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Functional analysis of free N-glycans involved in regulation of plant differentiation.

Research Project

Project/Area Number 21580414
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Applied molecular and cellular biology
Research InstitutionOkayama University

Principal Investigator

KIMURA Yoshinobu  岡山大学, 大学院・自然科学研究科, 教授 (70195387)

Project Period (FY) 2009 – 2011
Project Status Completed (Fiscal Year 2011)
Budget Amount *help
¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2011: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2010: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2009: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
Keywords植物タンパク質 / 遊離N-グリカン / ENGase(endo-β-N-acetyl glucosaminidase) / PNGase(Peptide : N-glycanase) / 遺伝子発現抑制 / トランスジェニック植物 / 糖鎖機能 / endo-β-N-acetylglucosaminidase / Peptide : N-glycanase / 植物糖タンパク質 / peptide : N-glycanase / α-mannosidase / 遺伝子クローニング / 遺伝子発現解析 / 分子モデリング / ERAD / 遺伝子ノックアウト
Research Abstract

In developing plant tissues, free N-glycan that are enzymatically liberated from glycoproteins occur ubiquitously at micromolar level. As a part of study to elucidate the physiological function(s) of these free N-glycans involved in plant development, we constructed knock-out or knock-down plants, in which the expressions of ENGase or PNGase genes were suppressed. The structural features of free N-glycans expressed in those transgenic plants were clearly distinguishable from those expressed in wild type plants. Although significant difference in phenotype between the transgenic plants and wild type was not observed, a specific difference in the fruit-ripening process was observed.

Report

(4 results)
  • 2011 Annual Research Report   Final Research Report ( PDF )
  • 2010 Annual Research Report
  • 2009 Annual Research Report
  • Research Products

    (58 results)

All 2011 2010 2009

All Journal Article (25 results) (of which Peer Reviewed: 11 results) Presentation (31 results) Book (2 results)

  • [Journal Article] Double Knockout of Putative Endo-β-N-acetylglucosaminidase(ENGase) genes in Arabidopsis thaliana : Loss of ENGase activity induced accumulation of high-mannose type free N-glycans bearing N, N'-acetylchitobiosyl unit2011

    • Author(s)
      Kimura, Y., Takeoka, Y., Maeda, M., Fujiyama, K.
    • Journal Title

      Biosci. Biotechnol. Biochem.

      Volume: 74 Pages: 1019-1021

    • NAID

      10028273128

    • Related Report
      2011 Final Research Report
  • [Journal Article] Purification and characterization of plantβ-xylosidase involved in turnover of plant complex-type N-glycans2011

    • Author(s)
      Yokouchi, D., Ono, N., Nakamura, K., and Kimura, Y.
    • Journal Title

      Glycoconjugate, J.

      Volume: 28 Pages: 316-316

    • Related Report
      2011 Final Research Report
  • [Journal Article] Changes of substrate specificity of plant ENGase(Endo-Os) by site-directed mutagenesis2011

    • Author(s)
      Okamoto, N., Maeda, M., Kimura, M., and Kimura, Y.
    • Journal Title

      Glycoconjugate, J.

      Volume: 28 Pages: 317-317

    • Related Report
      2011 Final Research Report
  • [Journal Article] Molecular characterization and expression analysis ofα-fucosidase from tomato fruits2011

    • Author(s)
      Fujishige, M., Ono, N., Nakamura, K., and Kimura, Y.
    • Journal Title

      Glycoconjugate, J.

      Volume: 28 Pages: 317-317

    • Related Report
      2011 Final Research Report
  • [Journal Article] A New Lectin from the Tuberous Rhizome of Kaempferia rotunda : Isolation, Characterization, Antibacterial and Antiproliferative Activities2011

    • Author(s)
      Kabir S. R., Hossen, M. A., Zubair, M. A., Alom, M. J., Islam, M. F., Hossain, M. A., Kimura, Y.
    • Journal Title

      Protein Pept Lett.

      Volume: 18 Pages: 1140-1149

    • Related Report
      2011 Final Research Report
  • [Journal Article] Purification and Characterization of a Ca^<2+>-dependent novel lectin from nymphaea nouchali tuber with antiproliferative activities2011

    • Author(s)
      Kabir SR, Zubair MA, Nurujjaman M, Haque MA, Hasan I, Islam MF, Hossain MT, Hossain MA, Rakib MA, Alam MT, Shaha RK, Hossain MT, Kimura Y, Absar N.
    • Journal Title

      Biosci Rep.

      Volume: 31 Pages: 465-475

    • Related Report
      2011 Final Research Report
  • [Journal Article] N-Glycans linked to glycoproteins in edible beans(Zatsu-mame): Natural resources for bioactive oligosaccharides2011

    • Author(s)
      Kimura, M., Hara, T., and Kimura, Y.
    • Journal Title

      Biosci. Biotechnol. Biochem.

      Volume: 74 Pages: 155-158

    • NAID

      10027897088

    • Related Report
      2011 Final Research Report
  • [Journal Article] Double-Knockout of Putative Endo-.BETA.-N-acetylglucosaminidase (ENGase) Genes in Arabidopsis thaliana: Loss of ENGase Activity Induced Accumulation of High-Mannose Type Free N-Glycans Bearing N,N'-Acetylchitobiosyl Unit2011

    • Author(s)
      Kimura, Y., et al
    • Journal Title

      Bioscience, Biotechnology, and Biochemistry

      Volume: 75 Issue: 5 Pages: 1019-1021

    • DOI

      10.1271/bbb.110148

    • NAID

      10028273128

    • ISSN
      0916-8451, 1347-6947
    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] N-Glycans Linked to Glycoproteins in Edible Beans (Zatsu-mame): Natural Resources for Bioactive Oligosaccharides2011

    • Author(s)
      Kimura, M., et al
    • Journal Title

      Bioscience, Biotechnology, and Biochemistry

      Volume: 75 Issue: 1 Pages: 155-158

    • DOI

      10.1271/bbb.100594

    • NAID

      10027897088

    • ISSN
      0916-8451, 1347-6947
    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] A New Lectin from the Tuberous Rhizome of Kaempferia rotunda : Isolation, Characterization, Antibacterial and Antiproliferative Activities2011

    • Author(s)
      Kabir S.R.
    • Journal Title

      Protein Pept Lett

      Volume: 18 Pages: 1140-1149

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Purification and Characterization of a Ca^<2+>-dependent novel lectin from nymphaea nouchali tuber with antiproliferative activities2011

    • Author(s)
      Kabir S.R.
    • Journal Title

      Bioscience Reports

      Volume: 31 Pages: 465-475

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] N-Glycans linked to glycoproteins in edible beans : Natural Resources for bioactive oligosaccharides2011

    • Author(s)
      Mariko Kimura
    • Journal Title

      Biosci.Biotechnol, Biochem.

      Volume: 75 Pages: 155-158

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Intracellular and extracellular free N-glycans produced by plant cells : Occurrence of unusual plant complex-type free N-glycans in extracellular spaces2010

    • Author(s)
      Maeda, M, Mariko, K., and Kimura, Y.
    • Journal Title

      J. Biochem.

      Volume: 148 Pages: 681-692

    • NAID

      10027879797

    • Related Report
      2011 Final Research Report
  • [Journal Article] Molecular identification and characterization of an acidic peptide : N-glycanase(PNGase) from tomato fruits2010

    • Author(s)
      Hossain, M. A., Nakano, R., Nakamura, K., and Kimura, Y.
    • Journal Title

      J. Biochem.

      Volume: 147 Pages: 157-165

    • Related Report
      2011 Final Research Report
  • [Journal Article] Molecular characterization of plant acidicα-mannosidase, a member of glycosylhydrolase family 38, involved in the turnover of N-glycans during tomato fruit ripening2010

    • Author(s)
      Hossain, M. A., Nakano, R., Nakamura, K., Hossain, M. T., and Kimura, Y.
    • Journal Title

      J. Biochem.

      Volume: 148 Pages: 603-616

    • Related Report
      2011 Final Research Report
  • [Journal Article] Identification of a royal jelly glycoprotein that carries unique complex-type N-glycans harboring the T-antigen(Galβ1-3GalNAc) unit2010

    • Author(s)
      Kimura, Y., Nagai, H., Miyamoto, M., Kimura, M., and Yonekura, M.
    • Journal Title

      Biosci. Biotechnol. Biochem.

      Volume: 74 Pages: 2148-2150

    • NAID

      10027560979

    • Related Report
      2011 Final Research Report
  • [Journal Article] Molecular characterization of plant acidic α-mannosidase, a member of glycosylhydrolase family 38, involved in the turnover of N-glycans during tomato fruit ripening2010

    • Author(s)
      Md.Anowar Hossain
    • Journal Title

      J.Biochem

      Volume: 148 Pages: 603-616

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Intracellular and extracellular free N-glycans produced by plant cells : occurrence of unusual plant complex type free N-glycans in extracellular spaces2010

    • Author(s)
      Megumi Maeda
    • Journal Title

      J.Biochem

      Volume: 148 Pages: 681-692

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Identification of a Royal Jelly Glycoprotein that Carries Unique Complex-Type N-Glycans Harboring the T-Antigen (Galβ1-3GalNAc) Unit.Biosci.Biotechnol.Biochem., 74, 2148-21502010

    • Author(s)
      Yoshinobu Kimura
    • Journal Title

      Biosci.Biotechnol, Biochem.

      Volume: 74 Pages: 2148-2150

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Molecular identification and characterization of an acidic peptide : N-glycanase from tomato(Lycopersicum esculentum)fruits.2010

    • Author(s)
      Anowar Md.Hossain
    • Journal Title

      J. Biochem., 147巻

      Pages: 157-165

    • Related Report
      2009 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Molecular cloning and gene expression analysis of tomato endo-β-N-acetylglucosaminidase, an endoglycosidase involved in the production of high-mannose type free N-glycans during tomato fruit ripening2009

    • Author(s)
      Nakamura, K., Inoue, M., Nakano, R., Hosoi, K., and Kimura, Y.
    • Journal Title

      Biosci. Biotechnol. Biochem.

      Volume: 73 Pages: 461-464

    • NAID

      10027537895

    • Related Report
      2011 Final Research Report
  • [Journal Article] α-Mannosidase involved in turnover of plant complex type N-glycans in tomato fruits2009

    • Author(s)
      Hossain, Md, A., Nakamura, K., and Kimura, Y
    • Journal Title

      Biosci. Biotechnol. Biochem.

      Volume: 73 Pages: 140-146

    • Related Report
      2011 Final Research Report
  • [Journal Article] Predominant occurrence of truncated complex type N-glycans among glycoproteins in mature-red tomato2009

    • Author(s)
      Nakamura, K., Inoue, M., and Kimura, Y.
    • Journal Title

      Biosci. Biotechnol. Biochem.

      Volume: 73 Pages: 221-223

    • NAID

      10027536646

    • Related Report
      2011 Final Research Report
  • [Journal Article] α-Mannosidase involved in turnover of plant complex type N-glycans in tomato(Lycopersicumesculentum)fruits.2009

    • Author(s)
      Anowar Md.Hossain
    • Journal Title

      Biosci. Biotechnol. Biochem., 73

      Pages: 140-146

    • Related Report
      2009 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Molecular cloning and gene expression analysis of tomato endo-β-N-acetylglucosaminidase, an endoglycosidase involved in the production of high-mannose type free N-glycans during tomato fruit ripening.2009

    • Author(s)
      Kosuke Nakamura
    • Journal Title

      Biosci. Biotechnol. Biochem., 73

      Pages: 461-464

    • Related Report
      2009 Annual Research Report
    • Peer Reviewed
  • [Presentation] Changes of substrate specificity of plant ENGase(Endo-Os) by site-directed mutagenesis2011

    • Author(s)
      Naoko Okamoto, (Yoshinobu KIMURA)
    • Organizer
      International Symposium on Glycoconjugate XXI
    • Place of Presentation
      Vienna(Austria)
    • Year and Date
      2011-08-25
    • Related Report
      2011 Final Research Report
  • [Presentation] Purification and characterization of plantβ-xylosidase involved in turnover of plant complex-type N-glycans2011

    • Author(s)
      Daisuke Yokouchi(Yoshinobu Kimura)
    • Organizer
      International Symposium on Glycoconjugate XXI
    • Place of Presentation
      Vienna(Austria)
    • Year and Date
      2011-08-25
    • Related Report
      2011 Final Research Report
  • [Presentation] Molecular characterization and expression analysis ofα-fucosidase from tomato fruits2011

    • Author(s)
      Makoto Fujishige(Yoshinobu Kimura)
    • Organizer
      International Symposium on Glycoconjugate XXI
    • Place of Presentation
      Vienna(Austria)
    • Year and Date
      2011-08-25
    • Related Report
      2011 Final Research Report
  • [Presentation] Structural features and immunological activity of N-glycans of sea weed glycoprot eins2011

    • Author(s)
      Takeo Yoshiie(Yoshinobu Kimura)
    • Organizer
      International Symposium on Glycoconjugate XXI
    • Place of Presentation
      Vienna(Austria)
    • Year and Date
      2011-08-25
    • Related Report
      2011 Final Research Report
  • [Presentation] Changes of substrate specificity of plant ENGase (Endo-Os) by site-directed mutagenesis2011

    • Author(s)
      Okamoto, N., et al
    • Organizer
      International Symposium on Glycoconjugate XXI (Glyco 21)
    • Place of Presentation
      ウィーン市/オーストリア
    • Year and Date
      2011-08-25
    • Related Report
      2011 Annual Research Report
  • [Presentation] Purification and characterization of plant β-xylosidase involved in turnover of plant complex-type N-glycans2011

    • Author(s)
      Yokouchi, D., et al
    • Organizer
      International Symposium on Glycoconjugate XXI (Glyco 21)
    • Place of Presentation
      ウィーン市/オーストリア
    • Year and Date
      2011-08-25
    • Related Report
      2011 Annual Research Report
  • [Presentation] Molecular characterization and expression analysis of α-fucosidase from tomato fruits2011

    • Author(s)
      Fujishige, M., et al
    • Organizer
      International Symposium on Glycoconjugate XXI (Glyco 21)
    • Place of Presentation
      ウィーン市/オーストリア
    • Year and Date
      2011-08-25
    • Related Report
      2011 Annual Research Report
  • [Presentation] Structural features and immunological activity of N-glycans of sea weed glycoproteins2011

    • Author(s)
      Yoshiie, T., et al
    • Organizer
      International Symposium on Glycoconjugate XXI (Glyco 21)
    • Place of Presentation
      ウィーン市/オーストリア
    • Year and Date
      2011-08-25
    • Related Report
      2011 Annual Research Report
  • [Presentation] 植物ERADで機能する糖鎖遺伝子の機能解析2011

    • Author(s)
      江原愛美(木村吉伸)
    • Organizer
      日本農芸化学会中四国支部第29回講演会
    • Place of Presentation
      徳島大学
    • Year and Date
      2011-01-22
    • Related Report
      2011 Final Research Report
  • [Presentation] トマト複合型N-グリカンの代謝に関わるα-Fucosidaseの遺伝子同定と発現系構築2011

    • Author(s)
      藤重誠(木村吉伸)
    • Organizer
      日本農芸化学会中四国支部第29回講演会
    • Place of Presentation
      徳島大学
    • Year and Date
      2011-01-22
    • Related Report
      2011 Final Research Report
  • [Presentation] 植物エンドグリコシダーゼの糖転移活性と部位特異的変異導入による活性残基同定2011

    • Author(s)
      岡本尚子(木村吉伸)
    • Organizer
      日本農芸化学会中四国支部第29回講演会
    • Place of Presentation
      徳島大学
    • Year and Date
      2011-01-22
    • Related Report
      2011 Final Research Report
  • [Presentation] 植物複合型N-グリカンの代謝に関わるβ-Xylosidaseの精製と発現系構築2011

    • Author(s)
      横内大輔(木村吉伸)
    • Organizer
      日本農芸化学会中四国支部第29回講演会
    • Place of Presentation
      徳島大学
    • Year and Date
      2011-01-22
    • Related Report
      2011 Final Research Report
  • [Presentation] ローヤルゼリー糖タンパク質に結合するT-抗原ユニット含有糖鎖の生合成に関わるGalTとGalNAcTの発現2011

    • Author(s)
      金澤雅人
    • Organizer
      日本農芸化学会 中四国支部第29回講演会
    • Place of Presentation
      徳島大学
    • Year and Date
      2011-01-22
    • Related Report
      2010 Annual Research Report
  • [Presentation] 植物ERADで機能する糖鎖遺伝子の機能解析2011

    • Author(s)
      江原愛美
    • Organizer
      日本農芸化学会 中四国支部第29回講演会
    • Place of Presentation
      徳島大学
    • Year and Date
      2011-01-22
    • Related Report
      2010 Annual Research Report
  • [Presentation] トマト複合型N-グリカンの代謝に関わるα-Fucosidaseの遺伝子同定と発現系構築2011

    • Author(s)
      藤重誠
    • Organizer
      日本農芸化学会 中四国支部第29回講演会
    • Place of Presentation
      徳島大学
    • Year and Date
      2011-01-22
    • Related Report
      2010 Annual Research Report
  • [Presentation] 植物エンドグリコシダーゼの糖転移活性と部位特異的変異導入による活性残基同定2011

    • Author(s)
      岡本尚子
    • Organizer
      日本農芸化学会 中四国支部第29回講演会
    • Place of Presentation
      徳島大学
    • Year and Date
      2011-01-22
    • Related Report
      2010 Annual Research Report
  • [Presentation] 植物複合型N-グリカンの代謝に関わるβ-Xylosidaseの精製と発現系構築2011

    • Author(s)
      横内大輔
    • Organizer
      日本農芸化学会 中四国支部第29回講演会
    • Place of Presentation
      徳島大学
    • Year and Date
      2011-01-22
    • Related Report
      2010 Annual Research Report
  • [Presentation] Molecular characterization and molecular modeling of plantα-mannosidase2010

    • Author(s)
      Md. Anowar Hossain(木村吉伸)
    • Organizer
      日本農芸化学会平成22年度中四国支部大会
    • Place of Presentation
      香川大学
    • Year and Date
      2010-09-25
    • Related Report
      2011 Final Research Report
  • [Presentation] Molecular characterization and molecular modeling of plant α-mannosidase2010

    • Author(s)
      Md.Anowar Hossain
    • Organizer
      日本農芸化学会 平成22年度中四国支部大会
    • Place of Presentation
      香川大学
    • Year and Date
      2010-09-25
    • Related Report
      2010 Annual Research Report
  • [Presentation] Molecular characterization of acidicα-mannosidase involved in turnover of glycoproteins in tomato(Lycopersicum esculentum)2010

    • Author(s)
      Md. Anowar Hossain(Yoshinobu Kimura)
    • Organizer
      25th International Carbohydrate Symposium
    • Place of Presentation
      Tokyo
    • Year and Date
      2010-08-05
    • Related Report
      2011 Final Research Report
  • [Presentation] Molecular characterization of acidic α-mannosidase involved in turnover of glycoproteins in tomato (Lycopersicum esculentum)2010

    • Author(s)
      Md.Anowar Hossain
    • Organizer
      International Carbohydrate Symposium
    • Place of Presentation
      Tokyo
    • Year and Date
      2010-08-05
    • Related Report
      2010 Annual Research Report
  • [Presentation] イネエンド-β-N-アセチルグルコサミニダーゼの糖転移酵素活性と変異体作成2010

    • Author(s)
      岡本尚子(木村吉伸)
    • Organizer
      第51回日本生化学会中四国支部例会
    • Place of Presentation
      山口大学
    • Year and Date
      2010-05-15
    • Related Report
      2011 Final Research Report
  • [Presentation] イネエンド-β-N-アセチルグルコサミニダーゼの糖転移酵素活性と変異体作成2010

    • Author(s)
      岡本尚子
    • Organizer
      第51回 日本生化学会 中四国支部例会
    • Place of Presentation
      山口大学
    • Year and Date
      2010-05-15
    • Related Report
      2010 Annual Research Report
  • [Presentation] 新生タンパク質の品質管理系に関与する植物α-グルコシダーゼの精製と性質2010

    • Author(s)
      京谷優, 木村吉伸
    • Organizer
      日本農芸化学学会中四国支部講演会
    • Place of Presentation
      松山
    • Year and Date
      2010-01-23
    • Related Report
      2011 Final Research Report
  • [Presentation] 機能性糖鎖を多価有する人工糖鎖ポリマーの作成2010

    • Author(s)
      山西真緒美, 木村吉伸
    • Organizer
      日本農芸化学学会中四国支部講演会
    • Place of Presentation
      松山
    • Year and Date
      2010-01-23
    • Related Report
      2011 Final Research Report
  • [Presentation] 肝がん患者由来の血清糖タンパク質糖鎖及び遊離型糖鎖の構造特性2010

    • Author(s)
      三輪昇(木村吉伸)
    • Organizer
      日本農芸化学学会中四国支部講演会
    • Place of Presentation
      愛媛大学
    • Year and Date
      2010-01-23
    • Related Report
      2011 Final Research Report
  • [Presentation] 新生タンパク質の品質管理系に関与する植物α-グルコシダーゼの精製と性質2010

    • Author(s)
      京谷優(木村吉伸)
    • Organizer
      日本農芸化学学会 中四国支部講演会
    • Place of Presentation
      松山
    • Year and Date
      2010-01-23
    • Related Report
      2009 Annual Research Report
  • [Presentation] Correlation analysis between tomato-ripening mutants and N-glycan metabolism in ERAD2009

    • Author(s)
      Megumi Ehara(Yoshinobu KIMURA)
    • Organizer
      15th European Carbohydrate Symposium
    • Place of Presentation
      Vienna(Austria)
    • Year and Date
      2009-07-23
    • Related Report
      2011 Final Research Report 2009 Annual Research Report
  • [Presentation] The Transglycosylation Activity of Endo-β-N-Acetylglucosaminidase from Oriza sativa L2009

    • Author(s)
      Naoko Okamoto(Yoshinobu KIMURA)
    • Organizer
      15th European Carbohydrate Symposium
    • Place of Presentation
      Vienna(Austria)
    • Year and Date
      2009-07-23
    • Related Report
      2011 Final Research Report
  • [Presentation] Suppression of Endo-β-N-acetylglucosaminidase and Peptide : N-glycanase Activity in Arabidopsis thaliana2009

    • Author(s)
      Yuki Takeoka(Yoshinobu KIMURA)
    • Organizer
      15th European Carbohydrate Symposium
    • Place of Presentation
      Vienna(Austria)
    • Year and Date
      2009-07-23
    • Related Report
      2011 Final Research Report 2009 Annual Research Report
  • [Presentation] The Transglycosylation Activity of Endo-β-N-Acetylglucosaminidase from Oriza saliva L.2009

    • Author(s)
      Naoko Okamoto(Yoshinobu KIMURA)
    • Organizer
      15th European Carbohydrate Symposium
    • Place of Presentation
      Vienna(Austria)
    • Year and Date
      2009-07-23
    • Related Report
      2009 Annual Research Report
  • [Book] はじめて学ぶ生命科学の基礎: 3章生体分子II-糖質,脂質-2011

    • Author(s)
      木村吉伸
    • Publisher
      化学同人
    • Related Report
      2011 Final Research Report
  • [Book] はじめて学ぶ生命科学の基礎:3章生体分子II-糖質,脂質-2011

    • Author(s)
      木村吉伸
    • Total Pages
      28
    • Publisher
      化学同人
    • Related Report
      2011 Annual Research Report

URL: 

Published: 2009-04-01   Modified: 2016-04-21  

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