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1996 Fiscal Year Final Research Report Summary

The development of a method for detection and quantification of cell wall components of bacteria and fungi.

Research Project

Project/Area Number 06554037
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section試験
Research Field 動物生理・代謝
Research InstitutionHOKKAIDO UNIVERSITY

Principal Investigator

ASHIDA Masaaki  Hokkaido Univ., The Institute of Low Temperature Science, Prof., 低温科学研究所, 教授 (50012422)

Co-Investigator(Kenkyū-buntansha) OCHIAI Masanori  Hokkaido Univ., The Institute of Low Temperature Science, Assistant Prof., 低温科学研究所, 助手 (10241382)
TSUCHIYA Masakazu  Research Institute of Wako Pure Chemical Industries, Ltd, Chief Researcher, 中央研究所, 研究員
Project Period (FY) 1994 – 1996
KeywordsPeptidoglycan / beta-1,3-Glucan / Phenol oxidase / cascade / Hemolymph / Lipopolysaccharide / Limulus test / SLP-reagent
Research Abstract

The end of the present project is to develop a method to detect and quantify minute amounts of peptidoglycan and beta-1,3-glucan. Although such method for lipopolysaccharide and beta-1,3-glucan has been developed by using hemocyte lysate supernatant of horseshoe crab (Limulus), peptidoglycan could not be detected by the method. Previously we reported that plasma fraction of silkworm hemolymph contains prophenol oxidase cascade that is triggered by minute amount of peptidoglycan or beta-1,3-glucan. The plasma fraction was studied if it could be used for detection and quantification of peptidoglycan and if it could be supplied commercially with minimal variation of its property. Toimprove the feasibility of the method, the mechanism of the activation of prophenol oxidase cascade with peptidoglycan or beta-1,3-glucan was also studied. The major results obtained during the term of the present project are as follows :
1) A method for processing silkworm hemolymph was developed for supplying plasma fraction for the detection and quantification of peptidoglycan and beta-1,3-glucan. The plasma fraction is now available commercially under a name of SLP-reagent.
2) Prophenol oxidase cascade of SLP-reagent could be triggered by bothof peptidoglycan and beta-1,3-glucan. SLP-reagent which is specifically triggered by peptidoglycan or by beta-1,3-glucan is desirable. We developed a method to make SLP-reagent specific to peptidoglycan or beta-1,3-glucan. This kind of method for raising the specificity of silkworm plasma has already been reported, but it had weak point in that prophenol oxidase cascade in plasma get start of activation during the manipulation of plasma. The newly developed method diminished the risk for the spontaneous activation. At present we succeeded in obtaining silkworm plasma of which prophenol oxidase cascade is 10,000 times more sensitive for being activated by peptidoglycan.

  • Research Products

    (22 results)

All Other

All Publications (22 results)

  • [Publications] Ashida, M.: "A target protease of serpins in insect hemolymph" Insect Biochem. & Molec. Biol.24. 1037-1041 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Brey, P. T.: "Tyrosinase-type prophenoloxidase distribution in the alimentary canal of strains of Anopheles gambiae refractory and susceptible to Plasmodium infection" Experimental Parasitol. 80. 654-664 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Katsumi, Y.: "A serine protease zymogen in insect plasma : Purification and activation by microbial cell wall components" Eur. J. Biochem.228. 870-877 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Yasuhara, Y.: "Re-examination of properties of prophenoloxidase isolated from larval hemolymph of the silkworm, Bombyx mori" Arch. Biochem. Biophys.320. 14-23 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kawabata, T.: "Molecular cloning of insect prophenoloxidase : A copper containing protein homologous to arthropod hemocyanin" Proc. Natl. Acad. Sci. U.S.A.92. 7774-7778 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ashida, M.: "Role of the integument in insect defense : Prophenoloxidase cascade in the cuticular matrix" Proc. Natl. Acad. Sci. U.S.A.92. 10698-10702 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Yoshida, H.: "Purification of a peptidoglycan recognition protein from hemolymph of the silkworm, Bombyx mori" J. Biol. Chem.271. 13854-13860 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tsuchiya, M.: "Detection of peptidoglycan and beta-glucan with silkworm larvae plasma test" FEMS Immunol. Med. Microbiol.15. 129-134 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ashida, M.: "Studies on the phenol oxidase system of insect" Zool. Sci.13. 3-4 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ashida, M.: "Recent advances on the research of the insect prophenoloxidase cascade" Molecular mechanisms of immune responses insects. (印刷中). (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Chen, C. C.: "Effects of inhibitors of serine protease, phenoloxidase and dopadecarboxylase on the melanization of Dirofilaria immitis microfilariae with Armigeres subalbatus hemolymph in vitro" Parasitology. (印刷中). (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ashida, M.: "A target protease of serpins in insect homolymph." Insect Biochem.& Molec.Biol. 24. 1037-1041 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Brey, P.T.: "Tyrosinase-type prophenoloxidase distribution in the alimentary canal of strains of Anopheles gambiae refractory and susceptible to Plasmodium infection." Experimental Parasitol. 80. 654-664 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Katsumi, Y.: "A serine protease zymogen in insect plasma : Purification and activation by microbial cell wall components." Eur.J.Biochem. 228. 870-877 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yasuhara, Y.: "Re-examination of properties of prophenoloxidase isolated from larval hemolymph, of the silkworm, Bombyx mori." Arch.Biochem.Biophys. 320. 14-23 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kawabata, T.: "Molecular cloning of insect prophenoloxidase : A copper containing protein homologous to arthropod hemocyanin." Proc.Natl.Acad.Sci.U.S.A.92. 7774-7778 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ashida, M.: "Role of the integument in insect defense ; Prophenoloxidase cascade in the cuticular matrix." Proc.Natl.Acad.Sci.U.S.A.92. 10698-10702 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yoshida, H.: "Purification of a peptidoglycan recognition protein from hemolymph of the silkworm, Bombyx mori." J.Biol.Chem.271. 13854-13860 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tsuchiya, M.: "Detection of peptidoglycan and beta-glucan with silkworm larvae plasma test." FEMS Immunology and Medical Microbiology. 15. 129-134 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ashida, M.: "Recent advances on the research of the insect prophenoloxidase cascade (Eds.Brey.P.T.and Hultmark, D.). In Molecular mechanisms of immune responses in insects." Chapman & Hall/London. (in press). (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Lee, W.-J.: "Molecular cloning and chromosomal localization of prophenoloxidase cDNA from the malaria vector Anopheles gambiae." Biochem.J.(in press). (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Chen, C.C.: "Effects of inhibitors of serine protease, phenoloxidase and dopa decarboxylase on the melanization of Dirofilaria immitis microfilariae with Armigeres subalbatus hemolymph in vitro" Parasitology. (in press). (1996)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1999-03-09  

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