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Establishment of axnic culture of non-invasive Entamoeba dispar and study on its biological characterization

Research Project

Project/Area Number 08670287
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 寄生虫学(含医用動物学)
Research InstitutionKeio University

Principal Investigator

KOBAYASHI Seiki  Research associate, School of Medicine, Keio University, 医学部, 助手 (70112688)

Co-Investigator(Kenkyū-buntansha) NOZAKI Tomoyoshi  Research associate, School of Medicine, Keio University, 医学部, 助手 (60198588)
Project Period (FY) 1996 – 1997
Project Status Completed (Fiscal Year 1997)
Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1996: ¥1,100,000 (Direct Cost: ¥1,100,000)
KeywordsEntamoeba dispar / axnic cultivation / growth promoting facoor / Pseudomonas sp. / Crithidia sp. / Entner-Doudoroff pathway / 6-phosphogluconate / dihydroxy acetone / 6ホスホグルコン酸 / hexokinase / fructose
Research Abstract

We developed an axnic cultivation system for Entamoeba dispar which is difficult to grow in usual axnic medium of Entamoeba histolytica without intestinal bacteria or some protozoa such as Crithidia fasciculata.
Since Clark (1995) reported successful axnic cultivation of strain SAW 760 RR clone A of E.dispar, other strains also have been tried for the axnic cultivation. However, E.dispar strain which could be adapted to axnic culture conditions is only the SAW 760 RR clone A,up to the present.
In this research project, we found one metabolic intermediate (6-phosphogluconate : 6PG) which could promote the growth of three strains, at least, of E.dispar. This finding followed our study on growth promoting activities in the extract of Pseudomonas aeruginosa. The basic monoxemic culture system of E.dispar with P.aeruginosa needed modification of the medium for axnic Entamoeba histolytica (BI-S-33 medium) as follows : the addition of acetone or dihydroxyacetone, removal or replacement of gluco … More se with maltose or hydrlized starch and sterilization by filtration. In this system, the number of E.dispar ameba isolates (over 12 strains) usually reached 2-3 x 10^5/ml at those growth peaks, constantly.
We tried modification of this system and design a new medium for cultivation of E.dispar axnically. Basic medium for the axnic culture was casein-free YI-S (Diamond, 1995) by which Clack (1995) reported successful axnic cultivation of E.dispar by supplementation of gastric mucin.
A E.dispar growth promoting activity could be found in the polysaccharide rich fraction of P.aeruginosa, and it was more effective in gluconate and dihydroxyacetone supplemented YI-S medium. Though gluconate kinase and glucose-6-phosphate dehydrogenase (G6PDH) for producing 6PG could not be detected in the extract of E.dispar, gluconate seemed to be metabolized by E.dispar in the presence of P.aeruginosa extract. And, interestingly, it was found that 6PG rich fraction made from glucose-6-phosphate (G6P), NADP and crude extract of Crithidia fasciculata which has extremely high G6PDH ativity and also gluconate kinase activity, has the growth promoting effect. It was further confirmed that this growth promoting effect was attributed to 6PG by using commercial G6PDH and G6P.These results are still preliminary but seem clear and reproducible, so we are now studying the role of 6PG in the axnic growth of E.dispar and the related glycolytic pathway such as Entner-Doudoroff pathway which was reported to be present in xenic E.histolytica (Hilker and White, 1959). Less

Report

(3 results)
  • 1997 Annual Research Report   Final Research Report Summary
  • 1996 Annual Research Report
  • Research Products

    (18 results)

All Other

All Publications (18 results)

  • [Publications] ASAO MAKIOKA: "Comparison of DNA polymerase between Entamoeba histolytica and E. dispar" Parasitology International. 46. 27-30 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] TSUTOMU TAKEUCHI: "Entamoeba dispar : cultivation without viable associate and its characterization" Arch. Med. Res.28. 108-109 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] JUNICHI SANUKI: "Identification of Entamoeba histolytica and E. dispar cysts in stool by polymerase chain reaction" Parasitology Research. 83. 96-98 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] HIROSHI TACHIBANA: "Preparation of monoclonal antibody specific for Entamoeba dispar and its ability to distinguish E. dispar from E. histolytica" Clinical and Diagnostic Laboratory Immundogy. 4. 409-414 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] HIROSHI TACHIBANA: "Differentiation of Entamoeba histolytica from E. dispar facilitated by monoclonal antibodies against a 150-KDasurface antigen" Parasitology Research. 83. 435-439 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] SEIKI KOBAYASHI: "Entamoeba dispar : cultivation with sterilized crithidia fasciculata." J. Euk. Microbiol.45. 173-178 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Asao Makioka: "Comparison of DNA polymerase between Entamoeba histolytica and E.dispar." Parasitology International. 46. 27-30 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Tsutomu Takeuchi: "Entamoeba dispar : Cultivation without viable associate and its characterization." Arch.Med.Res.28. 108-109 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Jun-ichi Sanuki: "Identification of Entamoeba histolytica and E.dispar cysts in stool by polymerase chain reaction." Parasitology Research.83. 96-98 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Hiroshi Tachibana: "Preparation of monoclonal antibody specific for Entamoeba dispar and its ability to distinguish E.dispar from E.histolytica." Clinical and Diagnostic Laborator Immunology.4. 409-414

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Hiroshi Tachibana: "Differentiation of Entamoeba histolytica from E.dispar facilitated by monoclonal antibodies against a 150-KDa surface antigen." Parasitology Research. 83. 435-439 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Seiki Kobayashi: "Entamoeba dispar : Cultivation with sterilized Crithidia fasciculata." J.Euk.Microbiol.45. 173-178 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] TSUTOMU TAKEUCHI: "Entamoeba dispar:cultivation without viable associate and its characterization" Arch.Med.Res.28. 108-109 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] JUNICHI SANUKI: "Identification of Entamoeba histolytica and E.dispar cysts in stool by polymerase chain reaction" Parasitology Research. 83. 96-98 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] HIROSHI TACHIBANA: "Preparation of monoclonal antibody specific for Entamoeba dispar and its ability to distinguish E.dispar from E.histolytica" Clinical and Diagnostic Laboratory Immunology. 4(4). 409-414 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] HIROSHI TACHIBANA: "Differentiation of Entamoeba histolytica from E,dispar facilitated by monoclonal antibodies against a 150-KDa surface antigen" Parasitology Research. 83. 435-439 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] SEIKI KOBAYASHI: "Entamoeba dispar:cultivation with sterilized crithidia fasciculata." J.Euk.Microbiol.45(2). 173-178 (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] Asao Makioka: "Comparison of DNA polymerase between Entamoeba histolytica and E.dispar" Pavasitology International. (in press). (1997)

    • Related Report
      1996 Annual Research Report

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Published: 1996-04-01   Modified: 2016-04-21  

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