenneth L. Rinehart
    JOURNAL OF NATURAL PRODUCTS 70 3 332 - 336 2007年03月 [査読有り][通常論文]
     
    Four novel polyketide-derived metabolites, myriaporones 1, 2, 3, and 4, have been isolated from the Mediterranean bryozoan Myriapora truncata. Their structures and stereochemistry have been assigned from the analysis of spectroscopic data. The inseparable equilibrium mixture of myriaporones 3 and 4 showed 88% inhibition of L1210 murine leukemia cells at 0.2 mu g/mL.
  • Makoto Sasaki, Koichi Tsubone, Muneo Shoji, Masato Okawa, Keiko Shimamoto, Ryuichi Sakai
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 16 22 5784 - 5787 2006年11月 [査読有り][通常論文]
     
    To enable studies to elucidate the detailed biological function of dysiherbaine and neodysiherbaine A, potent and subunit-selective agonists for ionotropic glutamate receptors, the derivative with a hydroxymethyl substituent at the C10 position has been developed. Preliminary biological evaluation of the analogue showed that a C10 hydroxymethyl substituent produced significant alterations in binding affinities for the ionotropic glutamate receptor subtypes. (c) 2006 Elsevier Ltd. All rights reserved.
  • Ryuichi Sakai, Hisao Kamiya
    JOURNAL OF ANTIBIOTICS 59 8 507 - 511 2006年08月 [査読有り][通常論文]
     
    Two 1-deoxynojirimycin derivatives, 1-deoxynojirimycin-6-phosphate (1) and N-methyl-ldeoxynojirimycin-6-phosphate (2) were isolated from an aqueous extract of Micronesian marine sponge Lendenfeldia chondrodes for the first time as natural products. Structures of these compounds were assigned on the basis of their spectral data and chemical degradation.
  • Shimamoto Keiko, Sakai Ryuichi, Shigeri Yasushi, Nakagawa Takayuki
    International Symposium on the Chemistry of Natural Products 2006 "P - 227" 天然有機化合物討論会 2006年07月23日
  • Muneo Shoji, Nobuyuki Akiyama, Koichi Tsubone, L. Leanne Lash, James M. Sanders, Geoffrey T. Swanson, Ryuichi Sakai, Keiko Shimamoto, Masato Oikawa, Makoto Sasaki
    JOURNAL OF ORGANIC CHEMISTRY 71 14 5208 - 5220 2006年07月 [査読有り][通常論文]
     
    [GRAPHICS] Dysiherbaine (1) and its congener neodysiherbaine A (2) are naturally occurring excitatory amino acids with selective and potent agonistic activity for ionotropic glutamate receptors. We describe herein the total synthesis of 2 and its structural analogues 3-8. Advanced key intermediate 16 was employed as a branching point to assemble a series of these analogues 3-8 with respect to the C-8 and C-9 functionalities, which would not have been accessible through manipulations of the natural product itself. The synthesis of key intermediate 16 features (i) stereocontrolled C-glycosylation to set the C-6 stereocenter, (ii) concise synthesis of the bicyclic ether skeleton through chemo- and stereoselective dihydroxylation of the exo-olefin and stereoselective epoxidation of the endo-olefin, followed by epoxide ring opening/5-exo ring closure, and (iii) catalytic asymmetric hydrogenation of enamide ester to construct the amino acid appendage. A preliminary biological evaluation of analogues for their in vivo toxicity against mice and binding affinity for glutamate receptors showed that both the type and stereochemistry of the C8 and C9 functional groups affected the subtype selectivity of dysiherbaine analogues for members of the kainic acid receptor family.
  • JM Sanders, OT Pentikainen, L Settimo, U Pentikainen, M Shoji, M Sasaki, R Sakai, MS Johnson, GT Swanson
    MOLECULAR PHARMACOLOGY 69 6 1849 - 1860 2006年06月 [査読有り][通常論文]
     
    Dysiherbaine (DH) and related molecules are high-affinity, subunit-selective kainate receptor (KAR) ligands originally isolated from a marine sponge. To elucidate why DH, an agonist, and MSVIII-19, a competitive antagonist, bind selectively to glutamate receptor (GluR) 5 but not to the KA2 KAR subunit, we used molecular dynamics simulations to generate binding models that were tested experimentally in radioligand binding and electrophysiological assays. Three candidate sites, Val685, Leu735, and Ser741 in GluR5, corresponding to Ile669, Phe719, and Met725 in KA2, were predicted to underlie the distinct binding profiles of the marine toxins. Single or multiple reciprocal mutations introduced into the receptor subunits produced a variety of effects on binding affinity. Most notably, mutation of Met725 to serine in KA2 increased the affinity of DH by 350-fold; in contrast, mutation of one or more of the residues in GluR5 did not markedly alter DH binding. MSVIII-19 affinity for the KA2 subunit was significantly increased in multiple site mutants, and reciprocal mutations in the GluR5 subunit produced substantial (700-fold) reductions in MSVIII-19 affinity. Physiological characterization of the double- and triple-mutant subunits demonstrated altered functional behavior consistent with the changes in binding affinity. The results provide experimental support for the importance of these three ligand binding domain (LBD) residues and suggest steric hindrance in the KA2 subunit LBD is largely responsible for the very low affinity for the two compounds. In this study, we identified the molecular basis for subunit selectivity of these marine-derived molecules on KARs, which could facilitate the rational design of selective ligands with distinct pharmacological profiles.
  • R Sakai, S Minato, K Koike, K Koike, M Jimbo, H Kamiya
    CELL AND TISSUE RESEARCH 322 3 491 - 502 2005年12月 [査読有り][通常論文]
     
    Polyclonal antibodies specific for the excitatory amino acid, kainic acid (KA), were raised in rabbits. The antibody recognized KA but did not cross-react with other structurally related amino acids, including glutamate. We used this anti-KA antibody to localize KA immunohistochemically in the KA-producing red alga Digenea simplex. KA immunoreactivity was most dense in the fine cylindrical thallus, which covers the middle to upper part of the alga. The cortical cells, but not the inner layers of the main axis, and cells of the rhizoid were also stained with this antibody. The presence of KA in cells that cover the surface of the alga might reflect its role in chemical defense. At the subcellular level, KA immunoreactivity was most intense in the nucleus, pit plugs, and the electron-dense areas denoted as "granule bodies", which were found only in the pericentral cells of the thallus.
  • R Sakai, T Nakamura, T Nishino, M Yamamoto, A Miyamura, H Miyamoto, N Ishiwata, N Komatsu, H Kamiya, N Tsuruzoe
    BIOORGANIC & MEDICINAL CHEMISTRY 13 23 6388 - 6393 2005年12月 [査読有り][通常論文]
     
    Four xanthocillins (1-4), including a new compound 4, were isolated from cultured marine fungus Basipetospora sp. as thrombopoietin (TPO) mimics. Compounds 1-4 promoted the proliferation of a TPO-sensitive human leukemia cell line, UT-7/TPO, and UT-7/EPO-mpl, genetically engineered to express c-Mpl, a receptor for TPO in dose-dependent manners. However, the proliferation of UT-7/EPO, a parental cell line of UT-7/EPO-mpl that was devoid of TPO receptor, was not affected by them. Thrombopoietic action of compound 1 was nearly as potent as that of TPO, inducing cell proliferation at a concentration ranging from 1 to 100 nM. Compound 1 also induced the phosphorylation of several proteins, including Janus kinase 2 (Jak2), signal transducers, and activators of transcription-3 (STAT3) and STAT5 in the UT-7/EPO-mpl cell line, but not in the UT-7/EPO cell line. These data indicated that xanthocillins are putative agonists for c-Mpl, as their cellular actions were analogous to those of TPO. (c) 2005 Elsevier Ltd. All rights reserved.
  • JM Sanders, K Ito, L Settimo, OT Pentikainen, M Shoji, M Sasaki, MS Johnson, R Sakai, GT Swanson
    JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 314 3 1068 - 1078 2005年09月 [査読有り][通常論文]
     
    Kainate receptors show a particular affinity for a variety of natural source compounds, including dysiherbaine (DH), a potent agonist derived from the marine sponge Dysidea herbacea. In this study, we characterized the pharmacological activity and structural basis for subunit selectivity of neodysiherbaine (neoDH) and MSVIII-19, which are natural and synthetic analogs of DH, respectively. NeoDH and MSVIII-19 differ from DH in the composition of two functional groups that confer specificity and selectivity for ionotropic glutamate receptors. In radioligand binding assays, neoDH displayed a 15- to 25-fold lower affinity relative to that of DH for glutamate receptor (GluR)5 and GluR6 kainate receptor subunits but a 7-fold higher affinity for kainate (KA)2 subunits, whereas MSVIII-19 displaced [H-3] kainate only from GluR5 subunits but not GluR6 or KA2 subunits. NeoDH was an agonist for kainate and alpha-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in patch-clamp recordings; in contrast, MSVIII-19 acted as a potent antagonist for homomeric GluR5 receptor currents with weaker activity on other kainate and AMPA receptors. Neither neoDH nor MSVIII-19 activated group I metabotropic GluRs. Homology modeling suggests that two critical amino acids confer the high degree of selectivity between the dysiherbaine analogs and the GluR5 and KA2 subunits. In summary, these data describe the pharmacological activity of two new compounds, one of which is a selective GluR5 receptor antagonist that will be of use for understanding native receptor function and designing more selective ligands for kainate receptors.
  • Mitsuru Jimbo, Kazuhiko Koike, Ryuichi Sakai, Koji Muramoto, Hisao Kamiya
    Biochemical and Biophysical Research Communications 330 1 157 - 162 2005年04月29日 [査読有り][通常論文]
     
    In the present study, the entire amino acid sequence and cDNA structure encoding the d-galactose-binding lectin, SLL-2, isolated from the octocoral Sinularia lochmodes, were determined. SLL-2 regulates the morphology of symbiotic dinoflagellates Symbiodinium spp. through unknown mechanisms. Here, three cDNAs that encode SLL-2 were cloned and characterized. All the SLL-2 cDNAs encoded 142 amino acids with high similarity to each other. The mature subunit of SLL-2 was found to be composed of 94 amino acids and to contain one putative glycosylation site common to all three SLL-2. N-Glycopeptidase F treatment of SLL-2 resulted in a protein band shift from 16.5 to 9.5 kDa in SDS-PAGE, confirming that SLL-2s are glycoproteins. Two-dimensional polyacrylamide gel electrophoresis analysis of the deglycosylated SLL-2 indicated a presence of three polypeptides as encoded in SLL-2 cDNAs. The deduced sequences of SLL-2 cDNAs had a similarity to the C-terminal region of discoidin I, the slime mold Dictyostelium discoideum lectin. © 2005 Elsevier Inc. All rights reserved.
  • K Koike, M Jimbo, R Sakai, M Kaeriyama, K Muramoto, T Ogata, T Maruyama, H Kamiya
    BIOLOGICAL BULLETIN 207 2 80 - 86 2004年10月 [査読有り][通常論文]
  • K Shimamoto, R Sakai, K Takaoka, N Yumoto, T Nakajima, SG Amara, Y Shigeri
    MOLECULAR PHARMACOLOGY 65 4 1008 - 1015 2004年04月 [査読有り][通常論文]
     
    Nontransportable blockers of the glutamate transporters are important tools for investigating mechanisms of synaptic transmission. DL-threo-beta-Benzyloxyaspartate (DL-TBOA) is a potent blocker of all subtypes of the excitatory amino acid transporters (EAATs). We characterized novel L-TBOA analogs possessing a substituent on their respective benzene rings. The analogs significantly inhibited labeled glutamate uptake, the most potent of which was (2S,3S)-3-{3-[4-(trifluoromethyl)benzoylamino]benzyloxy}aspartate (TFB-TBOA). In an uptake assay using cells transiently expressing EAATs, the IC50 values of TFB-TBOA for EAAT1, EAAT2, and EAAT3 were 22, 17, and 300 nM, respectively. TFB-TBOA was significantly more potent at inhibiting EAAT1 and EAAT2 compared with L-TBOA (IC50 values for EAAT1-3 were 33, 6.2, and 15 muM, respectively). Electrophysiological analyses revealed that TBOA analogs block the transport-associated currents in all five EAAT subtypes and also block leak currents in EAAT5. The rank order of the analogs for potencies at inhibiting substrate-induced currents was identical to that observed in the uptake assay. However, the kinetics of TFB-TBOA differed from the kinetics of L-TBOA, probably because of the strong binding affinity. Notably, TFB-TBOA did not affect other representative neurotransmitter transporters or receptors, including ionotropic and metabotropic glutamate receptors, indicating that it is highly selective for EAATs. Moreover, intracerebroventricular administration of the TBOA analogs induced severe convulsive behaviors in mice, probably because of the accumulation of glutamate. Taken together, these findings indicate that novel TBOA analogs, especially TFB-TBOA, should serve as useful tools for elucidating the physiological roles of the glutamate transporters.
  • R Sakai, K Suzuki, K Shimamoto, H Kamiya
    JOURNAL OF ORGANIC CHEMISTRY 69 4 1180 - 1185 2004年02月 [査読有り][通常論文]
     
    Three new betaines, dysibetaine PP (1), dysibetaine CPa (2), and dysibetaine CPb (3), were isolated from an aqueous extract of the marine sponge Dysidea herbacea collected in Yap state, Micronesia. The structure of 1 was determined by spectral methods as well as chemical degradation to be a novel dipeptide betaine, and those for 2 and 3 were determined to be unprecedented cyclopropane betaines. Compounds 2 and 3 showed weak affinity toward the N-methyl-D-aspartic acid-type and the kainic acid-type glutamate receptors, respectively, in a radioligand binding assay.
  • R Sakai, H Matsubara, K Shimamoto, M Jimbo, H Kamiya, M Namikoshi
    JOURNAL OF NATURAL PRODUCTS 66 6 784 - 787 2003年06月 [査読有り][通常論文]
     
    The bioassay-guided fractionation of the water-soluble extract of the marine sponge Cribrochalina olemda collected in Palau resulted in the isolation of a new amino acid cribronic acid (1): (2S,4R,5R)-5-hydroxy-4-sulfooxypiperidine-2-carboxylic acid. However, aqueous extracts of Stylotella aurantium and Axinella carteri collected in Yap State, Micronesia, afforded a known N-methyl-D-aspartic acid (NMDA)-type glutamate receptor agonist, (2S,4S)-4-sulfooxypiperidine-2-carboxylic acid (2), as a common active principle. Both 1 and 2 induced convulsive behaviors in mice upon intracerebroventricular (icv) injection with ED50 values of 29 +/- 3.0 and 20 +/- 2.8 pmol/mouse, respectively. Radioligand binding assay using rat cerebrocortical membrane demonstrated that I and 2 inhibit the binding of the labeled NMDA receptor ligand [H-3]CGP39653 at IC50 values of 83 +/- 15 and 214 +/- 20 nM, respectively. However, 1 and 2 did not displace [H-3]kainic acid or [H-3]AMPA. These data indicated that 1 is a selective NMDA-type glutamate receptor ligand with potent convulsant activity in mice.
  • R Sakai, T Koike, M Sasaki, K Shimamoto, C Oiwa, A Yano, K Suzuki, K Tachibana, H Kamiya
    ORGANIC LETTERS 3 10 1479 - 1482 2001年05月 [査読有り][通常論文]
     
    A new excitatory amino acid, neodysiherbaine A (2), was isolated as a minor constituent of the aqueous extract from the marine sponge Dysidea herbacea. The structure was deduced by spectroscopic methods and established unambiguously by the total synthesis. The present synthesis, including as a key step cross coupling of the 6/5-bicyclic core with an amino acid residue, is useful in constructing its structural analogues.
  • R Sakai, GT Swanson, K Shimamoto, T Green, A Contractor, A Ghetti, Y Tamura-Horikawa, C Oiwa, H Kamiya
    JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 296 2 650 - 658 2001年02月 [査読有り][通常論文]
     
    Dysiherbaine (DH) is a marine sponge-derived amino acid that causes seizures upon injection into mice. In this report we investigate the behavioral effects and characterize the pharmacological activity of DH. DH induced convulsive behaviors in mice with ED50 values of 13 pmol/mouse, i.c.v. and 0.97 mg/kg, i p. In rat brain synaptic membranes DH displaced binding of [H-3] kainic acid (KA) and [H-3] alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) with K-i values of 26 and 153 nM, respectively; in contrast, DH did not displace the N-methyl-D-aspartic acid (NMDA) receptor ligand [H-3] CGS-19755. DH displaced [H-3]KA from recombinant GluR5 and GluR6 kainate receptor subunits expressed in HEK293 cells with K-i values of 0.74 and 1.2 nM, respectively. In whole-cell voltage-clamp recordings from cultured rat hippocampal neurons, DH evoked inward currents from both AMPA and KA receptors with EC50 values of 9.7 muM and 210 nM, respectively. AMPA receptor currents were blocked by GYKI 53655, whereas KA receptor currents were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Surprisingly, in calcium imaging experiments we found that DH also activated recombinant mGluR5 receptors but did not activate mGluR1 receptors. DH did not activate glutamate transporters or gamma -aminobutyric acid A (GABA(A))receptors. These results indicate that DH is a potent non-NMDA-type agonist with very high affinity for KA receptors, as well as a subtype-selective mGluR agonist. DH possesses the most potent epileptogenic activity among the amino acids yet identified. This novel excitatory amino acid may prove useful for evaluating the physiological and pathological roles of non-NMDA receptors, especially KA receptors, in the central nervous system.
  • S Sato, R Sakai, M Kodama
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 10 16 1787 - 1789 2000年08月 [査読有り][通常論文]
     
    O-Sulfate group of gonyautoxin I and IV is transformed into methylene to form neosaxitoxin by thiols such as glutathione, a common cellular scavenger, in mild conditions. We isolated the intermediate of this conversion and propose that this reaction proceeds through formation of thiohemiketal, 1,2 shift to form stable thioether intermediate, and then redox exchange at sulfur atom to form the final product, (C) 2000 Elsevier Science Ltd. All rights reserved.
  • Jimbo M, Yanohara T, Koike K, Koike K, Sakai R, Muramoto K, Kamiya H
    Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 125 227 - 236 2 2000年02月 [査読有り][通常論文]
  • Yamaguchi M, Jimbo M, Sakai R, Muramoto K, Kamiya H
    Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 119 593 - 597 3 1998年03月 [査読有り][通常論文]
  • R Sakai, KL Rinehart, Kishore, V, B Kundu, G Faircloth, JB Gloer, Carney, JR, M Namikoshi, F Sun, RG Hughes, DG Gravalos, TG deQuesada, GR Wilson, RM Heid
    JOURNAL OF MEDICINAL CHEMISTRY 39 14 2819 - 2834 1996年07月 [査読有り][通常論文]
     
    Bioactivities of 42 didemnin congeners, either isolated from the marine tunicates Trididemnun solidum and Aplidium albicans or prepared synthetically and semisynthetically, have been compared. The growth inhibition of various murine and human tumor cells and plaque reduction of HSV-1 and VSV grown on cultured mammalian cells were used to assess cytotoxicity and antiviral activity. Biochemical assays for macromolecular synthesis (protein, DNA, and RNA) and enzyme inhibition (dihydrofolate reductase, thymidylate synthase, DNA polymerase, RNA polymerase, and topoisomerases I and II) were also performed to specify the mechanisms of action of each analogue. Immunosuppressive activity of the didemnins was determined using a mixed lymphocyte reaction (MLR) assay. These assays revealed that the native cyclic depsipeptide core is an essential structural requirement for most of the bioactivites of the didemnins, especially for cytotoxicities and antiviral activities. The linear side-chain portion of the peptide can be altered with a gain, in some cases, of bioactivities. In particular, dehydrodidemnin B, tested against several types of tumor cells and in in vivo studies in mice, as well as didemnin M, tested for the mixed lymphocyte reaction and graft vs host reaction in murine systems, showed remarkable gains in their in vitro and in vivo activities compared to didemnin B.
  • R SAKAI, KL RINEHART
    JOURNAL OF NATURAL PRODUCTS-LLOYDIA 58 5 773 - 777 1995年05月 [査読有り][通常論文]
     
    A new polyether acid, 14,15-dihydrodinophysistoxin-1 [1], was isolated along with dinophysistoxin-1 [2] and okadaic acid [3], from an unidentified species of the sponge genus Phakellia. The structure of 1 was assigned by comparison of its H-1- and C-13-nmr and fabms spectral data to those of 2 and 3, as well as interpretation of homonuclear 2D nmr data. Compounds 1-3 showed cytotoxicity against L-1210 leukemia cells with IC50 values of 3 ng/ml. The isolation, structure determination, and biological activities of these compounds are described (1).
  • Y GUAN, R SAKAI, KL RINEHART, AHJ WANG
    JOURNAL OF BIOMOLECULAR STRUCTURE & DYNAMICS 10 5 793 - 818 1993年04月 [査読有り][通常論文]
     
    Some members of marine alkaloid ecteinascidins (Et's), isolated from the Caribbean tunicate Ecteiriascidia turbinata, exhibit potent anticancer activity. The three dimensional structures of the N-12-formyl derivative of Et729 1a and the natural N-12-oxide of Et743 2 have been determined by x-ray crystallography at 0.9 angstrom resolution. Compounds 1a and 2 crystallize in the space groups P2(1)2(1)2(1) (a=23.214(9) angstrom, b=28.541(10) angstrom and c= 13.303(9) angstrom) and P2(1) (a=11.720(5) angstrom, b=13.230(4) angstrom, c=28.557(5) angstrom, beta=90.22(2)-degrees), respectively. Their crystal structures have been solved by the Patterson search method. which located the sulfur atoms permitting the phase extension. The final crystallographic R-factors are 0.059 and 0.069 for 1a and 2, respectively. There are two independent molecules, associated as a dimer, in the asymmetric unit of crystals of both 1a and 2. The structure determination allows an unequivocal assignment of the relative configuration of all the chiral centers. Assuming that ecteinascidins and safracin C (whose absolute configuration is known) have the same absolute configuration at C1 position, then the absolute configurations of various chiral positions in Ets are C1(R), N2(R), C3(R), C4(R), C11(R), C13(S), C21(S) and C22(R), respectively. The four independent Et molecules adopt two conformations in which the position of ring C relative to rings A & B is different.The molecules have a compact shape and they are conformationally strained due to a severe van der Waals clash between the sulfur atom and the aromatic ring A. By analogy to the related saframycin, the potent biological activity of Et's may be associated with their ability to form a covalent adduct to DNA using the reactive carbinolamine group. The covalent binding interaction between the Et and the N2 of guanine in the minor groove of the DNA double helix has been studied by computer modelling which suggests that rings A and B ''stack'' against the DNA backbone. While the bulky drug molecule makes numerous contacts with DNA, it does not significantly distort the conformation of the DNA double helix.
  • KL RINEHART, TG HOLT, NL FREGEAU, PA KEIFER, GR WILSON, TJ PERUN, R SAKAI, AG THOMPSON, JG STROH, LS SHIELD, DS SEIGLER, LH LI, DG MARTIN, CJP GRIMMELIKHUIJZEN, G GADE
    JOURNAL OF NATURAL PRODUCTS 53 4 771 - 792 1990年07月 [査読有り][通常論文]
  • KL RINEHART, R SAKAI, TG HOLT, NL FREGEAU, TJ PERUN, DS SEIGLER, GR WILSON, LS SHIELD
    PURE AND APPLIED CHEMISTRY 62 7 1277 - 1280 1990年07月 [査読有り][通常論文]
  • Rinehart KL, Kishore V, Bible KC, Sakai R, Sullins DW, Li KM
    Journal of natural products 51 624  3 1988年05月 [査読有り][通常論文]
  • KL RINEHART, KISHORE, V, KC BIBLE, R SAKAI, DW SULLINS, KM LI
    JOURNAL OF NATURAL PRODUCTS 51 1 1 - 21 1988年01月 [査読有り][通常論文]
  • R SAKAI, T HIGA
    CHEMISTRY LETTERS 1 127 - 128 1987年 [査読有り][通常論文]
  • Sakai Ryuichi, Higa Tatsuo, Kashman Yoel
    Chemistry Letters 9 1499 - 1502 The Chemical Society of Japan 1986年 
    A highly active antitumor macrolide, misakinolide-A has been isolated from the sponge Theonella sp. The gross structure was determined by extensive NMR studies including homo- and heteronuclear COSY experiments.
  • Ryuichi Sakai, Tatsuo Higa, Charles W. Jefford, GéRald Bernardinelli
    Helvetica Chimica Acta 69 1 91 - 105 1986年 [査読有り][通常論文]
     
    Four new halogenated chamigrenes have been isolated from the sea hare, the (E)‐ and (Z)‐9‐(bromomethylidene)‐1,2,5‐trimethylspiro[5.5]undeca‐1,7‐dien‐3‐ones (11 and 10, resp.) the structures of which were deduced by NMR spectroscopy and by comparison with a known relative, the (8R,9R)‐8‐bromo‐9‐chloro‐5,5,9‐trimethyl‐1‐methylidenespiro[5,5]undec‐3‐en‐2‐one (12), and its (11 R)‐11‐acetoxy‐substituted derivative 13. Their structures and absolute configurations were determined and deduced by X‐ray using the absolute‐structure parameter. The configurations were remarkable in being enantiomeric to those of the tertiary chlorochamigrenes isolated so far. Consequently, a more general scheme is proposed to account for their biogenesis. Copyright © 1986 Verlag GmbH & Co. KGaA, Weinheim

書籍

作品等

  • 海洋生物の産生する新規抗癌物質の探索
    2001年 -2003年
  • Dysiherbainのカイニン酸受容体に対する作用
    2000年 -2003年
  • 新規興奮性アミノ酸の生理作用
    1995年 -2002年
  • 海洋微生物の産生する生理活性物質

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  • 田中良和, 影山大夢, 辺ひろみ, 増渕菜弥, 中島寛也, 小野寺かこ, 大城拓未, 松井崇, 小寺義男, 小川智久, 横山武司, 小松則夫, 荒木真理人, 酒井隆一 日本生化学会大会(Web) 94th 2021年
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  • 酒井隆一 天然薬物の開発と応用シンポジウム講演要旨集 22nd 1‐4 2018年10月01日 [査読無し][通常論文]
  • 酒井 隆一, 松永智子 ポリアミン 5 (1) 3 -13 2018年04月 [査読無し][招待有り]
  • 飯田祐介, 中野宏治, 上田拓也, 北野雅也, 藤田雅紀, 酒井隆一 日本水産学会大会講演要旨集 2018 107 2018年03月26日 [査読無し][通常論文]
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  • 松村賢, 谷口透, REIMER James D., 野口峻太朗, 藤田雅紀, 酒井隆一 天然有機化合物討論会講演要旨集(Web) 60th 2018年
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  • 酒井 隆一 奨励研究報告書 2017 1 -11 2017年 [査読無し][通常論文]
  • Crystal structure of symbiosis related lectin SLL-2 in complex with tetrasaccharides
    A. Kita, M. Jimbo, R. Sakai, Y. Morimoto, R. Takeuchi, H. Tanaka, T. Takahashi, K. Miki 14th Conf. Asian Crystallographic Association 2016年12月 [査読有り][通常論文]
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  • 梅津 早希, 今井 一郎, 酒井 隆一, 藤田 雅紀 天然有機化合物討論会講演要旨集 58 (0) Poster32 2016年 [査読無し][通常論文]
     

    植物プランクトンの異常増殖によって引き起こされる赤潮は、養殖魚の大量斃死や二枚貝の毒化等により水産業に甚大な被害をもたらす。一方、海洋環境中には赤潮原因藻類を殺滅する活性を有する殺藻細菌の存在が知られており、また赤潮の消長と殺藻細菌の分布には相関が見られることから、殺藻細菌が実際に環境中で赤潮発生の抑止力となっていると推測されている。それら殺藻細菌の利用は赤潮の生物学的防除策として注目されているが、そのメカニズムについては未解明な点が多く、効率的な防除策の開発にはより多くの知見が必要である。

    そこで、赤潮発生域である広島湾海水から分離され、珪藻・渦鞭毛藻・ラフィド藻等の多様な赤潮原因藻類に対し殺滅活性を示すAlteromonas属細菌DおよびK株1（図１）を対象とし、赤潮殺滅機構、海洋環境中での赤潮抑制機能、および効果的な化学・生物学的防除法の開発を目標に、殺藻物質の同定と機能解析を試みた。

    1. 殺藻物質の精製と構造決定

    海洋細菌Alteromonas属D株を海水ベースの培地で培養後、培養上清に殺藻活性を確認した。活性物質を固相抽出し、各種クロマトグラフィーにより活性を指標に分画を進め、最終的に逆相HPLCにより440 nmに特徴的な吸収極大を示す化合物1を主要活性物質として得た。また、同様のUVスペクトルを示す3つのマイナー成分（化合物2-4）も合わせて精製した。

    高分解能質量分析 (HRMS) およびNMR解析から、化合物1の分子式をC12H8N2O2と決定し、さらにNMRスペクトルを文献値と比較し、化合物1を既知のフェノキサゾン誘導体である2-aminophenoxazin-3-one（APO）と同定した（図２）2。これはコムギやライムギ等の陸上高等植物が生産するアレロパシー物質としての報告があり3、またHalomonas属などの細菌から抗微生物剤としても報告されている4。

    図２．Alteromonas属由来殺藻物質APO (1) および新規誘導体2-4。

    化合物2の分子式はC12H7N2O2Brと決定され、化合物１の臭素置換体と考えられた。APO (1)とプロトンNMRスペクトルを比較したところ、化合物１の6.3-6.4ppmの二つのシングレットピークが化合物2では一つになっていることから1位もしくは4位がBrに置換されていると推測された。また、HMBCスペクトルにおいて6.4ppmの1Hから2位 (149.5 ppm) および12位 (145.6 ppm) に相関が見られたことから、1位が臭素化された構造と決定した（図２）。

    HRMSおよび一次元NMR測定の結果から化合物3および4の分子式はそれぞれC13H10N2O3SおよびC13H10N2O2Sと決定された。NMRスペクトル解析により、化合物2同様に1位が置換されており、さらに両化合物で新たなメチルシグナルが観測された。また、メチル基水素から1位の炭素へのHMBC相関が確認されたことから、これらの構造を化合物1のメチルスルホキシド体およびメチルスルフィド体と決定した(図２)。化合物2-4はいずれも新規化合物であった。

    ２. 生物活性

    化合物1および2について、赤潮原因藻類の一種であるラフィド藻Chattonella antiquaに対する生育阻害活性を評価した。C. antiquaを試験管中で培養し、培養開始1日目に化合物1を各濃度で

    (View PDFfor the rest of the abstract.)

  • 酒井隆一, 酒井隆一 トキシンシンポジウム予稿集 62nd 89 2015年06月15日 [査読無し][通常論文]
  • 酒井隆一, 市森大地, 小田悠太, 藤田雅紀, 今田千秋 マリンバイオテクノロジー学会大会講演要旨集 17th 51 2015年05月30日 [査読無し][通常論文]
  • 中島寛弥, 渥海航, 中村隆典, 藤田雅紀, 酒井隆一 日本水産学会大会講演要旨集 2015 178 2015年03月27日 [査読無し][通常論文]
  • 酒井隆一, 内枡肇, 津田正史, 熊谷慶子, 赤壁麻衣, 藤田雅紀 日本水産学会大会講演要旨集 2015 141 2015年03月27日 [査読無し][通常論文]
  • 藤田雅紀, 酒井隆一, 伊勢優史 日本水産学会大会講演要旨集 2015 139 2015年03月27日 [査読無し][通常論文]
  • 大八木悠, 佐々木真吾, 鈴木寛人, 大内仁志, 稲井誠, 浅川倫宏, 酒井隆一, 島本啓子, 濱島義隆, 菅敏幸 日本薬学会年会要旨集(CD-ROM) 135th ROMBUNNO.26G-AM07S 2015年 [査読無し][通常論文]
  • 内升肇, 松村賢, 津田正史, 熊谷慶子, 赤壁麻依, 藤田雅紀, 酒井隆一 天然有機化合物討論会講演要旨集(Web) 57th 369‐374(J‐STAGE) 2015年 [査読無し][通常論文]
  • 内升 肇, 松村 賢, 津田 正史, 熊谷 慶子, 赤壁 麻依, 藤田 雅紀, 酒井 隆一 天然有機化合物討論会講演要旨集 57 (0) PosterP31 2015年 [査読無し][通常論文]
     

    神経には多様なイオンチャンネル型受容体、代謝調節型受容体、そしてイオンチャンネルが存在し、神経伝達や恒常性の維持を担っている。脳をはじめとした中枢神経はこれらが集中する器官であるといえる。我々は、海洋生物の抽出物をマウスの脳室内に投与し行動変化を観察することで、受容体・イオンチャネルに作用する化合物を探索してきた。海綿・ホヤを中心とした501種の水抽出物のスクリーニングを行った結果、パラオ産Didemnidae科ホヤにマウスの自発行動を抑制する活性を見出した。そこでこの活性を指標に精製を進めたところ、新規ジヒドロイソキノリンアルカロイドであるオピオイド受容体アゴニストdopargimine（1）およびセロトニン受容体アンタゴニストmellpaladine（Mell）A-F（2-7）、既知化合物4-guanidino butyric acid（8）を得た（Fig. 1）。さらに、複数の硫黄原子が付加したドーパミン様構造を持つ既知化合物群であるlissoclibadin類（9）も多数確認した。本発表では、これらの単離・構造決定および生理活性、推定生合成経路について報告する。

    【Mellpaladine類及びDopargimineの単離】パラオ共和国で採取したDidemnidae科ホヤ（Pal 300）の冷凍保存試料を水で抽出し、抽出物を透析、低分子・高分子画分を得た。低分子画分にマウスに対する活性が認められたため、ゲルろ過カラム（Sephadex LH-20，H2O-0.05 %TFA，MeOH-0.05%TFA）、逆相カラム（Wako C18，H2O-0.05 %TFA，MeOH-0.05 %TFA）に供したのち、逆相HPLCで精製したところ、活性成分としてMell A-F（2-7）を得た。またMell類以外の新規活性成分としてdopargimine（1）を得た。また分離の過程で4-guanidino butyric acid（8）を得た。

    【Dopargimine（1）の構造決定】Dopargimine（1）の分子式はHR-ESI-MSおよび13C-NMRスペクトルによりC13H18N4O2と推定した。1H-NMRスペクトルの解析により2つのシングレット芳香族プロトン、１つのヘテロ原子に結合したプロトンなどを含む13個のプロトンが観測された。13C-NMRスペクトルでは8個のsp2炭素と5個のsp3炭素が観測された。HMBCスペクトルにおいて芳香族プロトンd 6.80（H-5）からd 116.1（C-8a）、d 145.7（C-7）、d 155.3（C-6）に、またd 7.44（H-8）からd132.8（C-4a）、d 145.7（C-7）、d155.3（C-6）に相関が観測されたこと、酸素原子が置換したと考えられる炭素がd 145.7（C-7）とd155.3（C-6）に観測されたことから2置換カテコール構造を持つと推定した。これはTLC上、１がFeCl3で呈色されたことからも支持された。さらに、COSY、HMBCスペクトルの解析の結果、3，4-ジヒドロイソキノリン骨格が構築されていると推定した。側鎖の構造はCOSYおよびHMBCスペクトルより3つの連続したメチレンCH2-9~11およびNH-12のイミンC-1のスピン系が推定された。また、1は坂口反応で赤色に呈色すること、およびアセチルアセトンと反応することでピリミジン誘導体1aを与えたことからC-13（d 157.6）はグアニジン基であると同定した。さらに1をNaBH4で還元したところジヒドロ体1b（C13H20N4O2）が生成し、C-1（d176.5）がd 53.1にシフトしたことよりイミンの存在を確認した。ジヒドロ体1bのNMRデータ

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  • 假屋唯香, 淺沼雄太, 稲井誠, 浅川倫宏, 濱島義隆, 江木正浩, 福田裕穂, 酒井隆一, 菅敏幸 中部化学関係学協会支部連合秋季大会講演予稿集 45th 215 2014年11月29日 [査読無し][通常論文]
  • 佐々木真吾, 鈴木寛人, 大内仁志, 浅川倫宏, 稲井誠, 酒井隆一, 島本啓子, 濱島義隆, 菅敏幸 反応と合成の進歩シンポジウム講演要旨集 40th 109 2014年10月22日 [査読無し][通常論文]
  • Takamasa Uekita, Satoko Fujii, Yuri Miyazawa, Reika Iwakawa, Mako Narisawa-Saito, Katsuhiko Nakashima, Koji Tsuta, Hitoshi Tsuda, Tohru Kiyono, Jun Yokota, Ryuichi Sakai MOLECULAR CANCER RESEARCH 12 (10) 1449 -1459 2014年10月 [査読無し][通常論文]
     
    Involvement of Ras in cancer initiation is known, but recent evidence indicates a role in cancer progression, including metastasis and invasion; however, the mechanism is still unknown. In this study, it was determined that human lung cancer cells with Ras mutations, among other popular mutations, showed significantly higher expression of CUB domain-containing protein 1 (CDCP1) than those without. Furthermore, activated Ras clearly induced CDCP1, whereas CDCP1 knockdown or inhibition of CDCP1 phosphorylation by Src-directed therapy abrogated anoikis resistance, migration, and invasion induced by activated-Ras. Activation of MMP2 and secretion of MMP9, in a model of Ras-induced invasion, was found to be regulated through induction of phosphorylated CDCP1. Thus, CDCP1 is required for the functional link between Ras and Src signaling during the multistage development of human malignant tumors, highlighting CDCP1 as a potent target for treatment in the broad spectrum of human cancers associated with these oncogenes. (C)2014 AACR.
  • 内舛肇, ENG Andrew, EKINS Merrick, HOOPER John, SWANSON Geoofrey T, 酒井隆一 日本水産学会大会講演要旨集 2014 121 2014年03月27日 [査読無し][通常論文]
  • 藤田雅紀, 時田学幸, 石川高史, 酒井隆一 日本水産学会大会講演要旨集 2014 121 2014年03月27日 [査読無し][通常論文]
  • 岸伶美, 酒井隆一 日本水産学会大会講演要旨集 2014 173 2014年03月27日 [査読無し][通常論文]
  • 大塚一憲, 石川裕一, 高見澤聡, 酒井隆一, 及川雅人 日本化学会講演予稿集 94th (4) 1468 2014年03月12日 [査読無し][通常論文]
  • Hideki Yamaguchi, Miho Takanashi, Nachi Yoshida, Yuumi Ito, Reiko Kamata, Kiyoko Fukami, Kazuyoshi Yanagihara, Ryuichi Sakai Cancer Science 105 (5) 528 -536 2014年 [査読無し][通常論文]
     
    Diffuse-type gastric carcinomas (DGC) exhibit more aggressive progression and poorer prognosis than intestinal-type and other gastric carcinomas. To identify potential therapeutic targets, we examined protein tyrosine phosphorylation in a panel of DGC and other gastric cancer cell lines. Protein tyrosine phosphorylation was significantly enhanced or altered in DGC cell lines compared with that in other gastric cancer cell lines. Affinity purification and mass spectrometry analysis of tyrosine-phosphorylated proteins identified Met as a protein that is preferentially expressed and phosphorylated in DGC cell lines. Unexpectedly, Met inhibitors blocked cell growth, Met downstream signaling and peritoneal dissemination in vivo in only a subset of cell lines that exhibited remarkable overexpression of Met. Likewise, only cell lines with overexpression of fibroblast growth factor receptor 2 (FGFR2) or phosphorylation of FRS2 were sensitive to an FGFR2 inhibitor. A Src inhibitor saracatinib impaired growth in cell lines that are insensitive to both Met and FGFR2 inhibitors. Saracatinib also effectively impaired peritoneal dissemination of Met-independent and FGFR2-independent SGC cells. Moreover, DGC cell lines exhibited nearly mutually exclusive susceptibility to Met, FGFR and Src inhibitors. These results suggest that DGC have distinct sensitivities to molecular target drugs and that targeting Src is beneficial in the treatment of DGC insensitive to Met and FGFR inhibition. © 2014 The Authors.
  • Hideki Yamaguchi, Nachi Yoshida, Miho Takanashi, Yuumi Ito, Kiyoko Fukami, Kazuyoshi Yanagihara, Masakazu Yashiro, Ryuichi Sakai PLOS ONE 9 (1) 2014年01月 [査読無し][通常論文]
     
    Scirrhous gastric carcinoma (SGC) has the worst prognosis of all gastric cancers, owing to its rapid expansion by invasion and frequent peritoneal dissemination. Due to the increased proliferation of stromal fibroblasts (SFs) that occurs within SGC lesions and the peritoneal metastatic sites, SFs have been proposed to support the progression of this disease. However, the biological and molecular basis and the pathological role of the intercellular interaction between SGC cells and SFs remain largely unknown. In this study, we investigated the role of SFs in the invasion of the extracellular matrix (ECM) by SGC cells. When SGC cells were cocultured with SFs derived from SGC tissue on three-dimensional (3D) Matrigel, they were attracted together to form large cellular aggregates that invaded within the Matrigel. Time-lapse imaging revealed that this process was associated with extensive contraction and remodeling of the ECM. Immunofluorescence and biochemical analysis showed that SGC cells stimulate phosphorylation of myosin light chain and actomyosin-mediated mechanical remodeling of the ECM by SFs. By utilizing this assay system for inhibitor library screening, we have identified several inhibitors that potently suppress the cooperation between SGC cells and SFs to form the invasive structures. Among them, a Src inhibitor dasatinib impaired the interaction between SGC cells and SFs both in vitro and in vivo and effectively blocked peritoneal dissemination of SGC cells. These results indicate that SFs mediate mechanical remodeling of the ECM by SGC cells, thereby promoting invasion and peritoneal dissemination of SGC.
  • 中島寛弥, 渥海航, 藤田雅紀, 酒井隆一 日本水産学会北海道支部大会講演要旨集 2014 41 2014年 [査読無し][通常論文]
  • 喜田昭子, 神保充, 酒井隆一, 森本幸生, 武内良太, 田中浩士, 高橋孝志, 高橋孝志, 三木邦夫 KURRI KR (193) 67 -70 2014年01月 [査読無し][通常論文]
  • 藤田 雅紀, 時田 学幸, 石川 高史, 酒井 隆一 天然有機化合物討論会講演要旨集 56 (0) Poster55 2014年 [査読無し][通常論文]
     

    シデロフォアは微生物が産生する鉄の獲得に関与する化合物群であり、生物利用可能な鉄が不足する環境中において、多くの微生物が多様なシデロフォアを産生する。分離培養が困難な環境微生物の中には、他種微生物が産生した外因性のシデロフォアを自身の成育に必須とするものが報告されている。1また、シデロフォアは微生物の宿主への感染・共生等の成立にも関与し、2さらには競合菌の遊泳阻害活性を持つものなども報告されている。3この様に、シデロフォアは微生物のケミカルコミュニケーションにおいて重要な働きを持つと考えられる。

    一方、海洋無脊椎動物由来の多様な生理活性物質の多くは共生微生物が産生していると考えられているが、その多くは分離培養が困難である。それら難培養共生菌が利用するシデロフォアを明らかにすることができれば、共生菌の生態の理解、さらには培養可能化につながると期待される。そこで、微生物を分離培養することなく、直接全てのゲノムDNAを取得し解析するメタゲノム法を用いて、海洋無脊椎動物中におけるシデロフォア生合成遺伝子の解析と、その生産物の同定を試みた。

    Figure 1. 主なカテコール型シデロフォアの構造。

    １．海洋メタゲノムライブラリの構築とシデロフォアスクリーニング

    日本沿岸各地で採集した海洋無脊椎動物を断片化後、バッファーあるいは海水中で組織を圧搾し滲出液を得た。遠心分離後、得られたペレットを溶菌バッファーで処理し、アルコール沈殿することで粗メタゲノムDNAを得た。その後、得られたDNAをフェノール/クロロホルム処理、CTAB処理等により精製し、アガロースゲル電気泳動により高分子量DNAを得た。その後、フォスミドベクターを用いクローニングする事で、平均インサートDNA長が約35 kbpで約50万クローンからなる海洋メタゲノムライブラリを構築した(Figure 2)。

    作成したメタゲノムライブラリに対して、Chrome Azurol Sを用いたシデロフォア活性スクリーニングを行ったところ、4合計52個のシデロフォア生産クローンを見出した(Figure 2)。得られた活性クローンについて、そのシデロフォア生合成遺伝子クラスターの一部を解析したところ、カテコール型、ヒドロキサム酸型、カルボキシレート型など様々なタイプのシデロフォア生合成遺伝子に相同性を示したことから、本手法によって多様なシデロフォア生合成遺伝子の取得と生産が可能であることが示された。しかしながら、既知の生合成遺伝子クラスターと全く同じものは存在せず、実際の生産物の同定は断片的な配列情報からだけからでは困難であった。

    Figure 2. 海洋無脊椎動物からのメタゲノムライブラリの構築とシデロフォアスクリーニングの概要。

    ２．海綿メタゲノム由来シデロフォア生合成遺伝子クラスターの解析

    生産物の同定を目的に、生合成遺伝子全体の解析と化合物の生産研究を行った。熊本県天草産の未同定海綿メタゲノム由来の活性クローンについて全長配列35,554 bpを決定したところ、得られたDNA配列は海洋細菌であるVibrio furnissiiのものと90%程度で一

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  • Takuya Shirakihara, Tomonori Kawasaki, Akihiko Fukagawa, Kentaro Semba, Ryuichi Sakai, Kohei Miyazono, Keiji Miyazawa, Masao Saitoh Cancer Science 104 (9) 1189 -1197 2013年09月 [査読無し][通常論文]
     
    Epithelial-mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair, and cancer progression in adult tissues. Transforming growth factor (TGF)-β induces EMT in mouse epithelial cells. During prolonged treatment, TGF-β successively induces myofibroblastic differentiation with increased expression of myofibroblast marker proteins, including smooth muscle α actin and calponin. We recently showed that fibroblast growth factor-2 prevented myofibroblastic differentiation induced by TGF-β, and transdifferentiated the cells to those with much more aggressive characteristics (enhanced EMT). To identify the molecular markers specifically expressed in cells undergoing enhanced EMT induced by the combination of TGF-β and fibroblast growth factor-2, we carried out a microarray-based analysis and found that integrin α3 (ITGA3) and Ret were upregulated. Intriguingly, ITGA3 was also overexpressed in breast cancer cells with aggressive phenotypes and its expression was correlated with that of δEF-1, a key regulator of EMT. Moreover, the expression of both genes was downregulated by U0126, a MEK 1/2 inhibitor. Therefore, ITGA3 is a potential marker protein for cells undergoing enhanced EMT and for cancer cells with aggressive phenotypes, which is positively regulated by δEF-1 and the MEK-ERK pathway. © 2013 Japanese Cancer Association.
  • 神保充, 國谷奈美, 竹内亮太, 谷本典加, 田中千瑛, 山下洋, 小池一彦, 酒井隆一 日本動物学会大会予稿集 84th 100 2013年08月12日 [査読無し][通常論文]
  • Masato Kasuga, Kohjiro Ueki, Naoko Tajima, Mitsuhiko Noda, Ken Ohashi, Hiroshi Noto, Atsushi Goto, Wataru Ogawa, Ryuichi Sakai, Shoichiro Tsugane, Nobuyuki Hamajima, Hitoshi Nakagama, Kazuo Tajima, Kohei Miyazono, Kohzoh Imai Cancer Science 104 (7) 965 -976 2013年07月 [査読無し][通常論文]
     
    In recent years, diabetes has been shown to be associated with cancer risk, and this has led to a joint committee being formed, enlisting experts from the Japan Diabetes Society and the Japanese Cancer Association to address this issue. Epidemiological data in Japan provides evidence to demonstrate that diabetes is associated with increased risk for cancers, especially colorectal, liver, and pancreatic cancers. The mechanisms through which diabetes is assumed to promote oncogenesis include insulin resistance and associated hyperinsulinemia, hyperglycemia, and inflammation. Common risk factors for type 2 diabetes and cancer include aging, male sex, obesity, physical inactivity, inappropriate diet (excessive red/processed meat intake, inadequate vegetable/fruit/dietary fiber intake), excessive alcohol drinking, and smoking. Given that inappropriate diet/exercise, smoking and excessive alcohol drinking are common risk factors for diabetes and cancer, diet/exercise therapy, smoking cessation and alcohol moderation may be associated with decreased risk for cancer in diabetic patients. There is as yet limited evidence as to whether any particular antidiabetic agents may influence cancer risk. © 2013 The Japanese Cancer Association and the Japan Diabetes Society.
  • Takamasa Uekita, Satoko Fujii, Yuri Miyazawa, Akinori Hashiguchi, Hitosi Abe, Michiie Sakamoto, Ryuichi Sakai Cancer Science 104 (7) 865 -870 2013年07月 [査読無し][通常論文]
     
    CUB (C1r/C1s, urchin embryonic growth factor, BMP1) domain-containing protein 1 (CDCP1) has been implicated in promoting metastasis of cancer cells through several mechanisms, including the inhibition of anoikis, which is cell death triggered by the loss of extracellular matrix interactions. However, the mechanism inhibiting cell death regulated by CDCP1 remains elusive. Inhibition of CDCP1 expression using small interfering RNA (siRNA) induced the cell death of suspended cancer cells without cleaving caspase-3, a marker of apoptosis cell death was not inhibited by a general caspase inhibitor, suggesting that the loss of CDCP1 induces caspase-independent cell death. In contrast, knockdown of CDCP1 as well as protein kinase Cδ (PKCδ), a downstream effector of CDCP1, in a suspension culture of lung cancer cells resulted in marked induction of membranous microtubule-associated protein 1 light chain 3 (LC3)-II protein, a hallmark of autophagy, and caused the formation of an autophagosome structure visualized using green fluorescent protein-tagged LC3-II. Expression and phosphorylation of exogenous CDCP1 by Fyn kinase reduced the formation of autophagosomes and inhibited phosphorylation of CDCP1 by PP2, a Src kinase inhibitor or inhibited PKCδ by rottlerin, stimulating autophagosome formation. Moreover, death of suspended lung cancer cells induced by CDCP1 siRNA or by PKCδ siRNA was reduced by the autophagy inhibitor 3-methyladenine. These results indicate that CDCP1-PKCδ signaling plays a critical role in inhibiting autophagy, which is responsible for anoikis resistance of lung cancer cells. © 2013 Japanese Cancer Association.
  • M. Kasuga, K. Ueki, N. Tajima, M. Noda, K. Ohashi, H. Noto, A. Goto, W. Ogawa, R. Sakai, S. Tsugane, N. Hamajima, H. Nakagama, K. Tajima, K. Miyazono, K. Imai Journal of the Japan Diabetes Society 56 (6) 374 -390 2013年06月01日 [査読無し][通常論文]
  • Yuri Miyazawa, Takamasa Uekita, Yuumi Ito, Motoharu Seiki, Hideki Yamaguchi, Ryuichi Sakai MOLECULAR CANCER RESEARCH 11 (6) 628 -637 2013年06月 [査読無し][通常論文]
     
    Complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing protein 1 (CDCP1) is a transmembrane protein that regulates anchorage-independent growth and cancer cell migration and invasion. Expression of CDCP1 is detected in a number of cancer cell lines and tissues and is closely correlated with poor prognosis. Invadopodia are actin-based protrusions on the surface of invasive cancer cells that promote the degradation of the extracellular matrix (ECM) via localized proteolysis, which is mainly mediated by membrane type 1 matrix metalloproteinase (MT1-MMP). MT1-MMP is accumulated at invadopodia by targeted delivery via membrane trafficking. The present study shows that CDCP1 is required for ECM degradation by invadopodia in human breast cancer and melanoma cells. CDCP1 localized to caveolin-1-containing vesicular structures and lipid rafts and was detected in close proximity to invadopodia. Further biochemical analysis revealed that substantial amounts of CDCP1 existed in the Triton X-100 insoluble lipid raft fraction. CDCP1 was coimmunoprecipitated with MT1-MMP and colocalized with MT1-MMP at the vesicular structures. The siRNA-mediated knockdown of the CDCP1 expression markedly inhibited MT1-MMP-dependent ECM degradation and Matrigel invasion and reduced the accumulation of MT1-MMP at invadopodia, as shown by immunofluorescence analysis. These results indicate that CDCP1 is an essential regulator of the trafficking and function of MT1-MMP- and invadopodia-mediated invasion of cancer cells. (C)2013 AACR.
  • 上田拓弥, FREYMANN DM, FOCIAL PJ, 中村友香, SMITH Caleb, 井上昌, 尾島孝男, 松永智子, SWANSON GT, 酒井隆一 日本水産学会大会講演要旨集 2013 102 2013年03月26日 [査読無し][通常論文]
  • 藤田雅紀, 中野宏治, 酒井隆一 日本水産学会大会講演要旨集 2013 100 2013年03月26日 [査読無し][通常論文]
  • 松永智子, 酒井隆一 日本水産学会大会講演要旨集 2013 96 2013年03月26日 [査読無し][通常論文]
  • 境倫宏, 田中健斗, 石川裕一, 酒井隆一, 及川雅人 日本化学会講演予稿集 93rd (4) 1220 2013年03月08日 [査読無し][通常論文]
  • Mitsuru Jimbo, Yuya Suda, Kazuhiko Koike, Sachiko Nakamura-Tsuruta, Junko Kominami, Masugu Kamei, Jun Hirabayashi, Ryuichi Sakai, Hisao Kamiya JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY 439 129 -135 2013年01月 [査読無し][通常論文]
     
    We previously demonstrated that the lectin SLL-2, isolated from the octocoral Sinularia lochmodes, binds to the symbiotic microalgae Symbiodinium within the coral. Upon binding SLL-2, Symbiodinium cells transform from a flagellated swimming form into a non-flagellated coccoid form, the latter morphologically similar to the symbiotic stage found in corals. However, the site recognized by the lectin on the surface of Symbiodinium cells and the transformation mechanism have yet to be elucidated. We found that the ability of SLL-2 to induce the morphological change in Symbiodinium cells can be attenuated by pretreating the cells with glycosidases or by adding the SLL-2 binding inhibitor N-acetyl-D-galactosamine to the culture medium. These results suggest that p-galactose-containing glycoconjugates on the Symbiodinium cell surface are the key ligand through which SLL-2 induces morphological transformation. We also found that SLL-2 binds with high affinity to the Forssman antigen, and masking the binding site on Symbiodinium cells by addition of anti-Forssman glycosphingolipid antibody inhibits the binding of SLL-2 to the cells. The antibody itself or other Forssman antigen-binding proteins, such as Helix pomatia agglutinin, can transform Symbiodinium cells into the coccoid stage in the absence of SLL-2. A neutral lipid fraction prepared from cultured Symbiodinium cells reacted with the anti-Forssman glycosphingolipid antibody, supporting the hypothesis that a Forssman antigen-like glycosphingolipid on the surface of Symbiodinium cells is involved in their morphological transformation induced by the lectin SLL-2. (C) 2012 Elsevier B.V. All rights reserved.
  • 藤田雅紀, 中野宏治, 酒井隆一 天然有機化合物討論会講演要旨集(Web) 55th 621‐626(J‐STAGE) 2013年 [査読無し][通常論文]
  • 藤田 雅紀, 中野 宏治, 酒井 隆一 天然有機化合物討論会講演要旨集 55 (0) PosterP -56 2013年 [査読無し][通常論文]
     

    シデロフォアは微生物が産生する鉄キレート能を有する化合物群である。生物利用可能な鉄が不足している自然環境において、微生物はシデロフォアを用いて必須元素である鉄を獲得している。1シデロフォアには医薬品として利用されるものもあり、また土壌や水質の改良剤としても期待されている。一方、微生物の中には他種の微生物が産生した外因性シデロフォアを自身の生育に必要とする種が数多く存在し、シデロフォアを介した微生物間の多様なコミュニケーションが存在する。2

    本研究グループでは環境中から微生物遺伝資源を網羅的に取得するメタゲノム法を用いて、シデロフォア生合成遺伝子の探索と異宿主生産を試み、その結果N-hydroxy-N-succinyl cadaverine (HSC, 1) を構成単位とするシデロフォアの一つ、bisucaberin (2) の生合成遺伝子の取得と大腸菌での生産を報告している。3これまでにHSC型シデロフォアは複数知られており(Figure 1)、免疫抑制活性、マクロファージによるガン細胞溶解活性の増強等の様々な生物活性が報告されており、またそのいくつかは生合成遺伝子も取得されている。4しかしながら、生合成における縮合回数および大環状化の制御機構などは全く不明であり、その解明は酵素によるポリマーを含む望む多量体の構築、大環状化合物の生産を行うための基盤情報となる。

    その様な中、パラオ産海綿由来細菌がこれまで微生物産物としては報告されていないHSC型シデロフォアを生産している事が示唆された。そこで、HSC型シデロフォアの構造多様化機構に関する知見を得ることを目的に活性物質の同定、およびその生合成遺伝子のクローニングを行ったので報告する。

    Figure 1. N-hydroxy-N-succinyl cadaverine (1) と1を構成単位とするシデロフォア。

    １：Bisucaberin Bの単離と構造決定

    パラオ産海綿から分離した細菌に対して金属呈色試薬であるChrome Azurol Sを用い、シデロフォア生産活性を指標にスクリーニングを行ったところ、顕著な活性を示す株を見出した。得られた細菌の16S rRNA配列1444 bpを解析したところ、バクテロイデス門に属するTenacibaculum mesophilum NBRC16307 (accession number, AB681058) と完全に一致したため、同属同種であると判断した。

    T. mesophilumを海水をベースとした培地で振とう培養後、シデロフォア活性を指標に培養上清の分画を進めたところ、単一の活性成分としてbisucaberin B (2, 38.9 mg/L) を得た。化合物2の1Hおよび13C NMRスペクトルを解析したところ、多くのメチレンシグナルを確認し、またメチレン炭素以外はカルボニル炭素のみ存在する事から、本化合物はHSC (1) を構成単位としたシデロフォアである事が示唆された(Table 1)。Bisucaberin (3) やdesferrioxamine E (4) のような大環状HSC型シデロフォアは、対称構造から単量体様のスペクトルを示すのに対して、化合物2は二組のHCSシグナルが分離して観測された事から、非対象構造を有していると考えられた。また、高分解能ESIMSにより決定したその分子式は環状二量体である化合物3よりH2O分大きい事から、本物質の構造を化合物3 のアミド結合が加水分解した非環状二量体であると推

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  • 海野 昌喜, 佐々木 誠, 酒井 隆一, 齋藤 正男 日本結晶学会誌 54 (6) 338 -344 2012年12月31日 [査読無し][通常論文]
     
    Kainate receptors (KARs) are members of the ionotropic glutamate receptors (iGluR) that play variety of roles in the mammalian brain. Because KARs are comprised of five different isoform proteins, isoform selective compounds are indispensable tools in physiological researches. Dysiherbaine (DH) and neodysiherbaine A (NDH), natural toxins found from marine sponges, were shown to be potent and isoform-selective agonists for KARs. To understand structure-activity relationship of DH and NDH, numbers of analogues are synthesized, and their pharmacological activities have been evaluated in detail. Here, we determined the crystal structures of human KAR isoforms, GluK1 and GluK2, ligand-binding domain in complex with the DH analogues at high resolution. From these structures, we elucidated the relationships between the binding affinity and the molecular structure of the compounds. We also demonstrated differential recognition by some of the DH analogues of closely related isoforms GluK1 and GluK2. Further, we found that neither degree of the domain closure nor twist motion of domains directly correlated to agonist efficacy of the ligands.
  • 海野 昌喜, 佐々木 誠, 酒井 隆一, 齋藤 正男 日本結晶学会誌 54 (6) 338 -344 2012年12月31日 [査読無し][通常論文]
     
    Kainate receptors (KARs) are members of the ionotropic glutamate receptors (iGluR) that play variety of roles in the mammalian brain. Because KARs are comprised of five different isoform proteins, isoform selective compounds are indispensable tools in physiological researches. Dysiherbaine (DH) and neodysiherbaine A (NDH), natural toxins found from marine sponges, were shown to be potent and isoform-selective agonists for KARs. To understand structure-activity relationship of DH and NDH, numbers of analogues are synthesized, and their pharmacological activities have been evaluated in detail. Here, we determined the crystal structures of human KAR isoforms, GluK1 and GluK2, ligand-binding domain in complex with the DH analogues at high resolution. From these structures, we elucidated the relationships between the binding affinity and the molecular structure of the compounds. We also demonstrated differential recognition by some of the DH analogues of closely related isoforms GluK1 and GluK2. Further, we found that neither degree of the domain closure nor twist motion of domains directly correlated to agonist efficacy of the ligands.
  • Masato Oikawa, Shota Sasaki, Michihiro Sakai, Yuichi Ishikawa, Ryuichi Sakai European Journal of Organic Chemistry (29) 5789 -5802 2012年10月 [査読無し][通常論文]
     
    The syntheses of the marine sponge-derived γ-amino carboxylic acid dysibetaine CPa and five analogs in their racemic forms were successfully performed by taking advantage of an electron-withdrawing N-(4-nitrophenyl) group in the cyclopropanation reaction, the reductive ring opening of an imide, and the ethanolysis of an N-Boc-protected imide. © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
  • 神保充, 國谷奈美, 武内良太, 田中浩士, 高橋孝志, 小池一彦, 酒井隆一, 神谷久男 日本水産学会大会講演要旨集 2012 79 2012年09月14日 [査読無し][通常論文]
  • Sonia Bhangoo, Bryan Copits, Yuka Nakamura, Shanti Frausto, John Robbins, Martin B. Gill, Caleb Smith, Tomohisa Ogawa, Koji Muramoto, Ryuichi Sakai, Geoffrey T. Swanson FASEB JOURNAL 26 2012年04月 [査読無し][通常論文]
  • Binding and Selectivity of the Marine Toxin Neodysiherbaine A, and its Synthetic Analogues, to GluK1 and GluK2 Kainate Receptors
    Masaki Unno, Ryuichi Sakai, Makoto Sasaki, Masao Ikeda-Saito Photon Factory Activity Reprot 2011, Part B 29 320 2012年04月 [査読無し][通常論文]
  • 境倫宏, 佐々木翔太, 石川裕一, 酒井隆一, SWANSON G.T, 及川雅人 日本化学会講演予稿集 92nd (4) 1182 2012年03月09日 [査読無し][通常論文]
  • 常盤一弥, 菅俣祐太郎, 石川裕一, 酒井隆一, 及川雅人 日本化学会講演予稿集 92nd (4) 1180 2012年03月09日 [査読無し][通常論文]
  • 菅俣祐太郎, 常盤一弥, 加曽利祐基, 片山理佐, 村上悦子, 石川裕一, 酒井隆一, 及川雅人 日本化学会講演予稿集 92nd (4) 1181 2012年03月09日 [査読無し][通常論文]
  • 酒井隆一 日本薬学会年会要旨集 132nd (1) 175 2012年03月05日 [査読無し][通常論文]
  • 海野 昌喜, 佐々木 誠, 酒井 隆一, 齋藤 正男 日本結晶学会誌 54 (6) 338 -344 2012年 [査読無し][通常論文]
     
    Kainate receptors (KARs) are members of the ionotropic glutamate receptors (iGluR) that play variety of roles in the mammalian brain. Because KARs are comprised of five different isoform proteins, isoform selective compounds are indispensable tools in physiological researches. Dysiherbaine (DH) and neodysiherbaine A (NDH), natural toxins found from marine sponges, were shown to be potent and isoform-selective agonists for KARs. To understand structure-activity relationship of DH and NDH, numbers of analogues are synthesized, and their pharmacological activities have been evaluated in detail. Here, we determined the crystal structures of human KAR isoforms, GluK1 and GluK2, ligand-binding domain in complex with the DH analogues at high resolution. From these structures, we elucidated the relationships between the binding affinity and the molecular structure of the compounds. We also demonstrated differential recognition by some of the DH analogues of closely related isoforms GluK1 and GluK2. Further, we found that neither degree of the domain closure nor twist motion of domains directly correlated to agonist efficacy of the ligands.
  • 上田 拓弥, 酒井 隆一, DM Freymann, PJ Focial, 中村 友香, Smith Caleb, 井上 昌, 尾島 孝男, 松永 智子, GT Swanson 天然有機化合物討論会講演要旨集 54 (0) 585 -590 2012年 [査読無し][通常論文]
     
    Here we report the bioactivity-guided isolation of novel galectins from the marine sponge Cinachyrella sp., collected from Iriomote Island, Japan. The lectin proteins, which we refer to as the Cinachyrella galectins (CchGs), were identified as the active principles in an aqueous sponge extract that modulated the function of mammalian ionotropic glutamate receptors. Aggregation of rabbit erythrocytes by CchGs was competed most effectively by galactosides but not mannose, a profile characteristic of members of the galectin family of oligosaccharide-binding proteins. The lectin activity was remarkably stable, with only a modest loss in hemagglutination after exposure of the protein to 100℃ for 1 h, and showed little sensitivity to calcium concentration. CchG-1 and -2 appeared as 16 and l8kDa in SDS-PAGE, respectively, whereas MALDI-TOF MS indicated broad ion clusters centered at 16,216 and 16,423 respectively. The amino acid sequences of the CchGs were deduced using a combination of Edman degradation and cDNA cloning and revealed that the proteins were distant orthologues of animal prototype galectins and that multiple isolectins comprised the CchGs. The biochemical properties of the CchGs as well as their unexpected activity on mammalian excitatory amino acid receptors suggest that further analysis of these new members of the galectin family will yield further glycobiological and neurophysiological insights.
  • Takamasa Uekita, Ryuichi Sakai CANCER SCIENCE 102 (11) 1943 -1948 2011年11月 [査読無し][通常論文]
     
    Tumor metastasis is a complex multistep process by which cells from the primary tumor invade tissues, move through the vasculature, settle at distant sites and eventually grow to form secondary tumors. Altered tyrosine phosphorylation signals in cancer cells contribute to a number of aberrant characteristics involved in tumor invasion and metastasis. CUB domain-containing protein 1 (CDCP1) is a substrate of Src family kinases and has been shown to regulate anoikis resistance, migration and matrix degradation during tumor invasion and metastasis in a tyrosine phosphorylation-dependent manner. Knockdown of CDCP1 blocks tumor metastasis or peritoneal dissemination in vivo, without significantly affecting cell proliferation. Moreover, expression levels of CDCP1 are of prognostic value in several cancers. Here, we summarize the studies on CDCP1, focusing on structure and signal transduction, to gain insight into its role in cancer progression. Understanding the signaling pathways regulated by CDCP1 could help establish novel therapeutic strategies against the progression of cancer. (Cancer Sci 2011; 102: 1943-1948)
  • 上田拓弥, 井上昌, 中村友香, 松永智子, 尾島孝男, SWANSON Geffrey T, 酒井隆一 日本水産学会大会講演要旨集 2011 115 2011年09月28日 [査読無し][通常論文]
  • 松永智子, 酒井隆一, GILL Martin B, LASH‐VAN WYHE, L. Leanne, SWANSON Geoffrey T 天然有機化合物討論会講演要旨集 53rd 409-413 -413 2011年09月02日 [査読無し][通常論文]
  • 境倫宏, 佐々木翔太, 石川裕一, 酒井隆一, SWANSON G. T, 及川雅人 天然有機化合物討論会講演要旨集 53rd 643-648 -648 2011年09月02日 [査読無し][通常論文]
  • 浅川倫宏, 大内仁志, 鈴木寛人, 磯部洋一郎, 東匠, 岡崎優子, 脇本敏幸, 古田巧, 島本啓子, 酒井隆一, 濱島義隆, 菅敏幸 天然有機化合物討論会講演要旨集 53rd 205-210 -210 2011年09月02日 [査読無し][通常論文]
  • Masato Oikawa, Shota Sasaki, Michihiro Sakai, Ryuichi Sakai Tetrahedron Letters 52 (34) 4402 -4404 2011年08月 [査読無し][通常論文]
     
    The cyclopropane-containing amino acid, dysibetaine CPa, isolated from Micronesian marine sponge, has been synthesized in 4.53% total yield over 12 steps starting from maleic anhydride to study the biological function in detail, by taking advantage of electron-withdrawing 4-nitrophenyl group. © 2011 Elsevier Ltd. All rights reserved.
  • Hideki Yamaguchi, Hideki Yamaguchi, Hideki Yamaguchi, Shuhei Yoshida, Emi Muroi, Emi Muroi, Nachi Yoshida, Nachi Yoshida, Masahiro Kawamura, Zen Kouchi, Yoshikazu Nakamura, Ryuichi Sakai, Kiyoko Fukami Journal of Cell Biology 193 (7) 1275 -1288 2011年06月 [査読無し][通常論文]
     
    Invadopodia are extracellular matrix-degrading protrusions formed by invasive cancer cells that are thought to function in cancer invasion. Although many invadopodia components have been identified, signaling pathways that link extracellular stimuli to invadopodia formation remain largely unknown. We investigate the role of phosphoinositide 3-kinase (PI3K) signaling during invadopodia formation. We find that in human breast cancer cells, both invadopodia formation and degradation of a gelatin matrix were blocked by treatment with PI3K inhibitors or sequestration of D-3 phosphoinositides. Functional analyses revealed that among the PI3K family proteins, the class I PI3K catalytic subunit p110α, a frequently mutated gene product in human cancers, was selectively involved in invadopodia formation. The expression of p110α with cancerous mutations promoted invadopodiamediated invasive activity. Furthermore, knockdown or inhibition of PDK1 and Akt, downstream effectors of PI3K signaling, suppressed invadopodia formation induced by p110α mutants. These data suggest that PI3K signaling via p110α regulates invadopodia-mediated invasion of breast cancer cells.
  • Chikako Ozeki, Yuichiro Sawai, Tatsuhiro Shibata, Takashi Kohno, Koji Okamoto, Jun Yokota, Fumio Tashiro, Sei-ichi Tanuma, Ryuichi Sakai, Tatsuya Kawase, Issay Kitabayashi, Yoichi Taya, Rieko Ohki JOURNAL OF BIOLOGICAL CHEMISTRY 286 (20) 18251 -18260 2011年05月 [査読無し][通常論文]
     
    The common polymorphism of p53 at codon 72, either encoding proline or arginine, has drawn attention as a genetic factor associated with clinical outcome or cancer risk for the last 2 decades. We now show that these two polymorphic variants differ in protein structure, especially within the N-terminal region and, as a consequence, differ in post-translational modification at the N terminus. The arginine form (p53-72R) shows significantly enhanced phosphorylation at Ser-6 and Ser-20 compared with the proline form (p53-72P). We also show diminished Mdm2-mediated degradation of p53-72R compared with p53-72P, which is at least partly brought about by higher levels of phosphorylation at Ser-20 in p53-72R. Furthermore, enhanced p21 expression in p53-72R-expressing cells, which is dependent on phosphorylation at Ser-6, was demonstrated. Differential p21 expression between the variants was also observed upon activation of TGF-beta signaling. Collectively, we demonstrate a novel molecular difference and simultaneously suggest a difference in the tumor-suppressing function of the variants.
  • 櫻田剛史, GILL Martin B, FRAUSTO Shanti, COPITS Bryan, 野口恵一, 島本啓子, SWANSON Geoffrey T, 酒井隆一 日本水産学会大会講演要旨集 2011 91 2011年03月27日 [査読無し][通常論文]
  • 松永智子, 酒井隆一 日本水産学会大会講演要旨集 2011 92 2011年03月27日 [査読無し][通常論文]
  • 佐々木翔太, 境倫宏, 酒井隆一, 及川雅人 日本化学会講演予稿集 91st (4) 1179 2011年03月11日 [査読無し][通常論文]
  • R. Yagi, M. Tanaka, K. Sasaki, R. Kamata, Y. Nakanishi, Y. Kanai, R. Sakai ONCOGENE 30 (12) 1413 -1421 2011年03月 [査読無し][通常論文]
     
    During the analysis of phosphotyrosine-containing proteins in scirrhous gastric carcinoma cell lines, we observed an unusual expression of Arf-GAP with Rho-GAP domain, ankyrin repeat and PH domain 3 (ARAP3), a multimodular signaling protein that is a substrate of Src family kinases. Unlike other phosphotyrosine proteins, such as CUB domain-containing protein 1 (CDCP1) and Homo sapiens chromosome 9 open reading frame 10/oxidative stress-associated Src activator (C9orf10/Ossa), which are overexpressed and hyperphosphorylated in scirrhous gastric carcinoma cell lines, ARAP3 was underexpressed in cancerous human gastric tissues. In this study, we found that overexpression of ARAP3 in the scirrhous gastric carcinoma cell lines significantly reduced peritoneal dissemination. In vitro studies also showed that ARAP3 regulated cell attachment to the extracellular matrix, as well as invasive activities. These effects were suppressed by mutations in the Rho-GTPase-activating protein (GAP) domain or in the C-terminal two tyrosine residues that are phosphorylated by Src. Thus, the expression and phosphorylation state of ARAP3 may affect the invasiveness of cancer by modulating cell adhesion and motility. Our results suggest that ARAP3 is a unique Src substrate that suppresses peritoneal dissemination of scirrhous gastric carcinoma cells. Oncogene (2011) 30, 1413-1421; doi:10.1038/onc.2010.522; published online 15 November 2010
  • Crystal structures of symbiosis related lectin and its saccharide\nbound form
    Akiko Kita, Mitsuru Jimbo, Yukio Morimoto, Ryuichi Sakai, Hisao Kamiya, Kunio Miki IUCr2011, Congress of Internation Union of Crystallography 635 -0 2011年 [査読無し][通常論文]
  • 境 倫宏, 佐々木 翔太, 石川 裕一, 酒井 隆一, Swanson Geoffrey T., 及川 雅人 天然有機化合物討論会講演要旨集 53 (0) 643 -648 2011年 [査読無し][通常論文]
     
    Micronesian marine sponge Lendenfeldia chondrodes contains structurally diverse neuroactive metabolites such as dysiherbaine, neodysiherbaine, dysibetaine, dysibetaine CPa and CPb, and cribronic acid, which have received significant attention from the synthetic community as structural motifs for developing novel neuroactive compounds. It is also of our considerable interest to use these metabolites as a probe for investigating functions of synaptic receptors. In the present study, we established a synthetic route to dysibetaine CPa (1) in racemic form, which is amenable to the analog synthesis, to study the biological function as well as the structure-activity relationships. Dysibetaine CPa (1) is a betaine consists of a quaternary ammonium group and two carboxyl groups, located on a novel 1,2,3-trisubstituted cyclopropane ring. From the synthetic point of view, differentiation of the functional groups on the cyclopropane ring is essential for the total synthesis but proved to be challenging during our study because of the instability of the synthetic intermediates. Upon considerable experimentation, we discovered that the cyclopropane ring was readily constructed by the reaction of N-(4-nitrophenyl)maleimide with sulfonium ylide 3. Reductive opening of the cyclopropane-fused imide 22 with NaBH_4 proceeded chemoselectively, giving rise to hydroxyamide 23. The amide was converted to ethyl ester over two steps, and then quaternary ammonium group was introduced by way of bromide 6'. Finally, acidic hydrolysis of two esters was effected by hydrochloric acid to achieve the total synthesis of dysibetaine CPa ((±ア)-1). Total yield was 4.5% for 12 steps. Furthermore, synthesis of five analogs was performed in the present study. Our route for the racemates, thus established here, will be also expanded to the asymmetric synthesis to determine the absolute stereochemistry of 1. Preliminary study indicated that all synthetic compounds did not induce noticeable behavioral change when 20 μg was administrated in mice intracerebroventricularly, as was observed with natural product.
  • 松永 智子, 酒井 隆一, Gill Martin B., Lash-Van Wyhe Leanne, Swanson Geoffrey T. 天然有機化合物討論会講演要旨集 53 (0) 409 -413 2011年 [査読無し][通常論文]
     
    Aculeines (ACUs) are new peptide toxins isolated from the marine sponge A. aculeata collected at Iriomote, Okinawa. ACUs exhibited neurotoxicity through disrupting cell membrane and inducing robust influx of Ca^<2+> ions. ACUs are modified by long-chain polyamines (LCPAs) at the Nterminal amino acid. Amino acid sequence of the peptide portion of ACU-A was determined on the basis of Edman degradation, nucleotide sequence analysis, and peptide-mass mapping to be a 44-amino acid polypeptide. The peptide-mass mapping for ACU-B showed that it shares the same peptide portion with ACU-A. The nucleotide sequence analysis suggested that the Nterminal of ACU-A/B to be Trp, however; their structures were difficult to be elucidated because of a minute amount of peptides available and highly unusual modification by LCPA. In the present study, we isolated Nterminal fragments E and E' obtained from enzyme digest of ACU-A and B, respectively. We also found a novel LCPA derivative protoaculaine (1) that possibly represents the structures of the Nterminal portion of ACUs form the aqueous extract. Here we report the structure elucidation of those compounds.
  • 浅川 倫宏, 酒井 隆一, 濱島 義隆, 菅 敏幸, 大内 仁志, 鈴木 寛人, 磯部 洋一郎, 東 匠, 岡崎 優子, 脇本 敏幸, 古田 巧, 島本 啓子 天然有機化合物討論会講演要旨集 53 (0) 205 -210 2011年 [査読無し][通常論文]
     
    Recently, kainoids, such as kainic acid, have received significant attention due to their potent binding affinity for ionotropic glutamate receptors (iGluRs). iGluRs are involved in important neurophysiological processes, such as memory and learning. Although many synthetic investigations of kainoids have been reported to date, efficient synthetic methods are still strongly required. During the pioneering investigations on acromelic acid A, isolated by the Shirahama group, it was discovered that a synthetic derivative, methoxyphenyl kainic acid, possessed more potent activity than the natural compound. Inspired by this interesting structure-activity relationship, we launched an investigation into the development of efficient synthetic methods for achieving MFPA and phenylkainic acid. In our synthetic strategy, we envisioned that the three consecutive chiral centers were constructed based on the stereochemistry of the C4 position. We employed our asymmetric intermolecular C-H insertion reaction assisted by the chiral auxiliary using diazo ester and cyclohexadiene to afford the desired diene ester in high yield with good diastereoselectivity. After the conversion into the lactone by successive ozonolysis of cyclohexadiene, nitrogen was installed with Ns amide to give the corresponding hemiaminal. The reduction of the aminal and deprotection of the acetal induced the cyclization to construct a pyrrolidine ring with the correct stereochemistry. Introduction of two cyano groups were performed by diastereoselective Strecker-type reaction and Mitsunobu reaction. Finally, hydrolysis of the two cyano groups gave the synthetic kainoids. Furthermore, several investigations using the synthesized kainoids have elucidated that these compounds selectively bind to iGluRs and are equally effective for mice in vivo.
  • 海野昌喜, 海野昌喜, 海野昌喜, 篠原正将, 高山昴一郎, 田中秀治, 酒井隆一, 佐々木誠, 齋藤正男 PFシンポジウム要旨集 28th 97(7) 2011年 [査読無し][通常論文]
  • Hitoyasu Futami, Ryuichi Sakai CANCER LETTERS 297 (2) 220 -225 2010年11月 [査読無し][通常論文]
     
    Recently, gene amplification and gain-of-function mutations of ALK have been found in some neuroblastoma cell lines and clinical tumor samples. We have previously reported that knockdown of ALK by RNAi induced apoptosis in neuroblastoma cells with gene amplification of ALK. We report that all-trans retinoic acid (ATRA) downregulates ALK in neuroblastoma cell lines. Downregulation of ALK protein by ATRA was accompanied by apoptosis in neuroblastoma cells with gene amplification or gain-of-function mutation of ALK but not in neuroblastoma cells without these genetic alterations. These results suggest that ALK downregulation by ATRA might lead to apoptosis in neuroblastoma cells with activated ALK. (C) 2010 Elsevier Ireland Ltd. All rights reserved.
  • Tatsuya Tazaki, Takaaki Sasaki, Kenta Uto, Norimasa Yamasaki, Satoshi Tashiro, Ryuichi Sakai, Minoru Tanaka, Hideaki Oda, Zen-Ichiro Honda, Hiroaki Honda HEPATOLOGY 52 (3) 1089 -1099 2010年09月 [査読無し][通常論文]
     
    p130Cas, Crk-associated substrate (Cas), is an adaptor/scaffold protein that plays a central role in actin cytoskeletal reorganization. We previously showed that mice in which Cos was deleted (Cas(-/-)) died in utero because of early cardiovascular maldevelopment. To further investigate the in vivo roles of Gas, we generated mice with a hypomorphic Gas allele lacking the exon 2-derived region (Cas(Delta ex2/Delta ex2).) which encodes Src homology domain 3 (SH3) of Cas.Cas(Delta ex2/Delta ex2) mice again died as embryos, but they particularly showed progressive liver degeneration with hepatocyte apoptosis. Because Cas expression in the liver is preferentially detected in sinusoidal endothelial cells (SECs), the observed hepatocyte apoptosis was most likely ascribable to impaired function of SECs. To address this possibility, we stably introduced a Gas mutant lacking the SH3 domain (Gas ASH3) into an SEC line (NP31). Intriguingly, the introduction of Gas ASH3 induced a loss of fenestrae, the characteristic cell-penetrating pores in SECs that serve as a critical route for supplying oxygen and nutrients to hepatocytes. The disappearance of fenestrae in Gas ASH3-expressing cells was associated with an attenuation of actin stress fiber formation, a marked reduction in tyrosine phosphorylation of Gas, and defective binding of Gas to CrkII. Conclusion: Gas plays pivotal roles in liver development through the reorganization of the actin cytoskeleton and formation of fenestrae in SECs. (HEPATOLOGY 2010;52:1089-1099)
  • 櫻田剛史, GILL Martin B, FRAUSTO Shanti, COPITS Bryan, 野口恵一, 島本啓子, SWANSON Geoffrey T, 酒井隆一 天然有機化合物討論会講演要旨集 52nd 625-630 -630 2010年09月01日 [査読無し][通常論文]
  • 及川雅人, 及川雅人, 生駒実, 佐々木誠, 酒井隆一, SWANSON Geoffrey 天然有機化合物討論会講演要旨集 52nd 85-90 -90 2010年09月01日 [査読無し][通常論文]
  • M. B. Gill, S. Frausto, M. Ikoma, M. Sasaki, M. Oikawa, M. Oikawa, R. Sakai, G. T. Swanson British Journal of Pharmacology 160 (6) 1417 -1429 2010年07月 [査読無し][通常論文]
     
    Background and purpose: A new class of heterotricyclic glutamate analogues recently was generated by incorporating structural elements of two excitotoxic marine compounds, kainic acid and neodysiherbaine A. Rather than acting as convulsants, several of these 'IKM' compounds markedly depressed CNS activity in mice. Here, we characterize the pharmacological profile of the series with a focus on the most potent of these molecules, IKM-159. Experimental approach: The pharmacological activity and specificity of IKM compounds were characterized using whole-cell patch clamp recording from neurons and heterologous receptor expression systems, in combination with radioligand binding techniques. Key results: The majority of the IKM compounds tested reduced excitatory synaptic transmission in neuronal cultures, and IKM-159 inhibited synaptic currents from CA1 pyramidal neurons in hippocampal slices. IKM-159 inhibited glutamate-evoked whole-cell currents from recombinant GluA2- and GluA4-containing α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors most potently, whereas kainate and NMDA receptor currents were not reduced by IKM-159. Antagonism of steady-state currents was agonist concentration dependent, suggesting that its mechanism of action was competitive, although it paradoxically did not displace [ 3 H]-AMPA from rec eptor binding sites. IKM-159 reduced spontaneous action potential firing in both cultured hippocampal neurons in control conditions and during hyperactive states in an in vitro model of status epilepticus. Conclusions and implications: IKM-159 is an AMPA receptor-selective antagonist. IKM-159 and related nitrogen heterocycles represent structurally novel AMPA receptor antagonists with accessible synthetic pathways and potentially unique pharmacology, which could be of use in exploring the role of specific populations of receptors in neurophysiological and neuropathological processes. © 2010 The British Pharmacological Society.
  • Hideki Yamaguchi, Hideki Yamaguchi, Hideki Yamaguchi, Shuhei Yoshida, Emi Muroi, Masahiro Kawamura, Zen Kouchi, Yoshikazu Nakamura, Ryuichi Sakai, Kiyoko Fukami Cancer Science 101 (7) 1632 -1638 2010年07月 [査読無し][通常論文]
     
    Invadopodia are ventral cell protrusions formed in invasive cancer cells. Because invadopodia have extracellular matrix (ECM) degradation activity, they are thought to function in cancer invasion. In this study, we examined the roles of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] and PI(4,5)P 2 -producing enzymes in invadopodia formation in MDA-MB-231 human breast cancer cells. Immunofluorescence analysis showed that PI(4,5)P 2 accumulates at invadopodia on the ventral cell surface. Injection of an anti-PI(4,5)P 2 antibody inhibited invadopodia formation along with gelatin degradation activity. Sequestering of PI(4,5)P 2 by overexpression of the phospholipase C (PLC) δ1-pleckstrin homology (PH) domain, a specific probe for PI(4,5)P 2 , also blocked invadopodia formation, while a mutated PLCδ1-PH domain that lacks PI(4,5)P 2 -binding activity had no effect. Cellular PI(4,5)P 2 production is mainly mediated by type-I phosphatidylinositol 4-phosphate 5-kinase (PIP5KI) family proteins, which include PIP5KIα, Iβ, and Iγ. Real-time quantitative PCR analysis showed that PIP5KIα is a dominant isoform expressed in MDA-MB-231 cells. Knockdown of PIP5KIα by small-interfering RNA (siRNA) inhibited invadopodia formation and gelatin degradation. Immunofluorescence analysis revealed that endogenous PIP5KIα protein localizes at invadopodia, which is corroborated by the observation that exogenously expressed green fluorescent protein (GFP)-fused PIP5KIα protein also accumulates at gelatin degradation sites. These results indicate that localized production of PI(4,5)P 2 by PIP5KIα is required for invadopodia formation and ECM degradation by human breast cancer cells. © 2010 Japanese Cancer Association.
  • Yuri Miyazawa, Takamasa Uekita, Nobuyoshi Hiraoka, Satoko Fujii, Tomoo Kosuge, Yae Kanai, Yoshihisa Nojima, Ryuichi Sakai CANCER RESEARCH 70 (12) 5136 -5146 2010年06月 [査読無し][通常論文]
     
    CUB domain-containing protein 1 (CDCP1) is a membrane protein that is highly expressed in several solid cancers. We reported previously that CDCP1 regulates anoikis resistance as well as cancer cell migration and invasion, although the underlying mechanisms have not been elucidated. In this study, we found that expression of CDCP1 in pancreatic cancer tissue was significantly correlated with overall survival and that CDCP1 expression in pancreatic cancer cell lines was relatively high among solid tumor cell lines. Reduction of CDCP1 expression in these cells suppressed extracellular matrix (ECM) degradation by inhibiting matrix metalloproteinase-9 secretion. Using the Y734F mutant of CDCP1, which lacks the tyrosine phosphorylation site, we showed that CDCP1 regulates cell migration, invasion, and ECM degradation in a tyrosine phosphorylation-dependent manner and that these CDCP1-associated characteristics were inhibited by blocking the association of CDCP1 and protein kinase C delta (PKC delta). CDCP1 modulates the enzymatic activity of PKC delta through the tyrosine phosphorylation of PKC delta by recruiting PKC delta to Src family kinases. Cortactin, which was detected as a CDCP1-dependent binding partner of PKC delta, played a significant role in migration and invasion but not in ECM degradation of pancreatic cells. These results suggest that CDCP1 expression might play a crucial role in poor outcome of pancreatic cancer through promotion of invasion and metastasis and that molecules blocking the expression, phosphorylation, or the PKC delta-binding site of CDCP1 are potential therapeutic candidates. Cancer Res; 70(12); 5136-46. (C) 2010 AACR.
  • 小國 義之, 由田 奨, 酒井 隆一 ターンアラウンドマネージャー 6 (6) 49 -59 2010年06月 [査読無し][通常論文]
  • 喜田昭子, 神保充, 森本幸生, 酒井隆一, 神谷久男, 三木邦夫 日本蛋白質科学会年会プログラム・要旨集 10th 112 2010年05月15日 [査読無し][通常論文]
  • 久保田 浩文, 酒井 隆一, 八木 雄毅 ターンアラウンドマネージャー 6 (5) 52 -68 2010年05月 [査読無し][通常論文]
  • Mitsuru Jimbo, Hiroshi Yamashita, Kazuhiko Koike, Ryuichi Sakai, Hisao Kamiya FISHERIES SCIENCE 76 (2) 355 -363 2010年03月 [査読無し][通常論文]
     
    We report herein the presence of a lectin in the scleractinian coral Ctenactis (Fungia) echinata. The lectin bound preferentially to lactose, melibiose, and D-galactose. The purified lectin CecL was composed of several isolectins, and it was found to have a molecular mass of 67.4 kDa via gel filtration. Glycopeptidase F-treated CecL showed a single band at 32.5 kDa. The mass/ charge ratios of the reduced CecL peaks were equivalent to half those of the native peaks. These results suggest that CecL is composed of two glycosylated polypeptides linked by interchain disulfide bonds. In a biological activity test using a zooxanthellal culture (Dinoflagellate Symbiodinium) clonally isolated from Fungia cf. fungites, CecL transformed the flagellated motile form of Symbiodinium into the nonmotile coccoid form, a form equivalent to the symbiotic stage. The activity of CecL on Symbiodinium cells was concentration dependent, and 100 mu g/ml CecL arrested Symbiodinium cells in the coccoid form for 5 days. CecL also suppressed the growth of Symbiodinium cells, unlike the octocoral lectin derived from Sinularia lochmodes, which arrests Symbiodinium cells in the coccoid form but does not affect the growth of the coccoid. This result provides further evidence that coral lectins play a role in symbiont engagement and maintenance in zooxanthellae-coral symbiosis.
  • Masamitsu Tanaka, Reiko Kamata, Kazuyoshi Yanagihara, Ryuichi Sakai CANCER SCIENCE 101 (1) 87 -93 2010年01月 [査読無し][通常論文]
     
    Interaction of the Eph family of receptor protein tyrosine kinases and their ligands, ephrin family members, induces bidirectional signaling through cell-cell contacts. High expression of B-type ephrin is associated with high invasion potential of tumors, and we previously observed that signaling through the C-terminus of ephrin-B1 mediates the migration and invasion of cells, and is involved in the promotion of carcinomatous peritonitis in vivo. Here we show that the intracellular introduction of a synthetic peptide derived from ephrin-B1 C-terminus blocks ephrin-B1 mediated signaling in scirrhous gastric cancer cells. Treatment of cancer cells with a fusion peptide consisting of HIV-TAT and amino acids 331-346 of ephrin-B1 (PTD-EFNB1-C) suppressed the activation of RhoA, mediated by the association of ephrin-B1 with an adaptor protein Dishevelled, and also inhibited extracellular secretion of metalloproteinase. Moreover, injection of PTD-EFNB1-C peptide into the peritoneal cavity of nude mice suppressed carcinomatous peritonitis of intraperitoneally transplanted scirrhous gastric cancer cells. These results indicate the possible application of ephrin-B1 C-terminal peptide to develop novel protein therapy for scirrhous gastric carcinoma, especially in the stage of tumor progression, including peritoneal dissemination. (Cancer Sci 2009).
  • 櫻田 剛史, Gill Martin B, Frausto Shanti, Copits Bryan, 野口 恵一, 島本 啓子, Swanson Geoffrey T., 酒井 隆一 天然有機化合物討論会講演要旨集 52 (0) 625 -630 2010年 [査読無し][通常論文]
     
    Marine benthi organisms have yielded a variety of natural products with neuropharmacological applications. Here we describe the isolation and pharmacological characterization of four novel, neurologically active 8-oxoderivatives of purine, 1-4, isolated from Haplosclerida sponges collected in the Republic of Palau. The structure of 1 was determined based on spectral data and was confirmed by X-ray crystallography. The structures of 2-4 were determined similarly using spectral data by analogy of that of 1. Compound 1 induced convulsant behavior upon intracerebroventricular injection into mice, with a CD_<50> value of 2,4 nmol/mouse. Purines 2-4 were active in mouse bioassays at higher doses. The seizurogenic activity observed with 1 was correlated with inhibition of neruonal GABAergic transmission, with only a modest mpact of excitatory signaling, in electrophysiological recordings from hippoampal neurons in cultured and acute brain slice preparations. Despite having a purine template structure, the inhibitory activity of 1 was not prevented by a nonselective adenosine receptor antagonist. The natural product 1 therefore represents a novel substituted purine that elicits convulsions through its actions on inhibitory neurotransmission. The four 8-oxoisoguanine analogs comprise a new family of compounds closely related in structure to both important endogenous neurosignaling molecules and commonly used CNS stimulants.
  • 及川 雅人, 生駒 実, 佐々木 誠, 酒井 隆一, Swanson Geoffrey T. 天然有機化合物討論会講演要旨集 52 (0) 85 -90 2010年 [査読無し][通常論文]
     
    Ionotropic glutamate receptors (iGluRs) are involved in higher brain functions such as memory and learning, nociception, and a number of brain disorders. Here, we report the synthesis of twelve artificial glutamate analogs whose core structure was inspired by two marine-derived excitatory amino acids, dysiherbaine and kainia acid. Four 7-oxznorbornenes, 2a-2d, were prepared in two steps, starting from and Ugi four component coupling reaction followed by spontaneous Diels-Alder reaction between 2-furfural, 3-iodoacrylic acid, 4-methoxybenzylamine, and benzyl isocyanide. An unprecedented domino metathesis reaction with less reactive vinyl acetate as a cross metathesis substrate was then performed with the Hoveyda-Grubbs second-generation catalyst, to successfully deliver four heterotricycles 3a-3d in good yiels. After functional group transformations followed by diversification at the C-ring, twelve artificial glutamate analogs 6a-6d, 7a-7d, 8a-8d were synthesized in total 7.2-25.8% yield for 13-15 steps. Mice in vivo assays indicated that all analogs are biologically active; namely, 6b, 7a, 7b, and 8b produce hyperactivity in injected i.c.v., whereas other analogs induce hypoactiity in the animals. In vitro electrophysiological assays showed that some hypoactive analogs inhibit spontaneous excitatory synaptic currents in hippocampal neurons and glutamate-evoked currents from recombinant AMPA receptors. With these pharmacological profiles, synthesis of other analogs werer further performed, and pyrrolidone dicarboxylic acid analog IKM-159 was discovered as a more potent, AMPA receptor-selective antagonist.
  • 喜田昭子, 神保充, 森本幸生, 酒井隆一, 神谷久男, 三木邦夫 PFシンポジウム要旨集 27th 42 2010年 [査読無し][通常論文]
  • 櫻田剛史, GILL Martin B, FRAUSTO Shanti, COPITS Bryan, 野口恵一, 島本啓子, SWANSON Geoffrey T, 酒井隆一 日本水産学会北海道支部大会講演要旨集 2010 22 2010年 [査読無し][通常論文]
  • 喜田昭子, 神保充, 森本幸生, 酒井隆一, 神谷久男, 三木邦夫 日本結晶学会年会講演要旨集 2010 123 2010年 [査読無し][通常論文]
  • 酒井 隆一 化学と生物 47 (10) 674 -675 2009年10月01日 [査読無し][通常論文]
  • 松永智子, 酒井隆一, 神保充 日本水産学会大会講演要旨集 2009 87 2009年09月30日 [査読無し][通常論文]
  • 海野昌喜, 海野昌喜, 篠原正将, 高山昴一郎, 渡邉朋子, 田中秀治, 酒井隆一, 佐々木誠, 齋藤正男 生化学 ROMBUNNO.2T4A-6 2009年09月25日 [査読無し][通常論文]
  • 松永智子, 酒井隆一, 神保充, GILL Martin B, SWANSON Geoffey T, 神谷久男 天然有機化合物討論会講演要旨集 51st 599-604 -604 2009年09月01日 [査読無し][通常論文]
  • Hitoyasu Futami, Ryuichi Sakai CANCER SCIENCE 100 (6) 1034 -1039 2009年06月 [査読無し][通常論文]
     
    The receptor tyrosine kinase RET is expressed in a number of neuroblastoma tissues and cell lines, but its role in neuroblastoma remains to be determined. In this study, we examined the roles of RET protein in neuroblastoma by the RNA interference technique using the NB-39-nu neuroblastoma cell line. NB-39-nu neuroblastoma cells show high expression and elevated tyrosine phosphorylation of RET, although short interfering RNA against RET (RET siRNA) did not significantly inhibit cell proliferation or suppression of basal levels of phosphorylation of extracellular regulated kinase (ERK)1/2 or protein kinase B (AKT). By the addition of glial cell line-derived neurotrophic factor (GDNF), both the expression and phosphorylation of RET and the phosphorylation of ERK1/2 and AKT were further increased, whereas cell proliferation was not stimulated under normal culture conditions. However, proliferation of cells cultured under non-adherent conditions was significantly increased by GDNF. The increased proliferation was suppressed by RET siRNA, which also caused inhibition of the phosphorylation of ERK1/2 and AKT. These results suggest that RET signaling plays an important role in GDNF-induced enhancement of non-adherent proliferation of NB-39-nu cells, which might contribute to the metastasis of neuroblastoma. (Cancer Sci 2009; 100: 1034-1039).
  • Kotaro Azuma, Tornohiko Urano, Kuniko Horie-Inoue, Shin-ichi Hayashi, Ryuichi Sakai, Yasuyoshi Ouchi, Satoshi Inoue CANCER RESEARCH 69 (7) 2935 -2940 2009年04月 [査読無し][通常論文]
     
    Estrogen receptor alpha (ER alpha) is a nuclear receptor that functions as a ligand-activated transcription factor. Besides its genomic action in nuclei, ER alpha could exert nongenomic actions at the plasma membrane. To investigate the mechanism underlying the nongenomic action of ER alpha in breast cancer cells, we generated a construct of membrane-targeted ER alpha (memER), an expression vector of ER alpha without the nuclear localizing signal and including instead the membrane-targeting sequence of Src kinase. MemER was stably expressed in human breast cancer MCF-7 cells. Cell migration test and tumorigenic assay in nude mice revealed that the in vitro motility and the in vivo proliferation activity of MCF-7 cells expressing memER were significantly enhanced compared with those of vector-transfected cells. Interestingly, the acetylation level of tubulin in memER-overexpressing cells was lower than that in control cells. We found that histone deacetylase (HDAC) 6 translocated to the plasma membrane shortly after estrogen stimulation, and rapid tubulin deacetylation subsequently occurred. We also showed that memER associated with HDAC6 in a ligand-dependent manner. Although tamoxifen is known for its antagonistic role in the ER alpha genomic action in MCF-7 cells, the agent showed an agonistic function in the memER-HDAC6 association and tubulin deacetylation. These findings suggest that ER alpha ligand dependently forms a complex with HDAC6 and tubulin at the plasma membrane. Estrogen-dependent tubulin deacetylation could provide new evidence for the nongenomic action of estrogen, which potentially contributes to the aggressiveness of ER alpha-positive breast cancer cells. [Cancer Res 2009;69(7):2935-40]
  • 中村友香, SWANSON Geffrey T, LASH Lianne L, 酒井隆一 日本水産学会大会講演要旨集 2009 131 2009年03月27日 [査読無し][通常論文]
  • 松永智子, 酒井隆一, 神保充, 神谷久男 日本水産学会大会講演要旨集 2009 131 2009年03月27日 [査読無し][通常論文]
  • 渡邉朋子, 局興一, 及川雅人, 佐々木誠, 酒井隆一, 海野昌喜, 齋藤正男 日本化学会講演予稿集 89th (2) 1440 2009年03月13日 [査読無し][通常論文]
  • 大平菜穂, 局興一, 及川雅人, 佐々木誠, 酒井隆一 日本化学会講演予稿集 89th (2) 1440 2009年03月13日 [査読無し][通常論文]
  • Jun-ichiro Ikeda, Tomofumi Oda, Masayoshi Inoue, Takamasa Uekita, Ryuichi Sakai, Meinoshin Okumura, Katsuyuki Aozasa, Eiichi Morii CANCER SCIENCE 100 (3) 429 -433 2009年03月 [査読無し][通常論文]
     
    CUB domain containing protein (CDCP1), a transmembrane protein with intracellular tyrosine residues which are phosphorylated upon activation, is supposed to be engaged in proliferative activities and resistance to apoptosis of cancer cells. Expression level of CDCP1 was examined in lung adenocarcinoma, and its clinical implications were evaluated. CDCP1 expression was immunohistochemically examined in lung adenocarcinoma from 200 patients. Staining intensity of cancer cells was categorized as low and high in cases with tumor cells showing no or weak and strong membrane staining, respectively. MIB-1 labeling index was also examined. There were 113 males and 87 females with median age of 63 years. Stage of disease was stage I in 144 cases (72.0%), II in 19 (9.5%), and III in 37 (18.5%). Sixty of 200 cases (30.0%) were categorized as CDCP1-high, and the remaining as CDCP1-low. Significant positive correlation was observed between CDCP1-high expression and relapse rate (P < 0.0001), poor prognosis (P < 0.0001), MIB-1 labeling index (P < 0.0001), and occurrence of lymph node metastasis (P = 0.0086). There was a statistically significant difference in disease-free survival (DFS) (P < 0.0001) and overall survival (OS) rates (P < 0.0001) between patients with CDCP1-high and CDCP1-low tumors. Univariate analysis showed that lymph node status, tumor stage, and CDCP1 expression were significant factors for both OS and DFS. Multivariate analysis revealed that only CDCP1 expression was an independent prognostic factor for both OS and DFS. CDCP1 expression level is a useful marker for prediction of patients with lung adenocarcinoma (Cancer Sci 2009; 100: 429-433).
  • 酒井 隆一 遺伝 63 (2) 60 -64 2009年03月 [査読無し][通常論文]
  • 酒井隆一 生物の科学 遺伝 63 (2) 60-64 -64 2009年03月01日 [査読無し][通常論文]
  • I. Miyake, M. Ohira, A. Nakagawara, R. Sakai ONCOGENE 28 (5) 662 -673 2009年02月 [査読無し][通常論文]
     
    The biological and clinical heterogeneityof neuroblastoma is closely associated with signaling pathways that control cellular characteristics such as proliferation, survival and differentiation. The Shc family of docking proteins is important in these pathways by mediating cellular signaling. In this study, we analysed the expression levels of ShcA and ShcC proteins in 46 neuroblastoma samples and showed that a significantly high er level of ShcC protein is observed in neuroblastomas with poor prognostic factors such as advanced stage and MYCN amplification (P < 0.005), whereas the expression level of ShcA showed no signi. cant association with these factors. Using TNB1 cells that express a high level of ShcC protein, it was demonstrated that knockdown of ShcC by RNAi caused elevation in the phosphorylation of ShcA, which resulted in sustained extracellular signal-regulated kinase activation and neurite outgrowth. The neurites induced by ShcC knockdown expressed several markers of neuronal differentiation suggesting that the expression of ShcC potentially has a function in inhibiting the differentiation of neuroblastoma cells. In addition, marked suppression of in vivo tumorigenicity of TNB1 cells in nude mice was observed by stable knockdown of ShcC protein. These findings indicate that ShcC is a therapeutic target that might induce differentiation in the aggressive type of neuroblastomas.
  • Masamitsu Tanaka, Kazuki Sasaki, Reiko Kamata, Yukari Hoshino, Kazuyoshi Yanagihara, Ryuichi Sakai MOLECULAR AND CELLULAR BIOLOGY 29 (2) 402 -413 2009年01月 [査読無し][通常論文]
     
    During the process of tumor progression and clinical treatments, tumor cells are exposed to oxidative stress. Tumor cells are frequently resistant to such stress by producing antiapoptotic signaling, including activation of Src family kinases (SFKs), although the molecular mechanism is not clear. In an attempt to identify the SFK-binding proteins selectively phosphorylated in gastric scirrhous carcinoma, we identified an uncharacterized protein, C9orf10. Here we report that C9orf10 ( designated Ossa for oxidative stress-associated Src activator) is a novel RNA-binding protein that guards cancer cells from oxidative stress-induced apoptosis by activation of SFKs. Exposure to oxidative stress such as UV irradiation induces the association of Ossa/C9orf10 with regulatory domains of SFKs, which activates these kinases and causes marked tyrosine phosphorylation of C9orf10 in turn. Tyrosine-phosphorylated Ossa recruits p85 subunits of phosphatidylinositol 3-kinase (PI3-kinase) and behaves as a scaffolding protein for PI3-kinase and SFKs, which activates the Akt-mediated antiapoptotic pathway. On the other hand, the carboxyl terminus of Ossa has a distinct function that directly binds RNAs such as insulin-like growth factor II (IGF-II) mRNA and promotes the extracellular secretion of IGF-II. Our findings indicate that Ossa is a dual-functional protein and might be a novel therapeutic target which modulates the sensitivity of tumors to oxidative stress.
  • 酒井 隆一 ターンアラウンドマネージャー 5 (1) 100 -104 2009年01月 [査読無し][通常論文]
  • 松永 智子, 酒井 隆一, 神保 充, Gill Martin B., Swanson Geoffrey T., 神谷 久男 天然有機化合物討論会講演要旨集 51 (0) 599 -604 2009年 [査読無し][通常論文]
     
    In our quest for neurotoxic copounds in marine benthic organisms, we discovered novel functionalized peptide toxin aculeine A (Acu-A) from a sponge Axinyssa aculeata. Acu-A is a 45 amino acid-residue peptide with novel post translational modification with long chain polypropanamine attached to the N-terminus. Aculeines were proconvulsant in mice after contral administration. Acu-A was shown to induce calcium influx in cultures HEK293 cells and cultured rat hippocampal cells in an external Ca^<2+> dependent manner suggesting membrane disrupting function of the molecule. Partial amino acid sequence of Acu-A was determined by combination of Edman detradation and MADLDI-TOFMS analysis of the enzyme digests. The whole amino acid sequence for Acu A was deduced from nucleic acid sequence determined by 3' and 5'-RACE agreeing well with the above amino acid sequence where six cysteine residues were arranged like typical cystine knots such as conotoxin. These data suggested that Acu-A was a novel class of polyamine-modified cystine knots.
  • 喜田昭子, 神保充, 森本幸生, 酒井隆一, 神谷久男, 三木邦夫 PFシンポジウム要旨集 26th 96 2009年 [査読無し][通常論文]
  • 田中秀治, 海野昌喜, 海野昌喜, 篠原正将, 高山昂一郎, 渡邉朋子, 酒井隆一, 佐々木誠, 齊藤正男 東北大学多元物質科学研究所研究発表会講演予稿集 9th 142 2009年 [査読無し][通常論文]
  • 海野昌喜, 海野昌喜, 篠原正将, 高山昴一郎, 渡邉朋子, 田中秀治, 酒井隆一, 佐々木誠, 齋藤正男 日本結晶学会年会講演要旨集 2009 32 2009年 [査読無し][通常論文]
  • Tetsuya Nakamoto, Sachiko Seo, Ryuichi Sakai, Takayuki Kato, Haruo Kutsuna, Mineo Kurokawa, Masaki Noda, Nobuyuki Miyasaka, Seiichi Kitagawa JOURNAL OF CELLULAR BIOCHEMISTRY 105 (1) 121 -128 2008年09月 [査読無し][通常論文]
     
    Crk-associated substrate lymphocyte type (Cas-L) protein, also known as human enhancer of filamentation 1 (Hef1) or neural precursor cell-expressed, developmentally down-regulated gene 9 (Nedd9), belongs to the Cas family of adapter proteins, which are involve(] in integrin signaling. Previous reports showed that Cas-L is expressed preferentially in lymphocytes and epithelial cells. Cas-L mediates signals from integrins, T-cell receptors, B-cells receptors, and transforming growth factor beta, leading to cell movement and cell division. Here, we report the expression of Cas-L in neutrophils. Cas-L was tyrosine-phosphorylated when human neutrophils were stimulated by fMLP, tumor necrosis factor-alpha (TNF), or lipopolysaccharide. The tyrosine phosphorylation of Cas-L in fMLP- or TNF-stimulated neutrophils was further enhanced by adhesion of the cells to their substrates. Cas-L was found to be localized at focal adhesions in stimulated neutrophils based on immunofluorescence microscopy. These findings suggest that Cas-L is one of the targets of inflammatory cytokines and is also modulated by cell adhesion process in neutrophils.
  • 海野昌喜, 篠原正将, 高山昴一郎, 酒井隆一, 佐々木誠, 齋藤正男 天然有機化合物討論会講演要旨集 50th 173-178 -178 2008年09月01日 [査読無し][通常論文]
  • Takamasa Uekita, Masamitsu Tanaka, Misato Takigahira, Yuri Miyazawa, Yukihiro Nakanishi, Yae Kanai, Kazuyoshi Yanagihara, Ryuichi Sakai AMERICAN JOURNAL OF PATHOLOGY 172 (6) 1729 -1739 2008年06月 [査読無し][通常論文]
     
    CUB-domain-containing protein 1 (CDCP1) is a type-I transmembrane protein that is highly expressed in colon, breast, and lung cancers. We recently revealed that CDCP1 is associated with and phosphorylated by Src family kinases and is involved in the regulation of anchorage independence of certain lung cancer cell lines. in this study, we examined whether CDCP1 is involved in the regulation of tumor progression of scirrhous gastric cancer, which is a diffusely infiltrative carcinoma with high invasion potential. Expression and phosphorylation levels of CDCP1 correlated with the invasive potential of scirrhous; gastric cancers. Reduction of CDCP1 expression by siRNA suppressed migration, invasion, and anchorage independence without affecting the proliferation of highly invasive scirrhous gastric cancer cells. However, CDCP1 overexpression promoted gastric cancer cell migration with low potential of invasion. Loss of CDCP1 suppressed invasion and dissemination of cancer cells that were orthotopically implanted in the gastric wall of nude mice. Expression and phosphorylation of CDCP1 were also detected in cancer cells of surgically resected tissues of human scirrhous gastric cancer by immunohistochemical analysis. Our results suggest that CDCP1 promotes invasion and peritoneal dissemination of cancer cells through the regulation of cell migration and anchorage independence. Therefore, it is both a potential prognostic and therapeutic target in certain types of gastrointestinal cancers, and suppression of its phosphorylation might be a useful strategy for modulating cancer metastasis.
  • Lin Jia, Takamasa Uekita, Ryuichi Sakai MOLECULAR CANCER RESEARCH 6 (4) 654 -662 2008年04月 [査読無し][通常論文]
     
    Cortactin is frequently overexpressed in cancer cells, and changes of the levels of its tyrosine phosphorylation have been observed in several cancer cells. However, how the expression level and phosphorylation state of cortactin would influence the ultimate cellular function of cancer cells is unknown. In this study, we analyzed the role of cortactin in gastric and breast cancer cell lines using RNA interference technique and found that knockdown of cortactin inhibited cell migration in a subset of gastric cancer cells with a lower level of its tyrosine phosphorylation, whereas it greatly enhanced cell migration and increased tyrosine phosphorylation of p130Cas in other subsets of cells with hyperphosphorylated cortactin. Consistent results were obtained when hyperphosphorylation of cortactin was induced in MCF7 breast cancer cells by expressing Fyn tyrosine kinase. Additionally, immunostaining analysis showed that knockdown of hyperphosphorylated cortactin resulted in the recruitment of p130Cas to focal adhesions. These results suggest that cortactin hyperphosphorylation suppresses cell migration possibly through the inhibition of membrane localization and tyrosine phosphorylation of p130Cas.
  • 町田平, 神保充, 酒井隆一, 神谷久男 日本水産学会大会講演要旨集 2008 65 2008年03月27日 [査読無し][通常論文]
  • 局興一, 青木邦衛, 及川雅人, 酒井隆一, 島本啓子, 佐々木誠 日本化学会講演予稿集 88th (2) 1041 2008年03月12日 [査読無し][通常論文]
  • Tatsuya Tazaki, Kazuko Miyazaki, Eiso Hiyama, Tetsuya Nakamoto, Ryuichi Sakai, Norimasa Yamasaki, Zen-ichiro Honda, Masaki Noda, Nobuyuki Miyasaka, Taijiro Sueda, Hiroaki Honda GENES TO CELLS 13 (2) 145 -157 2008年02月 [査読無し][通常論文]
     
    p130Cas (Cas, Crk-associated substrate) is an adaptor molecule composed of a Src homology 3 (SH3) domain, a substrate domain (SD) and a Src binding domain (SBD). The SH3 domain of Cas associates with focal adhesion kinase (FAK), but its role in cellular function has not fully been understood. To address this issue, we established and analyzed primary fibroblasts derived from mice expressing a truncated Cas lacking exon 2, which encodes the SH3 domain (Cas Delta exon 2). In comparison to wild-type cells, Cas exon 2(Delta/Delta) cells showed reduced motility, which could be due to impaired tyrosine-phosphorylation of FAK and Cas, reduced FAK/Cas/Src/CrkII binding, and also impaired localization of Cas Delta exon 2 to focal adhesions on fibronectin. In addition, to analyze downstream signaling pathways regulated by Cas exon 2, we performed microarray analyses. Interestingly, we found that a deficiency of Cas exon 2 up-regulated expression of CXC Chemokine Receptor-4 and CC Chemokine Receptor-5, which may be regulated by I kappa B alpha phosphorylation. These results indicate that the SH3-encoding exon of Cas participates in cell motility, tyrosine-phosphorylation of FAK and Cas, FAK/Cas/Src/CrkII complex formation, recruitment of Cas to focal adhesions and regulation of cell motility-associated gene expression in primary fibroblasts.
  • Crystal Structure of a Lectin from the Octocoral
    A. Kita, M. Jimbo, Y. Morimoto, R. Sakai, H. Kamiya, K. Miki XXI Congress of the Int. Union of Crystallography 64 -0 2008年 [査読無し][通常論文]
  • シゲミカタトサカレクチン SLL-2の糖結合性と結晶構造
    神保充, 喜田昭子, 小池一彦, 亀井麻直, 中村(鶴田)祥子, 平林淳, 三木邦夫, 酒井隆一, 神谷久男 日本サンゴ礁学会第11回大会 2008年 [査読無し][通常論文]
  • 海野 昌喜, 篠原 正将, 高山 昴一郎, 酒井 隆一, 佐々木 誠, 齋藤 正男 天然有機化合物討論会講演要旨集 50 (0) 173 -178 2008年 [査読無し][通常論文]
     
    Dysiherbaine (DH) and its congener neodysiherbaine A (NDH) are naturally occurring excitatory amino acids with high-affinity and subunit-selectivity for kainate type ionotropic glutamate receptors, especially GluR5 and GluR6 subunits. To elucidate why DH and NDH bind selectively to GluR5, we have determined the crystal structures of human GluR5 ligand-binding core in complexes with DH and NDH, in addition to the glutamate-complex. DH and NDH form unique hydrogen-bonding and hydrophobic interactions with the amino acid residues in the binding-cleft by excluding the water molecules, which mediate hydrogen bonding interactions with glutamate. The domain openings upon binding DH and NDH are much smaller than that found in the structures of partial-agonist- or antagonist-bound forms of GluR5. Here, we propose that the efficacy and ability of an agonist are not simply in inverse proportion to the extent of domain separation, but appear to be related to the stability of the "closed" conformation of the ligand-binding core. This structural information provides the basis for rational development of a series of novel synthetic analogues as useful probes for studying iGluRs in central nervous system and as potential therapeutic leads.
  • 喜田昭子, 神保充, 森本幸生, 酒井隆一, 神谷久男, 三木邦夫 生化学 4P-0107 2008年 [査読無し][通常論文]
  • 生駒実, 及川雅人, 佐々木誠, 酒井隆一 有機合成シンポジウム講演要旨集 93rd 65-68 -68 2008年 [査読無し][通常論文]
  • 及川雅人, 生駒実, 佐々木誠, 酒井隆一 Abstr Conf Comb Chem Jpn 27th 88-91 2008年 [査読無し][通常論文]
  • Takamasa Uekita, Lin Jia, Mako Narisawa-Saito, Jun Yokota, Tohru Kiyono, Ryuichi Sakai MOLECULAR AND CELLULAR BIOLOGY 27 (21) 7649 -7660 2007年11月 [査読無し][通常論文]
     
    Malignant tumor cells frequently achieve resistance to anoikis, a form of apoptosis induced by detachment from the basement membrane, which results in the anchorage-independent growth of these cells. Although the involvement of Src family kinases (SFKs) in this alteration has been reported, little is known about the signaling pathways involved in the regulation of anoikis under the control of SFKs. In this study, we identified a membrane protein, CUB-domain-containing protein 1 (CDCP1), as an SFK-binding phosphoprotein associated with the anchorage independence of human lung adenocarcinoma. Using RNA interference suppression and overexpression of CDCP1 mutants in lung cancer cells, we found that tyrosine-phosphorylated CDCP1 is required to overcome anoikis in lung cancer cells. An apoptosis-related molecule, protein kinase C delta, was found to be phosphorylated by the CDCP1-SFK complex and was essential for anoikis resistance downstream of CDCP1. Loss of CDCP1 also inhibited the metastatic potential of the A549 cells in vivo. Our findings indicate that CDCP1 is a novel target for treating cancer-specific disorders, such as metastasis, by regulating anoikis in lung adenocareinoma.
  • 神保充, 本橋詳子, 酒井隆一, 神谷久男 日本動物学会大会要旨集 78th 41 2007年08月31日 [査読無し][通常論文]
  • 局興一, 秋山伸之, 青木邦衛, 及川雅人, 酒井隆一, 島本啓子, 佐々木誠 天然有機化合物討論会講演要旨集 49th 691-696 -696 2007年08月24日 [査読無し][通常論文]
  • Makoto Sasaki, Nobuyuki Akiyama, Koichi Tsubone, Muneo Shoji, Masato Oikawa, Ryuichi Sakai TETRAHEDRON LETTERS 48 (32) 5697 -5700 2007年08月 [査読無し][通常論文]
     
    An efficient total synthesis of dysiherbaine, a potent and subtype-selective agonist for ionotropic glutamate receptors, has been achieved. An advanced key intermediate in the previous synthesis of neodysiherbaine A and its analogues was selected as the starting point, and cis-oriented amino alcohol functionality on the tetrahydropyran ring was installed by using an intramolecular S(N)2 cyclization of N-Boc-protected amino alcohol. The amino acid appendage was constructed by catalytic asymmetric hydrogenation of enamide ester. (c) 2007 Elsevier Ltd. All rights reserved.
  • Masamitsu Tanaka, Reiko Kamata, Misato Takigahira, Kazuyoshi Yanagihara, Ryuichi Sakai AMERICAN JOURNAL OF PATHOLOGY 171 (1) 68 -78 2007年07月 [査読無し][通常論文]
     
    Interaction of the Eph family of receptor protein tyrosine kinase and its ligand ephrin family induces bidirectional signaling via cell-cell contacts. High expression of B-type ephrin is frequently found in various cancer cells, and their expression levels are associated with high invasion of tumors and poor prognosis. However, whether ephrin-B1 actually promotes invasion of cancer cells in vivo has not been shown. We investigated the involvement of ephrin-B1 in regulating the invasiveness of scirrhous gastric cancer, which is a diffusely infiltrative carcinoma with high invasion potential. Reduction of ephrin-B1 expression by short interfering RNA or overexpression of phosphorylation-defective mutant suppressed migration and invasion of scirrhous gastric cancer cells in vitro without affecting tumor cell proliferation and apoptosis. Blocking of tyrosine phosphorylation of ephrin-B1 attenuates not only dissemination of cancer cells injected intraperitoneally but also local invasion and dissemination of orthotopically implanted cancer cells in the gastric wall of nude mice. Furthermore, blocking of ephrin-B1 phosphorylation attenuated the activation of Rac1 GTPase in these invasive gastric cancer cells. Our results suggest that tyrosine phosphorylation of ephrin-B1 promotes invasion of cancer cells in vivo and is a potential therapeutic target in some types of gastrointestinal cancers.
  • Masamitsu Tanaka, Kazuki Sasaki, Reiko Kamata, Ryuichi Sakai JOURNAL OF CELL SCIENCE 120 (13) 2179 -2189 2007年07月 [査読無し][通常論文]
     
    Interaction of the Eph family of receptor protein tyrosine kinases and their ligands, ephrin family members, induces bi-directional signaling via cell-cell contacts. High expression of B-type ephrin is associated with high invasion potential of tumors, however, the mechanism by which ephrin-B promotes cancer cell invasion is poorly understood. We show that interaction of ephrin-B1 with the Eph receptor B2 (EphB2) significantly enhances processing of the extracellular domain of ephrin-B1, which is regulated by the C-terminus. Matrix metalloproteinase-8 (MMP-8) is the key protease that cleaves ephrin-B1, and the C-terminus of ephrin-B1 regulates activation of the extracellular release of MMP-8 without requirement of de novo protein synthesis. One possible mechanism by which ephrin-B1 regulates the exocytosis of MMP-8 is the activation of Arf1 GTPase, a critical regulator of membrane trafficking. In support of this hypothesis, activation of ephrin-B1 increased GTP-bound Arf1, and the secretion of MMP-8 was reduced by expression of a dominant-negative mutant of Arf1. Expression of ephrin-B1 promoted the invasion of cancer cells in vivo, which required the C-terminus of ephrin-B1. Our results suggest a novel function of the C-terminus of ephrin-B1 in activating MMP-8 secretion, which promotes the invasion of cancer cells.
  • 酒井隆一 マリンバイオテクノロジー学会大会講演要旨集 10th 63 2007年05月26日 [査読無し][通常論文]
  • 酒井隆一, 松永智子, 神保充, 神谷久男 日本水産学会大会講演要旨集 2007 117 2007年03月28日 [査読無し][通常論文]
  • 神保充, 安部聡一郎, 山下洋, 石明大, 母家大樹, 岩尾研二, 小池一彦, 酒井隆一, 神谷久男 日本水産学会大会講演要旨集 2007 275 2007年03月28日 [査読無し][通常論文]
  • 木村敦子, 酒井隆一, 吉田和史, 小池一彦, 小池香苗, 小檜山篤志, 神保充, 神谷久男 日本水産学会大会講演要旨集 2007 117 2007年03月28日 [査読無し][通常論文]
  • 松永智子, 酒井隆一, 神保充, 神谷久男 日本水産学会大会講演要旨集 2007 117 2007年03月28日 [査読無し][通常論文]
  • 秋山伸之, 局興一, 青木邦衛, 庄司宗生, 酒井隆一, 及川雅人, 佐々木誠 日本化学会講演予稿集 87th (2) 1187 2007年03月12日 [査読無し][通常論文]
  • 生駒実, 及川雅人, 酒井隆一, 佐々木誠 日本化学会講演予稿集 87th (2) 1187 2007年03月12日 [査読無し][通常論文]
  • 神保充, 土橋一貴, 巳城摩倫, 三宅裕志, 吉田尊雄, 佐藤孝子, 丸山正, 酒井隆一, 神谷久男 しんかいシンポジウム予稿集 23rd 58 2007年03月 [査読無し][通常論文]
  • Crystallographic studies of a lectin from the octocoral.
    A. Kita, M. Jimbo, Y. Morimoto, R. Sakai, H. Kamiya, K. Miki The 8th Conference of the Asian Crystallographic association 0 -0 2007年 [査読無し][通常論文]
  • 局 興一, 秋山 伸之, 青木 邦衛, 及川 雅人, 酒井 隆一, 島本 啓子, 佐々木 誠 天然有機化合物討論会講演要旨集 49 (0) 691 -696 2007年 [査読無し][通常論文]
     
    Dysiherbaine (DH, 1) and its congener neodysiherbaine A (2), isolated from the Micronesian marine sponge, Dysidea herbacea, are remarkable excitatory amino acids with potent convulsant activity. DH activates the AMPA and kainate classes of ionotropic glutamate receptors, with a higher affinity for the latter. Furthermore, it has been revealed that DH had extremely high affinity for GluR5 and GluR6 kainate receptor subunits. Due to these intriguing pharmacological properties to kainite receptors, DH and its designed analogues are anticipated to serve as useful tools for understanding the complex functions of glutamate receptors in the central nervous system. In this presentation, we describe an efficient total synthesis of dysiherbaine. In addition, we report preparation and preliminary biological evaluation of several DH analogues to elucidate the detailed structure-activity relationship profile. A key intermediate 15 was synthesized by two approaches. The first synthetic route utilized compound 6 as the starting point, and the cis-oriented amino alcohol functionality on the tetrahydropyran ring was installed by using an S_N2 cyclization of N-Boc protected amino alcohol 12. An alternative and even more efficient synthetic approach to 15 featured stereoselective introduction of an amino group at C8 by iodoaminocyclization prior to constructing the bicyclic ether skeleton. The amino acid appendage was efficiently constructed by a catalytic asymmetric hydrogenation of enamide ester 24. The synthetic route developed here provided access to several DH analogues, including 9-epi-dysiherbaine (26a), 9-deoxydysiherbaine (26b), 9-O-methyldysiherbaine (26c), and N-ethyldysiherbaine (26d). The preliminary biological evaluation of these variants will be presented.
  • 神保充, 佐藤心, 吉田尊雄, 丸山正, 三宅裕志, 酒井隆一, 神谷久男 生化学 4P-0032 2007年 [査読無し][通常論文]
  • 篠原正将, 海野昌喜, 照屋健太, 酒井隆一, 堂浦克美, 佐々木誠, 齋藤正男 生化学 1P-0039 2007年 [査読無し][通常論文]
  • H. Kamiya, R. Sakai, M. Jimbo Progress in molecular and subcellular biology 43 215 -239 2006年12月01日 [査読無し][通常論文]
     
    Sea hares, belonging to the order Opisthobranchia, subclass Gastropoda, are mollusks that have attracted many researchers who are interested in the chemical defense mechanisms of these soft and "shell-less" snails. Numbers of small molecules of dietary origin have been isolated from sea hares and some have ecologically relevant activities, such as fish deterrent activity or toxicity. Recently, however, greater attention has been paid to biomedically interesting sea hare isolates such as dolastatins, a series of antitumor peptide/macrolides isolated from Dolabella auricularia. Another series of bioactive peptide/macrolides, as represented by aplyronines, have been isolated from sea hares in Japanese waters. Although earlier studies indicated the potent antitumor activity of aplyronines, their clinical development has never been conducted because of the minute amount of compound available from the natural source. Recent synthetic studies, however, have made it possible to prepare these compounds and analogs for a structure-activity relationship study, and started to uncover their unique action mechanism towards their putative targets, microfilaments. Here, recent findings of small antitumor molecules isolated from Japanese sea hares are reviewed. Sea hares are also known to produce cytotoxic and antimicrobial proteins. In contrast to the small molecules of dietary origin, proteins are the genetic products of sea hares and they are likely to have some primary physiological functions in addition to ecological roles in the sea hare. Based on the biochemical properties and phylogenetic analysis of these proteins, we propose that they belong to one family of molecule, the "Aplysianin A family," although their molecular weights are apparently divided into two groups. Interestingly, the active principles in Aplysia species and Dolabella auricularia were shown to be L-amino acid oxidase (LAAO), a flavin enzyme that oxidizes an alpha-amino group of the substrate with molecular oxygen and liberates hydrogen peroxide, with a sequence similar to other known LAAOs, including snake venom. Possible antibacterial activity and cytotoxic activity mechanisms of these proteins are also discussed.
  • Yasuhiro Sawada, Masako Tamada, Benjamin J. Dubin-Thaler, Oksana Cherniavskaya, Ryuichi Sakai, Sakae Tanaka, Michael P. Sheetz CELL 127 (5) 1015 -1026 2006年12月 [査読無し][通常論文]
     
    How physical force is sensed by cells and transduced into cellular signaling pathways is poorly understood. Previously, we showed that tyrosine phosphorylation of p130Cas (Cas) in a cytoskeletal complex is involved in force-dependent activation of the small GTPase Rap1. Here, we mechanically extended bacterially expressed Cas substrate domain protein (CasSD) in vitro and found a remarkable enhancement of phosphorylation by Src family kinases with no apparent change in kinase activity. Using an antibody that recognized extended CasSD in vitro, we observed Cas extension in intact cells in the peripheral regions of spreading cells, where higher traction forces are expected and where phosphorylated Cas was detected, suggesting that the in vitro extension and phosphorylation of CasSD are relevant to physiological force transduction. Thus, we propose that Cas acts as a primary force sensor, transducing force into mechanical extension and thereby priming phosphorylation and activation of downstream signaling.
  • 秋山伸之, 庄司宗生, 酒井隆一, SWANSON G.T, 及川雅人, 佐々木誠 化学系学協会東北大会プログラムおよび講演予稿集 2006 105 2006年09月22日 [査読無し][通常論文]
  • 松永智子, 酒井隆一, 神保充, 神谷久男 天然有機化合物討論会講演要旨集 48th 307-312 -312 2006年09月15日 [査読無し][通常論文]
  • J Huang, R Sakai, T Furuichi MOLECULAR BIOLOGY OF THE CELL 17 (7) 3187 -3196 2006年07月 [査読無し][通常論文]
     
    Crk-associated substrate (Cas) is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of the actin cytoskeletal. organization and cell migration in fibroblasts. The function of Cas in neurons, however, is poorly understood. Here we report that Cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. During cerebellar development, Cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. Cas coinununoprecipitates with Src family protein tyrosine kinases, Crk, and cell adhesion molecules and colocalizes with these proteins in granule cells. The axon extension of granule cells is inhibited by either RNA interference knockdown of Cas or overexpression of the Cas mutant lacking the YDxP motifs, which are tyrosine phosphorylated and thereby interact with Crk. These findings demonstrate that Cas acts as a key scaffold that links the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation.
  • 越川阿紗美, 酒井隆一, 小池一彦, 山下洋, 神保充, 神谷久男 マリンバイオテクノロジー学会大会講演要旨集 9th 108 2006年05月27日 [査読無し][通常論文]
  • 木村敦子, 酒井隆一, 吉田和史, 小池一彦, 小池香苗, 神保充, 神谷久男 マリンバイオテクノロジー学会大会講演要旨集 9th 109 2006年05月27日 [査読無し][通常論文]
  • 松永智子, 酒井隆一, 神保充, 神谷久男 マリンバイオテクノロジー学会大会講演要旨集 9th 109 2006年05月27日 [査読無し][通常論文]
  • 神保充, 佐藤心, 酒井隆一, 佐藤孝子, 丸山正, 三宅裕志, 神谷久男 マリンバイオテクノロジー学会大会講演要旨集 9th 101 2006年05月27日 [査読無し][通常論文]
  • 松永智子, 酒井隆一, 神保充, 神谷久男 日本水産学会大会講演要旨集 2006 184 2006年03月30日 [査読無し][通常論文]
  • 神保充, 野中敦司, 井上知美, 三宅裕志, 佐藤孝子, 丸山正, 酒井隆一, 神谷久男 日本水産学会大会講演要旨集 2006 186 2006年03月30日 [査読無し][通常論文]
  • 本橋詳子, 神保充, 酒井隆一, 神谷久男 日本水産学会大会講演要旨集 2006 185 2006年03月30日 [査読無し][通常論文]
  • 神保充, 佐藤心, 酒井隆一, 佐藤孝子, 丸山正, 三宅裕志, 神谷久男 日本水産学会大会講演要旨集 2006 185 2006年03月30日 [査読無し][通常論文]
  • 酒井隆一, 中村隆典, 西野泰斗, 山本正雄, 宮村淳史, 宮本久恵, 石綿紀久, 小松則夫, 神谷久男, 水流添暢智 日本水産学会大会講演要旨集 2006 184 2006年03月30日 [査読無し][通常論文]
  • 庄司宗生, 秋山伸之, LASH L. L, SANDERS J. M, SWANSON G. T, 酒井隆一, 島本啓子, 及川雅人, 佐々木誠 日本化学会講演予稿集 86th (2) 1356 2006年03月13日 [査読無し][通常論文]
  • 庄司宗生, 局興一, 酒井隆一, 島本啓子, 及川雅人, 佐々木誠 日本化学会講演予稿集 86th (2) 1357 2006年03月13日 [査読無し][通常論文]
  • LL Lash, JM Sanders, M Shoji, M Sasaki, R Sakai, GT Swanson FASEB JOURNAL 20 (4) A687 -A687 2006年03月 [査読無し][通常論文]
  • Ryuichi Sakai, Geoffrey T. Swanson, Makoto Sasaki, Keiko Shimamoto, Hisao Kamiya Central Nervous System Agents in Medicinal Chemistry 6 (2) 83 -108 2006年 [査読無し][通常論文]
     
    The molecular diversity of marine secondary metabolites has been recognized for a number of years, and classic marine-derived excitatory amino acids (EAAs) such as kainic and domoic acid have been indispensable tools in neurobiological research. The recent discovery of the sponge-derived EAA dysiherbaine (DH, 1), a novel di-amino di-acid glutamate analogue with potent convulsant activity, underscores the relatively untapped potential of marine organisms to serve as sources of EAAs with unique structures and activities [1]. DH (1) has a number of pharmacological actions but binds with highest affinity to kainate receptors, a sub-family of non-N-methyl-D-aspartate (non-NMDA)-type GluRs, which are also the molecular targets of other potent EAA convulsants like domoic acid. The high affinity and selectivity of 1 towards certain kainate receptor subtypes made it a useful tool for exploring aspects of the biophysical function of these ion channels [2]. In combination with chemical syntheses and neurophysiological techniques, we have shown that 1 and its structural analogues can serve as unique biophysical and physiological probes of GluR function [3]. Current studies have begun to elucidate the critical moieties on 1 that confer activity and selectivity. We anticipate that 1 will serve as a useful template upon which to build molecules with novel pharmacological actions and potential therapeutic applications. In this review, we describe the chemical, pharmacological and behavioral profile of 1 and closely related analogues, with a particular emphasis on their actions on iGluRs, a family of ligand-gated ion channel critical for excitatory neurotransmission in the mammalian brain. © 2006 Bentham Science Publishers Ltd.
  • 松永 智子, 酒井 隆一, 神保 充, 神谷 久男 天然有機化合物討論会講演要旨集 48 (0) 307 -312 2006年 [査読無し][通常論文]
     
    Long chain polyamines (LCPA), which have been known exclusively in silica wall of diatoms in nature, are thought to play a central role in diatom's silica deposition. In the present study, however, we identified LCPA for the first time from the marine sponge Axinyssa aculeata and named LCPA-Aa. The LCPA-Aa was obtained as a mixture of oligo-propaneamine sulfates of 5 to 15 repeating units (ru). It has been reported that LCPA of diatoms mediate precipitation of silica in a multivalent anion-dependent manner, in that the multivalent anions are important in facilitating the formation of large aggregate of polyamines via electrostatic interaction. Eventually the aggregate grows large enough to give rise macroscopic phase separation. Interaction between the aggregate and oligosilicic acid is of a crucial step for further "hardening" of silica precipitation. Formation of large aggregate of LCPA-Aa was demonstrated by ^1H NMR titration experiment with Ba^<2+> as significant signal broadening was observed when the ratio of sulfate ion [SO_4^<2->] and the propane amine repeating unit [ru] became about 0.36 ([SO_4^<2->]/[ru]). This value was similar to the ratio where macroscopic phase separation of polyallylamine takes place. Above results suggested that LCPA-Aa possibly involve in precipitation of silica, i.e. spicule formation, in the sponge. In fact, LCPA was also isolated from the HF-treated spicules of this sponge. We thus tested the ability of LCPA-Aa to precipitate monosilicic acid into silica in vitro. Silica precipitation was formed at pH6.5, the physiological pH of sponge cellular extract, in the presence of LCPA-Aa while no precipitation formed in the solution without LCPA-Aa. Although biologically controlled silica deposition in sponges is known to be mediated by a protein called silicatein, our results allow proposing additional mechanism on the biomineralization process in the sponges.
  • M Tanaka, R Kamata, R Sakai JOURNAL OF BIOLOGICAL CHEMISTRY 280 (51) 42375 -42382 2005年12月 [査読無し][通常論文]
     
    Eph receptors and ephrin ligands are widely expressed in epithelial cells and mediate cell-cell interaction. EphA2 is expressed in various cancer tissues and cell lines. Although the mechanism of action of EphA2 is unknown, its expression correlates with progression of the malignant phenotype of cancerous tissues. Here, we have shown that EphA2 modulates the localization and function of claudin-4, a constituent of tight junctions. EphA2 associates with claudin-4 via their extracellular domains. This association, in turn, leads to phosphorylation of the cytoplasmic carboxyl terminus of claudin-4 at Tyr-208. The tyrosine phosphorylation of claudin-4 attenuates association of claudin-4 with ZO-1, decreasing integration of claudin-4 into sites of cell-cell contact and enhancing paracellular permeability. These results indicate that EphA2 moderates the function of tight junctions via phosphorylation of claudin-4.
  • R Sakai, S Minato, K Koike, K Koike, M Jimbo, H Kamiya CELL AND TISSUE RESEARCH 322 (3) 491 -502 2005年12月 [査読無し][通常論文]
     
    Polyclonal antibodies specific for the excitatory amino acid, kainic acid (KA), were raised in rabbits. The antibody recognized KA but did not cross-react with other structurally related amino acids, including glutamate. We used this anti-KA antibody to localize KA immunohistochemically in the KA-producing red alga Digenea simplex. KA immunoreactivity was most dense in the fine cylindrical thallus, which covers the middle to upper part of the alga. The cortical cells, but not the inner layers of the main axis, and cells of the rhizoid were also stained with this antibody. The presence of KA in cells that cover the surface of the alga might reflect its role in chemical defense. At the subcellular level, KA immunoreactivity was most intense in the nucleus, pit plugs, and the electron-dense areas denoted as "granule bodies", which were found only in the pericentral cells of the thallus.
  • R Sakai, T Nakamura, T Nishino, M Yamamoto, A Miyamura, H Miyamoto, N Ishiwata, N Komatsu, H Kamiya, N Tsuruzoe BIOORGANIC & MEDICINAL CHEMISTRY 13 (23) 6388 -6393 2005年12月 [査読無し][通常論文]
     
    Four xanthocillins (1-4), including a new compound 4, were isolated from cultured marine fungus Basipetospora sp. as thrombopoietin (TPO) mimics. Compounds 1-4 promoted the proliferation of a TPO-sensitive human leukemia cell line, UT-7/TPO, and UT-7/EPO-mpl, genetically engineered to express c-Mpl, a receptor for TPO in dose-dependent manners. However, the proliferation of UT-7/EPO, a parental cell line of UT-7/EPO-mpl that was devoid of TPO receptor, was not affected by them. Thrombopoietic action of compound 1 was nearly as potent as that of TPO, inducing cell proliferation at a concentration ranging from 1 to 100 nM. Compound 1 also induced the phosphorylation of several proteins, including Janus kinase 2 (Jak2), signal transducers, and activators of transcription-3 (STAT3) and STAT5 in the UT-7/EPO-mpl cell line, but not in the UT-7/EPO cell line. These data indicated that xanthocillins are putative agonists for c-Mpl, as their cellular actions were analogous to those of TPO. (c) 2005 Elsevier Ltd. All rights reserved.
  • M Tanaka, R Kamata, R Sakai EMBO JOURNAL 24 (21) 3700 -3711 2005年11月 [査読無し][通常論文]
     
    The interaction of the Eph family of receptor protein tyrosine kinase and its ligand ephrin family induces bidirectional signaling via the cell-cell contacts. Although most previous studies have focused on the function of Eph-ephrin pathways in the neural system and endothelial cells, this process also occurs in epithelial and cancer cells, of which the biological involvement is poorly understood. We show that ephrin-B1 creates an in vivo complex with adjacent claudin1 or claudin4 via the extracellular domains of these proteins. The cytoplasmic domain of ephrin-B1 was phosphorylated on tyrosine residues upon the formation of cell-cell contacts, possibly recognizing an intercellular adhesion of claudins. Phosphorylation of ephrin-B1 induced by claudins was abolished by the treatment with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, an inhibitor of the Src family kinases. Moreover, overexpression of ephrin-B1 triggered consequent change in the level of cell-cell adhesion depending on its phosphorylation. These results suggest that ephrin-B1 mediated the cell cell adhesion of epithelial and cancer cells via a novel Eph receptor-independent mechanism.
  • 庄司宗生, 塩原薫, 及川雅人, 酒井隆一, SWAMSON G. T, SANDERS J. A, 島本啓子, 佐々木誠 天然有機化合物討論会講演要旨集 47th 49-54 -54 2005年09月15日 [査読無し][通常論文]
  • S Seo, T Asai, T Saito, T Suzuki, Y Morishita, T Nakamoto, M Ichikawa, G Yamamoto, M Kawazu, T Yamagata, R Sakai, K Mitani, S Ogawa, M Kurokawa, S Chiba, H Hirai JOURNAL OF IMMUNOLOGY 175 (6) 3492 -3501 2005年09月 [査読無し][通常論文]
     
    The lymphocyte-specific Cas family protein Cas-L (Crk-associated substrate lymphocyte type) has been implicated to function in lymphocyte movement, mediated mainly by integrin signaling. However, its physiological role is poorly understood. In this study we analyzed the function of Cas-L in lymphocytes using gene-targeted mice. The mutant mice showed a deficit of marginal zone B (MZB) cells and a decrease of cell number in secondary lymphoid organs. An insufficient chemotactic response and perturbed cell adhesion were observed in Cas-L-deficient lymphocytes, suggesting that the aberrant localization was responsible for the deficit of MZB cells. Moreover, we found that lymphocyte trafficking was altered in Cas-L-deficient mice, which gave a potential reason for contraction of secondary lymphoid tissues. Thus, Cas-L affects homeostasis of MZB cells and peripheral lymphoid organs, which is considered to be relevant to impaired lymphocyte migration and adhesion.
  • JM Sanders, K Ito, L Settimo, OT Pentikainen, M Shoji, M Sasaki, MS Johnson, R Sakai, GT Swanson JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 314 (3) 1068 -1078 2005年09月 [査読無し][通常論文]
     
    Kainate receptors show a particular affinity for a variety of natural source compounds, including dysiherbaine (DH), a potent agonist derived from the marine sponge Dysidea herbacea. In this study, we characterized the pharmacological activity and structural basis for subunit selectivity of neodysiherbaine (neoDH) and MSVIII-19, which are natural and synthetic analogs of DH, respectively. NeoDH and MSVIII-19 differ from DH in the composition of two functional groups that confer specificity and selectivity for ionotropic glutamate receptors. In radioligand binding assays, neoDH displayed a 15- to 25-fold lower affinity relative to that of DH for glutamate receptor (GluR)5 and GluR6 kainate receptor subunits but a 7-fold higher affinity for kainate (KA)2 subunits, whereas MSVIII-19 displaced [H-3] kainate only from GluR5 subunits but not GluR6 or KA2 subunits. NeoDH was an agonist for kainate and alpha-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in patch-clamp recordings; in contrast, MSVIII-19 acted as a potent antagonist for homomeric GluR5 receptor currents with weaker activity on other kainate and AMPA receptors. Neither neoDH nor MSVIII-19 activated group I metabotropic GluRs. Homology modeling suggests that two critical amino acids confer the high degree of selectivity between the dysiherbaine analogs and the GluR5 and KA2 subunits. In summary, these data describe the pharmacological activity of two new compounds, one of which is a selective GluR5 receptor antagonist that will be of use for understanding native receptor function and designing more selective ligands for kainate receptors.
  • M Shoji, K Shiohara, M Oikawa, R Sakai, M Sasaki TETRAHEDRON LETTERS 46 (33) 5559 -5562 2005年08月 [査読無し][通常論文]
     
    Synthesis of dysiherbaine analogue 4, which corresponds to 8,9-epi-neodysiherbaine A, is described. The synthesis features a concise route to the bicyclic ether skeleton through stereoselective C-glycosylation to set the C-6 stereocenter and 5-exo ring-closure to form the tetrahydrofuran ring. The results of preliminary biological studies of 4 are also provided. (c) 2005 Elsevier Ltd. All rights reserved.
  • Y Osajima-Hakomori, Miyake, I, M Ohira, A Nakagawara, A Nakagawa, R Sakai AMERICAN JOURNAL OF PATHOLOGY 167 (1) 213 -222 2005年07月 [査読無し][通常論文]
     
    Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor originally identified as part of the chimeric nucleophosmin-ALK protein in the t(2;5) chromosomal rearrangement associated with anaplastic large cell lymphoma. We recently demonstrated that the ALK kinase is constitutively activated by gene amplification at the ALK locus in several neuroblastoma cell lines. Forming a stable complex with hyperphosphorylated ShcC, activated ALK modifies the responsiveness of the mitogen-activated protein kinase pathway to growth factors. In the present study, the biological role of activated ALK was examined by suppressing the expression of ALK kinase in neuroblastoma cell lines using an RNA interference technique. The suppression of activated ALX in neuroblastoma cells by RNA interference significantly reduced the phosphorylation of ShcC, mitogen-activated protein kinases, and Akt, inducing rapid apoptosis in the cells. By immunohistochemical analysis, the cytoplasmic expression of ALK was detected in most of the samples of neuroblastoma tissues regardless of the stage of the tumor, whereas significant amplification of ALK was observed in only 1 of 85 cases of human neuroblastoma. samples. These data demonstrate the limited frequency of ALK activation in the real progression of neuroblastoma.
  • K Azuma, M Tanaka, T Uekita, S Inoue, J Yokota, Y Ouchi, R Sakai ONCOGENE 24 (30) 4754 -4764 2005年07月 [査読無し][通常論文]
     
    To acquire information on signal alteration corresponding to the changes in metastatic potential, we analysed protein tyrosine phosphorylation of low- and high-metastatic human osteosarcoma HuO9 sublines, which were recently established as the first metastatic model of human osteosarcoma. Tyrosine phosphorylation of proteins around 60, 70, and 120 - 130 kDa was enhanced in high-metastatic sublines. Among these proteins, the protein around 70kDa, which was most remarkably phosphorylated, was identified as paxillin, a scaffold protein in integrin signaling. Activity of Src family kinase correlated well with metastatic potential, and a Src family kinase inhibitor, PP2, not only abolished tyrosine phosphorylation of paxillin but also impaired the motility of high-metastatic sublines. The expression of paxillin was also elevated in high-metastatic sublines, and knocking down of paxillin expression by RNAi method resulted in attenuated motility of high-metastatic cells. We also demonstrated that the phosphorylated form of paxillin is essential for the migration-promoting effect in human osteosarcoma. These findings suggest that enhanced activity of Src family kinases and overexpression of paxillin synergistically contribute to the high metastatic potential of human osteosarcoma through the hyperphosphorylation of paxillin.
  • Mitsuru Jimbo, Kazuhiko Koike, Ryuichi Sakai, Koji Muramoto, Hisao Kamiya Biochemical and Biophysical Research Communications 330 (1) 157 -162 2005年04月29日 [査読無し][通常論文]
     
    In the present study, the entire amino acid sequence and cDNA structure encoding the d-galactose-binding lectin, SLL-2, isolated from the octocoral Sinularia lochmodes, were determined. SLL-2 regulates the morphology of symbiotic dinoflagellates Symbiodinium spp. through unknown mechanisms. Here, three cDNAs that encode SLL-2 were cloned and characterized. All the SLL-2 cDNAs encoded 142 amino acids with high similarity to each other. The mature subunit of SLL-2 was found to be composed of 94 amino acids and to contain one putative glycosylation site common to all three SLL-2. N-Glycopeptidase F treatment of SLL-2 resulted in a protein band shift from 16.5 to 9.5 kDa in SDS-PAGE, confirming that SLL-2s are glycoproteins. Two-dimensional polyacrylamide gel electrophoresis analysis of the deglycosylated SLL-2 indicated a presence of three polypeptides as encoded in SLL-2 cDNAs. The deduced sequences of SLL-2 cDNAs had a similarity to the C-terminal region of discoidin I, the slime mold Dictyostelium discoideum lectin. © 2005 Elsevier Inc. All rights reserved.
  • Miyake, I, Y Hakomori, Y Misu, H Nakadate, N Matsuura, M Sakamoto, R Sakai ONCOGENE 24 (19) 3206 -3215 2005年04月 [査読無し][通常論文]
     
    ShcC is a family member of the Shc docking proteins that possess two different phosphotyrosine-binding motifs and conduct signals as Grb2-binding substrates of various receptor tyrosine kinases. We have recently shown that some neuroblastoma cell lines, such as NB-39-nu cells, express a protein complex of hyperphosphorylated ShcC and anaplastic lymphoma kinase (ALK), which is self-activated by gene amplification. Here, we demonstrate that the expression of a mutant ShcC lacking Grb2-binding sites, 3YF-ShcC, significantly impaired the survival, differentiation and motility of NB-39-nu cells by blocking the ERK and Akt pathways. On the other hand, cells overexpressing ShcC or 3YF-ShcC, but not a mutant ShcC that lacks SH2, showed decreased anchorage independency and in vivo tumorigenicity, suggesting a novel ShcC-specific suppressive effect through its SH2 domain on cell transformation. Notably, overexpression of ShcC suppressed the sustained phosphorylation of Src family kinase after cell detachment, which might be independent of phosphorylation of Grb2-binding site. It was indicated that the Src/Fyn-Cas pathway is modulated as a target of these suppressive effects by ShcC. Reciprocal change of ShcC expression and phosphorylation observed in malignant neuroblastoma cell lines might be explained by these phosphotyrosine-dependent and - independent functions of ShcC.
  • 木村敦子, 酒井隆一, 吉田和史, 小池香苗, 小池一彦, 神保充, 神谷久男 日本水産学会大会講演要旨集 2005 123 2005年04月01日 [査読無し][通常論文]
  • 酒井隆一, 平田泰子, 小池一彦, 神谷久男 日本水産学会大会講演要旨集 2005 116 2005年04月01日 [査読無し][通常論文]
  • 神保充, 尾野壮一, 酒井隆一, 神谷久男 日本水産学会大会講演要旨集 2005 119 2005年04月01日 [査読無し][通常論文]
  • 本橋詳子, 宮根敦子, 神保充, 酒井隆一, 神谷久男 日本水産学会大会講演要旨集 2005 118 2005年04月01日 [査読無し][通常論文]
  • 酒井 隆一 日本水産学会誌 71 (2) 232 -232 2005年03月15日 [査読無し][通常論文]
  • 庄司宗生, 塩原薫, 及川雅人, 佐々木誠, 酒井隆一 日本化学会講演予稿集 85th (2) 854 2005年03月11日 [査読無し][通常論文]
  • Y Miyamoto, L Chen, M Sato, M Sokabe, T Nabeshima, T Pawson, R Sakai, N Mori JOURNAL OF NEUROSCIENCE 25 (7) 1826 -1835 2005年02月 [査読無し][通常論文]
     
    N-Shc (neural Shc) (also ShcC), an adapter protein possessing two phosphotyrosine binding motifs [PTB (phosphotyrosine binding) and SH2 (Src homology 2) domains], is predominantly expressed in mature neurons of the CNS and transmits neurotrophin signals from the TrkB receptor to the Ras/mitogen-activated protein kinase (MAPK) pathway, leading to cellular growth, differentiation, or survival. Here, we demonstrate a novel role of ShcC, the modulation of NMDA receptor function in the hippocampus, using ShcC gene-deficient mice. In behavioral analyses such as the Morris water maze, contextual fear conditioning, and novel object recognition tasks, ShcC mutant mice exhibited superior ability in hippocampus-dependent spatial and nonspatial learning and memory. Consistent with this finding, electrophysiological analyses revealed that hippocampal long-term potentiation in ShcC mutant mice was significantly enhanced, with no alteration of presynaptic function, and the effect of an NMDA receptor antagonist on its expression in the mutant mice was notably attenuated. The tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B was also increased, suggesting that ShcC mutant mice have enhanced NMDA receptor function in the hippocampus. These results indicate that ShcC not only mediates TrkB-Ras/MAPK signaling but also is involved in the regulation of NMDA receptor function in the hippocampus via interaction with phosphotyrosine residues on the receptor subunits and serves as a modulator of hippocampal synaptic plasticity underlying learning and memory.
  • 庄司 宗生, 塩原 薫, 及川 雅人, 酒井 隆一, Sanders James M., Swanson Geoffrey T., 島本 啓子, 佐々木 誠 天然有機化合物討論会講演要旨集 47 (0) 49 -54 2005年 [査読無し][通常論文]
     
    Dysiherbaine (DH, 1) and its congener neodysiherbaine A (2), isolated from the Micronesian marine sponge, Dysidea herbacea, are novel excitatory amino acids with potent convulsant activity. DH activates non-NMDA type glutamate receptors [AMPA and kainic acid (KA) receptors] with considerable preference over KA receptors. Moreover, it has been shown that DH binds to the GluR5 and GluR6 KA receptor subunits with high affinity. Due to these intriguing pharmacological properties to KA receptors, DH and its designed analogues are anticipated to serve as useful tools for understanding the structure and functions of glutamate receptors in the central nervous system. In order to reveal the detailed structure-activity relationship profiles of DH, we undertook a diverted synthesis of structural analogues of DH. In this paper, we report the synthesis of simplified analogue 3,8,9-epi-neodysiherbaine A (4a), 8-epi-neodysiherbaine A (4b), 8-deoxyneodysiherbaine A (4c) and 9-deoxyneodysiherbaine A (4d) from a common intermediate 5. The synthesis of 5 started with C-glycosylation of allylsilane 9 with diacetyl-L-arabinal (10), which led to C-glycoside 8 as the sole product. Chemo- and stereoselective dihydroxylation using (DHQD)_2AQN and subsequent epoxidation delivered epoxy alcohol 7, which underwent epoxide opening/5-exo ring-closure during chromatography on silica gel, leading to bicyclic ether core 12. Stereoselective construction of the amino acid chain was efficiently realized by DuPHOS-mediated asymmetric hydrogenation of enamido ester 17 to generate key intermediate 5 via 18. Global deprotection of 5 by acid hydrolysis furnished analogue 4a. Selective deprotection of the acetonide group of 5 was achieved by using DDQ to afford diol 19, which was further converted to other analogues (3 and 4b-d) via cyclic sulfate 20 and thiocarbonate 24. The toxicity of analogues 4a-d were tested on mice. Intracerebral injection of analogue 4a did not induce any convulsant behavior even at higher dose (20μg/mouse), whereas that of 4b induced a sleeper effect. Analogues 4c and 4d were found to show convulsant activity although the potency was much weaker than that of the natural DH (1). In the radioligand binding assay using rat synaptic membrane preparation, 4b displaced [^3H]KA and [^3H]AMPA with IC_<50> values of 24.1±6.8μM and 9.7±2.3μM, respectively. In contrast, analogue 4a did not displace these radioligand from receptors even at 100μM. In addition, detailed pharmacological studies revealed that simplified analogue 3 is a selective antagonist for GluR5 KA receptors. Further neurophysiological studies of analogues are underway and will be described.
  • 佐藤心, 宮尾佳子, 神保充, 酒井隆一, 神谷久男, 佐藤孝子, 丸山正 日本水産学会北海道支部大会講演要旨集 2005 82 2005年 [査読無し][通常論文]
  • Kotaro Azuma, Kotaro Azuma, Kuniko Horie, Satoshi Inoue, Satoshi Inoue, Yasuyoshi Ouchi, Ryuichi Sakai FEBS Letters 577 (3) 339 -344 2004年11月 [査読無し][通常論文]
     
    There is accumulating evidence that the estrogen receptor (ER) can transduce specific signals at the plasma membrane. We tried to clarify the biological function of ER as a signaling molecule by identifying proteins that interact with the membrane-localized ER. The activation function 1 and 2 (AF-1 and AF-2) domains of ERα with or without the membrane-targeting sequence were stably expressed in the breast cancer cell line, MCF-7. The level of tyrosine phosphorylation of AF-2 was significantly elevated by the membrane localization. By mass-spectrometry analysis, α- and β-tubulins and heat shock protein 70 were identified as the AF-1-associated proteins. Of these, tubulins are associated only with membrane-targeted AF-1. © 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Ryuichi Sakai, Ryuichi Sakai Biotherapy 18 397 -404 2004年09月01日 [査読無し][通常論文]
     
    Src family kinases have been investigated intensively in relationship to cancer development and progression, although their precise functions in cancer remain unclear. Numerous studies on Src family kinases to date have revealed them to be key molecules regulating proliferation, metastasis and invasion of tumor cells and most promising targets for anticancer therapeutics.
  • 神保充, 酒井隆一, 神谷久男, 太田英司, 村本光二 日本水産学会大会講演要旨集 2004 198 2004年03月31日 [査読無し][通常論文]
  • 神保充, 江頭浩司, 高橋正興, 得能大輔, 酒井隆一, 神谷久男 日本水産学会大会講演要旨集 2004 199 2004年03月31日 [査読無し][通常論文]
  • 酒井隆一, 神谷久男, 鈴木克治 日本水産学会大会講演要旨集 2004 191 2004年03月31日 [査読無し][通常論文]
  • 湊早樹子, 酒井隆一, 小池香苗, 小池一彦, 神保充, 神谷久男 日本水産学会大会講演要旨集 2004 195 2004年03月31日 [査読無し][通常論文]
  • 佐藤心, 神保充, 酒井隆一, 神谷久男 日本水産学会大会講演要旨集 2004 196 2004年03月31日 [査読無し][通常論文]
  • JM Sanders, K Ito, M Shoji, M Sasaki, R Sakai, GT Swanson FASEB JOURNAL 18 (5) A962 -A962 2004年03月 [査読無し][通常論文]
  • M Tanaka, R Ohashi, R Nakamura, K Shinmura, T Kamo, R Sakai, H Sugimura EMBO JOURNAL 23 (5) 1075 -1088 2004年03月 [査読無し][通常論文]
     
    Bidirectional signals mediated by Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, play pivotal roles in the formation of neural networks by induction of both collapse and elongation of neurites. However, the downstream molecular modules to deliver these cues are largely unknown. We report here that the interaction of a Rac1-specific guanine nucleotide-exchanging factor, Tiam1, with ephrin-B1 and EphA2 mediates neurite outgrowth. In cells coexpressing Tiam1 and ephrin-B1, Rac1 is activated by the extracellular stimulation of clustered soluble EphB2 receptors. Similarly, soluble ephrin-A1 activates Rac1 in cells coexpressing Tiam1 and EphA2. Cortical neurons from the E14 mouse embryos and neuroblastoma cells significantly extend neurites when placed on surfaces coated with the extracellular domain of EphB2 or ephrin-A1, which were abolished by the forced expression of the dominant-negative mutant of ephrin-B1 or EphA2. Furthermore, the introduction of a dominant-negative form of Tiam1 also inhibits neurite outgrowth induced by the ephrin-B1 and EphA2 signals. These results indicate that Tiam1 is required for neurite outgrowth induced by both ephrin-B1-mediated reverse signaling and EphA2-mediated forward signaling.
  • R Sakai, K Suzuki, K Shimamoto, H Kamiya JOURNAL OF ORGANIC CHEMISTRY 69 (4) 1180 -1185 2004年02月 [査読無し][通常論文]
     
    Three new betaines, dysibetaine PP (1), dysibetaine CPa (2), and dysibetaine CPb (3), were isolated from an aqueous extract of the marine sponge Dysidea herbacea collected in Yap state, Micronesia. The structure of 1 was determined by spectral methods as well as chemical degradation to be a novel dipeptide betaine, and those for 2 and 3 were determined to be unprecedented cyclopropane betaines. Compounds 2 and 3 showed weak affinity toward the N-methyl-D-aspartic acid-type and the kainic acid-type glutamate receptors, respectively, in a radioligand binding assay.
  • Y Shigeri, R Sakai, K Takaoka, N Yumoto, T Nakajima, S Amara, K Shimamoto JOURNAL OF PHARMACOLOGICAL SCIENCES 94 163P -163P 2004年 [査読無し][通常論文]
  • Koike K, Jimbo M, Sakai R, Kaeriyama M, Muramoto K, Ogata T, Maruyama T, Kamiya H.Octocoral chemical signaling selects and controls dinoflagellate symbionts.
    Biol. Bull. 207, 80-86 2004年 [査読無し][通常論文]
  • K. Shimamoto, R. Sakai, K. Takaoka, N. Yumoto, T. Nakajima, S. G. Amara, and Y. Shigeri Characterization of Novel L-threo-b-benzyloxyaspartate Derivatives, High Affinity Blockers for the Glutamate Transporters
    Mol. Pharmacol. 65, 1108-1015. 2004年 [査読無し][通常論文]
  • 酒井 隆一, スワンソン ジェフリー, 島本 啓子, 神谷 久男 日本生理学会大会発表要旨集 2004 (0) S13 -S13 2004年 [査読無し][通常論文]
     
    Dysiherbaine (DH) is a novel marine sponge-derived amino acid and is highly epileptogenic in mice. Administration of DH induced long lasting convulsive behaviors with ED50 values of 13 pmol/mouse, i.c.v., and 0.97 mg/kg, i.p. which is 5-7 time more potent than that of domoic acid. In rat brain synaptic membranes DH displaced binding of [3H]kainic acid (KA) and [3H]AMPA with Ki values of 26 and 153 nM, respectively. In contrast, DH did not displace the NMDA receptor ligand [3H] CGS-19755. DH also displaced [3H]KA from the recombinant GluR5 and GluR6 at Ki value of each 0.74 and 1.2 nM, respectively. In whole-cell voltage clamp recordings from cultured rat hippocampal neurons DH evoked inward currents from both AMPA and KA receptors with EC50 values of 9.7 μM and 210 nM, respectively. Additionally DH activated mGlu5 but not mGluR1. In the heteromerically expressed KA receptors, GluR5-KA2, in HEK 293 cells DH evoked desensitizing inward current. However, the inward current arose again after removal of DH. Application of glutamate to this "activated" receptor can further elicit the desensitizing inward current. Surprisingly, non-desensitizing inward current was gated by CNQX, a classically defined antagonist. These results demonstrated that DH can activate only high affinity GluR5 site in the heteromerically assembled GluR5/KA2, and that for the first time the low affinity subunit, KA2, can gate channel currents individually upon application of the agonists in quite unexpected ways. [Jpn J Physiol 54 Suppl:S13 (2004)]
  • M Jimbo, F Nakanishi, R Sakai, K Muramoto, H Kamiya FISHERIES SCIENCE 69 (6) 1240 -1246 2003年12月 [査読無し][通常論文]
     
    L-amino acid oxidase (LAAO) activity, as well as mechanisms of antimicrobial action of aplysianin A, a sea hare-derived 340 kDa hornotetrameric protein, was determined. Spectrophotometric and High-performance liquid chromatography analyses of aplysianin A indicated that one flavin adenine dinucleoticle, a cofactor of LAAO, bound to each subunit of the homotetramer. Aplysianin A can specifically catalyze oxidation of basic amino acids (L-arginine and L-lysine), and is the first protein from marine invertebrate animals with LAAO activity. Substrate specificity of aplysianin A is markedly different from that of commonly known LAAO, such as snake venom LAAO, which prefer hydrophobic amino acids. Km value of aplysianin A was the smallest of those for all known LAAO reported. In the presence of catalase, the antibacterial activity of aplysianin A was inhibited as expected, indicating that the antibacterial action of aplysianin A results from hydrogen peroxide production during the reaction with substrates. Interestingly, aplysianin A acted as an antibacterial agent even in the presence of excess catalase. Antibacterial assays in various media suggested that this phenomenon was simply attributed to the consumption of amino acids required for bacterial growth in the media by aplysianin A.
  • 神保充, 江頭浩司, 高橋正興, 得能大輔, 飯島亮介, 山崎正利, 村本光二, 酒井隆一, 神谷久男 日本分子生物学会年会プログラム・講演要旨集 26th 583 2003年11月25日 [査読無し][通常論文]
  • JH Huang, T Asawa, T Takato, R Sakai JOURNAL OF BIOLOGICAL CHEMISTRY 278 (48) 48367 -48376 2003年11月 [査読無し][通常論文]
     
    Src family kinases are major regulators of various integrin-mediated biological processes, although their functional roles and substrates in cancer metastasis are unknown. We explored the roles of Src family tyrosine kinases in cell migration and the spread of K-1735 murine melanoma cell lines with low or high metastatic potential. Corresponding to elevated cell motility and spreading ability, Fyn was selectively activated among Src family kinases, and the cell motility was blocked by an inhibitor of Src family kinases. Significant tyrosine phosphorylation of cortactin, stable complex formation between activated Fyn and cortactin, and co-localization of cortactin with Fyn at cell membranes were all observed only in cells with high metastatic potential. Both integrin-mediated Fyn activation and hyperphosphorylation of cortactin were observed 2 - 5 h after stimulation in highly metastatic cells, and they required de novo protein synthesis. We demonstrate that cortactin is a specific substrate and cooperative effector of Fyn in integrin-mediated signaling processes regulating metastatic potential.
  • 酒井隆一, 吉田和文, 小池香苗, 神保充, 小池一彦, 湊早樹子, 神谷久男 天然有機化合物討論会講演要旨集 45th 629-634 -634 2003年09月01日 [査読無し][通常論文]
  • 神保充, 畠中聡, 飯島亮介, 酒井隆一, 山崎正利, 神谷久男 日本水産学会大会講演要旨集 2003 253 2003年04月01日 [査読無し][通常論文]
  • 吉田和史, 酒井隆一, 小池香苗, 小池一彦, 神保充, 神谷久男 日本水産学会大会講演要旨集 2003 247 2003年04月01日 [査読無し][通常論文]
  • 酒井隆一, 松原裕樹, 神保充, 神谷久男, 島本啓子, 浪越通夫 日本水産学会大会講演要旨集 2003 247 2003年04月01日 [査読無し][通常論文]
  • 湊早樹子, 酒井隆一, 小池香苗, 小池一彦, 神保充, 神谷久男 日本水産学会大会講演要旨集 2003 247 2003年04月01日 [査読無し][通常論文]
  • 酒井 隆一, 吉田 和文, 小池 香苗, 神保 充, 小池 一彦, 湊 早樹子, 神谷 久男 天然有機化合物討論会講演要旨集 45 (0) 629 -634 2003年 [査読無し][通常論文]
     
    Cellular localization of the marine sponge-derived excitatory amino acid dysiherbaine (1) was investigated using immunohistochemical methods. Dysiherbaine (1), isolated from a sponge Dysidea herbacea, selectively activates mammalian non-NMDA type glutamate receptors (GluRs) and induces seizure in mice. Recently 1 was found to have unusually high affinity towards GluR5 and 6, subtypes of kainite-type GluRs, and can activate these subtype receptors selectively in the heteromerically expressed receptor complex. These unusual actions of 1 to the neuronal receptors warrants its usefulness as a research tool, however, its production, biosynthesis, and roles in the natural environment are left to be investigated. Since microorganisms including a cyanobacterial, Oscillatoria spongeliae are known to be heavily associated with D. herbacea, actual producers of the secondary metabolites and their roles in the sponge and associated microorganisms are difficult problem to solve. In this study, we paved a way to approach this elusive problems by showing cellular localization of 1 using immnumohistochemical methods. We also show a presence of putative DH-binding protein of 111kD using DH-conjugated affinity chromatography and SDS-PAGE.
  • 酒井隆一, 鈴木克治, 石田真喜子, 神谷久男, 佐々木誠, 橘和夫, 小池達樹, 島本啓子, 波越通夫 天然有機化合物討論会講演要旨集 44th 295-300 -300 2002年09月01日 [査読無し][通常論文]
  • Miyake, I, Y Hakomori, A Shinohara, T Gamou, M Saito, A Iwamatsu, R Sakai ONCOGENE 21 (38) 5823 -5834 2002年08月 [査読無し][通常論文]
     
    She family of docking proteins, ShcA, Shell and ShcC, play roles in cellular signal transduction by binding to phosphotyrosine residues of various activated receptor tyrosine kinases. Both Shell and ShcC proteins are selectively expressed in the neural system of adult mouse tissues. In most of neuroblastoma cells, obvious tyrosine phosphorylation of ShcC was observed, whereas expression of ShcB was considerably low. Phosphoproteins associated with hyperphosphorylated ShcC were purified from neuroblastoma cell lines, and identified by mass-spectrometry. Anaplastic lymphoma kinase (ALK), which turned out to be one of these phosphoproteins, was constitutively activated and associated with the PTB domain of ShcC in three neuroblastoma cells. In vitro kinase assay revealed that ShcC is a potent substrate of the activated ALK kinase. The ALK gene locus was significantly amplified in both of these cell lines, suggesting that gene amplification leads to constitutive activation of the ALK kinase, which results in hyperphosphorylation of ShcC. Constitutive activation of ALK appeared to interfere with signals from other receptor tyrosine kinases. ALK-ShcC signal activation, possibly caused. by co-amplification with the N-myc gene, might give additional effects on malignant tumor progression of neuroblastoma.
  • JH Huang, H Hamasaki, T Nakamoto, H Honda, H Hirai, M Saito, T Takato, R Sakai JOURNAL OF BIOLOGICAL CHEMISTRY 277 (30) 27265 -27272 2002年07月 [査読無し][通常論文]
     
    The Crk-associated substrate Was) is a unique docking protein that possesses a repetitive stretch of tyrosine-containing motifs and an Src homology 3 (SH3) domain. Embryonic fibroblasts lacking Cas demonstrated resistance to Src-induced transformation along with impaired actin bundling and cell motility, indicating critical roles of Cas in actin cytoskeleton organization, cell migration, and oncogenesis. To gain further insight into roles of each domain of Cas in these processes, a compensation assay was performed by expressing a series of Cas mutants in Cas-deficient fibroblasts. The results showed that motifs containing YDxP were indispensable for actin cytoskeleton. organization and cell migration, suggesting that CrkII-mediated signaling regulates these biological processes. The C-terminal Src-binding domain played essential roles in cell migration and membrane localization of Cas, although it was dispensable in the organization of actin stress fibers. Furthermore, the Src-binding domain was also a prerequisite for Src transformation possibly, because of its crucial role in the phosphorylation of Cas during transformation. Overall, differential uses of the Cas domains in individual biological processes were demonstrated.
  • GT Swanson, T Green, R Sakai, A Contractor, W Che, H Kamiya, SF Heinemann NEURON 34 (4) 589 -598 2002年05月 [査読無し][通常論文]
     
    Neuronal kainate receptors are assembled from subunits with dissimilar specificities for agonists and antagonists. The composite biophysical behavior of heteromeric kainate receptors is determined by intersubunit interactions whose nature is unclear. Here we use dysiherbaine, a selective kainate receptor agonist, to show that GluR5 subunits assembled in heteromeric GluR5/KA-2 kainate receptor complexes can gate current without concomitant activation of their partner KA-2 subunits. A long-lasting interaction between dysiherbaine and GluR5 subunits elicits a tonic current from GluR5/KA-2 receptors; subsequent cooperative gating of KA-2 subunits can be elicited by both agonists, such as glutamate, and some classically defined antagonists, such as CNQX. This study demonstrates that each type of subunit within a heteromeric kainate receptor contributes a distinct conductance upon activation by agonist binding, and therefore provides insight into the biophysical function of ionotropic glutamate receptors.
  • 鈴木克治, 酒井隆一, 神谷久男 日本水産学会大会講演要旨集 2002 201 2002年03月30日 [査読無し][通常論文]
  • 本郷直人, 佐藤繁, 小藤田安希子, 酒井隆一, 児玉正昭 日本水産学会大会講演要旨集 2002 215 2002年03月30日 [査読無し][通常論文]
  • 成毛央人, 神保充, 酒井隆一, 村本光二, 神谷久男 日本水産学会大会講演要旨集 2002 213 2002年03月30日 [査読無し][通常論文]
  • Tetsuya Nakamoto, Takahiro Suzuki, Jinhong Huang, Tomoko Matsumura, Sachiko Seo, Hiroaki Honda, Ryuichi Sakai, Hisamaru Hirai Biochemical and Biophysical Research Communications 294 (3) 635 -641 2002年 [査読無し][通常論文]
     
    p130Cas (Cas) is a docking protein that becomes tyrosine phosphorylated in v-Src- or v-Crk-transformed cells and in integrin-stimulated cells. Cas -/- fibroblasts show defects in stress fiber formation, cell spreading, cell migration, and transformation by activated Src. To further characterize the role of Cas in signaling, we compared the expression profile in Cas -/- fibroblasts with that in Cas-re-expressing fibroblasts using the microarray methods. In Cas -/- fibroblasts, the expression of heme oxygenase 1 and caveolin-1 was reduced, but the expression of procollagen 1 α 1, procollagen 3 α 1, procollagen 11 α 1, elastin, periostin, TSC-36, and MARCKS was enhanced. The domains in Cas necessary for the change varied among these genes. Activated Src reduced the expression of most of these genes both in Cas -/- and in Cas +/+ fibroblasts. These results suggest the existence of signaling pathways that emanate from Cas to gene expression. © 2002 Elsevier Science (USA). All rights reserved.
  • Mitsuru Jimbo, Fumie Nakanishi, Ryuichi Sakai, Koji Muramoto, Hisao Kamiya Fisheries Science 68 1635 -1636 2002年 [査読無し][通常論文]
     
    cDNA encoding the sea hare-derived antitumour-antibacterial protein Aplysianin A (APA) was expressed on Escherichia coli. APA displays sequence similarity to L-amino acid oxidase (LAAO) found in the snake {Crotalus atrox) venom. Spectrophotometric analysis along with thin layer chromatography of APA indicated that one flavin adenine dinucleotide, a cofactor of LAAO, bound to each subunit of the homotetramer. APA also displayed LAAO activity. Specifically, treatment of L-phenylalanine with APA resulted in the generation of hydrogen peroxide. This finding indicated that the antibacterial activity of APA is mainly caused by hydrogen peroxide production. © 2002, The Japanese Society of Fisheries Science. All rights reserved.
  • Hiroshi Yako, Mitsuru Jimbo, Ryuichi Sakai, Nobuhiko Takamatsu, Tadayoshi Shiba, Koji Muramoto, Hisao Kamiya Fisheries Science 68 1127 -1130 2002年 [査読無し][通常論文]
     
    Megabalanus rosa contained the C-type lectins as the major constituents of hemolymph proteins. BRA-1 (330 kDa) and BRA-2 (140 kDa) were composed of identical subunits (173 amino acids), and BRA-3 (64 kDa) of four same subunits (138 amino acids). These lectins were present in various tissues other than the hemolymph. However, only BRA-2 was detected in the organic matrix of shell by western blotting. Both BRA-2 and BRA-3 showed high association constants to calcium ions and inhibitory activity to CaCO3 crystallization. The immobilized BRA-2 was found to promote the CaCO3 crystallization. M. rosa lectins also showed opsonic activity when checked with murine macrophage. The gene structures of BRA-2 and BRA-3 were different from each other. These results suggest the participation in defense as well as biomineralization and the allotted biological role of M. rosa humoral lectins. © 2002, The Japanese Society of Fisheries Science. All rights reserved.
  • 酒井 隆一, 鈴木 克治, 石田 真喜子, 神谷 久男, 佐々木 誠, 橘 和夫, 小池 達樹, 島本 啓子, 波越 通夫 天然有機化合物討論会講演要旨集 44 (0) 295 -300 2002年 [査読無し][通常論文]
     
    Excitatory amino acids bind and activate ionotropic glutamate receptors (iGluR) in central nervous system (CNS). Since iGluR is a highly diverse class of the synaptic receptors both structurally and functionally, highly selective agonists and antagonists are in demand as tools to understand their roles in the CNS. Here, we report isolation, structure, and biological activities of excitatory amino acids and related compounds from sponges collected in the Micronesian waters. Dysidea herbacea collected in Yap state afforded isolations of neodysiherbaine A (2), a related compound of dysiherbaine (1), dysibetaine PP (3), dysibetaines CPa (4) and b(5). Compound 2 induced convulsion in mice when injected intracerebroventricular route with ED_<50> value of 16pmol/mouse. In radioligand binding assay 2 was shown to selectively displace labeled kainite and AMPA receptor ligands with IC_<50> values of 104±9.2,161±21nM, respectively. Dysibetaine PP (3) is a dipeptide betaine composed of N-methyl proline and pipecolic acid connected both by peptide bond and N,N-aminal linkage. Dysibetaines CPa (4) is a cyclopropane di-acid substituted with a (trimethylammonium)methyl group. Although 4 did not induce convulsion in mice, it showed moderate affinity both toward kinate and NMDA binding site with IC_<50> values of 13, 10μM, respectively. On the other hand 5 has affinity only toward KA binding site(IC_<50>=4.9±2.3μM). Spectral data indicated that planer structure of 5 corresponds with a mono-amide of 4. Determination of stereochemistry of 5 is now underway. Cribronic acid (2S, 4R, 5R)-4,5-dihydroxypipecolic acid-4-O-sulfate, 6) is isolated from a Palauan sponge Cribrochalina olemda as a potent convulsant in mice. On thither hand, (2S, 4S)-4-hydroxypipecolic acid-4-O-sulfate (7) was isolated as a common convulsant of Axinella carteri and Stylotella aurantium. Both 6 and 7 selectively displaced labeled NMDA ligand in the radioligand binding assay.
  • TM Saxton, AM Cheng, SH Ong, Y Lu, R Sakai, JC Cross, T Pawson CURRENT BIOLOGY 11 (9) 662 -670 2001年05月 [査読無し][通常論文]
     
    Background: The mammalian Grb2 adaptor protein binds pTyr-X-Asn motifs through its SH2 domain, and engages downstream targets such as Sos1 and Gab1 through its SH3 domains. Grb2 thereby couples receptor tyrosine kinases to the Ras-MAP kinase pathway, and potentially to phosphatidylinositol (PI) 3 ' -kinase, By creating a null (Delta) allele of mouse Grb2, we have shown that Grb2 is required for endoderm differentiation at embryonic day 4.0. Results: Grb2 likely has multiple embryonic and postnatal functions. To address this issue, a hypomorphic mutation, first characterized in the Caenorhabditis elegans Grb2 ortholog Sem-5, was engineered into the mouse Grb2 gene. This mutation (E89K) reduces phosphotyrosine binding by the SH2 domain. Embryos that are compound heterozygous for the null and hypomorphic alleles exhibit defects in placental morphogenesis and in the survival of a subset of migrating neural crest cells required for branchial arch formation. Furthermore, animals homozygous for the hypomorphic mutation die perinatally because of clefting of the palate, a branchial arch-derived structure. Analysis of E89K/Delta Grb2 mutant fibroblasts revealed a marked defect in ERK/MAP kinase activation and Gab1 tyrosine phosphorylation following growth factor stimulation. Conclusions: We have created an allelic series within mouse Grb2, which has revealed distinct functions for phosphotyrosine-Grb2 signaling in tissue morphogenesis and cell viability necessary for mammalian development. The placental defects in E89K/Delta mutant embryos are reminiscent of those seen in receptor tyrosine kinase-, Sos1-, and Gab1-deficient embryos, consistent with the finding that endogenous Grb2 is required for efficient RTK signaling to the Ras-MAP kinase and Gab1 pathways.
  • R Sakai, T Koike, M Sasaki, K Shimamoto, C Oiwa, A Yano, K Suzuki, K Tachibana, H Kamiya ORGANIC LETTERS 3 (10) 1479 -1482 2001年05月 [査読無し][通常論文]
     
    R. Sakai, T. Koike, M. Sasaki, K. Shimamoto, C. Oiwa, A. Yano, K. Suzuki, K. Tachibana, and H. Kamiya, Isolation, Structure Determination, and Synthesis of Neodysiherbaine A, a New Excitatory Amino Acid from a Marine Sponge, Org. Letters, 2001, 3, 1479-1482.
  • R Sakai, T Koike, M Sasaki, K Shimamoto, C Oiwa, A Yano, K Suzuki, K Tachibana, H Kamiya ORGANIC LETTERS 3 (10) 1479 -1482 2001年05月 [査読無し][通常論文]
     
    A new excitatory amino acid, neodysiherbaine A (2), was isolated as a minor constituent of the aqueous extract from the marine sponge Dysidea herbacea. The structure was deduced by spectroscopic methods and established unambiguously by the total synthesis. The present synthesis, including as a key step cross coupling of the 6/5-bicyclic core with an amino acid residue, is useful in constructing its structural analogues.
  • 神保充, 佐藤愛, 酒井隆一, 神谷久男 日本水産学会大会講演要旨集 2001 33 2001年04月01日 [査読無し][通常論文]
  • 小池竜樹, 佐々木誠, 橘和夫, 酒井隆一, 大岩智恵, 神谷久男 日本化学会講演予稿集 79th (2) 1023 2001年03月15日 [査読無し][通常論文]
  • R Sakai, GT Swanson, K Shimamoto, T Green, A Contractor, A Ghetti, Y Tamura-Horikawa, C Oiwa, H Kamiya JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 296 (2) 650 -658 2001年02月 [査読無し][通常論文]
     
    Dysiherbaine (DH) is a marine sponge-derived amino acid that causes seizures upon injection into mice. In this report we investigate the behavioral effects and characterize the pharmacological activity of DH. DH induced convulsive behaviors in mice with ED50 values of 13 pmol/mouse, i.c.v. and 0.97 mg/kg, i p. In rat brain synaptic membranes DH displaced binding of [H-3] kainic acid (KA) and [H-3] alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) with K-i values of 26 and 153 nM, respectively; in contrast, DH did not displace the N-methyl-D-aspartic acid (NMDA) receptor ligand [H-3] CGS-19755. DH displaced [H-3]KA from recombinant GluR5 and GluR6 kainate receptor subunits expressed in HEK293 cells with K-i values of 0.74 and 1.2 nM, respectively. In whole-cell voltage-clamp recordings from cultured rat hippocampal neurons, DH evoked inward currents from both AMPA and KA receptors with EC50 values of 9.7 muM and 210 nM, respectively. AMPA receptor currents were blocked by GYKI 53655, whereas KA receptor currents were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Surprisingly, in calcium imaging experiments we found that DH also activated recombinant mGluR5 receptors but did not activate mGluR1 receptors. DH did not activate glutamate transporters or gamma -aminobutyric acid A (GABA(A))receptors. These results indicate that DH is a potent non-NMDA-type agonist with very high affinity for KA receptors, as well as a subtype-selective mGluR agonist. DH possesses the most potent epileptogenic activity among the amino acids yet identified. This novel excitatory amino acid may prove useful for evaluating the physiological and pathological roles of non-NMDA receptors, especially KA receptors, in the central nervous system.
  • R Sakai, JT Henderson, JP O'Bryan, AJ Elia, TM Saxton, T Pawson NEURON 28 (3) 819 -833 2000年12月 [査読無し][通常論文]
     
    Shc proteins possess SH2 and PTB domains and serve a scaffolding function in signaling by a variety of receptor tyrosine kinases. There are three known mammalian She genes, of which ShcB and ShcC are primarily expressed in the nervous system. We have generated null mutations in ShcB and ShcC and have obtained mice lacking either ShcB or ShcC or both gene products. ShcB-deficient animals exhibit a loss of peptidergic and nonpeptidergic nociceptive sensory neurons, which is not enhanced by additional loss of ShcC. Mice lacking both ShcB and ShcC exhibit a significant loss of neurons within the superior cervical ganglia, which is not observed in either mutant alone. The results indicate that these She family members possess both unique and overlapping functions in regulating neural development and suggest physiological roles for ShcB/ShcC in TrkA signaling.
  • 神保充, 佐藤愛, 矢野原泰士, 小池一彦, 酒井隆一, 村本光二, 神谷久男 日本分子生物学会年会プログラム・講演要旨集 23rd 327 2000年11月25日 [査読無し][通常論文]
  • 中西文恵, 大山智樹, 神保充, 酒井隆一, 神谷久男 日本水産学会大会講演要旨集 2000 104 2000年09月27日 [査読無し][通常論文]
  • 臼井梨香, 角井陽子, 西尾寛明, 神保充, 酒井隆一, 村本光二, 神谷久男 日本水産学会大会講演要旨集 2000 104 2000年09月27日 [査読無し][通常論文]
  • S Sato, R Sakai, M Kodama BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 10 (16) 1787 -1789 2000年08月 [査読無し][通常論文]
     
    O-Sulfate group of gonyautoxin I and IV is transformed into methylene to form neosaxitoxin by thiols such as glutathione, a common cellular scavenger, in mild conditions. We isolated the intermediate of this conversion and propose that this reaction proceeds through formation of thiohemiketal, 1,2 shift to form stable thioether intermediate, and then redox exchange at sulfur atom to form the final product, (C) 2000 Elsevier Science Ltd. All rights reserved.
  • M Sasaki, T Koike, R Sakai, K Tachibana TETRAHEDRON LETTERS 41 (20) 3923 -3926 2000年05月 [査読無し][通常論文]
     
    A total synthesis of (-)-dysiherbaine, a neuroexcitotoxic amino acid isolated from the Micronesian marine sponge Dysidea herbacea, starting from a carbohydrate precursor, is described. The present synthesis features a palladium(0)-catalyzed cross-coupling of the 6/5-bicyclic core with an amino acid residue. (C) 2000 Elsevier Science Ltd. All rights reserved.
  • 矢野原泰士, 神谷久男, 神保充, 酒井隆一, 村本光二 日本水産学会大会講演要旨集 2000 167 2000年04月01日 [査読無し][通常論文]
  • 酒井隆一, 神谷久男, 日景 日本水産学会大会講演要旨集 2000 165 2000年04月01日 [査読無し][通常論文]
  • T Nakamoto, T Yamagata, R Sakai, S Ogawa, H Honda, H Ueno, N Hirano, Y Yazaki, H Hirai MOLECULAR AND CELLULAR BIOLOGY 20 (5) 1649 -1658 2000年03月 [査読無し][通常論文]
     
    p130(cas) (Cas) is a docking protein that contains an SH3 domain and multiple tyrosine residues. p130(cas) is located at focal adhesions, is tyrosine phosphorylated in response to integrin stimulation, and is thought to transmit signals, via c-Crk and other proteins, for the remodeling of actin stress fibers and cell movement. In a search for the ligands of the SH3 domain of p130(cas) by far-Western screening,,ve cloned a novel protein named CIZ (for Gas-interacting zinc finger protein). CIZ consists of the following: a putative leucine zipper; a serine/threonine-rich region; a proline-rich sequence; five, six, or eight Kruppel-type C2H2 zinc fingers; and the glutamine-alanine repeat. CIZ binds Gas in cells and is located in the nucleus and at focal adhesions. We showed that CIZ is a nucleocytoplasmic shuttling protein, by using the transient interspecies heterokaryon formation assay. In order to search for the targets of CIZ in nucleus, we determined the DNA binding consensus of CIZ as (GIC)AAAAA(A) by cyclic amplification and selection of targets analysis. The consensus-like sequences are found in several promoters of matrix metalloproteinases (MMPs), which are the enzymes used to degrade the extracellular matrix proteins. CIZ binds to a consensus-like sequence in the MMP-1 (collagenase) promoter. Overexpression of CIZ upregulates the transcriptions from MMP-1, MMP-3 (stromelysin), and MMP-7 (matrilysin) promoters, and this transactivation was enhanced in the presence of Gas. Furthermore, the stable overexpression of CIZ promoted the production of MMP-7 in culture medium, In summary, CIZ, a novel zinc finger protein, binds Cas, is a nucleocytoplasmic shuttling protein, and regulates the expression of MMPs.
  • M Jimbo, T Yanohara, K Koike, K Koike, R Sakai, K Muramoto, H Kamiya COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 125 (2) 227 -236 2000年02月 [査読無し][通常論文]
     
    A D-galactose binding lectin (SLL-2) was isolated from Sinularia lochmodes, an octocoral, by a combination of affinity chromatography on acid-treated agarose and FPLC on Superdex 200. SLL-2 agglutinated rabbit and horse erythrocytes while SLL-1, a minor component, reacted only with rabbit erythrocytes. SLL-2 is a glycoprotein with a molecular mass of 122 kDa and is composed of eight identical subunits (15 kDa). The sequence of the amino terminal region of SLL-2 did not show any apparent homology to the sequences of other animal and plant lectins. D-Galactose, N-acetyl-D-galactosamine, lactose, and melibiose were moderate inhibitors to the agglutination of rabbit erythrocytes. In contrast, horse erythrocytes were much more susceptible to agglutination by SLL-2, which was inhibited by sugars and glycoproteins such as D-galactose, N-acetyl-D-galactosamine, lactose, melibiose, and porcine stomach mucin. SLL-2 showed considerable tolerance to heating and kept its activity after heating at 80 degrees C for 60 min. In immuno-histochemical studies using an anti-SLL-2 antiserum and protein A gold conjugate, SLL-2 was found to be present in high amounts in the nematocysts. SLL-2 was also detected on the surface of symbiotic dinoflagellate, Symbiodinium sp. cells irrespective whether they were surrounded with or without host cells. These observations suggest the presence of lectin-mediated interaction between symbiotic dinoflagellates and S. lochmodes. (C) 2000 Elsevier Science Inc. All rights reserved.
  • GT Swanson, R Sakai, T Green, A Contractor, H Kamiya, SF Heinemann EUROPEAN JOURNAL OF NEUROSCIENCE 12 393 -393 2000年 [査読無し][通常論文]
  • 児玉正昭, 小瀧裕一, 酒井隆一, 佐藤繁 食肉に関する助成研究調査成果報告書 17 326-331 -331 1999年12月 [査読無し][通常論文]
  • Ryuichi Sakai, Chie Oiwa, Kojiroh Takaishi, Hisao Kamiya, Michito Tagawa Tetrahedron Letters 40 (38) 6941 -6944 1999年09月 [査読無し][通常論文]
     
    A new α,α-disubstituted α-amino acid derivative, dysibetaine (1), was isolated from an aqueous extract of the marine sponge Dysidea herbacea collected in Yap, Micronesia. The structure of 1, (2R*,4R*)-2-(trimethylammonium)methyl-4-hydroxy-5-oxoprolinate, was determined by spectral methods and X-ray crystallography.
  • 佐藤繁, 酒井隆一, 児玉正昭 天然有機化合物討論会講演要旨集 41st 403-408 -408 1999年09月01日 [査読無し][通常論文]
  • H Honda, T Nakamoto, R Sakai, H Hirai BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 262 (1) 25 -30 1999年08月 [査読無し][通常論文]
     
    p130(Cas) (Cas) is an adaptor molecule which becomes tyrosine phosphorylated by v-Src- or v-Crk-triggered transformation and several physiological stimuli, such as cell attachment to fibronectin. We previously generated mice lacking Cas and demonstrated that Cas functions as an assembling molecule of actin filaments. To further explore Cas role in cellular function, we established Cas-deficient and Gas-re-expressing fibroblasts and compared their behaviors in response to several biological stimuli. We found that Cas-deficient fibroblasts showed significant defects in cell movement after mechanical wounding and in cell migration toward fibronectin as compared with Cas-reexpressing cells. In addition, when plated on fibronectin-coated dishes, Cas-deficient cells exhibited a significant delay in cell spreading as compared with Gas-re-expressing cells albeit that protein-tyrosine phosphorylation was similarly induced. These results demonstrated that Cas functions as a molecule promoting cell movement, cell migration, and cell spreading and suggest that Cas would be implicated in various physiological and pathological processes, such as would healing, chemotaxis, and tumor invasion. (C) 1999 Academic Press.
  • M Sasaki, T Maruyama, R Sakai, K Tachibana TETRAHEDRON LETTERS 40 (16) 3195 -3198 1999年04月 [査読無し][通常論文]
     
    Synthesis of dysiherbaine model compound 2 and its diastereomer 3 is described. The structurally simplified model compound 2, lacking the hydroxyl and N-methyl groups on the tetrahydropyran ring, induced convulsive behavior in mice upon intracerebral injections; (C) 1999 Elsevier Science Ltd. All rights reserved.
  • 矢野原泰士, 神保充, 小池一彦, 酒井隆一, 神谷久男, 村本光二 日本水産学会大会講演要旨集 1999 189 1999年04月01日 [査読無し][通常論文]
  • 佐藤繁, 酒井隆一, 坂本節子, 児玉正昭 日本水産学会大会講演要旨集 1999 185 1999年04月01日 [査読無し][通常論文]
  • 神谷久男, 矢野原泰士, 神保充, 小池一彦, 小池香苗, 酒井隆一, 村本光二 日本水産学会大会講演要旨集 1999 188 1999年04月01日 [査読無し][通常論文]
  • M Yamaguchi, M Jimbo, R Sakai, K Muramoto, H Kamiya COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 122 (3) 363 -363 1999年03月 [査読無し][通常論文]
  • 酒井 隆一 化学と生物 37 (1) 7 -9 1999年01月25日 [査読無し][通常論文]
  • M. Yamaguchi, M. Jimbo, R. Sakai, K. Muramoto, H. Kamiya Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology 122 (3) 363 1999年 [査読無し][通常論文]
  • 佐藤 繁, 酒井 隆一, 児玉 正昭 天然有機化合物討論会講演要旨集 41 (0) 403 -408 1999年 [査読無し][通常論文]
     
    Gonyautoxins 1-4 (GTXs) are 11-O-sulfated analogues of saxitoxins (STXs). These compounds block voltage-gated sodium channels at very low concentration and cause paralytic shellfish poisonings. GTXs have been shown to be transformed reductively into STXs by homogenates of accumulating organisms or by bacteria. On the other hand, mild chemical treatment of GTXs with thiol reagents such as glutathione or 2-mercaptoethanol also results in the formation of STXs. Although the machanism of these transformations have risen intriguing questions, no detailed study has been conducted. Herein, we describe for the first time, isolation and characterization of stable intermediates, 11-sulfide of STXs, formed in the course of reductive transformations of GTXs by thiols, and propose the mechanism of these unusual conversion to be "two step" reactions. These steps include (a) replacement of O-sulfate by a thiol reagent to give a thioether inter mediate, and (b) attack of another thiol at the sulfur atom of the inter mediate to give the products, STX and a disulfide. The proposed mechanism in the above reductive transformation is reminiscent of several enzymatic reduction processes, such as reductive cleavage of carbon-nitrogen bond catalyzed by glycine reductase, proline reductase or reduction of tetrachlorohydr oquinone (TCHQ) by TCHQ dehalogenase. Discovery of stable 11-thiol derivatives allowed us to develop biochemical probes, which would facilitate not only the study of the more detailed mechanisms of the transformation and dynamics of these toxins in nature, but also to confirm STX binding mechanisms in the voltage-gated sodium channel.
  • AM Cheng, TM Saxton, R Sakai, S Kulkarni, G Mbamalu, W Vogel, CG Tortorice, RD Cardiff, JC Cross, WJ Muller, T Pawson CELL 95 (6) 793 -803 1998年12月 [査読無し][通常論文]
     
    Proteins with SH2 and SH3 domains link tyrosine kinases to intracellular pathways. To investigate the biological functions of a mammalian SH2/SH3 adaptor, we have introduced a null mutation into the mouse gene for Grb2. Analysis of mutant embryonic stem cells, embryos, and chimeras reveals that Grb2 is required during embyrogenesis for the differentiation of endodermal cells and formation of the epiblast. Grb2 acts physiologically as an adaptor, since replacing the C terminus of the Ras activator Sos1 with the Grb2 SH2 domain yields a fusion protein that largely rescues the defects caused by the Grb2 mutation. Furthermore, Grb2 is rate limiting for mammary carcinomas induced by polyomavirus middle T antigen. These data provide genetic evidence for a mammalian Grb2-Ras signaling pathway, mediated by SH2/SH3 domain interactions, that has multiple functions in embryogenesis and cancer.
  • 神谷久男, 神保充, 酒井隆一, 井田斎, 村本光二 日本水産学会大会講演要旨集 1998 135 1998年09月 [査読無し][通常論文]
  • 松原裕樹, 神谷久男, 酒井隆一 日本水産学会大会講演要旨集 1998 136 1998年09月 [査読無し][通常論文]
  • 大岩智恵, 神谷久男, 酒井隆一 日本水産学会大会講演要旨集 1998 136 1998年09月 [査読無し][通常論文]
  • M Toda, M Jimbo, K Muramoto, R Sakai, H Kamiya FISHERIES SCIENCE 64 (4) 638 -642 1998年08月 [査読無し][通常論文]
     
    The hemolymph of the acorn barnacle Balanus rostratus showed hemagglutinating activity and inhibition of calcium carbonate crystallization. A lectin having D-galactose-binding specificity was isolated from the hemolymph by a combination of affinity chromatography on acid-treated agarose gel and HPLC. The purified lectin was dependent on the presence of calcium ion for hemagglutinating activity. In SDS-PACE, it gave one protein band (25 kDa) under a reduced condition, while it gave two components (35 and 95 kDa) without a reducing agent. The molecular mass of the intact lectin was estimated to be 120 kDa by HPLC on TSK G-3000SW. Isolectric point was determined to be pH 4.4. The same amino-terminal sequence, Tyr-Val-Ser-Asn-Gln-Ser-Val-Glu-Pro-Asp-Thr-Ala, was obtained with the purified lectin as well as the components observed in SDS-PAGE. The purified lectin did not inhibit calcium carbonate crystallization at 1 mg/30 ml, although the hemolymph inhibited the crystallization at 0.1 mg protein/30 ml. The inhibitory factor(s) was an acidic and small molecular-weight compound(s).
  • H Honda, H Oda, T Nakamoto, Z Honda, R Sakai, T Suzuki, T Saito, K Nakamura, K Nakao, T Ishikawa, M Katsuki, Y Yazaki, H Hirai NATURE GENETICS 19 (4) 361 -365 1998年08月 [査読無し][通常論文]
     
    p130(Cas) (Cas), the protein encoded by the Crkas gene (also known as Gas), is an adaptor molecule with a unique structure that contains a Src homology (SH)-3 domain followed by multiple YXXP motifs and a proline-rich region(1). Cas was originally cloned as a highly tyrosine-phosphorylated protein in cells transformed by v-Src (refs 2,3) or v-Crk (ref, 4) and has subsequently been implicated in a variety of biological processes including cell adhesion(5), cell migration(6), growth factor stimulation(7-9), cytokine receptor engagement(10,11) and bacterial infection(12,13). To determine its role in vivo, we generated mice lacking Gas. Cas-deficient embryos died in utero showing marked systemic congestion and growth retardation. Histologically, the heart was poorly developed and blood vessels were prominently dilated. Electron microscopic analysis of the heart revealed disorganization of myofibrils and disruption of Z-disks. In addition, actin stress fiber formation was severely impaired in Cas-deficient primary fibroblasts. Moreover, expression of activated Src in Cas-deficient primary fibroblasts did not induce a fully transformed phenotype, possibly owing to insufficient accumulation of actin cytoskeleton in podosomes. These findings have defined Cas function in cardiovascular development, actin filament assembly and Src-induced transformation.
  • 酒井隆一, 神谷久男, 島本啓子, 堀川義子, 宮前丈明 日本水産学会大会講演要旨集 1998 177 1998年04月 [査読無し][通常論文]
  • 神谷久男, 神保充, 酒井隆一, 小池一彦, 村本光二 日本水産学会大会講演要旨集 1998 149 1998年04月 [査読無し][通常論文]
  • M Yamaguchi, M Jimbo, R Sakai, K Muramoto, H Kamiya COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 119 (3) 593 -597 1998年03月 [査読無し][通常論文]
     
    Microcystis aeruginosa, strain M228, a laboratory culture of freshwater cyanobacterium, showed hemagglutinating activity against rabbit, horse and human ABO erthrocytes. Crossed absorption tests revealed the presence of a single type of lectin in the extract of M228 strain cells. The lectin, termed MAL, was purified in combination with the affinity chromatography on acid-treated agarose gel and the gel permeation chromatography in an electrophoretically pure form. MAL was a glycoprotein containing 7.8% neutral sugars and was composed of a single polypeptide having a molecular weight of 57 kDa. Isoelectric point was estimated to be pH 6.4. Hemagglutinating activity of the lectin was inhibited effectively by N-acetyl-D-galactosamine and by glycoproteins. D-galactose and lactose also showed moderate inhibitory activity. The destruction of the hemagglutinating activity by a 2-mercaptoethanol treatment suggests the presence of intra-chain disulfide bond(s) essential for the activity in the molecule. The sequence of the amino-terminal region of MAL was determined as Val-Lys-Ala-Ser-Lys-Val-Ser-Thr-Ser-Gln-Ala-Gly-S er-Lys-Glu-Lys-Lys-Ala. (C) 1998 Elsevier Science Inc. All rights reserved.
  • T. Yamagata, J. Nishida, R. Sakai, T. Tanaka, Y. Yazaki, H. Hirai Leukemia 11 501 -502 1997年08月07日 [査読無し][通常論文]
     
    Interleukin-5 (IL-5) is produced by T lymphocytes and known to support B cell growth and eosinophilic differentiation of the progenitor cells. Using ATL-16T cells which express IL-5 mRNA, we have identified a region, within the human IL-5 gene promoter, that regulates IL-5 gene transcription. This cis-acting sequence contains the core binding motif, (A/T)GATA(A/G), for GATA-binding family proteins and thus suggests the involvement of these family members. In this report, we describe the cloning of human GATA-4 (hGATA-4) and show that hGATA-4 selectively interacts with the -70 GATA site within the IL-5 proximal promoter region. By promoter deletion and mutation analyses, we established this region as a positive regulatory element. Cotransfection experiments revealed that both hGATA-4 and PMA/A23187 stimulation are necessary for the IL-5 promoter activation. The requirement of another regulatory element called CLEO, which lies downstream of the -70 GATA site, was also demonstrated. ATL-16T cells express mRNA of three GATA-binding proteins, hGATA-2, hGATA-3 and hGATA-4 and each of them has a potential to bind to the consensus (A/T)GATA(G/A) motif. However, using ATL-16T nuclear extract, we demonstrated that GATA-4 is the only GATA-binding protein that forms specific DNA-protein complex with the -70 GATA site. The electrophoretic mobility shift assay with extracts of COS cells expressing GATA-binding proteins showed that GATA-4 has the highest binding affinity to the -70 GATA site among the three GATA-binding proteins. When the transactivation ability was compared among the three, GATA-4 showed the highest activity. These results demonstrate the selective role of GATA-4 in the transcriptional regulation of the IL-5 gene in a circumstance where multiple members of the GATA-binding proteins are expressed.
  • T Nakamoto, R Sakai, H Honda, S Ogawa, H Ueno, T Suzuki, S Aizawa, Y Yazaki, H Hirai MOLECULAR AND CELLULAR BIOLOGY 17 (7) 3884 -3897 1997年07月 [査読無し][通常論文]
     
    p130(cas) (Gas) is an adapter protein that has an SH3 domain followed by multiple SH2 binding motifs in the substrate domain. It also contains a tyrosine residue and a proline-rich sequence near the C terminus, which are the binding sites for the SH2 and SH3 domains of Src kinase, respective?v. Cas was originally identified as a major tyrosine-phosphorylated protein in v-Crk- and v-Src-transformed cells. Subsequently, Cas was shown to be inducibly tyrosine phosphorylated upon integrin stimulation; it is therefore regarded as one of the focal adhesion proteins, Using an immunofluorescence study, rye examined the subcellular localization of Cas and determined the regions required for its localization to focal adhesions, In nontransformed cells, Cas was localized predominantly to the cytoplasm and partially to focal adhesions, However, in 527F-c-Src-transformed cells, Cas was localized mainly to podosomes, where the focal adhesion proteins are assembled, The localization of Cas to focal adhesions was also observed in cells expressing the kinase-negative 527F/295M-c-Src. A series of analyses with deletion mutants expressed in various cells revealed that the SH3 domain of Cas is necessary for its localization to focal adhesions in nontransformed cells while both the SH3 domain and the C-terminal Src binding domain of Cas are required in 527F-c-Src-transformed cells and fibronectin-stimulated cells. In addition, the localization of Cas to focal adhesions was abolished in Src-negative cells, These results demonstrate that the SH3 domain of Cas and the association of Cas with Src kinase play a pivotal role in the localization of Cas to focal adhesions.
  • R Sakai, H Kamiya, M Murata, K Shimamoto JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 119 (18) 4112 -4116 1997年05月 [査読無し][通常論文]
     
    A new amino acid, dysiherbaine (1), was isolated from a Micronesian sponge Dysidea herbacea., The structure was determined by using FABMS, ESIMS, FABMS/CID/MS, and one- and two-dimensional NMR experiments of 1 and its dimethyl derivative 3 to be a navel diamino dicarboxylic acid, which consisted of a cis-fused hexahydrofuro[3,2-b]pyran ring substituted with a 3-[2-aminopropanoic acid] side chain. The relative configuration of the bicyclic portion of 1 was determined by (3)J(H,H) analysis and difference NOE experiments, and that of the acyclic side chain was assigned by additional (2,3)J(C,H) analysis, measured by hetero half-filtered TOCSY (HETLOC) and phase sensitive HMBC experiments. Systemic administration of 1 induced neurotoxic symptoms in mice which were reminiscent of neuroexcitatory amino acids such as domoic acid. Dysiherbaine inhibited bindings of [H-3]-kainic acid (KA) and [H-3]-1-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), but not [H-3]-CGS-19755, an N-methyl-D-asparatic acid (NMDA) receptor antagonist, on rat brain synaptic membranes, suggesting that 1 is a selective agonist of non-NMDA type glutamate receptors in the central nervous system.
  • 山口政人, 礁保充, 酒井隆一, 村本光二, 神谷久男 日本水産学会大会講演要旨集 1997 186 1997年04月 [査読無し][通常論文]
  • M Yamaguchi, M Jinbo, R Sakai, K Muramoto, H Kamiya EUROPEAN JOURNAL OF CELL BIOLOGY 74 44 -44 1997年 [査読無し][通常論文]
  • Ryuichi Sakai, Tetsuya Nakamoto, Keiya Ozawa, Shin-Ichi Aizawa, Hisamaru Hirai Oncogene 14 (12) 1419 -1426 1997年 [査読無し][通常論文]
     
    The cellular transformation by v-Src or v-Crk induces tyrosine phosphorylation of a common substrate molecule, p130(Cas), which tightly binds these oncoproteins in vivo. From its structure, Cas is deduced to be an ideal substrate for Src family kinases and Abl kinase. The tyrosine kinase activity associated with Cas was analysed using mouse variant fibroblasts lacking at least one of tyrosine kinases. In normal fibroblasts, Cas is associated with a significant level of tyrosine kinase activity which efficiently phosphorylates Cas in vitro. The Cas-associated tyrosine kinase activity was remarkably elevated in Csk(-/-) cells, which resulted in hyperphosphorylation of cellular Cas. The associated kinase activity was slightly increased in Src(-/-) cells whereas not significantly changed in Abl(-/-) nor Fak(-/-) cells. On the contrary, the Cas-associated kinase activity was remarkably decreased in Fyn(-/-) cells. At the same time, association of Cas with Fyn kinase in vitro was most obviously detected in normal fibroblasts as well as Csk(-/-) cells. Transient expression of v-Crk induced elevation of the Cas-associated kinase activity in all of these cell lines except the primary culture of Fyn(-/-) fibroblasts. These results indicate that Fyn kinase plays an essential role in v-Crk-mediated phosphorylation of Cas.
  • Akira Gomi, Souji Shinoda, Ryuichi Sakai, Hisamaru Hirai, Keiya Ozawa, Toshio Masuzawa Biochemical and Biophysical Research Communications 227 (2) 558 -563 1996年10月 [査読無し][通常論文]
     
    We investigated the expression of DNA polymerase β (β-pol) and O 6 -methylguanine-DNA methyltransferase (MGMT) in human glioma cells with acquired resistance to 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3- nitrosourea (ACNU) and in the parent cells. ACNU-resistant T430 (T430R) and A172 (A172R) glioma cell lines were established following repeated exposure to ACNU. The level of MGMT mRNA expression was elevated in T430R, but not in A172R. In contrast, the level of β-pol mRNA expression and the level of β-pol protein were elevated in A172R, compared with the parent cells. While the mechanism of MGMT repair has been considered to be important in the drug resistance of human brain tumors to ACNU, our present results demonstrate that β-pol may also play an important role in the acquisition of tumor cell resistance to ACNU in human gliomas.
  • 山口政人, 酒井隆一, 神保充, 神谷久男 日本水産学会大会講演要旨集 1996 110 1996年10月 [査読無し][通常論文]
  • Y. Nojima, T. Mimura, N. Morino, K. Hamasaki, H. Furuya, R. Sakai, T. Nakamoto, Y. Yazaki, H. Hirai Human cell : official journal of Human Cell Research Society 9 (3) 169 -174 1996年09月01日 [査読無し][通常論文]
     
    Integrins comprise the major class of receptors used by cells to interact with the extracellular matrix. Integrin/matrix interactions play a critical role in a variety of biological processes, including embryonic development, wound healing, tumor metastasis, cell growth and differentiation. It is now evident that integrins can transduce biochemical signals across the plasma membrane to the cell interior. Protein tyrosine phosphorylation has attracted much attention as an important regulator for integrin-mediated signal transduction. Recently, we have identified a novel signaling molecule, p130Cas, which participates in integrin-mediated signal transduction. p130Cas was originally identified as a protein hyperphosphorylated in cells expressing transforming gene products p47v-crk (v-Crk) and p60v-crk (v-Src). In this brief review, we will discuss about a role of p130Cas in signal transduction triggered by cell adhesion and transformation.
  • R Sakai, EA JaresErijman, Manzanares, I, MVS Elipe, KL Rinehart JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 118 (38) 9017 -9023 1996年09月 [査読無し][通常論文]
     
    New bioactive ecteinascidins (Et's) 597 (1), 583 (2), 594 (3), and 596 (4)-putative biosynthetic precursors of previously described Et's [e.g., Et 743 (5)]-were isolated from the Caribbean tunicate Ecteinascidia turbinata. Structures assigned to these compounds based on spectroscopic data represent a novel series of Et's with L-cysteine or its alpha-oxo analog as unit C. The absolute configuration of the L-Cys unit of 1 was assigned by chiral GC, while a 2D POESY (rotating-frame Overhauser enhancement spectroscopy) spectrum of its acetyl derivative 1a completed the assignment of the stereochemistry of 1 as 1R,2R,3R,4R,11R,13S,21S,1'R.
  • 酒井隆一, 神谷久男, 原山重明, 大西知子 研究基盤施設合同シンポジウム講演予稿集 1996 79-84 -84 1996年09月 [査読無し][通常論文]
  • 酒井隆一, 神谷久男, 村田道雄 天然有機化合物討論会講演要旨集 38th 463-468 -468 1996年09月 [査読無し][通常論文]
  • T Yamagata, J Nishida, T Tanaka, R Sakai, K Mitani, M Yoshida, T Taniguchi, Y Yazaki, H Hirai MOLECULAR AND CELLULAR BIOLOGY 16 (4) 1283 -1294 1996年04月 [査読無し][通常論文]
     
    We have isolated a novel cDNA clone encoding interferon (IFN) consensus sequence-binding protein in adult T-cell leukemia cell lines or activated T cells (ICSAT); this protein is the human homolog of the recently cloned Pip/LSIRF. ICSAT is structurally most closely related to the previously cloned ICSBP, a member of the IFN regulatory factor (IRF) family of proteins that binds to interferon consensus sequences (ICSs) found in many promoters of the IFN-regulated genes. Among T-cell lines investigated, ICSAT was abundantly expressed in human T-cell leukemia virus type 1 (HTLV-1)-infected T cells. When the HTLV-1 tax gene was expressed or phorbol myristate acetate-A23187 stimulation was used, ICSAT expression was induced in Jurkat cells which otherwise do not express ICSAT. When the binding of ICSAT to four different ICSs was tested, the relative differences in binding affinities for those ICSs were determined. To study the functional role of ICSAT, we performed cotransfection experiments with the human embryonal carcinoma cell line N-Tera2. ICSAT was demonstrated to possess repressive function over the gene activation induced by IFN stimulation or by IRF-1 cotransfection. Such repressive function is similar to that seen in IRF-2 or ICSBP. However, we have found that ICSAT has a different repressive effect from that of IRF-2 or ICSBP in some IFN-responsive reporter constructs. These results suggest that a novel mechanism of gene regulation by ''differential repression'' is used by the multiple members of repressor proteins with different repressive effects on the IFN-responsive genes.
  • 神谷久男, 坂本節子, 善方容子, 酒井隆一, 栃本武良 日本水産学会大会講演要旨集 1996 161 1996年03月 [査読無し][通常論文]
  • 酒井隆一, 神里貴紀, 矢葺渉, 米津亜矢子, 高山昌彦, 神谷久男 日本水産学会大会講演要旨集 1996 163 1996年03月 [査読無し][通常論文]
  • 山口政人, 村本光二, 酒井隆一, 神谷久男 日本水産学会大会講演要旨集 1996 161 1996年03月 [査読無し][通常論文]
  • 酒井 隆一, 神谷 久男, 村田 道雄 天然有機化合物討論会講演要旨集 38 (0) 463 -468 1996年 [査読無し][通常論文]
     
    A new amino acid dysiherbaine (1), was isolated from a Micronesian sponge Dysidea herbacea. The structure was determined spectroscopically by using FABMS, ESIMS, FABMS/CID/MS, and one- and two-dimensional NMR experiments of 1 and its dimethyl derivative 2 to be a novel di-amino di-carboxylic acid, which consisted of a cis fused-hexahydrofuro[3,2-b] pyran ring substituted with a 3-[2-amino propanoic acid] side chain. The relative configuration of the bicyclic portion of 1 was determined by 3^J_analysis and deference NOE experiments, and that of acyclic side chain was assigned by additional ^<2.3>J_analysis, measured by conventional proton NMR and newer hetero half-filtered TOCSY (HETLOC) and phase sensitive HMBC experiments. A systemic administration of 1 induced typical neurotoxic symptoms in mice which were reminiscent of neuroexcitatory amino acids, such as domoic acid. Dysiherbaine inhibited bindings of [^3H]kainic acid (KA) and [^3H]1-amino-3-hydroxy-5-methyl-4-isoxazol-propionic acid (AMPA), but not not [^3H]CGS-19755, an NMDA receptor agonist, to the rat brain synaptic membranes, suggesting that 1 is a specific agonist of KA/AMPA class glutamate receptors in the central nervous system.
  • Naoto Hirano, Naoto Hirano, Futoshi Shibasaki, Ryuichi Sakai, Tomoyuki Tanaka, Junji Nishida, Yoshio Yazaki, Tadaomi Takenawa, Hisamaru Hirai, Hisamaru Hirai European Journal of Biochemistry 234 (1) 336 -342 1995年11月 [査読無し][通常論文]
     
    Recently it was shown that putative phospholipase C‐α cDNA does not code for an isotype of the phospholipase C superfamily but for one of the glucose‐regulated proteins (GRPs), ERp57/GRP58. We have isolated human ERp57/GRP58 cDNA from human placenta. Sequence analysis showed that ERp57/GRP58 has two Trp‐Cys‐Gly‐His‐Cys‐Lys motifs completely conserved among the mammals. Bacterially expressed recombinant ERp57/GRP58 protein contained a thiol‐dependent reductase activity which was completely abolished when Ser residues were substituted for Cys residues in both of the two motifs. Furthermore, we have identified a soluble form of ERp57/GRP58 by Western blotting and biosynthetic labeling. In v‐onc transformants of normal rat kidney cells, the expression level of ERp57/GRP58 was elevated at the protein level. In NIH3T3 cells transformed with v‐src, activated c‐src (Y527F) or c‐src, the expression level of ERp57/GRP58 was upregulated in proportion to their transforming abilities. These results indicate that a soluble form of ERp57/GRP58 exists and that this protein may control both extracellular and intracellular redox activities through its thiol‐dependent reductase activity. Moreover, it is likely that ERp57/GRP58 is involved in the oncogenic transformation. Copyright © 1995, Wiley Blackwell. All rights reserved
  • R SAKAI, JG STROH, DW SULLINS, KL RINEHART JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 117 (34) 8885 -8885 1995年08月 [査読無し][通常論文]
  • Y NOJIMA, N MORINO, T MIMURA, K HAMASAKI, H FURUYA, R SAKAI, T SATO, K TACHIBANA, C MORIMOTO, Y YAZAKI, H HIRAI JOURNAL OF BIOLOGICAL CHEMISTRY 270 (25) 15398 -15402 1995年06月 [査読無し][通常論文]
     
    p130(Cas) (Cas) has been recently identified as a 130-kDa protein that is highly phosphorylated on tyrosine residues and is stably associated with p47(v-crk) (v-Crk) and p60(v-src) (v-Src) oncogene products in cells transformed by the respective genes. Cas is a novel signaling molecule having a single Src homology (SH) 3 domain and a cluster of multiple SH2-binding motifs. While the tight association of Cas with v-Crk and v-Src is strongly suggestive of a significant role in regulating cellular transformation, the function of Cas in normal untransformed cells is totally unknown. We report here that cell adhesion to fibronectin rapidly promotes tyrosine phosphorylation of Cas in human and rat fibroblast cell lines. The response was equally induced by cell adhesion to plates coated with vitronectin, laminin, and collagen but not by cell attachment to nonspecific substrate poly-L-lysine. The kinetic profile of Cas phosphorylation was almost identical with that of tyrosine phosphorylation of focal adhesion kinase pp125(FAK) (Fak), which is well known to be activated subsequent to integrin-mediated cell adhesion. Adhesion-dependent Cas phosphorylation was completely inhibited by treating cells with cytochalasin D, an agent that disrupts polymerization of actin stress fibers. These results suggest that tyrosine phosphorylation of Cas is stimulated by normal cell adhesion in close association with Fak phosphorylation and the formation of actin stress fibers. In v-Src- or v-Crk-transformed cells, however, the tyrosine phosphorylation of Cas is markedly increased in an adhesion-independent manner that is insensitive to treatment with cytochalasin D. Thus, Cas plays a role in signaling pathways mediated by cell adhesion as well as by transformation. We propose that Cas may amplify and propagate integrin-mediated signals by interacting with SH2-containing molecule(s).
  • 山口政人, 伊藤晃一, 坂本節子, 酒井隆一, 神谷久男, 渡辺真理代 日本水産学会大会講演要旨集 1995 316 1995年04月 [査読無し][通常論文]
  • 坂本節子, 伊藤晃一, 山口政人, 酒井隆一, 神谷久男, 渡辺真利代, 渡辺真之 日本水産学会大会講演要旨集 1995 315 1995年04月 [査読無し][通常論文]
  • 戸田道寿, 今井利樹, 八子博, 坂本節子, 酒井隆一, 村本光二, 神谷久男 日本水産学会大会講演要旨集 1995 79 1995年04月 [査読無し][通常論文]
  • Bruce J. Mayer, Hisamaru Hirai, Ryuichi Sakai Current Biology 5 (3) 296 -305 1995年 [査読無し][通常論文]
     
    Background: Non-receptor protein-tyrosine kinases often contain at least one Src homology 2 (SH2) domain, a protein module that binds with high affinity to tyrosine-phosphorylated peptides. Because SH2 domains would be predicted to bind with high affinity to proteins phosphorylated by the kinase, but not to the unphosphorylated substrate, their presence in tyrosine kinases has been puzzling. An important role for the SH2 domain of the Abl tyrosine kinase was suggested by work showing that Abl requires an intact SH2 domain in order to malignantly transform cells, and that replacement of the Abl SH2 domain with heterologous SH2 domains alters the spectrum of proteins phosphorylated detectably by Abl in vivo. Results We have used purified wild-type and mutant Abl kinases to examine the roles of the Abl's SH2 and catalytic domains in phosphorylation of p130CAS, a model substrate that has multiple potential phosphorylation sites. We find that an SH2 domain is required for efficient hyperphosphorylation of p130 in vitro. We use chimeric mutants with heterologous SH2 domains to demonstrate that the SH2 domain of the oncogenically transforming adaptor protein Crk, which is the SH2 domain predicted to bind with highest affinity (of those tested) to potential phosphorylation sites in p130, is best able to facilitate hyperphosphorylation. This is the case whether the catalytic domain of the kinase is derived from Abl or from its distant relative, Src. These studies also reveal a role for binding of Crk to Abl in mediating phosphorylation by the kinase. Using purified proteins, we demonstrate that association with Crk strikingly enhances the ability of Abl to hyperphosphorylate p130. There is an excellent correlation between the ability of mutant Crk proteins to promote hyperphosphorylation of p130 by Abl and their ability to transform rodent fibroblasts. Conclusion Our data suggest that, ultimately, the substrate specificity of a non-receptor tyrosine kinase is dependent on the binding specificity of its associated SH2 domain. The SH2 domain binds tightly to a subset of proteins phosphorylated by the catalytic domain, leading to processive phosphorylation of those proteins. Substrate specificity can be broadened by an association between the kinase and proteins, such as Crk, that contain additional SH2 domains this may play a role in malignant transformation by Crk. © 1995 Elsevier Science Ltd. All rights reserved.
  • Ryuichi Sakai, Justin G. Stroh, David W. Sullins, Kenneth L. Rinehart Journal of the American Chemical Society 117 3734 -3748 1995年01月01日 [査読無し][通常論文]
     
    Seven new didemnins—didemnins M (1), N (2), X (3), and Y (4), nordidemnin N (5), epididemnin A 1 (6), and acyclodidemnin A (7)—were isolated from an extract of the Caribbean tunicate Trididemnum solidum. The structures of these compounds were assigned, based on FABMS, high-field NMR data, and chemical degradation studies. Biological activities of these peptides are also described. © 1995, American Chemical Society. All rights reserved.
  • Akira Gomi, Ryuichi Sakai, Seishi Ogawa, Souji Shinoda, Hisamaru Hirai, Toshio Masuzawa Japanese Journal of Cancer Research 86 (4) 342 -346 1995年 [査読無し][通常論文]
     
    Studies have shown that homozygous deletion of the cyclin‐dependent kinase‐4 inhibitor (CDK41) gene, which is mapped to chromosome 9p21, is frequently observed in various types of human cancers. Here we report that the CDK4I gene was deleted in gliomas. Eight cell lines derived from glioblastomas and samples from 14 patients with various grades of gliomas were examined by Southern blot analysis. We found that the CDK4I gene was deleted in 7 of 8 (87.5%) cell lines and 7 of 9 (78%) samples from high‐grade glioma patients, whereas it was deleted in 1 of 5 (20%) low‐grade glioma samples. These results suggested that inactivation of the CDK4I gene may play an important role in the progression of human glioma. Copyright © 1995, Wiley Blackwell. All rights reserved
  • Tetsuya Yamagata, Junji Nishida, Ryuichi Sakai, Tomoyuki Tanaka, Hiroaki Honda, Naoto Hirano, Hiroyuki Mano, Yoshio Yazaki, Hisamaru Hirai Molecular and Cellular Biology 15 (7) 3830 -3839 1995年 [査読無し][通常論文]
     
    Interleukin-5 (IL-5) is produced by T lymphocytes and known to support B- cell growth and eosinophilic differentiation of the progenitor cells. Using ATL-16T cells which express IL-5 mRNA, we have identified a region within the human IL-5 gene promoter that regulates IL-5 gene transcription. This cis- acting sequence contains the core binding motif, (A/T)GATA(A/G), for GATA- binding family proteins and thus suggests the involvement of this family members. In this report, we describe the cloning of human GATA-4 (hGATA-4) and show that hGATA-4 selectively interacts with the -70 GATA site within the IL-5 proximal promoter region. By promoter deletion and mutation analyses, we established this region as a positive regulatory element. Cotransfection experiments revealed that both hGATA-4 and phorbol-12-myristate-13-acetate (PMA)-A23187 stimulation are necessary for IL-5 promoter activation. The requirement for another regulatory element called CLE0, which lies downstream of the -70 GATA site, was also demonstrated. ATL-16T cells express mRNAs of three GATA-binding proteins, hGATA-2, hGATA-3, and hGATA-4, and each of them has a potential to bind to the consensus (A/T)GATA(G/A) motif. However, using ATL-16T nuclear extract, we demonstrated that GATA-4 is the only GATA- binding protein that forms a specific DNA-protein complex with the -70 GATA site. An electrophoretic mobility shift assay with extracts of COS cells expressing GATA-binding proteins showed that GATA-4 has the highest binding affinity for the -70 GATA site among the three GATA-binding proteins. When the transactivation abilities were compared among the three, GATA-4 showed the highest activity. These results demonstrate the selective role of GATA-4 in the transcriptional regulation of the IL-5 gene in a circumstance where multiple members of the GATA-binding proteins are expressed.
  • Journal of Americium Chemical Society (117) 1995年 [査読無し][通常論文]
  • R SAKAI, A IWAMATSU, N HIRANO, S OGAWA, T TANAKA, J NISHIDA, Y YAZAKI, H HIRAI JOURNAL OF BIOLOGICAL CHEMISTRY 269 (52) 32740 -32746 1994年12月 [査読無し][通常論文]
     
    The transforming gene v-crk found in CT10 and ASV-1 avian sarcoma viruses induces marked phosphorylation of several proteins in cells expressing p47(v-crk) (v-Crk), In this work, the main tyrosine-phosphorylated proteins in ASV-1-infected chicken cells and v-crk transfected rat cells were characterized biochemically, Both these proteins have a molecular mass of about 130 kDa and are tightly associated with v-Crk in vivo, Two-dimensional gel electrophoresis revealed that they are both essentially single proteins (p130) with modifications that result in a broad spot in an acidic region, The broad band of semi-purified p130 became sharp at an elevated position in the gel upon treatment with orthovanadate in vivo or with c-Src kinase produced using a baculovirus vector in vitro, whereas it shifted at a lower position upon treatment with alkaline phosphatase in vitro, These results suggest multiple phosphorylation states of p130, which result in a broad band of p130. Two procedures of immunoaffinity purification were used to purify p130 from SY1 cells transfected with v-crk. Approximately 30 pmol of purified p130 was obtained in an immobilized form on a filter starting from 3 x 10(10) cells, Peptide mapping of p130 digested in situ by peptidase revealed that the purity and quantity of the final material were enough for peptide sequencing, Several stretches of partial amino acid sequences were determined, and they indicated that p130 is a novel protein.
  • Naoto Hirano, Naoto Hirano, Futoshi Shibasaki, Hiroyuki Kato, Ryuichi Sakai, Tomoyuk Tanaka, Tomoyuk Tanaka, Junji Nishida, Yoshio Yazaki, Tadaomi Takenawa, Hisamaru Hirai Biochemical and Biophysical Research Communications 204 (1) 375 -382 1994年10月 [査読無し][通常論文]
     
    We have isolated bovine phospholipase C (PLC)-α cDNA from bovine thymus. Sequence analysis showed that PLC-α is highly conserved among rat, mouse, and calf and that it has two Trp-Cys-Gly-His-Cys-Lys motifs completely conserved in the mammals. Southern blot analysis revealed that bovine PLC-α is derived from a single gene. When PLC-α cDNA was stably transfected in NIH3T3 cells, there was no increase in PLC activity. PLC-α is supposed to be a member not of PLC superfamily but of Trp-Cys-Gly-His-Cys-Lys motif-containing proteins consisting of protein disulfide isomerase, P5, ERp72, and thioredoxin. PLC-α should be redesignated ERp57 (ER-resided p57). © 1994 Academic Press, Inc.
  • R SAKAI, A IWAMATSU, N HIRANO, S OGAWA, T TANAKA, H MANO, Y YAZAKI, H HIRAI EMBO JOURNAL 13 (16) 3748 -3756 1994年08月 [査読無し][通常論文]
     
    p47(v-crk) (v-Crk), transforming gene product containing Src homology (SH)-2 and -3 domains, induces an elevated level of tyrosine phosphorylation of several cellular proteins. Among these proteins, a 125-135 kDa protein (p130) shows marked phosphorylation at tyrosines and tight association with v-Crk, suggesting a direct signal mediator of v-Crk. Here we report the molecular cloning of rat p130 by immunoaffinity purification. The p130 is a novel SH3-containing signaling molecule with a cluster of multiple putative SH2-binding motifs of v-Crk. Immunochemical analyses revealed that p130 is highly phosphorylated at tyrosines during transformation by p60(v-src) (v-Src), as well as by v-Crk, forming stable complexes with these oncoproteins. The p130 behaves as an extremely potent substrate of kinase activity included in the complexes and it is a major v-Src-associated substrate of the Src kinase by partial peptidase mapping. Subcellular fractionation demonstrated that the cytoplasmic p130 could move to the membrane upon tyrosine phosphorylation. The p130 (designated Cas for Crk-associated substrate) is a common cellular target of phosphorylation signal via v-Crk and v-Src oncoproteins, and its unique structure indicates the possible role of p130(Cas) in assembling signals from multiple SH2-containing molecules.
  • S OGAWA, H TOYOSHIMA, H KOZUTSUMI, K HAGIWARA, R SAKAI, T TANAKA, N HIRANO, H MANO, Y YAZAKI, H HIRAI ONCOGENE 9 (6) 1669 -1678 1994年06月 [査読無し][通常論文]
     
    We have isolated the mouse c-crk cDNA from a mouse liver cDNA library. It encodes 304 amino acids and consists mainly of SH2/SH3 regions. In Northern blot analysis, the mouse c-crk mRNA is expressed ubiquitously in every tissue and organ, suggesting that the c-Crk protein may be a common signal transducing molecule among tissues. In contrast to the v-Crk protein, which has a single SH3 domain, the c-Crk protein contains two, the more N-terminal SH3(1) domain and the C-terminal SH3(2) domain. To elucidate functions of these SH3 domains, we have constructed two c-crk mutants, B-cuk and D-crk which lack the SH3(2) and the SH3(1) domain, respectively. These mutants were expressed in rat 3Y1 cells, and examined for their transforming ability in terms of morphological phenotypes and for tyrosine phosphorylation profiles of cells expressing the mutant proteins. Morphological alteration and increased tyrosine phosphorylation of 130-140 kDa proteins, the major component of which is the Crk-associated p130, were observed in cells expressing B-Crk as well as those expressing v-Crk, but little in cells expressing c-Crk even at a similar level of expression. Although a highly tyrosine-phosphorylated form of the p130 was coimmunoprecipitated with c-Crk as well as B-Crk, the relative level of tyrosine phosphorylation of the p130, which is normalized to the amount of Crk protein immunoprecipitated, was 10 to 20 times higher in B-Crk-expressing cells than in c-Crk- or D-Crk-expressing cells. The present results indicate that the SH3(2) domain of mouse c-Crk protein negatively regulates tyrosine phosphorylation of the p130, and that lack of the SH3(2) domain in B-Crk and v-Crk may contribute, at least partly, to their morphological alteration or transforming ability through increasing tyrosine phosphorylation of the p130.
  • M NAMIKOSHI, BW CHOI, R SAKAI, F SUN, KL RINEHART, WW CARMICHAEL, WR EVANS, P CRUZ, MHG MUNRO, JW BLUNT JOURNAL OF ORGANIC CHEMISTRY 59 (9) 2349 -2357 1994年05月 [査読無し][通常論文]
     
    A general method has been developed for assigning the structures of nodularin, a potent hepatotoxin, tumor promoter, and protein phosphatase inhibitor, and minor components isolated from a cultured and a bloom sample of the cyanobacterium Nodularia spumigena. It consists of (1) FABMS analysis (determination of molecular weight and molecular formula), (2) H-1 NMR spectroscopy on the parent compound and chiral GC analysis of an acid hydrolyzate (identification and stereochemistry of amino acid components), (3) ozonolysis followed by NaBH4 reduction (conversion to a linear peptide), and (4) FABMS/CID/MS analyses of the linear peptide and the parent compound (sequence analysis). The method has been employed in assigning structures to three new nodularins (2-4) and can be applied to other cyclic peptides containing alpha,beta-dehydroamino acid unit(s), especially the related microcystins, cyclic heptapeptide hepatotoxins. Two nodularins, [DMAdda(3)]nodularin (2) and [(6Z)-Adda(3)] nodularin (3), were obtained from a bloom sample collected from Lake Ellesmere (New Zealand), and [D-Asp(1)] nodularin (4) was isolated from cultured cells (strain L-575). The LD50s of 2 and 4 were 150 and 75 mu g/kg (ip, mice), respectively, but 3 did not show apparent toxicity at 2.0 mg/kg.
  • M NAMIKOSHI, WW CARMICHAEL, R SAKAI, EA JARESERIJMAN, AM KAUP, KL RINEHART ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 205 252 -ORGN 1993年03月 [査読無し][通常論文]
  • M NAMIKOSHI, WW CARMICHAEL, R SAKAI, EA JARESERIJMAN, AM KAUP, KL RINEHART JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 115 (6) 2504 -2505 1993年03月 [査読無し][通常論文]
  • EA JARESERIJMAN, R SAKAI, A INGRUM, CARNEY, JR, KL RINEHART ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 205 250 -ORGN 1993年03月 [査読無し][通常論文]
  • EA JARESERIJMAN, CP BAPAT, AL BERTELLONI, KL RINEHART, R SAKAI ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 205 251 -ORGN 1993年03月 [査読無し][通常論文]
  • Elizabeth A. Jares-Erijman, Chintamani, P. Bapat, Anna Lithgow-Bertelloni, Kenneth L. Rinehart, Ryuichi Sakai Journal of Organic Chemistry 58 5732 -5737 1993年01月01日 [査読無し][通常論文]
     
    Crucigasterins 277, 275, and 225, three new polyunsaturated amino alcohols, 10–12, were isolated from the Mediterranean tunicate Pseudodistoma crucigaster. The structures of these compounds were assigned based on NMR and FABMS data. Absolute stereochemistry of the amino alcohol portion in 10 was assigned to be 2R,3S based on chiral GC comparison of 3-hydroxy-4-aminopentanoic acid 13d, a chemical degradation product of 10, with a synthetic sample prepared from l-alanine. Compounds 10–12 exhibited moderate cytotoxicity and antimicrobial activity. © 1993, American Chemical Society. All rights reserved.
  • Elizabeth A. Jares-Erijman, April L. Ingrum, John R. Carney, John R. Carney, Kenneth L. Rinehart, Ryuichi Sakai Journal of Organic Chemistry 58 4805 -4808 1993年01月01日 [査読無し][通常論文]
     
    The absolute stereochemistry of the pentacyclic guanidine moieties of crambescidin 816 (1) 1 and of 13,14,15-isocrambescidin 800 (4), a new member of this family, was determined, based on chiral GC analysis of a derivative of 2-hydroxybutanoic acid, an ozonolysis product of the crambescidins. Significantly less antiviral activity and cytotoxicity were observed for 4. © 1993, American Chemical Society. All rights reserved.
  • R SAKAI, KL RINEHART, Y GUAN, AHJ WANG PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 89 (23) 11456 -11460 1992年12月 [査読無し][通常論文]
     
    Ecteinascidins (Ets), isolated from the Caribbean tunicate Ecteinascidia turbinata, protect mice in vivo against P388 lymphoma, B16 melanoma, M5076 ovarian sarcoma, Lewis lung carcinoma, and the LX-1 human lung and MX-1 human mammary carcinoma xenografts. Crystal structures of two tris(tetrahydroisoquinoline) Ets were investigated with single crystals of the 21-O-methyl-N-12-formyl derivative of Et 729 and the natural N-12 -oxide of Et 743. Representatives of an additional class of Ets, Et 722 and Et 736, isolated from the same organism, were assigned tetrahydro-beta-carboline-substituted bis(tetrahydroisoquinoline) structures by NMR and fast atom bombardment MS spectra.
  • T TANAKA, F SHIBASAKI, M ISHIKAWA, N HIRANO, R SAKAI, J NISHIDA, T TAKENAWA, H HIRAI BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 187 (2) 1022 -1028 1992年09月 [査読無し][通常論文]
     
    Actins are major cytoskeletal components and highly conserved in evolution. In mammals, there are six actin isoforms, a pair of which shows at least 93% identity in the amino acid sequence. We have cloned cDNA for a bovine protein that is distantly related to members of the mammalian actin isotypes. The predicted amino acid sequence (418 residues long, calculated molecular mass 47369) shows that this protein, which we have named actin2, exhibits 36% identity to mammalian actins and 60% identity to the yeast actin-like protein, act2. We have concluded that actin2 defines a new class of mammalian actin-like proteins. It was also revealed that actin2 messenger RNA is expressed in a broad range of tissues. © 1992.
  • K SUGIMOTO, H TOYOSHIMA, R SAKAI, K MIYAGAWA, K HAGIWARA, F ISHIKAWA, F TAKAKU, Y YAZAKI, H HIRAI BLOOD 79 (9) 2378 -2383 1992年05月 [査読無し][通常論文]
     
    The p53 gene is currently considered to function as a tumor-suppressor gene in various human malignancies. In hematologic malignancies, alterations in the p53 gene have been shown in some human leukemias and lymphomas. Although mutations in the p53 gene are infrequent in acute myelogenous leukemia (AML) patients, we show in this report that alterations in the p53 gene are frequent in myeloid leukemia cell lines. We studied alterations of the p53 gene in nine human myeloid leukemia cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR), single-strand conformation polymorphism (SSCP) analysis, and direct sequencing. Expression of the p53 gene was not detected at all by RT-PCR in two of the nine cell lines. In these two cell lines, Southern blot analysis showed gross rearrangements and deletions in both of the p53 alleles. Six of the nine cell lines were found to express only mutant p53 mRNA by RT-PCR/SSCP analysis and direct sequencing, and wild-type p53 mRNA was not detected. Two of the mutant p53 mRNAs were shown to be products of abnormal splicing events induced by intronic point mutations. Taken together, eight of nine human myeloid leukemia cell lines expressed no or an undetectable amount of wild-type p53 mRNA. Three of the eight cell lines were growth factor-dependent. Our results suggest that inactivation of the p53 gene may be a common feature in myeloid leukemia cell lines and may play an important role in the establishment of these cell lines. © 1992 by The American Society of Hematology.
  • KL RINEHART, R SAKAI, KISHORE, V, DW SULLINS, KM LI JOURNAL OF ORGANIC CHEMISTRY 57 (11) 3007 -3013 1992年05月 [査読無し][通常論文]
     
    The eight possible stereoisomers of isostatine, (3S,4R,5S)-4-amino-3-hydroxy-5-methylheptanoic acid, have been synthesized from the four isomeric D- and L-isoleucinals and D- and L-allo-isoleucinals and ethyl lithioacetate. The eight isomers have been compared for the GC retention times of their bis(trifluoroacetyl) methyl ester derivatives and the H-1 NMR properties of the gamma-lactams derived from them. The natural isomer was shown to be the 3S,4R,5S isomer.
  • M NAMIKOSHI, KL RINEHART, R SAKAI, RR STOTTS, AM DAHLEM, VR BEASLEY, WW CARMICHAEL, WR EVANS JOURNAL OF ORGANIC CHEMISTRY 57 (3) 866 -872 1992年01月 [査読無し][通常論文]
     
    Eleven minor components were isolated, together with microcystin-LR, (LR, I, Scheme I) as the principal toxin (ca. 90% of the toxic components), from Microcystis cyanobacteria (blue-green algae) collected from Homer Lake (Illinois) in the summer of 1988. The components were characterized by amino acid analysis and HRFABMS, FABMS/MS, H-1 NMR, and UV spectroscopic methods as microcystins-RR (2) and -YR (3) (Scheme I) and nine new microcystins. The structures of seven new microcystins were assigned as [DMAdda5]microcystin-LR (4), [Dha7]microcystin-LR (5), microcystin-FR (6), microcystin-AR (7), microcystin-M(O)R (8), [Mser7]microcystin-LR (9), and microcystin-WR (12). Compound 4 is the first microcystin containing 9-O-demethyl-Adda, while phenylalanine, N-methylserine, and tryptophan are also new variations in amino acid components of microcystins. Compound 11 was deduced to be a (C3H7O) monoester of the alpha-carboxyl on the Glu unit of LR (1). New microcystin 11 caused no apparent toxic effects in mice dosed ip at 1 mg/kg, while the others had LD50's of 90-800-mu-g/kg.
  • EA JARESERIJMAN, R SAKAI, KL RINEHART JOURNAL OF ORGANIC CHEMISTRY 56 (19) 5712 -5715 1991年09月 [査読無し][通常論文]
  • K SUGIMOTO, H TOYOSHIMA, R SAKAI, K MIYAGAWA, K HAGIWARA, H HIRAI, F ISHIKAWA, F TAKAKU BLOOD 77 (6) 1153 -1156 1991年03月 [査読無し][通常論文]
     
    p53 is currently considered to be a tumor suppressor gene product, and its alterations are suggested to be involved in several human malignancies. Here we show evidence of the possible involvement of p53 gene mutations in lymphoid leukemias studied by reverse transcriptase-polymerase chain reaction, single strand conformation polymorphism analysis, and nucleotide sequencing. Fourteen patients with various leukemias were examined and two with acute lymphoblastic leukemia and one with Waldenström's macroglobulinemia were identified to have mutations in the coding region of the p53 gene. These mutations included point mutation, triplet deletion, and single nucleotide insertion. Furthermore, expression of the wild-type p53 mRNA was not detected in the samples from these three patients. In one of them, chromosome 17p was deleted, suggesting the absence of the nonmutated p53 gene, whereas in the other two patients, chromosome 17p seemed to be intact by cytogenetic analysis. Our results suggest that alterations of the p53 gene may have a role in the genesis of some leukemias. © 1997 by The American Society of Hematology.
  • Gang‐Hong ‐H Lee, Ryuichi Sakai, Minako Nagao, Tomoyuki Kitagawa International Journal of Cancer 47 (1) 60 -65 1991年01月 [査読無し][通常論文]
     
    The nature of 15 immortal mouse liver epithelial cell (MLEC) lines established from normal C3H mice was investigated specifically in terms of ras oncogene activation. Neither transforming activity nor point mutation within codon 61 of c‐H‐ras could be demonstrated in any of the cell lines by DNA transfection in a NIH/3T3 cell system or by the direct sequencing method after polymerase chain reaction, respectively. Acrylamide gel migration analysis of ras p2l products did not show any shift from the normal. Transplantation experiments demonstrated only 2 out of the 15 lines to be tu‐morigenic in nude mice. When 4 of the non‐tumorigenic MLEC lines were transfected with cloned activated c‐H‐ras containing a point mutation within codon 61, they all became tumorigenic, the resultant neoplasms being hepatocellular carcinomas often associated with albumin mRNA expression. Our results thus indicate that ras activation is not necessary for immortalization or even for transformation of mouse liver cells in culture, and that ras activation may be an event during the progression process in mouse hepatocarcinogene‐sis in vivo. Copyright © 1991 Wiley‐Liss, Inc., A Wiley Company
  • Toshikazu Ushijima, Masahiro Tsutsumi, Masahiro Tsutsumi, Ryuichi Sakai, Ryuichi Sakai, Yukihito Ishizaka, Fumimaro Takaku, Fumimaro Takaku, Yoichi Konishi, Yoichi Konishi, Michihito Takahashi, Michihito Takahashi, Takashi Sugimura, Minako Nagao Japanese Journal of Cancer Research 82 (9) 965 -968 1991年 [査読無し][通常論文]
     
    Five pancreatic carcinomas induced by N‐nitrosobis(2‐hydroxypropyl)amine in Syrian golden hamsters were analyzed for activation of Ki‐ras at codons 12 and 13, using the polymerase chain reaction and direct sequencing. The Ki‐ras gene was shown to be activated in four of the five carcinomas, and the results were further confirmed by subcloning and sequencing. All the mutations involved a G‐to‐A transition at the second position of codon 12, which resulted in a change at the amino acid level from glycine to aspartic acid. This mutation is identical with that reported for pancreatic tumors of Syrian hamsters induced by N‐nitrosobis(2 ‐oxopropyl)amine. Copyright © 1991, Wiley Blackwell. All rights reserved
  • M NAMIKOSHI, KL RINEHART, R SAKAI, K SIVONEN, WW CARMICHAEL JOURNAL OF ORGANIC CHEMISTRY 55 (25) 6135 -6139 1990年12月 [査読無し][通常論文]
     
    Three new hepatotoxic cyclic heptapeptides in the microcystin class were isolated from the cyanobacterium (blue-green alga) Nostoc sp. strain 152 and assigned structures based on their high-resolution FABMS, FABMS/MS, 1 H and 13 C NMR spectra, amino acid analysis, and GC on a chiral capillary column. All three toxins (1–3) have 9-acetoxy-3-amino-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid as an unusual structural component (Scheme I) instead of the corresponding 9-methoxyl derivative (Adda) found in the microcystins. © 1990, American Chemical Society. All rights reserved.
  • T. Tahira, M. Shiraishi, M. Shiraishi, Y. Ishizaka, I. Ikeda, R. Sakai, T. Sugimura, M. Nagao Nucleic Acids Research 18 (24) 7472 -7472 1990年12月01日 [査読無し][通常論文]
  • T HIGA, R SAKAI, S SAKEMI, T ICHIBA, J TANAKA ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 200 77 -AGFD 1990年08月 [査読無し][通常論文]
  • M. Nagao, R. Sakai, Y. Kitagawa, I. Ikeda, K. Sasaki, H. Shima, T. Sugimura Princess Takamatsu symposia 20 177 -184 1989年12月01日 [査読無し][通常論文]
     
    Many oncogene products are protein kinases and signals are transduced via phosphorylation of proteins. Similarly, protein-dephosphorylation may play a critical role in malignant cell transformation. We have cloned two catalytic subunits of ser/thr protein phosphatase (PP) type 2A, PP2A alpha, and PP2A beta, from a rat liver cDNA library. Both cDNAs encode peptides of 309 amino acids with a difference of only 8 amino acids between the two. All primary hepatocellular hyperplastic nodules or carcinomas, which were induced by a food carcinogen, 2-amino-3-methylimidazo[4,5-f]quinoline, showed up-regulation of expression of the mRNAs of both PP2A alpha and PP2A beta. NIH3T3 cell transformants obtained by introducing activated c-raf, ret-II or Ki-ras oncogenes also showed high levels of PP2A alpha transcripts. Okadaic acid, a non-TPA type tumor promoter, was found to be a potent inhibitor of PP1 and PP2A. Its IC50 for PP1 was much higher than that for PP2A with phosphorylase a as a substrate. When raf and ret-II transformants were cultured with okadaic acid at 8 ng/ml for 2 days, both transformants became flattened and showed strict contact inhibitions. This flat cell morphology was stable for at least one month in the presence of okadaic acid, but in its absence, the cells reverted to their original transformed shape within 7-10 days. Colony formation by raf and ret-II transformants in soft agar was inhibited dose-dependently by okadaic acid; very few colonies grew in the presence of the acid at 8 ng/ml. Okadaic acid had less effect on a transformant of the Ha-ras gene, causing only 50% inhibition of colony formation at 8 ng/ml. The role of protein phosphatases in cellular transformation by certain oncogenes is suggested.
  • R SAKAI, IKEDA, I, H KITANI, H FUJIKI, F TAKAKU, U RAPP, T SUGIMURA, M NAGAO PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 86 (24) 9946 -9950 1989年12月 [査読無し][通常論文]
     
    Okadaic acid is a non-phorbol 12-myristate 13-acetate (PMA)-type tumor promoter on mouse skin and known to be a potent inhibitor of serine/threonine protein phosphatases. Contrary to expectation from its tumor-promoting activity, okadaic acid was shown to have a potential to revert the phenotype of cells transformed by raf and ret-II to that of normal cells. Two to 3 days after addition of 8 ng of okadaic acid per ml to the culture medium, raf and ret-II transformants changed to flat cells and gained contact inhibition. The amount of fibronectin, which was decreased in malignant transformed cells, was increased in the flat revertants. Moreover, okadaic acid caused a dose-dependent loss of ability to grow in soft agar. The morphology of the cells reverted to malignant phenotype within 1 week after removal of okadaic acid. The levels of mRNA and protein of activated c-raf in flat revertants were similar to those in parental transformed cells. The level of mRNA of ret-II was also not changed by flat reversion. No induction of flat reversion was observed with okadaic acid tetramethyl ether, an inactive compound, or a phorbol ester, PMA. As okadaic acid is a potent inhibitor of protein phosphatases 1 and 2A, the possibility that these phosphatases are involved in signal transduction from the raf and ret-II oncogenes is suggested.
  • R SAKAI, KL RINEHART ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 197 171 -ORGN 1989年04月 [査読無し][通常論文]
  • F. Ishikawa, R. Sakai, M. Ochiai, T. Sugimura, M. Nagao Nucleic acids symposium series (19) 39 -42 1988年12月01日 [査読無し][通常論文]
     
    Activation mechanism of raf oncogene was studied by applying in vitro mutagenesis to its cDNA. Previous studies suggested the presence of an activation suppressing sequence in the amino-terminal half of c-raf product. Loss of the sequence by genetic rearrangement was presumed to convert c-raf to possess transforming activity. To identify such sequence, we prepared cDNA mutants by random linker insertion. Synthetic oligonucleotide linker was inserted into the plasmid containing cDNA at a single and random site. Coupling two different mutants, in-frame deletion mutants were constructed systematically. Analysis of these deletion mutants revealed a region, the loss of which made c-raf activated.
  • F ISHIKAWA, R SAKAI, M OCHIAI, F TAKAKU, T SUGIMURA, M NAGAO ONCOGENE 3 (6) 653 -658 1988年12月 [査読無し][通常論文]
     
    Our previous study revealed that the rat c-raf was activated by a rearrangement leading to replacement of the amino-terminal half of the product. Therefore, we suggested that some sequences present in the amino-terminal half might prevent c-raf from becoming an active oncogene. To examine this possibility, we constructed a series of deletion mutants of c-raf cDNA by the random linker insertion method. By transfection of NIH3T3 cells with these mutants, a region whose deletion resulted in activation of c-raf was identified. This region is located at amino acid residues 245 to 261, immediate upstream of the kinase domain of the c-raf product and is rich in serine and threonine residues. This region includes a sequence of six amino acids, RSTSTP, which is conserved in the products of normal raf gene families of various species. This sequence is the best candidate for suppressing transforming activity of c-raf.
  • Yoshinori Kitagawa, Ryuichi Sakai, Tomoko Tahira, Hiroyuki Tsuda, Nobuyuki Ito, Takashi Sugimura, Minako Nagao Biochemical and Biophysical Research Communications 157 (2) 821 -827 1988年12月 [査読無し][通常論文]
     
    A cDNA clone coding for an isotype of the catalytic subunit of rat phosphoprotein phosphatase 2A was isolated. The deduced amino acid sequence of the clone was different at 8 positions from that of rat phosphatase 2Aα determined in a previous study. The deduced amino acid sequence of the clone was, however, identical to that of human phosphatase 2Aβ and differed only at one position from that of rabbit 2Aβ. Thus, the isolated cDNA was identified as a clone coding for rat phosphatase 2Aβ. Using a 2Aβ specific probe, two kinds of transcripts were detected in rat liver: a major 2.0 kb mRNA transcript and a minor 1.4 kb mRNA transcript. These transcripts were both greatly increased in rat liver tumors induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) regardless of the carcinoma or hyperplastic nodule. © 1988 Academic Press, Inc.
  • NB PERRY, JW BLUNT, MHG MUNRO, T HIGA, R SAKAI JOURNAL OF ORGANIC CHEMISTRY 53 (17) 4127 -4128 1988年08月 [査読無し][通常論文]
  • T ICHIBA, R SAKAI, S KOHMOTO, G SAUCY, T HIGA TETRAHEDRON LETTERS 29 (25) 3083 -3086 1988年 [査読無し][通常論文]
     
    Manzamines E (2) and F (3) have been isolated as cytotoxic constituents of a sponge of the genus Xestospongia and the structures elucidated by the application of 2D-NMR tecniques and spectroscoplc comparison with manzamine A (1). Manzamine F was shown to be identical with keramamine B (4), the structure of which should therefore be revised as 3. © 1988.
  • Isuzu Ikeda, Yukihito Ishizakz, Masako Ochiai, Ryuichi Sakai, Masayuki Itabashi, Masahiko Onda, Takashi Sugimura, Minako Nagao Japanese Journal of Cancer Research 79 (6) 674 -676 1988年 [査読無し][通常論文]
     
    The correlations of the restriction fragment length polymorphism (RFLP) pattern of L‐myc with the progressive state of cancer and metastases to lymph nodes or other organs were examined in 35 cases of human colorectal cancer by χ 2 analysis. No significant correlation was found. Copyright © 1988, Wiley Blackwell. All rights reserved
  • KL RINEHART, KISHORE, V, S NAGARAJAN, RJ LAKE, JB GLOER, FA BOZICH, KM LI, RE MALECZKA, WL TODSEN, MHG MUNRO, DW SULLINS, R SAKAI JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 109 (22) 6846 -6848 1987年10月 [査読無し][通常論文]
  • R SAKAI, S KOHMOTO, T HIGA TETRAHEDRON LETTERS 28 (45) 5493 -5496 1987年 [査読無し][通常論文]
     
    The title compounds were isolated from a marine sponge collected off Okinawa. Their structures were determined by X-ray and shown to be 1-β-carbolines. Manzamine C was the 2-ethyl-N-azacycloundec-6-ene derivative, whereas manzamine B was more complex being the epoxy isomer of the free base of dihydromanzamine A. © 1987.
  • Y KATO, N FUSETANI, S MATSUNAGA, K HASHIMOTO, R SAKAI, T HIGA, Y KASHMAN TETRAHEDRON LETTERS 28 (49) 6225 -6228 1987年 [査読無し][通常論文]
     
    Two antitumor macrodiolides, bistheonellides A and B, were isolated from a marine sponge Theonella sp. Bistheonellide A was found to be identical with misakinolide A, whose structure has been revised from the previously proposed monomeric macrolide (3) to the dimeric macrodiolide (1). Similarly, bistheonellide B was deduced to be the related structure 2. © 1987.
  • 加藤 裕子, 伏谷 伸宏, 松永 茂樹, 橋本 周久, 酒井 隆一, 比嘉 辰雄, Kashman Yoel 天然有機化合物討論会講演要旨集 29 (0) 301 -308 1987年 [査読無し][通常論文]
     
    During the course of our search for bioactive metabolites from Japanese marine invertebrates, we found that methanol extract of a sponge Theonella sp. collected in Hachijo-jima island revealed strong activity both in the echinoderm egg assay and in the cytotoxicity test. Fran this sponge we have isolated two active components, named bistheonellides A and B (1 and 2, respectively), which inhibited development of starfish embryos at 0.1 and 0.2μg/ml, respectively as well as growth of tumor cells at low concentrations. Bistheonellide A exhibited spectral data, except for molecular weight, identical with those for misakinolide A which had been reported from an Okinawan Theonella sponge. Direct comparison showed that both compounds were identical. On the basis of FABMS, molecular weight determination by vapor pressure method, and NMR analyses of octaacetyl and p-bromobenzoyl derivatives, bistheonellide A (misakinolide A) was revised from the previously proposed monomeric macrolide (3) to its dimeric macrodiolide (1). This was also confirmed by chemical degradation work. Similarly, bistheonellide B was found to have structure 2 by spectral and chemical analyses.
  • Ryuichi Sakai, Tatsuo Higa, Charles W. Jefford, Gérald Bernardinelli Journal of the American Chemical Society 108 (20) 6404 -6405 1986年 [査読無し][通常論文]
  • Ryuichi Sakai, Tatsuo Higa, Charles W. Jefford, Gérald Bernardinelli Journal of the American Chemical Society 108 (20) 6404 -6405 1986年 [査読無し][通常論文]
  • 酒井 隆一, 比嘉 辰雄 琉球大学理学部紀要 (36) p99 -104 1983年09月 [査読無し][通常論文]
  • 阿波島清, 井上理人, 岡本静江, 酒井隆一, 柳瀬彦三 南大阪病院医学雑誌 29 (2/3) 212 -215 1981年12月01日 [査読無し][通常論文]
  • W. Mori, H. Asakawa, T. Taguchi, R. Sakai Acta Haematologica Japonica 41 1309 -1317 1978年12月01日 [査読無し][通常論文]
     
    Our recent studies on ferritin have revealed, at least, two interesting points concering its close relation to malignant neoplasms; frequent elevation of ferritin level in cancer patients' serum, and existence of biochemical as well as immunological heterogeneities between several isoferritins of human origin. The former indicates some possibility of its usefulness as a laboratory test for cancer diagnosis, and the latter suggests indispensable necessity of selection of the most suitable human isoferritin as the material for the test. Our tentative conclusion at present is that human placental ferritin seems to be the most suitable material for producing the antiserum to be used for the test of cancer diagnosis from various points of view, our studies therefore have been carried out mainly using anti-human placental ferritin antiserum. In such a way we have obtained some meaningful results on the ferritin test of patients' serum, which we would like to review with some other, more fundamental data on human ferritin, very briefly here in this paper. A short discussion including future problems in this field of study is also to be made at the conclusion.
  • Ryuichi Sakai, Elizabeth A. Jares-Elijman, Ignacio Manzanares, Maria Victoria Silva Elipe and Kenneth L. Rinehart "Ecteinascidins: Putative Biosynthetic Precursors and Absolute Stereochemistry" J. Am. Chem. Soc. 1996, 118, 9017-9023.
    [査読無し][通常論文]
  • R. Sakai, K. L. Rinehart, V. Kishore, B. Kundo, G. Faircloth, J. B. Gloer, J. R. Carney, M. Namikoshi, F. Sun, R. G. Hughes Jr., D. G. Gravalos, T. G. deQueseda, G. R. Wilson, R. M. Heid. "Structure-Activity relationships of the Didemnins" J・・・
    [査読無し][通常論文]
     
    R. Sakai, K. L. Rinehart, V. Kishore, B. Kundo, G. Faircloth, J. B. Gloer, J. R. Carney, M. Namikoshi, F. Sun, R. G. Hughes Jr., D. G. Gravalos, T. G. deQueseda, G. R. Wilson, R. M. Heid. "Structure-Activity relationships of the Didemnins" J. Med. Chem. 1996, 39, 2819-2834.
  • R. Sakai, H. Kamiya, M. Murata, K. Shimamoto "Dysiherbaine: A New Neurotoxic Amino Acid from the Micronesian Marine Sponge Dysidea herbacea" J. Am. Chem. Soc. 1997, 119, 4112-4116.
    [査読無し][通常論文]
  • R. Sakai, G.T. Swanson, K. Shimamoto, A. Contractor, A. Ghetti, Y. Tamura-Horikawa, C. Oiwa and H. Kamiya. Pharmacological Properties of the Potent Epileptogenic Amino Acid Dysiherbaine, a Novel Glutamate Receptor Agonist Isolated from the Marine Spong・・・
    [査読無し][通常論文]
     
    R. Sakai, G.T. Swanson, K. Shimamoto, A. Contractor, A. Ghetti, Y. Tamura-Horikawa, C. Oiwa and H. Kamiya. Pharmacological Properties of the Potent Epileptogenic Amino Acid Dysiherbaine, a Novel Glutamate Receptor Agonist Isolated from the Marine Sponge Dysidea herbacea．J. Pharm. Exp. Ther. 2001, 296：655-663.
  • G. T. Swanson, T. Green, R. Sakai, A. Contractor, W. Che, H. Kamiya and S. F. Heinemann. Diferential activation of individual subunits in heteromeric kainite receptors. Neuron 2002, 34, 589-598.
    [査読無し][通常論文]
  • R. Sakai, H. Matsubara, K. Shimamoto, H. Kamiya, M. Namikoshi Isolations of N-methyl-D-aspartic acid-type glutamate receptor ligands from Micronesian sponges. J Nat Prod, 2003, 66, 784-787.
    [査読無し][通常論文]
  • M. Jimbo, F. Nakanishi, R. Sakai, K. Muramoto, H. Kamiya Characterization of L-amino acid oxidase and antimicrobial activity of Aplysianin A, a sea hare-derived antitumor-antimicrobial protein Fish. Sci. 69, 1240-1246(2003).
    [査読無し][通常論文]

特許

  • 酒井 隆一, 中野 宏治  国立大学法人北海道大学  202103015771026891
  • 酒井 隆一, 神谷 久男, 村田 道雄, 島本 啓子  サントリー株式会社  200903001742833242
  • amino acid dysiherbaine
    United States Patent 6,147,230
  • Antiviral guanidine derivatives and compositions therefor
    United States Patent 4,772,609
  • Antiviral guanidine derivatives compositions and their methods of use
    United States Patent 4,851,441
  • Misakinolide compositions and their derivatives
    United States Patent 4,859,782
  • Novel Cytotoxic cyclic depsipeptides from the tunicate trididemnum solidum
    United States Patent 4,948,791
  • Antitumor Alkaloids
    United States Patent 4,895,854
  • Antitumor Alkaloids
    U.S. Patent 4,895,853
  • Antitumor Alkaloids
    U.S. Patent 4,895,852
  • Ecteinascidins (ET 729, 743 analogues and derivatives of ET 729 and 743)
    U.S. Patent #5,478,932
  • Ecteinascidins
    U.S. Patent #5,654,426
  • Compositions Comprising Ecteinascidins and a Method of Treating Herpes Simplex Virus infections Therewith
    U.S. Patent # 5,256,663
  • Ecteinascidins 736 and 722
    U.S. Patent #5,149,804

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    研究期間 : 2021年04月 -2025年03月 
    代表者 : 山下 洋, 酒井 隆一, 井上 菜穂子, 神保 充, 新里 宙也, 小池 一彦, 横田 秀夫
     
    海の熱帯雨林とも称されるサンゴ礁では、刺胞動物のサンゴや二枚貝のシャコガイが褐虫藻と呼ばれる微細な藻類と共生して繁栄している。本研究では、観察により得られた「現象」に関与する「物質」の詳細を明らかにし、それら物質の共生体内での「局在・分布・遷移」を可視化することで、生命現象が複雑に絡み合う褐虫藻共生システムを体系的に理解することを目的とする。令和3年度は主にシャコガイ類を用いて実験を実施した。シャコガイの生殖腺からDNAを抽出して次世代シーケンサーによる新規ゲノム解読に着手し、現在150を超えるコンティグを得ている。代謝物の解析はシャコガイの外套膜、生殖腺、中腸腺、腎臓、エラ、筋肉をそれぞれ個別に採取し、液体クロマトグラフ質量分析計や核磁気共鳴装置等の機器を用いて実施した。その結果、各部位ごとに特徴的な代謝物の存在が明らかとなったため、組織中での実際の局在をイメージング質量分析顕微鏡で確認した。また、外套膜由来の抽出物をゲル濾過によりサイズ分画してシャコガイから単離した褐虫藻培養株に添加した。添加後、褐虫藻の光合成活性を二次元イメージングクロロフィル蛍光測定装置により測定したところ、いくつかの画分で活性や増殖に影響が見られた。さらに、シャコガイ内で褐虫藻が共生する特殊な器官である共生藻管の分布を明らかにするため、小型のシャコガイを用いて３次元内部構造顕微鏡観察を実施した。シャコガイ内の褐虫藻の多くは外套膜に分布していたが、貝柱周辺やエラ基部などにも褐虫藻が存在することを確認した。
  • 日本学術振興会：科学研究費助成事業 挑戦的研究(萌芽)
    研究期間 : 2021年07月 -2024年03月 
    代表者 : 山下 洋, 酒井 隆一, 井上 菜穂子, 馬久地 みゆき, 山口 智史
     
    本研究ではサンゴ礁生態系が本来の生息域であるが、近年九州の岩礁生態系へと分布域が拡大しつつあるハタ科魚類のスジアラを用いて、生物の分布域拡大メカニズムの研究に「体組成の変化」という新たな視点を提供することを目的とする。令和３年度はまず、沖縄県の八重山周辺と九州長崎県の五島列島・対馬周辺でそれぞれ採取されたスジアラの代謝物の分析を実施した。薄層クロマトグラフィーによる解析の結果、本来の生息域である沖縄のスジアラではリン脂質、九州のスジアラでは遊離脂肪酸が多く含まれていた。ガスクロマトグラフィー質量分析の結果、沖縄と九州のスジアラそれぞれに特徴的な脂肪酸が見られた。また、イメージング質量分析顕微鏡により、筋肉組織中のリン脂質・中性脂肪・アミノ酸の分布を可視化することにも成功した。腸内細菌叢の解析に関してはスジアラの採取後すぐに試料を得る必要があるため、当初予定では九州に出張してサンプリングを行う予定であった。しかし、新型コロナウイルス感染症の拡大により県外への出張が困難となったため、九州のスジアラの腸内細菌叢解析は延期し、令和３年度は沖縄のスジアラの腸内細菌叢のみ実施した。次世代シーケンサーを用いてアンプリコンシーケンス解析の結果、沖縄のスジアラの腸内細菌はProteobacteriaが優占していたが、個体によって組成がやや異なっていた。また、令和４年度から実施するスジアラ仔稚魚を用いた水槽実験のため、水槽やエサの準備と使用する褐虫藻培養株の選定を実施して拡大培養を始めた。
  • 日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2020年04月 -2022年03月 
    代表者 : 酒井 隆一
     
    前年度までの研究で、シロボヤモドキおよびヨーロッパザラボヤよりその生態制御に関与していると思われる化合物を単離した。本年度はこれらの成分の生理的な役割を調べた。シロボヤモドキの卵、幼生、幼若体、成体の成分を比較したところ、二次代謝物は主に成体に含まれており、幼生や幼若体にはほとんど見られなかった。一方、紫外線防御物質であるマイコスポリン類が卵と幼生のみに含まれており、これらの化合物が卵や孵化後の幼生の保護に関与している可能性が示唆された。ヨーロッパザラボヤからは長鎖アルキル硫酸ジエステル化合物 (ADS) およびそのモノエステル体 (AMS)を単離したが、LC-MSおよびイメージングMSによる解析から、AMSがフンに、ADSが内蔵、特に腸管に局在することが分かった。また、ホヤの飼育水中にはAMSのみが放出されていた。生育ステージでの成分を調べた結果、幼生にはADSのみが含まれており、成長とともにAMSが増加した。AMSは幼生の変態時の尾部吸収を促進したことから、ホヤの生態制御に関与している可能性が示唆された。近縁種の探索の結果、長鎖アルキル硫酸エステルは、マボヤ目には含まれず、マメボヤ目およびマンジュウボヤ目のホヤに特有の代謝物である可能性を見出し、広くホヤの生理・生態に関与している可能性が示唆された。海綿Haliclona sp.組織の顕微鏡観察の結果、共生微生物が多数観察された。またメタゲノム解析を行ったところプロテオバクテリア門ついで未記載のシアノバクテリア2種を主に含んでいた。フローサイトメーターにより共生微生物を分離したところ、得られた一種の細胞にPap/Halが含まれることが分かった。これらの結果からPap/Halは共生微生物により産生され、海水中に放出されることで海綿のケミカルコミュニケーション物質としまたて機能している可能性が示唆された。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2019年04月 -2022年03月 
    代表者 : 酒井 隆一, 田中 良和, 及川 雅人, 松永 智子
     
    海綿より得られたアーキュレイン―A (Acu―A ) の細胞バリア攻略能を評価した。HeLa細胞にAcu-Aと蛍光ラベル化したデキストランを加えると、細胞内、特に核内の顆粒に強い蛍光が観察された。この結果はAcu-Aがナノ粒子を細胞内そして核まで送達できることを示した。そこで次にCell penetrating peptide（CPP） として市販されているHIV-Tat等を用いて同じ実験を行ったところ、細胞内に弱いながらも蛍光が観察されたが、核への送達は確認できなかった。これらのペプチドで細胞を処理し、細胞外液と細胞質の質量分析を行ったところ、両者とも細胞質に取り込まれていることが確認できたが、Acu-Aのみは細胞外液に残余していなかったことから、CPP以上に細胞に取り込まれ易いことが示唆されAcu-Aが天然のCPPであることを示した。今回、pAcu-Bの全合成に成功したので、そのNMRスペクトルを天然物と比較したところ一致しなかった。そこで、天然物の構造を再検討しモデル化合物を合成したところ、候補構造が提出された。また、４４残基ペプチドを固相合成で作成しモデルN末端残基と結合することで合成アーキュレインモデル化合物を作成した。さらに天然より類縁体を得た。これらの生理活性から、ペプチドのみでは活性を示さず、ポリアミンが必須であることが分かった。またペプチド鎖の短い天然物は極弱い活性を示した。パラオの海洋生物2種の抽出物より細胞膜を透過し小胞体に局在するタンパク質が得られた。また、ソリテシジン（SOR）の発現実験では発現たんぱく質に毒性は確認できなかった。現在、SORのアミノ酸変異体を作成しその細胞到達能を調べている。低分子化合物で細胞攻略能を示すと考えられるアルカロイドの単離・精製を行った。またトロンボポエチン様のタンパク質ThCと類縁化合物の評価・探索を進めた。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2018年04月 -2021年03月 
    代表者 : 山下 洋, 酒井 隆一, 神保 充, 新里 宙也, 鈴木 豪, 福岡 弘紀
     
    本研究ではサンゴなどの共生藻として知られる褐虫藻の代謝産物を指標としてサンゴ礁生態系の食物網に新たなルートを提案しようとするものである。また，その過程で褐虫藻とサンゴ等宿主動物の間で行われる物質のやり取りについてもより詳細な知見の蓄積が期待される。本年度はまず、5株の褐虫藻培養株を大量に培養し、代謝産物の網羅的な取得を行った。培養株の内訳は、サンゴから単離したもの2株とシャコガイから単離したもの2株、加えて動物とは共生しない自由生活型のもの1株である。代謝産物の詳細な解析は現在も進行中であるが，大まかな解析結果としては，自由生活型褐虫藻は他の培養株と比べて特異な代謝産物プロファイルを示した。また，新規化合物を含む褐虫藻に特徴的な物質も複数得られた。予備的にサンゴ礁海域の動物プランクトンの持つ物質のプロファイルと比較したところ、褐虫藻由来が疑われる物質がいくつか検出されたため、来年度はさらに詳細な実験と解析を実施する。物質の生合成経路の確認に利用する褐虫藻のタンパク質解析を実施したが，褐虫藻ゲノムから推定されたタンパク質の一部しか検出できなかった。褐虫藻は動物から離し，単独で培養すると日周的に形態変化を行うので，より多くのタンパク質を検出するためには形態変化を考慮した試料の採取と解析が必要であった。サンゴ幼生を用いた遺伝子解析の結果，特定の褐虫藻が共生した時のみ発現量が変化する遺伝子を複数同定することに成功した。また、来年度以降に実施予定のサンゴ礁海域の動物プランクトンの代謝産物解析にむけ，動物プランクトンの採取場所と対象種を決定した。
  • 褐虫藻を起点としたサンゴ礁生態系の新しい食物網ルートの提案
    文部科学省：基盤研究B
    研究期間 : 2018年04月 -2021年03月 
    代表者 : 山下 洋, 国立研究開発法人, 水産研究, 教育機構
  • 日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2018年04月 -2020年03月 
    代表者 : 酒井 隆一
     
    西表島産海綿より強い抗カビ作用を持つ既知脂溶性成分パプアミン類を単離した。パプアミン類は主にアルコール抽出物に含まれているが、水抽出物のタンパク質画分をメタノール抽出することによっても得られたことから、パプアミンが海綿のタンパク質と結合していることが示唆された。また、西表産海綿Axinyssa aculeataより新規のポリアミンペプチド、アーキュレインDを単離・構造決定した。さらにパラオ産の海綿2種よりポリアルキルピリジン（PAP）の分離を行い、分子サイズの異なるPAPを得た。パラオのスナギンチャクEpizoanthus illoricatusに含まれるトリスグアニジンアルカロイドKB343および類縁化合物の構造と活性を検討した。西表・石垣島、およびパラオ生物の観察と採集を行い、150種の底生生物を採集した。また石垣島でタイマイ一頭を捕獲し、本年度の行動実験に備え飼育している。西表産の海洋生物の抽出物については、ヤドカリと海水魚（Pempheris）を用いて行動実験を行った結果、ポリカチオンを含む海綿はヤドカリに摂食阻害作用を示したが、試験魚は忌避行動を引き起こさず、24時間以内に死亡した。単離化合物については細胞毒性、抗カビ性、溶血性の評価を行うとともに、細胞内に蛍光ナノ粒子を送達するか否かを調べた。またA3班との共同でKB343の活性を変異型酵母によるケミカルゲノミクス解析を行ないKB343 が特定の遺伝子欠損株に対して強く活性を示すことを見出した。ポリカチオン化合物により、細胞内に10kDa程度のナノ粒子を送達できることを観察した。また、西表産の海綿Spongosoritesに含まれる毒素タンパク質ソリテシジンに関する研究も進め、論文化した。これは化学コミュニケーション物質としてタンパク質も重要であることを示したものである。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2016年04月 -2020年03月 
    代表者 : 笠井 久会, 酒井 隆一
     
    卵膜軟化症は，サケ増殖事業における卵期の主な減耗要因の一つである。本年度は，恒常的に卵膜軟化症が発症する施設と発症歴のない施設に卵を収容し，経時的に卵膜タンパク質を観察することで卵膜軟化症の動態を検討するとともに，両事業所の飼育水成分を比較することで，発症環境に特有の物質を探索した。加えて，カテキンおよび各種ポリフェノールに予め卵を浸漬することよる卵膜軟化症防除効果を比較した。 SDS-PAGEやLC/MS/MS分析により得られたトータルイオンスペクトルの比較により，孵化時のような卵膜の分解が早期の段階で起きていること，さらに本病による卵膜の分解が孵化時の変化と類似するが同一ではないことを明らかにした。加えて，発症卵膜およびふ化後卵膜由来のバンドにおけるN末端アミノ酸配列を明らかにし，得られた切断部位の情報から，卵膜の溶解に関与しうる酵素を推定した。さらに，LC/MS/MSを用いて飼育水中における低分子物質を検出し，発症環境に特異的な成分を抽出することが可能であることが明らかとなった。卵膜軟化症の防除においては，カテキン2種（EGCおよびEGCg）のうち分子内にガロイル基を含むEGCgがより予防効果が高いものの，ガロイル基単体物質である没食子酸には予防効果を認めなかった。タンニン酸およびテアフラビンはカテキンよりも低濃度の条件下において高い防除効果を発揮したことから，ガロイル基とこれを含むポリフェノール構造の違いが防除効果の程度に影響を及ぼすことを明らかにした。
  • 魚類卵膜軟化症の発症・促進機構の解明に基づく防除技術の確立
    文部科学省：基盤研究B
    研究期間 : 2016年04月 -2020年03月 
    代表者 : 笠井 久会
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2015年04月 -2019年03月 
    代表者 : 酒井 隆一, 東田 千尋, 及川 雅人
     
    細胞膜表面に埋め込まれたイオンチャンネルや受容体、脂質、糖鎖等は細胞間の情報交換を担う窓口であり、多細胞生物の恒常性を維持する「生命装置」といえる．本研究では海洋生物の水溶性抽出物より、シナプス受容体であるセロトニン受容体、オピオイド受容体、グルタミン酸受容体等に作用する新規化合物を見出した。これに加え水溶性にもかかわらず細胞膜を透過して細胞内に侵入するペプチド毒、タンパク質毒そして低分子化合物を見出し、海洋生物の水溶性抽出物が優れた神経活性物質の探索源であるのみならず、細胞のバリアである生体膜を攻略する分子を含んでいることを示した．これらの分子は、薬物送達システムの開発に資する可能性が高い．
  • 海洋生物のポリカチオンコミュニケーション戦略の研究
    文部科学省：新学術領域研究(研究領域提案型)
    研究期間 : 2018年04月 -2019年03月 
    代表者 : 酒井 隆一
  • 細胞表面の「生命装置」に作用する海洋天然物の探索
    文部科学省：基盤研究B
    研究期間 : 2015年04月 -2018年03月 
    代表者 : 酒井 隆一
  • 日本学術振興会：科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2015年04月 -2017年03月 
    代表者 : 酒井 隆一, 田中 良和, 及川 雅人
     
    本研究では、まず海綿レクチンCchGを精製、そしてリガンドとしてCchGとの親和性が高いラクトース（Lac）を原料として、アミノ酸と混合するだけで容易に結合するプローブ分子である１－フルオロ-２，４-ジニトロベンゼン―５-（１－アミノラクトシド）（LacDNFB）を合成した。次に、ラクトース、LacDNFBおよびLacDNFBとアラニンを結合したLacDNFB－AlaとCchGの共結晶の作成を試みたところ、LacおよびLacDNFBとの共結晶が得られた。これらのX線構造解析を行ったところ、ラクトースーCChG複合体の構造が得られ、ラクトースの+１位に未知分子を導入する余地があることを確認した。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2014年04月 -2017年03月 
    代表者 : 及川 雅人, 酒井 隆一
     
    イオンチャネル型グルタミン酸レセプター（iGluR）は、脊椎動物の中枢シナプスの神経伝達において中心的な役割を担い、創薬ターゲットとして注目されている。本研究では、申請者が見いだした抑制化合物IKM-159の活性の強さと選択性の向上に取り組みつつ、そのパーシャルアゴニズムあるいはアロステリックな作用機構を構造生物学を含むタンパク質レベルから個体レベルまでの評価系で解明し、神経性疾患の治療薬へと発展が可能なリガンドの創製を目指した。 まず、そのC環部類縁体の多様合成法を開発し、さらにIKM-154の構造活性相関を明らかにして、AMPA受容体の活性を自在に調節する鍵化合物の開発に成功した。
  • レクチン―糖相互作用を利用した低分子化合物の直接構造決定
    文部科学省：挑戦的萌芽研究
    研究期間 : 2015年04月 -2017年03月 
    代表者 : 酒井 隆一
  • 日本学術振興会：科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2013年04月 -2015年03月 
    代表者 : 及川 雅人, 酒井 隆一
     
    本研究ではイオンチャネル型グルタミン酸受容体 (iGluR) のゲーティングメカニズムの解明に役立てることを目的とし海洋天然物ダイシハーベイン (DH) のプローブの合成化学的開発を試みた。具体的には、出発原料にD-riboseを用いて、まずそのアノマー位にビニル基を導入し、それに対するジヒドロキシ化を行った。アミノ酸側鎖は、アルデヒドに対するHWE反応によりエナミドを得たのちにBophozを用いた不斉水素化により構築した。本研究ではプローブの比較対照に用いるための類縁体の保護体までを得ることができた。また、研究分担者の酒井はマウスを用いた合成化合物の活性評価法の予備的な検討を行った。
  • 日本学術振興会：科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2012年04月 -2014年03月 
    代表者 : 酒井 隆一, 今田 千秋
     
    近年カリブ海産のホヤTrididemnum solidumより見出された抗腫瘍性ペプチド、ジデムニンが世界各地で採集された海洋細菌Tistrella mobilisより相次いで発見された。しかしホヤにおける真の生産者や生合成に関する知見は皆無である。そこで、培養T.mobilis及びカリブ海産のT. solidumについて、化学的・生化学的な比較分析を行った。その結果、ホヤと細菌ではジデムニン類の生産パターンが異なり、また、ホヤには共生生物として大量のシアノバクテリアが含まれるがT.mobilisは極僅か検出されたのみであったことから、ホヤと細菌での生合成経路は異なっていることが示唆された。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2010年04月 -2014年03月 
    代表者 : 酒井 隆一
     
    海洋生物毒・生理活性物質は神経伝達やその調整を司る受容体やチャンネルの宝庫である。本研究では海綿やホヤなどの無脊椎動物より新規の神経毒ペプチドであるアーキュレイン、グルタミン酸受容体の糖鎖を認識して機能を変調する海綿ガレクチンCchG、新規NMDA型グルタミン酸受容体のアゴニストであるHDDA、神経疾患薬のターゲットであるＧ－タンパク質共役型受容体（ＧＰＣＲ）に作用する新規のアルカロイドを見出し、それらの構造決定を行うとともに生理活性について詳細に検討した。
  • イオンチャネル・受容体の動的構造変化追跡を可能にするケミカルプローブの創製
    文部科学省：挑戦的萌芽研究
    研究期間 : 2013年04月 -2014年03月 
    代表者 : 及川 雅人
  • 日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2011年04月 -2013年03月 
    代表者 : 酒井 隆一
     
    海綿は特徴的な生理活性物質を含むが、その生合成研究は容易ではない。本研究では、海綿Lendenfeldia chondrodesより見出された興奮性アミノ酸ダイシハーベイン（DH）をモデルケースに海綿由来化合物の生合成研究の足がかりを得ることを目的とした。本海綿にはラン藻Synechocystisが共生しており、DHはこのラン藻に局在していることがわかっている。また、DH産生型、非産生型の海綿も存在する。本研究ではDH産生型の海綿に特有のタンパク質（DHP）の情報を手がかりに研究を進めることとした。海綿に共生するSynechocystisの分離を試みたところ、パーコール法でSynechocystisの細胞を濃縮することができた。分離した細胞にDHPが含まれることが確認できたのでそれを分離してN-末端アミノ酸配列を得た。また分離した細胞のゲノム解析を行い、ドラフト配列を得ることができた。その中にはDHPのN-末端アミノ酸配列に相同性の高い配列も見出された。一方で、海綿に含まれる水溶性代謝物を分離しその代謝物のパターンを調べたところ、微生物の適合溶質として見出されたグリセリル化糖が見出された。また、海綿には多くのマイコスポリン類が含まれていたが、分離した細胞では主に2種のマイコスポリンが確認された。これらの化合物は共生藍藻の代謝物である可能性が高いので、その生合成遺伝子のゲノム情報が得られると期待される。また、新規のベタインも得られたのでその構造決定を行った。これらの結果から本海綿の水溶性成分は細胞保護作用を示す適合溶質やベタイン等を多く含むことがわかり、これらの成分が共生微生物の細胞保護に資するものと推測された。生合成機構に関する情報が多いマイコスポリンや糖、そしてDHPが得られたことからこれらの情報を参考にしてDHの生合成遺伝子をゲノム中から見出されることが期待される。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2009年 -2011年 
    代表者 : 中尾 洋一, 堀 寛治, 酒井 隆一, 伏谷 伸宏, 松岡 俊二, 福沢 世傑, 脇本 敏幸, 藤田 雅紀, 高田 健太郎
     
    ミクロネシア、ヴェトナム、およびインドネシアの3か国で海洋生物の採集を行い、合計515検体のサンプル採集が達成できた。これらのサンプルをもとに、可能な範囲で混合抽出物、共生微生物、遺伝子を取り出し、それぞれのライブラリーとして保存した。現在、本ライブラリーをもとに有用生物活性および活性本体のデータベース化を行っている。また、調査活動を通じて現地の研究機関との強固な共同研究体制も築くことができた。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2009年 -2011年 
    代表者 : 及川 雅人, 酒井 隆一
     
    ほ乳類動物の中枢神経系のシナプス受容体に作用するリガンド化合物の開発は、複雑な脳機能を分子レベルで理解し制御するためにきわめて重要である。本研究ではイオンチャネル型グルタミン酸受容体のサブタイプであるAMPA受容体を特異的に阻害するIKM-159を見いだし、その構造活性相関を合成化学的に展開した。その結果、(2R)-体がその活性を担うことを明らかにし、さらにGluA2二量体との複合体の結晶構造およびそれらの相互作用をX線結晶構造解析により明らかにした。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2007年 -2009年 
    代表者 : 茂里 康, 酒井 隆一
     
    中性アミノ酸トランスポーターの一種であるアラニン、セリン、システイン輸送機構(alanine-serine-cysteine transporter, ASCT1,2)の阻害剤スクリーニングのためのハイスループットアッセイ系の開発のために、内因性のASCT1,2が高発現していないホスト細胞を検索した。そこでCHO細胞、HEK293細胞、3T3細胞等を用いて遺伝子レベル、タンパクレベルでの実験を実施したが、内因性のASCT1,2の発現量が高く、ASCT1,2のアッセイ用のホスト細胞としては不向きであることがわかった。しかしヒト型、ラット型ASCT1,2の遺伝子をクローニングし、哺乳類及びアフリカツメガエル卵母発現系に使用できるベクターに組み込みを完了した。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2005年 -2007年 
    代表者 : 酒井 隆一, 神保 充, 小池 一彦
     
    本研究では、神経細胞およびシナプスの受容体に対し興味深い活性を示す新規ポリアミンオリゴマー(LCP-Aa),ポリアミンにより修飾を受けたペプチドアーキュレイン、神経毒作用を示す新規プリン誘導体クリブロプリン、新規タンパク質毒ソリテシジンを単離した。LCPA-Aaについては、NMDA受容体に対してリガンドの結合を促進する作用およびマウスに対して遅発性の痙攣誘発作用を持つほかにも、海綿において骨片の形成に関与している可能性を見出した。クリブロプリン酸は海馬の神経細胞に対し自発的な痙攣様の電流を引き起こした。また、パラオ産海綿Raspailiaspに含まれるラスピロールが既知化合物ではあるがマウスに対して痙攣誘発作用を示すことを初めて見出した。一方で、ミクロネシア産海綿Lendenfeldia chondrodesに含まれる興奮性アミノ酸ダイシハーベインが海綿に共生する藍藻Synechocystisの一種に局在することを解明し、藍藻がダイシハーベインの生産に重要な役割を果たしている可能性を提示した。また同海綿から新規のデオキシノジリマイシン誘導体を得た。さらに、西表産の海綿Cinachyrella spに含まれる神経シナプス受容体の増強作用物質(AMPAKine様活性)に関しては、マウスアッセイを指標に活性成分を特定した。また本海綿に含まれるレクチンについても分離を行った。その結果、マウスに対して活性を示す化合物は低分子であり、受容体の増強物質は新規のレクチンである可能性が示唆された。本研究で得られた化合物の構造、生理活性をさらに明らかにしてゆくことでシナプスの受容体の動態を含めた未知の機能が見出されてゆくと期待される。
  • 日本学術振興会：科学研究費助成事業 特定領域研究
    研究期間 : 2004年 -2007年 
    代表者 : 佐々木 誠, 及川 雅人, 不破 春彦, 酒井 隆一, 島本 啓子
     
    グルタミン酸受容体は高等動物の中枢神経シナプスにおいて興奮性神経伝達の中心的役割を司るのみならず、記憶や学習などの高次脳機能、種々の脳疾患における神経細胞死に深く関与している。グルタミン酸受容体は数多くのサブタイプに細分化されているが、個々の生理機能は未だ完全には明らかにされていない。本研究では、グルタミン酸受容体サブタイプ選択的なアゴニストである海綿由来の興奮性アミノ酸ダイシハーベインおよびネオダイシハーベインAとそれらの類縁体の効率的全合成と構造展開により、サブタイプ選択的な生体機能分子の創製と機能解析を目的とした。 ネオダイシハーベインA類縁体全合成における重要中間体から、N-Boc保護1,2-アミノアルコールの分子内S_N2環化反応により6員環上の官能基導入を行い、ダイシハーベインの全合成を達成した。さらに、ヨードアミノ環化によるC8位アミノ基の立体選択的導入法を開発し、より効率的な全合成ルートを確立した。 ダイシハーベインおよびネオダイシハーベインAとGluR5カイニン酸受容体リガンド結合ドメインとの複合体のX線結晶構造解析に初めて成功し、原子レベルでその結合様式を明らかにした。 上記合成ルートを利用して、ダイシハーベイン類縁体を合成し、構造活性相関研究の結果からC9α水酸基が受容体との結合に重要であることが示唆された。また、ネオダイシハーベインAの構造類縁体について詳細な生物活性評価を行い、2,4-エピ体がGluR5およびGluR6カイニン酸受容体に対する機能的アンタゴニストとして機能する初めての化合物であることを明らかにした。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2003年 -2005年 
    代表者 : 神谷 久男, 酒井 隆一, 神保 充, 小池 一彦, 丸山 正
     
    SLL-2のメラニン化を防ぎ、高収率の精製法を確立した。構造を精査した結果、SLL-2は分子量122Kの糖タンパク質で、それぞれ等モルのサブユニット2a、2b、2c9個から分子が構成されていることがわかった。フロンタルアフィニティークロマトグラフィーにより、SLL-2の各種糖鎖に対する結合性を調べた。その結果、SLL-2はGal α 1-3(Fuc α 1-2)Gal β 1-4(Fuc α 1-3)Glcに高いKa値を示した。また、GalNAc α 1-3 GalNAcをもつForssman抗原に結合性を示したほか、Gal α 1-3 Gal構造をもつ糖鎖との結合性を示した。これらのSLL-2と高い結合性を示す糖鎖はいずれも糖脂質由来のものであり、SLL-2は共生藻表面糖鎖の糖脂質と結合するものと推測された。二次元電気泳動法によりSLL-2処理して得られたSymbiodinium培養細胞のタンパク構成成分を調べたところ、培養細胞の日周変化により形成される栄養細胞のパターンと類似しており、遊泳細胞とは泳動パターンが異なることがわかった。また、SLL-2処理によって誘導された栄養細胞には培養細胞とは異なるタンパク成分が存在することが認められた。これらのタンパク質成分の本体について現在検討を加えているので、宿主内の共生藻が栄養型のまま留める機構についての知見が得られる可能性がある。 一方、六放サンゴ類についてもレクチン活性と形態制御活性を検索した結果、トゲクサビライシのレクチンもSLL-2と同様の形態制御活性が示すことがわかった。また、Theonella属海綿に認められたSymbiodinium JCUCS細胞の形態制御物質の本体は既知のデプシペプチド類(TP類)であることを確認した。TP I dおよびTP1436は濃度依存的に遊泳細胞から栄養細胞へと形態を転換させ、細胞増殖の抑制を示すことが明らかとなった。この活性は可逆的で、処理JCUCS細胞は洗浄することによって正常な日周的な形態変化、増殖を回復した。そこで、各種細胞阻害剤の形態制御活性を調べたところ、Latrunculin Aなどのアクチン制御成分がTPと似た遊泳細胞の栄養細胞への形態変化を示素ことを確認できた。また、プロテインキナーゼ阻害剤であるBisindolylmaleimide Iも同様の活性を示した。いずれも洗浄によって正常な増殖過程に回復した。このことからSymbiodinium遊泳細胞から栄養細胞への転換にはミクロフィラメント構造の変化が密接に関連することが示唆された。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2003年 -2004年 
    代表者 : 酒井 隆一, 神保 充, 小池 一彦
     
    ダイシハーベイン(DH)はミクロネシア連邦ヤップ州産海綿Dysidea herbaceaより得られる興奮性アミノ酸で、既存のアミノ酸には無い特異な構造を持つことから、機能・構造の両面で注目されている。これまでの研究で、DHは海綿内の間充織に存在する直径10μm程度の球状細胞に存在することがわかっている。本研究ではこの球状細胞の機能と起源を多面的に比較・解析した。 形態観察:電子顕微鏡、蛍光顕微鏡観察の結果、球状細胞は光合成補助色素フィコエリトリンを含み、3重の細胞壁を持つこと、また核やオルガネラは存在しないことを明らかにした。これらの形態的特徴は藍藻と類似するものであるが、藍藻類に発達するチラコイド膜構造も観察されなかった。またこの細胞のDAPI染色を行ったところ、核の染色は認められなかったものの、分裂途中にある細胞においてのみ蛍光の局在が観察された。この様なきわめて特異な性状を持つ細胞が、本種はもとより海綿の共生生物として報告された例は無い。 分子生物学的解析:次に、海綿より球状細胞および共生藍藻Oscillatoria spongeliaeを分離し、それらの細胞を直接鋳型としてリボゾームDNAのPCR解析を行った。その結果、球状細胞のrDNA配列はホストの海綿と類縁のD.avaraより報告されているものと類似するものであった。一方、共生藍藻のrDNAは既報のO.spongeliaeのものとよく一致した。これらの結果は、ダイシハーベインの産生・貯蔵に深く関わっていると考えられるこの細胞は、藍藻と海綿の特徴を具有している可能性を示すものである。
  • 海洋生物に含まれる生理活性アミノ酸の機能と起源の解明
    研究期間 : 2000年 -2004年
  • 文部科学省：科学研究費補助金(基盤研究(B))
    研究期間 : 2000年 -2003年 
    代表者 : 児玉 正昭, 品川 邦汎, 酒井 隆一, 佐藤 繁
     
    麻ひ性貝毒(PSP)の測定には多くの方法が開発されているが、わが国を含む多くの国では貝の安全性を調べるための公定法としてマウス試験法を採用している。本方法は信頼できる優れた方法であるが、動物試験に伴う多くの問題を抱えており、これに代わる毒の簡易測定法の開発が期待されている。このような簡易測定法の一つとして、毒に対する抗体を用いるELISA法が考えられる。ELISAの開発には毒に対する抗体が必要となるが、PSPのような低分子物質に対する抗体を得るにはタンパクなどの高分子物質に毒を結合したハプテン抗原を作製する必要がある。最近我々はGTX2,3のような11位に硫駿エステルをもつ成分がチオール化合物によりSTXに還元される際、両者が結合した複合体を形成することを発見した。複合体はPSPの11位の炭素がチオールの硫黄原子と共有結合した安定な化合物である。そこで試みに牛血清アルブミン(BSA)にPSPをグルタチオン(GSH)を介して結合させた抗原により抗体を得た。本抗体はこれまで報告されている抗体が全く認識しなかったCトキシン群を含む種々の毒成分にほぼ同様の親和性を示したが、ELISA作成に適した力価を示さなかった。そこでチオールにエタンジチオール(EDT)を用いてSH基をSTXに導入し、マレイミド基を導入したBSAと反応させBSA-EDT-STXを得た。これを免疫して得た抗体は高い力...
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2001年 -2002年 
    代表者 : 酒井 隆一, 小池 一彦, 神保 充
     
    海綿Dysidea.herbaceaおよび紅藻マクリ、Digenea simplexを材料にそれぞれに含まれる興奮性アミノ酸であるダイシハーベイン(DH)およびカイニン酸の生体内における局在性を免疫組織化学的に検討した。 それぞれの低分子アミノ酸を特異的に認識するポリクローナル抗体の作成に成功したので、それらを用いて固定組織切片の染色を行った。その結果、Dysidea.herbacea直径10μm程度の球状細胞が強く染色されるという結果が得られた。一方で、海綿の組織および本海綿の共生生物として知られている藍藻Oscillatoria spongeliaeは全く染色されなかった。さらに藍藻の色素であるフコビリンの抗体を用いて同様の染色を行ったところO. spongeliaeと共に球状細胞も染色された。一方、Digenea simplexについても同様の手法で局在性を検討したところ、藻体の表層細胞が強く染色された。 次にDHを固層化したアフィニティーカラムを作成し、DH結合タンパクの有無を検討したところ海綿の界面活性剤抽出物にカラムに特異的に保持される111kDのタンパクの存在が明らかとなった。 また、グルタミン酸受容体の高度に保存されたアミノ酸配列をもとにプライマーを作成しD. herbaceaのRTPCRを行った。得られたcDNAについてクローニングを行いその塩基配列を調べた。その結果、いくつかのクローンにグルタミン酸受容体と相同な部分をもつアミノ酸配列が確認された。この結果は、海綿内にグルタミン酸受容体様タンパクが含まれ、何らかの生理機能を有することを示唆するものである。 カイニン酸はマクリの藻体の表層および剛毛様の短条と呼ばれる部分の細胞に局在していた。細胞内では一様に染色が認められたものの、周心細胞に点在する顆粒状の構造にとくに強い染色が認められた。
  • 日本学術振興会：科学研究費助成事業 萌芽研究
    研究期間 : 2001年 -2002年 
    代表者 : 神谷 久男, 小池 一彦, 神保 充, 酒井 隆一
     
    共生渦鞭毛藻Symbiodiniumの自由生活ステージから共生ステージへの形態変化を引き起こす物質を検索するために、様々な海洋生物抽出物のスクリーニングを行った結果、海綿Theonella sp.の抽出物に、Symbiodinium CS-156培養株の増殖に影響を与えずに形態を共生ステージに変化させ、その状態を持続させる活性を見出した。今年度はTheonella中の活性成分の性状解析を目指し、(1)分子量分画、(2)加熱安定性、(3)SephadexLH-20ゲルクロマトグラフィー上の挙動、(4)タンパク分解酵素耐性、(5)有機溶媒分画を行い、活性成分の挙動を精査した。 セントリコン(分子量分画限界20,000ダルトン)を用いる透限外ろ過で分画し、形態変化を誘起する活性成分の挙動を追跡したところ、活性は高分子画分にのみ認められた。また、高分子画分にはウサギ血球に対する凝集活性と溶血活性とが認められた。一方、抽出物を80℃、2時間加熱した後でも、活性が残存することが確認された。そこで抽出物を蒸留水を展開剤とするSephadex LH-20ゲルろ過クロマトグラフィーに付したところ、Symbiodinium形態制御活性は血球に対する活性を示さない低分子溶出画分にのみ見出された。また、トリプシンなど3種のプロテアーゼで37℃、1時間処理したが、とくに活性の変化は認められなかった。次いで抽出物をアセトンあるいはメタノール可溶性画分と沈澱画分とに分画して活性を調べたところ、活性はすべて有機溶媒可溶性画分に認められ、高分子画分からなると考えられる沈澱は活性を示さなかった。これらの結果から、Theonellaの活性本体はシゲミカタトサカレクチンSLL-2のような高分子成分ではなく、限外濾過膜を通過しないものの低分子成分であると推測せれた。現在、メタノール可溶性画分をSephadx LH-20によるカラムクロマトグラフィーで分画し、各画分の活性とNMR測定を行い、活性本体の性状検索を継続中である。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2000年 -2002年 
    代表者 : 神谷 久男, 小池 一彦, 神保 充, 酒井 隆一, 丸山 正, 村本 光二
     
    シゲミカタトサカレクチンSLL-2のSymbiodinium以外の微細藻に対する形態変化能を調べたところ、SLL-2に対する反応は種類によって様々で、なんら影響を受けないもの、細胞が破壊されるもの、細胞が凝集し増殖が抑制されるものとがあった。Cy2結合SLL-2を作成して調べたところ、Symbiodiniumその他の微細藻のいずれの場合でも藻体表面にSLL-2が結合することを確認した。一方、Con-A、UEA-IのいずれもSymbiodinium細胞に対する結合性は無いか、ごく弱いものと判定された。また、両レクチンは遊泳細胞を栄養細胞に変化させる効果を示したが、その反応性および持続性はSLL-2よりはるかに弱かった。また、Symbiodinium細胞壁成分中にはCy2-SLL-2と結合し、D-ガラクトースで阻害されるリガンドが存在することを確認した。 天然SLL-2の分子量とcDNAから推測されるアミノ酸配列との差、3Kに相当する成分の本体を確認できなかったが、チロシナーゼによって紫外部吸収が変化することからSLL-2分子中にDopaの存在が推測された。この結果からアミノ酸配列中のTyr残基が修飾を受けてSLL-2ではDopaとなっており、さらに付加物が結合して3K成分を形成しているものと推測される。 一方、沖縄で採集した八放サンゴ類から直ちに抽出液を作成して、抗SLL-2血清とのウェスタンブロット解析を行ったところ、すべての八放サンゴ類でバンドが検出されたが、いずれも100kDa以上の高分子成分であった。この結果は、抗血清に反応する抗原が様々な種に存在していることを示しているが、SLL-2とは異なる挙動を示した。これらの結果はSLL-2がシゲミカタトサカなどSinularia属に特異的なレクチンであることを示唆している。
  • 文部科学省：科学研究費補助金(萌芽的研究)
    研究期間 : 2000年 -2001年 
    代表者 : 児玉 正昭, 酒井 隆一, 佐藤 繁
     
    毒化したホタテガイ中腸腺より水溶性タンパクおよび膜結合タンパク画分を得、両タンパク画分をサキシトキシン(STX)に対する抗体を用いたwestem blot分析にかけたところ、両者共に抗体と反応する複数のタンパクバンドを観察した。抗体と反応するバンドは膜結合タンパク画分に強く認められた。westem blotにおける抗体との反応を指標にこれらタンパクの精製を試みたが、精製過程でタンパクのバンドが変化し、タンパクの分解が起こっていることが考えられた。この原因は現在のところ不明で、ホタテガイ試料からは毒が結合したタンパクの精製が困難であった。そこで市販のメタロチオネインに毒を結合させた標品を用い、タンパクと毒との結合様式を検討した。まず毒を結合させたメタロチオネインをゲルろ過および抗STX抗体をリガンドとするアフィニティクロマトグラフィーで精製し、これをproteaseで分解した。分解物をGSHと毒の複合体(GS-STXs)分析用に開発したHPLCに付したところ、GS-STXsとは異なる位置に1本のピークを与えた。用いたproteaseは非特異的なものであるので、peptideは完全に加水分解されたと考えられ、HPLCで検出された分解物はシステインと毒の複合体であることが考えられた。これに対し、PSPの代謝研究においてシステインと結合した毒は速やかに分解されることが明らかにされてい...
  • 日本学術振興会：科学研究費助成事業 奨励研究(A)
    研究期間 : 1997年 -1997年 
    代表者 : 酒井 隆一
     
    本研究では、ダイシハーベイン(DH)およびその関連化合物の構造、生理活性、絶対構造そして構造-活性相関について検討を行なった。DHはnon-NMDAタイプのグルタミン酸受容体に結合して、アゴニストとして作用することが示されたきたが、本研究では、さらにその薬理活性を詳しく評価するために、マウスを用いたin vivoでの活性、培養神経細胞およびラットのシナプス膜標本におけるin vitroでの挙動を既知の興奮性アミノ酸と詳しく比較した。その結果、DHはマウスに対して既知の興奮性アミノ酸中最強の、興奮性活性を示すことが明らかになった。一方、ラットのシナプス膜標本に対してDHはカイニン酸,/AMPA受容体に選択的ではあるが弱い結合を示したにとどまった。このような活性パターンをもつ興奮性アミノ酸は知られておらず、DHが受容体とのユニークな相互作用を有することが示唆された。 我々はさらにDHの絶対構造、構造活性相関、生合成機構に関して有用な知見を得るためにD.herbaceaよりDH誘導体の作製および関連化合物の単離を試みた。DHの誘導体として、メチルエステルおよびFmoc化誘導体が得られたが。絶対構造情報を得るための誘導体作製の条件は現在検討中である。一方、DH関連化合物の探索の結果、新規アミノ酸誘導体であるダイシベタイン(DB)の単離、構造決定に成功した。DBは4-hydoroxy-5-oxoprolineのα位に(trimethylammonium)methyl基が置換した新しい骨格を有するベタイン化合物であるが、その構造単位にはDHのそれと重なる4-hydoroxyglutamateが含まれており、生合成上の関連性が示唆された.DBはマウスに対して、弱い興奮性の活性を示したことから中枢神経に何らかの作用があると推定されるが、詳しい薬理学的性質については現在検討中である。DHの詳しい構造活性相関に関しては今後さらに検討を加えていく予定である。
  • 日本学術振興会：科学研究費助成事業 奨励研究(A)
    研究期間 : 1996年 -1996年 
    代表者 : 酒井 隆一
     
    ミクロネシア連邦ヤップ島の推進5〜10mにおいて採集された海綿Dysidea herbaciaの水抽出物はマウスに対して腹腔内投与で興奮性神経毒特有の症状を引き起こすことが見出されたので、Sephadex LH20,C18逆相HPLC等を用いて活性化合物の単離を行った。その結果、200gの海綿から約7mgの新規アミノ酸Dysiherbaineが単離された。Dysiherbaineは、分子式C_<12>H_<20>N_2O_7のdi-amino,di-acidであり、その構造は各種NMR,質量スペクトル等を解析することによってcis-hexahydrofuro[3,2-b]pyran環を中心とした新規骨格を持つ化合物であると決定された。またその相対立体配置は、NOE実験に加えて、HETLOC及びPSHMBCスペクトルを用いて測定した^<2,3>J_を解析することにより決定された。Dysiherbaineはラット脳より調製されたシナプス膜標品に対する[^3H]kainic acid および[^3H]AMPAの結合をそれぞれlC_<50>=59,224nMの濃度で阻害したが、NMDAレセプターに対するアゴニストである[^3H]CGS-19755の結合は阻害しなかった。また腹腔内投与によりマウスは、脇腹をむやみに掻く行動("scratching"、20μg/kg)、テンカン様の痙攣(1.3mg/kg)を示した。さらに投与量を上げると激しい発作の後死亡した(6.5mg/kg)。これらのin vivoおよびin vitroアッセイの結果DysiherbaineはKainateおよびAMPAサブタイプのグルタミン酸受容体の選択的なアゴニストであることを強く示唆した。
  • 文部科学省：科学研究費補助金(奨励研究(A))
    研究期間 : 1995年 -1995年 
    代表者 : 酒井 隆一
     
    カリブ海産のホヤEcteinascidia turbinataは現在米国で臨床試験が行われているエクテナシディン43をはじめとする抗癌性アルカロイドを産生することが知られている。本研究ではこのホヤの水溶性抽出物が強い抗菌性を示すことから、この抗菌性を指標に水溶性の生理活性物質を単離精製することを目的として研究を進めた。E. turbinataの体液を遠心分離で固形分と分離し、水溶性の抽出物を得た。この抽出物に硫酸アンモニウムを加え、タンパク成分を沈殿除去した後上清を限外濾過しその分子量1000以上の画分を逆相シリカゲルを担体としたHPLCにより分離して、4つの活性画分を得た。最も強い活性を示したEt1-131-1は枯草菌に対して39ng/discという強い抗菌活性を示した。この画分の各種癌細胞に対する細胞毒性を調べた。その結果、マウスの白血病細胞L1210, P388に大して細胞毒性はそれぞれIC_<50>1.5、25ng/mLであった。現在、さらに構造的知見を得るために活性化合物の分離精製を行っている。
  • 海洋生物の生理活性物質：分離、構造、生理作用、役割の解明
    海洋生物に含まれる中枢シナプス制御物質の探索
  • 海洋生物の産生する生理活性物質の分離と構造決定
  • Isolation and structure determination of Bioactive Marine natural products.

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