Studies on the Mechanisms of Lipopolysaccharide-induced Degranulation of Hemocytes of Invertebrate Animals (Horseshoe Crabs).
| Project/Area Number |
09680597
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | KYUSHU UNIVERSITY |
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Principal Investigator |
MUTA Tatsushi Kyushu Univ.Sch.Med., Biochem., Associate Professor, 医学部, 助教授 (60222337)
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| Co-Investigator(Kenkyū-buntansha) |
KAWABATA Shun-ichiro Kyushu Univ., Biology, Associate Professor, 理学部, 助教授 (90183037)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| 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)
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| Keywords | Lipopolysaccharide / Pattern Recognition / Coagulation / Degranulation / Horseshoe Crab / Signal Transduction / Receptor / Innate Immunity |
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| 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.
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Report
(3 results)
Research Products
(10 results)
