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Studies on the Mechanisms of Lipopolysaccharide-induced Degranulation of Hemocytes of Invertebrate Animals (Horseshoe Crabs).

Research Project

Project/Area Number 09680597
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Structural biochemistry
Research InstitutionKYUSHU UNIVERSITY

Principal Investigator

MUTA Tatsushi  Kyushu Univ.Sch.Med., Biochem., Associate Professor, 医学部, 助教授 (60222337)

Co-Investigator(Kenkyū-buntansha) KAWABATA Shun-ichiro  Kyushu Univ., Biology, Associate Professor, 理学部, 助教授 (90183037)
Project Period (FY) 1997 – 1998
Project Status Completed (Fiscal Year 1998)
Budget Amount *help
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1998: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1997: ¥1,900,000 (Direct Cost: ¥1,900,000)
KeywordsLipopolysaccharide / Pattern Recognition / Coagulation / Degranulation / Horseshoe Crab / Signal Transduction / Receptor / Innate Immunity
Research Abstract

When washed horseshoe crab hemocytes were stimulated with lipopolysaccharide (LPS), degranulation of the cells was found to occur under microscopic observation. Therefore, plasma components are not required for the degranulation. Although the degranulation of the cells is induced by a calcium ionophore, the degranulation was observed even in the presence of extracellular EDTA.Thus, it was suggested that the degranulation does not require calcium influx from outside of the cells, but is induced by calcium release from the intracellular calcium pool.
We also analyzed LPS-binding of the hemocyte by using flow cytometry utilizing FITC- or biotin-labeled LPS . The hemocytes showed a strong LPS binding ability, which was even stronger than those found on the LPS -responsive mammalian cell. The binding was competed with excess amounts of intact LPS and was reduced by protease treatments of the cells. Furthermore, the binding was observed with LPS derived from Salmonella minnesota R595, which lacks the O-antigen polysaccharide. These results strongly indicated the presence of a protein(s) that specifically recognizes and bind to lipid A, which has most of the biological activity of LPS.
Although we tried to clone the cDNA for this protein by expression cloning on mammalian cells, we could not obtain any real positive signals. We had prepared several monoclonal antibodies against the hemocyte plasma membrane in order to isolate the binding protein by screening these antibodies. Thus far, we found that some of the antibodies were shown to inhibit the LPS-binding of the hemocytes. We are currently examining if this binding is specific and if they inhibit the degranulation of the hemocytes.

Report

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

    (10 results)

All Other

All Publications (10 results)

  • [Publications] Iwanaga,S.: "New Types of Clotting Factors and Defense Molecules Found in Horseshoe Crab Hemolymph:Their Structures and Functions." Journal of Biochemistry. 123,(1). 1-15 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Nanbo,A.: "Lipopolysaccharide stimulates HepG2 human hepatoma cells in the presence of lipopolysaccharide-binding protein via CD14" European Journal of Biochemistry. 発表予定.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Iwanaga, S., et al.: "New Types of Clotting Factors and Defense Molecules Found in Horseshoe Crab Hemolymph : Their Structures and Functions." Journal of Biochemistry. 123, (1). 1-15 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Nanbo, A., et al.: "Lipopolysaccharide stimulates HepG2 human hepatoma cells in the presence of lipopolysaccharide-binding protein via CD14" European Journal of Biochemistry. (in press). (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Iwanaga, S.: "New Types of Clotting Factors and Defense Molecules Found in Horseshoe Crab Hemolymph: Their Structures and Functions." Journal of Biochemistry. 123,(1). 1-15 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Nanbo, A.: "Lipopolysaccharide stimulates HepG2 human hepatoma cells in the presence of lipopolysaccharide-binding protein via CD14" European Journal of Biochemistry. (発表予定).

    • Related Report
      1998 Annual Research Report
  • [Publications] Takaki,Y.: "A Peptidyl-prolyl cis/trans Isomerase(Cyclophilin G)in Regulated Secretory Granules." J.Biol.Chem.272・45. 28615-28621 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Bergner,A.: "Horseshoe Crab Coagulogen Is an Invertebrate Protein with a Nerve Growth Factor-like Domain." Biol.Chem.Hoppe Seyler. 378. 283-287 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Iwanaga,S.: "New Types of Clotting Factors and Defense Molecules Found in Horseshoe Crab Hemolymph: Their Structures and Functions." J.Biochem.123・1. 1-15 (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] Muta,T.: "Methods Mol.Biol.,Vol.78: Antibacterial Peptide Protocols" Humana Press Inc., 259 (1997)

    • Related Report
      1997 Annual Research Report

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

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