| Project/Area Number |
08456145
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Basic veterinary science/Basic zootechnical science
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| Research Institution | Obihiro University of Agriculture and Veterinary Medicine |
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Principal Investigator |
SHINAGAWA Morikazu Obihiro University of Agriculture and Veterinary Medicine・Veterinary Medicine, Professor, 畜産学部, 教授 (00001537)
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| Co-Investigator(Kenkyū-buntansha) |
KUWAYAMA Hideto Obihiro University of Agriculture and Veterinary Medicine・Veterinary Medicine, A, 畜産学部, 助教授 (40125399)
ISHIGURO Naotaka Obihiro University of Agriculture and Veterinary Medicine・Veterinary Medicine, A, 畜産学部, 助教授 (00109521)
堀内 基広 帯広畜産大学, 原虫病分子免疫研究センター, 助教授 (30219216)
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| Project Period (FY) |
1996 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥7,500,000 (Direct Cost: ¥7,500,000)
Fiscal Year 1998: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1997: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1996: ¥3,200,000 (Direct Cost: ¥3,200,000)
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| Keywords | prion amvloid / in vitro structural conversion / recombinant PrP^c / proteinase K resistance / CD spectrum / スクレイピー / コンゴーレッド結合 / プリオン / PrPC / in vitr o assay |
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| Research Abstract |
As a final goal of this study is in vitro formation of infectious prion amyloid using mouse PrP^C, in vitro structural conversion of PrP^C using a small amount of mouse prion was carried out. To eliminate the effects of unknown mouse protein contaminating in PrP^C, mouse recombinant PrP^C which possessed a histidine tag at N-terminus of mature PrP^C was used. The recombinant PrP^C converted to a proteinase K (PK) resistant form in the presence of one-thousandth amounts of mouse scrapie prion at pH 5.2 and room temperature for one day and after 14 days the PK-resistance increased more. A decrease of alpha-helix contents estimated by CD spectrum and an increase of beta-sheet contents estimated by Congo red binding were observed in the PK-resistant recombinant PrP^C, while the recombinant PrP^C incubated for 14 days without mouse prion showed a slight increase of PK-resistance and no change in alpha-helix and beta-sheet contents. These facts indicate that structural conversion occurred in the recombinant PrP^C in the presence of mouse prion. The conversion was also induced by adding one-hundredth amounts of the PK-resistant recombinant PrP^C, but was inhibited by adding 40 mM of a synthetic peptide corresponding to mouse prion codons 113-141. The molecular weight of the PK-resistant recombinant PrP^C did not change after PK-treatment but that of prion polypeptide decreases after PK-treatment in polyacrylamide gel electrophoresis. This indicates that the structure of the PK-resistant recombinant PrP^C differed from that of prion. In addition to PrP^C and prion, some unknown factors may be required to form prion amyloid. Other studies in relation to this theme also have been done.
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