| Project/Area Number |
09670275
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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 |
Bacteriology (including Mycology)
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| Research Institution | FACULTY OF AGRICULTURE,TOHOKU UNIVERSITY |
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Principal Investigator |
TOMITA Toshio TOHOKU UNIVERSITY,FACULTY OF AGRICULTURE,ASSOCIATE PROFESSOR, 農学部, 助教授 (00126129)
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| Co-Investigator(Kenkyū-buntansha) |
KAMIO Yoshiyuki TOHOKU UNIVERSITY,FACULTY OF AGRICUTURE,PROFESSOR, 農学部, 教授 (00109175)
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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: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | Staphylococcus aureus / bi-component cytolysin / staphylococcal gamma-hemolysin / staphylococcal leukocidin / pore-forming toxin / complex formation / ring structure / erythrocyte / neutrophil / リング状構造 |
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| Research Abstract |
Staphylococcal gamma-hemolysin consists of Hlg1 (or Luk F) and Hlg2, which cooperatively lyze human erythrocytes. Since gamma-hemolysin caused swelling of human erythrocytes before lysis, we studied pore-forming nature of the toxin by use of polyethylene glycols as osmotic protectants and determined the functional diameter of the pore. To elucidate the molecular architecture of the membrane pore formed by gamma-hemolysin, we solubilized the pore complex with 2 % sodium dodecyl sulfate, purified it by sucrose gradient centrifugation, and observed the purified pore complex under an electron microscope. Our data showed that the Hlg1 and the Hlg2 components assemble into a ring-shaped 200 kDa complex in a molar ratio of 1 : 1, forming a membrane pore with a functional diameter of 2.1-2.4 nm.
Staphylococcal leukocidin consists of LukS and LukF, which lyse human and rabbit polymorphonuclear leukocytes and rabbit erythrocytes. Here we studied the pore-forming properties of leukocidin and the molecular architecture of the leukocidin pore. (1) Leukocidin caused an efflux ofpotassium ions from rabbit erythrocytes and swelling of the cells before lysis. However, ultimate lysis of the swollen cells did not occur when polyethylene glycols with hydrodynamic diameters of <greater than or equal> 2.1 nm were present in the extracellular space. (2) Electron microscopy showed that the leukocidin formed a ring-shaped structure with outer and inner diameters of 9 and 3 nm, respectively, on human polymorphonuclear leukocytes and rabbit erythrocytes. (3) Ring-shaped structures of the same dimensions were isolated from the target cells, and they contained LukS and LukF in a molar ratio of 1 : 1. (4) A single ring-shaped toxin complex had a molecular size of 205 kDa. These results indicated that LukS and LukF assemble into a ring-shaped oligomer of approximately 200 kDa on the target cells, forming a membrane pore with a functional diameter of approximat
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