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Biosynthetic mechanism of informational lipid molecules in the signal transduction of mammalian cells and its molecular regulation by food functional agents

Research Project

Project/Area Number 09660139
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 食品科学・栄養科学
Research InstitutionShimane University

Principal Investigator

YOKOTA Kazushige  Shimane University Faculty of Life and Environmental Science Professor, 生物資源科学部, 教授 (90158361)

Co-Investigator(Kenkyū-buntansha) JISAKA Mitsuo  Shimane University Faculty of Life and Environmental Science Associate Professor, 生物資源科学部, 助教授 (60243424)
滝波 弘一  島根大学, 生物資源科学部, 教授 (40243422)
Project Period (FY) 1997 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1999: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1998: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1997: ¥1,300,000 (Direct Cost: ¥1,300,000)
Keywordsarachidonic acid / cyclooxygenase / reverse transcriptase-polymerase chain reaction / lipoxygenase / 8-lipoxygenase / 15-lipoxygenase / chimera / Madin-Darby canine kidney cell line / エイコサノイド / RT-PCR / アポトーシス / アラキドン酸カスケード / 不飽和化反応 / 必須脂肪酸 / エイコサペンタエン酸 / プロスタグランジン / プロスタノイド / アラキド酸 / アイソフォーム / 逆転写・ポリメラーゼ連鎖反応 / Maclin-Darbyイヌ腎臓上皮性細胞株 / リノール酸
Research Abstract

Arachidonate metabolites function as informational lipid molecules involved in the signal transduction of mammalian cells. We studied the regulatory mechanism of the arachidonate cascade by food-derived functional agents. As an interacting point of the food agents, we focused the regulation of gene expression of two isoforms of cyclooxygenase (COX), an rate-limiting enzyme in the biosynthetic pathway of prostanoids. To determine their levels specifically with higher sensitivity, the method using reverse transcriptase-polymerase chain reaction was developed with cRNA as internal standards. This method enabled us to determine the change in the levels of 2-type isoform in Madin-Darby canine kidney cells and mouse Swiss 3T3 cells. Tumor-promoting phorbol diesters caused an increase in the level of COX-2. The modification of the membrane phospholipids with n-6 and n-3 essential fatty acids resulted in the distinct regulation of the gene expression of COX isoforms depending on the types of unsaturated fatty acids.
The structures and function of phorbol diester-inducible novel lipoxygenase (LOX)s were studied by the recombinant DNA technology and protein engineering. The exchange of the C-terminal one-third of the 8S-LOX for the corresponding human homologue, 15S-LOX-2 produced a chimeric enzyme with exclusive 15S-lipoxygenase activity. The stereospecificity was found to be S configuration. These findings suggested that the C-terminal regions were determinant for the positional specificity of the lipoxygenases. By using the in situ chimeragenesis in Escherichia coli followed by the site-directed mutagenesis, a pair of amino acids, Tyrosine-603 and Histidine-604 was identified as the positional determinant. The mutation of those in 8S-LOX to the corresponding 15S-LOX-2 residues converted to the enzyme expressing almost 15S-lipoxygenase activity.

Report

(4 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • 1997 Annual Research Report
  • Research Products

    (40 results)

All Other

All Publications (40 results)

  • [Publications] 横田一成: "n-6及びn-3系列必須脂肪酸によるMadin-Darbyイヌ腎臓上皮性細胞株の膜修飾と細胞応答の制御"日本農芸化学会誌. 71. 285 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 横田一成: "Modification of membrane phospholipids with n-6 and n-3 essential fatty acid regulates the gene expression of prostaglandin endoperoxide synthase isoforms upon agonist-stimulation"Prostaglandins, Leukotrienes and Essential Fatty Acids. 57. 187 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 横田一成: "n-6及びn-3系列必須脂肪酸での膜リン脂質の修飾と細胞応答によるシクロオキシゲナーゼアイソフォーム遺伝子の発現調節"生化学. 69. 938 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 横田一成: "Modification of membrane phospholipids with n-6 and n-3 essential fatty acid regulation of arachidonate cascades in cultured Madin-Darby canine kidney cells"Proceedings of International Conference on Frontiers in Biotechnology, Trivandrum,. 48-49 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 横田一成: "必須脂肪酸による細胞膜リン脂質の修飾とII型シクロオキシゲナーゼアイソフォーム遺伝子の発現調節"日本農芸化学会誌. 72. 187 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 横田一成: "Madin-Darbyイヌ腎臓細胞株の膜修飾でのn-6及びn-3系列必須脂肪酸代謝の用量依存性と必須脂肪酸代謝"日本農芸化学会誌. 72. 177 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 地阪光生: "8-S及び15-Sリポキシゲナーゼ2の反応位置特異性の解析"生化学. 70. 1063 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 横田一成: "Molecular cell biology of arachidonate cascades and their dietary regulation focusing cyclooxygenase isoforms"Recent Res. Devel. in Agricultural and Biological Chem.. 2. 333-344 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 地阪光生: "Molecular cloning of a second human 15S-lipoxygenase and its murine homologue, an 8S lipoxygenase"Advance in Experimental Medicine and Biology. 447. 29-36 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 横田一成: "ジホモ-γ-リノレン酸を前駆体とするプロスタグランジンE_1に特異的なモノクローン抗体の作成と免疫特異性に関する研究"日本農芸化学会誌. 74巻臨時増刊. 157 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 地阪光生: "Identification of amino acid determinants of the positional specificity of mouse 8S-lipoxygenase and human 15S-lipoxygenases"J. Biol. Chem.. 275. 1287-1293 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 横田一成: "Essential Fatty Acids and Eicosanoids"AOCS Press, Champaign, USA. 6 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 横田一成: "Advances in Biotechnology, ed. by A. Pandey"Educational Publishers and Distributors, New Delhi. 19 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K.Yokota: "Modification of cell membranes in Madin-Darby canine kidney cells with n-6 and n-3 essential fatty acids and its regulation of cell responses"Nippon Nogei Kagaku Kaishi. 71. 285 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K.Yokota: "Modification of membrane phospholipids with n-6 and n-3 essential fatty acids regulates the gene expression of prostaglandin endoperoxide synthase isoforms upon agonist-stimulation"Prostaglandins, Leukotrienes and Essential Fatty Acids. 57. 187 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K.Yokota: "Modification of membrane phospholipids with n-6 and n-3 essential fatty acids and the regulation of gene expression of cyclooxygenase isoforms in cell resonses"Seikagaku. 69. 938 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K.Yokota: "Modification of membrane phospholipids with n-6 and n-3 essential fatty acids and the regulation of arachidonate cascades in cultured Madin-Darby canine kidney cells"Proceeding of International Conference on Frontiers in Biotechnology, Trivandrum. 48-49 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K.Yokota: "Modification of membrane phospholipids with essential fatty acids and the regulation of cyclooxygenase isoform-II"Nippon Nogei Kagaku Kaishi. 72. 187 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K.Yokota: "Dose-dependence of n-6 and n-3 essential fatty acids for the modification of membrane phospholipids in Madin-Darby canine kidney cells and the metabolism of essential fatty acids"Nippon Nogei Kagaku Kaishi. 72. 177 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] M.Jisaka: "Analysis of the positional specificity of the reaction of 8-S and 15-S lipoxygenase-2"Seikagaku. 70. 1063 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K.Yokota: "Molecular cell biology of arachidonicate cascades and their dietary regulation focusing cyclooxygenase isoforms"Recent Res. Devel. in Agricultural and Biological Chem.. 2. 333-344 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] M.Jisaka: "Molecular cloning of a second human 15S-lipoxygenase and its murine homologue, an 8S-lipoxygenase"Advance in Experimental Medicine and Biology. 447. 29-36 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K.Yokota: "Preparation of monoclonal antibodies against prostaglandin EィイD21ィエD2 derived from dihomo-γ-linolenic acid and their specificity"Nippon Nogei Kagaku Kaishi. 74 Supplement. 157 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] M.Jisaka: "Identification of amino acid determinants of the positional specificity of mouse 8S-lipoxygenase and human15S-lipoxygenases"J. Biol. Chem.. 275. 1287-1293 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K.Yokota: "Essential Fatty Acids and Eicosanoids"AOCS Press, Champaign. 6 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K.Yokota: "Advances in Biotechnology"Educational Publishers and Distributors. 19 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 横田一成: "ジホモ-γ-リノレン酸を前駆体とするプロスタグランジンEX_1に特異的なモノクローン抗体の作製と免疫反応特異性に関する研究"日本農芸化学会誌. 74臨時増刊. 157 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] 地阪光生: "Molecular cloning of a second human 15S-lipoxyglnese and its murine humologue,an 8S lipoxygenase"Advance in Experimental Medicine and Biology. 447. 29-36 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 地阪光生: "Identification of amino acid determinants of the positional specificity of mouse 8S-lipoxygenase and human 15S-lipoxygenase"J.Bial.Chem.. 275. 1287-1293 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Kazushige Yokota: "Recent Res.Devel.in Agricultural&Biological chem, vol 2"Research Signpost, Trivandrum. 12 (1998)

    • Related Report
      1999 Annual Research Report
  • [Publications] Kazushige Yokota: "Advandes in Biotechnology, ed.by A.Pandey"Educational Publishers&Distributors, New Delhi. 19 (1998)

    • Related Report
      1999 Annual Research Report
  • [Publications] 横田一成: "必須脂肪酸による細胞膜リン脂質の修飾とII型シクロオキシゲナーゼアイソフォーム遺伝子の発現調節" 日本農芸化学会誌. 72. 187 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] 浜口剛生: "Madin-Darbyイヌ肝臓細胞株の膜修飾でのn-6及びn-3系列必須脂肪酸代謝の用量依存性と必須脂肪酸代謝" 日本農芸化学会誌. 72. 177 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] 地阪光生: "8-S及び15-Sリポキシゲナーゼ2の反応位置特異性の解析" 生化学. 70. 1063 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] K.Yokota: "Modification of membrane phospholipids with n-6 and n-3 essential fatty acids regulates the expression of prostaglandin endoperoxide synthase isoforms upon agonist stimulation" AOCS Press,Champaign,USA, 6 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] 横田一成: "n-6及びn-3系列必須脂肪酸によるMaclin-Darbyイヌ腎臓上皮性細胞株の膜修飾と細胞応答の制御" 日本農芸化学会誌. 71. 285 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 横田一成: "Madification of membrane phosholipids with n-6 and n-3 essential fatly acids regulates the gene erpression of prestaglandclin endoperoxide synthase isoforms upon agonist-stimufation" Prostaglandins,denkotienes and Essential Fatty Acids. 57. 187 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 横田一成: "n-6及びn-3系列必須脂肪酸での脱リン脂質の修飾と細胞応答によるシクロオキシゲナーゼアイソフォーム遺伝子の発現調節" 生化学. 69. 938 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 横田一成: "Modification of membrane phosholipids with n-6 and n-3 essential fatty acids and regulation of arachidonate cascades in cultured Modin-Darby canine kidnisy cells" Proceedings of International Conference on Frontiers in Biotechnology,Trirandrum,India. 48-49 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 横田一成: "Recent REsearch Developments in Agricultural and Biological Chemistry" Research Signpost,Trirandrum(発表予定), (1998)

    • Related Report
      1997 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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