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    <ArticleTitle>磁気記録再生装置の電磁界数値解析に関する研究</ArticleTitle>
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    <ArticleTitle>SENSITIVITY ANALYSIS IN COVARIANCE STRUCTURE ANALYSIS</ArticleTitle>
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    <ArticleTitle>コガネムシ類の飛翔筋多型発現機構と移動分散</ArticleTitle>
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    <ArticleTitle>Exploration and Applications of Asymmetric Synthesis Directed toward Chiral Molecules Beadng Nitrogen Functionality</ArticleTitle>
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    <ArticleTitle>Exact distributions of some statistics related to random sequences and their applications</ArticleTitle>
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  <Article>
    <Journal>
      <PublisherName/>
      <JournalTitle>Acta Medica Okayama</JournalTitle>
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      <Volume/>
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        <Year>1994</Year>
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    <ArticleTitle>エンドウ褐紋病菌サプレッサーの構造と作用機構</ArticleTitle>
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    <AuthorList>
      <Author>
        <FirstName EmptyYN="N">Toshiaki</FirstName>
        <LastName>Kato</LastName>
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    <Abstract>植物は諸々の微生物による攻撃から身を守るために様々な防御機構を備えているにも拘らず、ある種の微生物は特定の植物の防御機構を乗り越えて寄生する。植物の防御反応は、菌の細胞壁の分解物など、菌が植物に感染しようとする際に必然的に生じる"引金"物質を植物が認識して能動的に誘起きれる。奥ら(1977)は、エンドウ褐紋病菌について、"引金"物質が存在するにも拘らず、菌がエンドウの防御反応の起動を抑制し、感染に成功していることを突き止め、そのような役割を担う物質をサプレッサーと定義した。その後、エンドウ褐紋病菌以外の植物病原菌についてもサプレッサーの存在が報告され、サプレッサーが病原性決定因子として重要な役割を担っていることが広く認められるようになった。病原性決定因子としては、宿主特異的毒素(HST)が知られているが、その生産菌はAlternaria属菌とHelminthosporium 属菌に限られ、他の多くの植物病原菌の宿主特異性を扱うサプレッサーを生産しているものと考えられている。しかしながら、それらの構造や詳細な作用機構については解明されてはいなかった。エンドウ褐紋病菌サプレッサーの生理作用については、1)菌が寄生できる植物種に限って効果が認められ、2)ファイトアレキシンと呼ばれる抗菌性物質の蓄積阻害などにより、本来病原性のない菌の感染をも許すようになること、3)抵抗性反応に関与する一連の酵素の遺伝子発現を抑制(遅延)することが明らかにされており、サプレッサーは、単独処理した場合でも宿主組織に壊死を起こすことはなく、抵抗反応の抑制や感受化をもたらすという点でHSTとは大きく異なっている。サプレッサーの植物側の作用点としては、4)原形質膜ATPaseである可能性が高いことが推定されていた。本論文においては、エンドウ褐紋病菌胞子発芽液よりサプレツサーを単離・精製し、その化学構造を決定し、サプレッサー活性を担う化学構造の特定とサプレツサーの作用機作の解明について研究した。（1）褐紋病菌サプレッサーの精製および構造解析　エンドウ褐紋病菌胞子発芽液より、限外ろ過、ゲルろ過、SepPAK-C(18)、逆相カラム、イオン配位子カラムなどを用いて、2種類のサプレツサー(Supprescin A, B)を単離し、アミノ酸分析(組成および配列)、糖組成分析、核磁気共鳴法(NMR)にて化学構造をGalNAc-Ser-Ser-G ly, Gal-GalN Ac-Ser-Ser-Gly-Asp-Glu-Thrと決定し、各々Supprescin AおよびBと命名した。（2）掲紋病菌サプレッサーの生理活性　両Supprescinともエリシターによって誘導されるエンドウのファイトアレキシン(ピサチン)蓄積を抑制したが、原形質膜ATPase活性阻害(in vitro, in situ)と非病原菌の感染促進効果はSupprescin Bにのみ認められた。Supprescin Aは、Supprescin BによるATPase活性阻害の程度を低減する効果がみられたことより、両Supprescinに共通した化学構造がATPase分子への結合に深く関していることが示唆された。（3）サプレッサーとしての活性を担う化学構造とその作用機構　1) Supprescinのペプチド部分に関する検討：Supprescinのアミノ酸配列に準じて、化学合成した2～6残基の11種類のペプチドに関して、ピサチン蓄積抑制効果、原形質膜ATPase活性阻害効果(in vitro)、エンドウ褐紋病菌の感染に及ぼす効果を調べた結果、Supprescin Bのペプチド部分であるSer-Ser-Gly-Asp-Glu-Thrについては上記3つのサプレッサー活性が認められたことから、本ペプチド部分がサプレッサーとしての活性を担っていることが判明した。しかしながら、もとのSupprescin Bに比べて弱い効果であったことから、糖鎖部分の重要性が示唆された。また、両Supprescinに共通したペプチド部分であるSer-Ser-Glyは、ピサチン蓄積抑制の他にATPase活性阻害の効果が認められたことから、Supprescin AのATPase分子への結合を裏付ける結果であった。ATPase活性に関するカイネティクス解析の結果、Ser-Ser-Glyを含むペプチドは括抗型の阻害、Asp-Gluを含むペプチドは非拮抗型の阻害であることを明らかにし、後者は酸性ホスファターゼの活性をも阻害したことから、ATPase酵素分子のホスファターゼ部位へ作用していることが推定された。Supprescinないしその部分ペプチドが、界面活性剤存在下で超音波処理したエンドウ原形質膜画分におけるATPase活性を阻害したことは、それらが原形質膜ATPaseを直接阻害していることを示している。2) Supprescinの糖部分に関する検討：Supprescinを構成するGalおよびGalN Acは、サプレッサーによるピサチン蓄積の抑制を低減させる効果が認められ、さらに、GalN Acについてはエンドウ褐紋病菌の感染を阻害する効果も認められたことなどから、GalやGalN Acはサプレッサーの作用点への結合において、重要な役割を担っていることが示唆された。（4）エンドウ原形質膜ATPase遺伝子の解析　サプレッサーの標的分子と考えられたエンドウ原形質膜ATPaseについて、その遺伝子解析を実施した。既報の他種高等植物の原形質膜H(+)-ATPase遺伝子の塩基配列に基づいて設計したDNAプライマーセットを用いて、エンドウcDNAを鋳型としたPCR[Polymerase chain reaction]法により増幅したDNA断片をクローニングし、コード領域の約60%に相当する1.7kbpの塩基配列を決定した。その結果、他種高等植物の原形質膜ATPase遺伝子とは塩基配列で73～76 % 、アミノ酸配列で79～86%の高い相向性が認められた。塩基配列を決定した範囲に存在するATPaseの活性部位とみられるドメインのアミノ酸配列は完全に保存されていた。さらに、ATPaseの活性部位(特にATP結合部位およびホスファターゼ部位)には、GluないしAsp残基が重要であるとされていることから、Supprescin Bに存在するAsp-Glu配列がATP分子と直接結合するか、あるいはATPase分子の活性ドメインの機能を撹乱することによって活性阻害を引き起こしている可能性が考えられた。このように、植物病原菌はサプレッサーを生産して、宿主の防御反応を抑制することによって感染に成功している。サプレッサーの標的分子としてはいくつかの可能性が考えられたが、原形質膜ATPaseがその1つであることを明らかにした。今後、エンドウ細胞における原形質膜ATPaseの役割やその制御機構を明らかにすると共に、サプレッサーが結合する標的分子を直接的に同定することが重要な課題であろう。</Abstract>
    <CoiStatement>No potential conflict of interest relevant to this article was reported.</CoiStatement>
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      <PublisherName/>
      <JournalTitle>Acta Medica Okayama</JournalTitle>
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        <Year>1994</Year>
        <Month/>
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    <ArticleTitle>FUNDAENTAL STUDIES ON RECLAMATION AND IMPROVEMENT OF PROBLEM SOILS IN RELATION TO RICE PRODUCTION</ArticleTitle>
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    <ArticleTitle>Molecular characterization of cryptic flagellin genes and flagellar regulons in strains of the genus Shigella</ArticleTitle>
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