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In Vitro studies on the formation of prion amyloid

Research Project

Project/Area Number 08456145
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Basic veterinary science/Basic zootechnical science
Research InstitutionObihiro University of Agriculture and Veterinary Medicine

Principal Investigator

SHINAGAWA Morikazu  Obihiro University of Agriculture and Veterinary Medicine・Veterinary Medicine, Professor, 畜産学部, 教授 (00001537)

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)
Project Period (FY) 1996 – 1998
Project Status Completed (Fiscal Year 1998)
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)
Keywordsprion 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
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.

Report

(4 results)
  • 1998 Annual Research Report   Final Research Report Summary
  • 1997 Annual Research Report
  • 1996 Annual Research Report
  • Research Products

    (25 results)

All Other

All Publications (25 results)

  • [Publications] Grathwohl, K.-U.D.et al.: "Sensitive enzyme-linked immunosorbent assay for detection of PrP^<Sc> in crude tissue extracts from scrapie-affected mice." J Virol Methods. 64. 205-216 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Horiuchi, M.et al.: "Alternative usage of exon 1 of bovine PrP mRNA." Biochem Biophy Res Commun. 233. 650-654 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Komatsu, Y.et al.: "Characterization of the sheep apolipoprotein E Gene (Apo E) gene and allelic variations of the ApoE gene in scrapie Suffolk sheep." Gene. 208. 131-138 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Horiuchi, M.et al.: "Genomic structure of the bovine PrP gene and complete nucleotide sequence of bovine PrP cDNA." Animal Genetics. 29. 37-40 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Ishiguro, N.et al.: "Rapid analysis of allelic variants of the sheep PrP gene by oligonucleotide probes." Microbiol Immunol. 42. 579-582 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Nemoto, T.et al.: "Detection methods of possible prion contaminants in collagen and gelatin." Arch Virol. 144. 177-184 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Laplanche, J.-L.et al.: "In Prion Biology and Discases (Prusiner, S.B.ed)" Cold Spring Harbor Laboratory Press, (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Grathwohl, K-U.D., Horiuchi, M., Ishiguro, N., Shinagawa, M.: "Improvement of PrPSc-detection in mouse spleen early at the preclinical stage of scrapie with collagenase-completed tiusse homogenization and Sarkosyl-NaCl extraction of PrP^<Sc>." Arch.Virol.141. 1863-1874 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Inoue S., Tanaka, M., Horiuchi, M., Ishiguro, N., Shinagawa, M.: "Characterization of the bovine prion protein gene : The expression requires interecation between the promoter and intron." J.Vet.Med.Sci.59. 175-183 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Grathwohl, K-U.D., Horiuchi, M., Ishiguro, N., Shinagawa, M.: "Sensitive enzyme-linked immunisorbent assay for detenction of PrP^<Sc> in crude tissue extracts from scrapie-affected mice." J.Virol.Methods. 64. 205-216 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Horiuchi, M., Ishiguro, N., Nagasawa, H., Toyoda, Y.and Shinagawa, M.: "Alternative usage of exon 1 of bovine PrP mRNA." Biochem.Biophy.Res.Commum. 233. 650-654 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Komatsu, Y., Horiuchi, M., Ishiguro, N., Matsui, T., Shinagawa, M.: "Characterization of the sheep apolipoprotein E Gene(Apo E)gene and allelic variations of the Apo E gene in scrapie Suffolk sheep." Gene. 208. 131-138 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Horiuchi, M., Ishiguro, N., Nagasawa, H., Toyoda, Y., Shinagawa, M.: "Genomic structure of the bovine PrP gene and complete nucleotide sequence of bovine PrP cDNA." Animal Genetics. 29. 37-40 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Ishiguro, N., Shinagawa, M., Onoe, S., Yamanouchi, K., Saito, T.: "Rapid analysis of allelic variants of the sheep PrP gene by oligonucleotide probes." Microbiol.Immunol. 42. 579-582 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Nemoto, T., Horiuchi, M., Ishiguro, N., Shinagawa, M.: "Detection methods of possible prion contaminants in collagen and gelatin." Arch.Virol.144. 177-184 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Laplanche, J-L., Hunter, N., Shinagawa, M., Williams, E.In Prion Biology and Diseases(Prusiner, S.B.ed): Scrapie, Chronic Wasting Disease, and Transmissible Mink Encephalopathy.Cold Spring Harbor Laboratory Press(in press),

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Grathwohl,K.-U.D.et al.: "Sensitive enzyme-linked immunosorbent assay for detection of PrP^<Sc> in crude tissue extracts from scrapie-affected mice." J Virol Methods. 64. 205-216 (1997)

    • Related Report
      1998 Annual Research Report
  • [Publications] Horiuchi,M.et al.: "Alternative usage of exon 1 of bovine PrP mRNA." Biochem Biophy Res Commun. 233. 650-654 (1997)

    • Related Report
      1998 Annual Research Report
  • [Publications] Komatsu,Y.et al.: "Characterization of the sheep apolipoprotein E Gene(Apo E)gene and allelic variations of the ApoE gene in scrapie Suffolk sheep." Gene. 208. 131-138 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Horiuchi,M.et al.: "Genomic structure of the bovine PrP gene and complete nucleotide sequence of bovine PrP cDNA." Animal Genetics. 29. 37-40 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Ishiguro,N.et al.: "Rapid analysis of allelic variants of the sheep PrP gene by oligonucleotide probes." Microbiol Immunol. 42. 579-582 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Nemoto,T.et al.: "Detection methods of possible prion contaminants in collagen and gelatin." Arch Virol. 144. 177-184 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] Laplanche,J.-L.et al.: "In Prion Biology and Diseases(Prusiner,S.B.ed)" Cold Spring Harbor Laboratory Press, (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] Inoue,S.et.al.: "Characterization of the bovine prion protein gene: The expression requires interaction between promoter and intron." J.Vet.Med Sci.59. 175-183 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] S.Inoue et al.: "Char acterization of the boving prion protein gene : the expression requires interaction between the promter and intron." J.Vet.Med.Sci.59(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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