In vitroプリオン増殖機構解析:PrP遺伝子欠損細胞を用いた改良PMCA法

2008 Fiscal Year Annual Research Report

• Project/Area Number: 20780219
• Research Institution: Osaka University
• Principal Investigator: 作道 章一 Osaka University, 微生物病研究所, 助教 (10397672)
• Keywords: 微生物 / 獣医学 / 感染症 / ウイルス / 神経科学 / プリオン / 人畜共通感染症

Research Abstract

本研究は、プリオン蛋白質(PrP)遺伝子欠損マウス神経細胞株に欠損変異マウスPrP遺伝子もしくは異種PrP(ハムスター、ウシ)を導入した細胞のライゼートを正常型PrP(PrPC)供給源として、マウススクレイピープリオンもしくは異種プリオン(それぞれハムスタースクレイピープリオン、Bovine spongiform encephalopathy (BSE)プリオン)存在下でprotein misfolding cyclic amplification(PMCA)を行い、異常型PrP(PrPSc)増幅の有無からPrPSc増幅に必要なPrPC領域の同定および異種PrPScの増幅をおこなうことを最終目的とする。本年度は、PrP遺伝子欠損マウス神経細胞株に欠損変異マウスPrP遺伝子欠損変異PrP遺伝子(PrPΔ#1, PrPΔ#2, PrPΔ#3)を導入した細胞、ハムスターPrP遺伝子を導入した細胞、ウシPrP遺伝子を導入した細胞を樹立した。さらに、これらの樹立した細胞より細胞ライゼートを調整し、抗PrP抗体を用いたウエスタンブロッティングによるPrPの検出を行った。その結果、PMCAに利用するに十分な量のPrPC供給が可能なPrP発現細胞の樹立と細胞ライゼートの調整ができていることがわかった。加えて、異種および同種PrP遺伝子発現細胞のアポトーシス感受性を血清除去誘導アポトーシスにより比較したところ、同種(マウス)PrPの場合のみPrPがアポトーシス抑制に働き、ハムスターやウシPrPにはアポトーシス抑制効果が見られないことが明らかとなった。

Research Products

• [Journal Article] Quantification of PrPC in bovine peripheral tissues : analysis in wild-type and PrPC-deficient cattle (2009)
  • Author(s): Kobayashi S, Ano Y, Sakudo A, Yukawa M, Sugiura K, Manabe N, Nakayama H, Onodera T.
  • Journal Title: Mol Med Rep (印刷中)
  • Peer Reviewed
• [Journal Article] Role of cellular prion proteins in the function of macrophages and dendritic cells (2009)
  • Author(s): Nitta K, Sakudo A, Masuyama J, Xue G, Sugiura K, Onodera T.
  • Journal Title: Protein Peptide Lett (印刷中)
  • Peer Reviewed
• [Journal Article] Uptake and dynamics of infectious prion protein in the intestine (2009)
  • Author(s): Ano Y, Sakudo A, Nakayama H, Onodera T.
  • Journal Title: Protein Peptide Lett (印刷中)
  • Peer Reviewed
• [Journal Article] Fundamentals of prion diseases and their involvement in the loss of function of cellular prion protein (2009)
  • Author(s): Sakudo A, Ikuta K
  • Journal Title: Protein Peptide Lett (印刷中)
  • Peer Reviewed
• [Journal Article] Intestinal uptake of amyloid β protein through columnar epithelium cells in suckling mice (2009)
  • Author(s): Ano Y, Nakayama H, Sakudo A, Sawano Y, Tanokura M, Itohara S, Onodera T
  • Journal Title: Histol Histopathol 24 Pages: 283-292
  • Peer Reviewed
• [Journal Article] Prion protein functions and dysfunction in prion diseases (2009)
  • Author(s): Sakudo A, Ikuta K
  • Journal Title: Curr Med Chem 16 Pages: 380-389
  • Peer Reviewed
• [Journal Article] Octapeptide repeat region of prion protein (PrP) is required at an early stage for production of abnormal prion protein in PrP-deficient neuronal cell line (2008)
  • Author(s): Sakudo A, Wu G, Onodera T, Ikuta K
  • Journal Title: Biochem Biophys Res Commun 365 Pages: 164-169
• [Journal Article] Cellular prion protein prevents brain damage after encephalomyocarditis virus infection in mice (2008)
  • Author(s): Nasu-Nishimura Y, Taniuchi Y, Nishimura T, Sakudo A, Nakajima K, Ano Y, Sugiura K, Sakaguchi S, Itohara S, Onodera T
  • Journal Title: Arch Virol 153 Pages: 1007-1012
• [Journal Article] PrPSc level and incubation time in a transgenic mouse model expressing Borna disease virus phosphoprotein after intracerebral prion infection (2008)
  • Author(s): Sakudo A, Onodera T, Ikuta K
  • Journal Title: Neurosci Lett 431 Pages: 81-85
• [Journal Article] Establishment of a new glial cell line from hippocampus of prion protein gene-deficient mice (2008)
  • Author(s): Nishimura T, Sakudo A, Xue G, Ikuta K, Yukawa M, Sugiura K, Onodera T
  • Journal Title: Riochem Bionhys Res Commun 377 Pages: 1047-1050
  • Peer Reviewed
• [Journal Article] Species-specific anti-apoptotic activity of cellular prion protein in a mouse PrP-deficient neuronal cell line transfected with mouse, hamster, and bovine Prnp (2008)
  • Author(s): Wu G, Nakajima K, Takeyama N, Yukawa M, Taniuchi Y, Sakudo A, Onodera T
  • Journal Title: Neurosci Lett 446 Pages: 11-15
  • Peer Reviewed
• [Journal Article] Incorporation of beta-amyloid protein through the bovine ileal epithelium before and after weaning : model for orally transmitted amyloidoses (2008)
  • Author(s): Ano Y, Nakayama H, Sakai Y, Sakudo A, Endo M, Ebisu S, Li J, Uetsuka K, Manabe N. Onodera T
  • Journal Title: Microbiol Immunol 52 Pages: 429-434
  • Peer Reviewed
• [Journal Article] Coordinate regulation of bovine prion protein gene promoter activity by two Sp1 binding site polymorphisms (2008)
  • Author(s): Xue G, Sakudo A, Kim CK, Onodera T.
  • Journal Title: Biochem Biophys Res Commun 372 Pages: 530-535
  • Peer Reviewed
• [Journal Article] Distinct immunohistochemieal localization in Kuru plaques using novel anti-prion protein antibodies (2008)
  • Author(s): Hosokawa T, Ono F, Tsuchiya K, Sato I, Takeyama N, Ueda S, Zanusso G, Takahashi H, Sata T, Sakudo A, Suguira K, Baj A, Toniolo A ; Yoshikawa Y, Oondera
  • Journal Title: Microbiol Immunol 52 Pages: 25-29
  • Peer Reviewed
• [Journal Article] Normal cytochrome c oxidase activity in prion proteingene-deficient mice (2008)
  • Author(s): Sakudo A ; Taniuchi Y, Kobayashi T, Onodera T, Ikuta K
  • Journal Title: Protein Peptide Lett 15 Pages: 250-254
  • Peer Reviewed
• [Journal Article] GPI-anchor-less human prion protein is secreted and glycosylated but lacks SOD activity (2008)
  • Author(s): Sakudo A, Nakamura I, Isuji S, Ikuta K
  • Journal Title: Int J Mol Med 21 Pages: 217-222
  • Peer Reviewed
• [Journal Article] A monoclonal antibody (1D12) defines novel distribution patterns of prion protein (PrP) as granules in nucleus (2008)
  • Author(s): Hosokawa T, Tsuchiya K, Sato I, Takeyama N, Ueda S, Tagawa Y, Kimura KM, Nakamura I, Wu G, Sakudo A, Casalone C, Mazza M, Caramelli M, Takahashi H, Sata T, Sugiura K, Baj A, Toniolo A, Onodera T
  • Journal Title: Biochem Biophys Res Commun 366 Pages: 657-663
  • Peer Reviewed
• [Presentation] スクレーピー感染マウス脳における酸化ストレス障害 (2009)
  • Author(s): 阿野泰久, 作道章一, 梶村佳史, 生田和良, 小野寺節
  • Organizer: 第147回日本獣医学会学術集会
  • Place of Presentation: 栃木
  • Year and Date: 20090402-20090404
• [Presentation] Uptake of amyloid protein in the murine and bovine intestines before and after weaning : a model for the enteric invasion of infectious prion protein (2008)
  • Author(s): Ano Y, Nakayama H, Sato Y, Sakudo A, Manabe N, Yukawa M, Onodera T
  • Organizer: Prion 2008
  • Place of Presentation: Madrid
  • Year and Date: 20081008-10
• [Presentation] 可視・近赤外分光法によるマウススクレーピー感染病態解析 (2008)
  • Author(s): 作道章一, 阿野泰久, 小林孝徳, 小野寺節, 生田和良
  • Organizer: 第146回日本獣医学会学術集会
  • Place of Presentation: 宮崎
  • Year and Date: 20080924-20080926
• [Presentation] DNA damage through oxidative stresses in the prion-infected mouse brain (2008)
  • Author(s): Ano Y, Sakudo A, He XJ, Sato Y, Yukawa M, Ikuta K, Yokoyama T, Nakayama H, Onodera T
  • Organizer: The 14th International Congress of Virology
  • Place of Presentation: Istanbul. Turkey
  • Year and Date: 20080810-15
• [Book] Molecular detection of Foodborne pathogens (Edited by Liu D)Chapter 43 : Slow viral disease (eg, prions)
  • Author(s): Onodera T, Sakudo A, Sugiura K
  • Publisher: Taylor and Francis Group(印刷中)