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1999 Fiscal Year Final Research Report Summary

Development of Real-time Imaging Method of Protein Tyrosine Phosphorylation

Research Project

Project/Area Number 09670115
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field General pharmacology
Research InstitutionNational Institute of Health Sciences

Principal Investigator

KAWANISHI Toru  National Institute of Health Sciences, Division of Biological Chemistry and Biologicals, Section Chief, 生物薬品部, 室長 (40124383)

Co-Investigator(Kenkyū-buntansha) WATABE Akiko  National Institute of Health Sciences, Division of Biological Chemistry and Biologicals, Researcher, 生物薬品部, 研究員 (50291117)
UCHIDA Eriko  National Institute of Health Sciences, Division of Biological Chemistry and Biologicals, Section Chief, 生物薬品部, 室長 (80176685)
KAWAI Hiroshi  National Institute of Health Sciences, Division of Biological Chemistry and Biologicals, Researcher, 生物薬品部, 研究員 (20321854)
Project Period (FY) 1997 – 1999
Keywordstyrosine phosphorylation / SH2 domain / fluorescence probe / Green Fluorescence Protein / FRET / fluorescence microscopy
Research Abstract

We have tried to develop fluorescence probes for imaging of protein tyrosine phosphorylation. First, the genes coding SH2 domains in Grb2/Ash and PLCγ1 were fused with pEGFP-N and -C vectors (Clonetech) and the pEGFP-SH2 vectors were produced for the expression of EGFP-SH2. Four conjugated proteins, showing the almost same fluorescence spectra as EGFP, have been expressed in E.Coli. The proteins could be expressed in Hela cell, RBL-2H3 cell, HegG2 cell, and so on. However, no reproducible change in the localization of the fluorescence has been observed after the treatment with the stimulants of tyrosine phosphorylation. Second, the fluorescence probes, whose fluorescence wavelength is changed by FRET between ECFP and EYFP, have been developed. The plasmids for the expression of CFP-SH2 (Grb2/Ash) -YFP, YFP-SH2 (Grb2/Ash) -CFP, and YFP-SH2 (PLCγ1) -CFP were produced. The peptides, which are recognized by the SH2 domains in EGF receptor and PDGF receptor β, and those, whose tyrosine are phosphorylated, are both chemically synthesized. The fluorescence spectrum is different between YFP-SH2 (PLCγ1) -CFP incubated with the synthesized peptide and that incubated with the phosphorylated peptide. The plasmids for the expression of the fused proteins of the polypeptide (which are designed from N-terminal and C-terminal of EGF receptor and PDGF receptor), whose tyrosine are phosphorylated, and ECFP or EYFP have been produced. The fluorescence spectrum of one (CFP- (20amino acids residue around the tyrosine residue phosphorylated in PDFG receptor) -YFP) of the fused proteins has been changed after the incubation with the homogenate debris of HeLa cell, RBL-2H3 cell and so on, in the presence of ATP and EGF (PDGF). Unfortunately, the significant change of the fluorescence of those probes following the stimulation of tyrosine phosphorylation has not been detected in the cells, yet.

  • Research Products

    (26 results)

All Other

All Publications (26 results)

  • [Publications] 川西 徹: "細胞内カルシウムイオンのイメージング"組織細胞工学. 24. 4-7 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hikaru Tanaka,Toshiyuki Sekina,Toru Kawanishi,Ryu Nakamura,and Koki Shigenobu:: "Intrasarcomere [Ca^<2+>] Gradients and Their Spatio-Temporal Relation to Ca^<2+> Sparks in Rat Cardiomyocytes"J.Physiol. 508. 145-158 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Isao Kasugai,Toru Kawanishi,Kazushige Morimoto,and Takao Hayakawa: "Establishment of a human leukemia HL-60 cell line that expresses high levels of M-CSF receptors"In Vitro Cell Dev.Biol.,-Animals. 34. 93-96 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Masayuki YAMAMOTO,Toru KAWANISHI,et al.: "Discrepant Intracellular pH Changes Following Intracellular Ca^<2+> Increase Induced by Glutamate and Ca^<2+> lonophores in Rat Hippocampal Neurons"Life Sci.. 63. 55-63 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 川西 徹: "イオンインジケータ"病理と臨床. 16. 1141-1147 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 川西 徹: "共焦点レーザー走査顕微鏡を用いた細胞内カルシウムイオンの高速高分解能画像化"日本薬理学雑誌. 112. 89-95 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Toru Kawanishi,Hiroki Asoh,Takashi Kato,Chikako Uneyama,Kazuhiro Toyoda,Reiko Teshima,Hideharu Ikebuchi,Hisayuki Ohata,Kazutaka Momose,Takao Hayakawa,and Michihito Takahashi: "Suppression of Calcium Oscillation by Tributylt Chloride in Cultured Rat Hepatocytes"Toxicol.Appl.Pharmacol.. 155. 54-61 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] M.Takahashi,Y.Misawa,N.Watanabe,T.Kawanishi,H.Tanaka,K.Shigenobu,and Y.Kobayashi: "Role of P-glycoprotein in human natural killer-like cell line-mediated cytotoxicity"Exp.Cell Res.. 253. 396-402 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Nana Kawasaki,Yuan C.Lee,Osamu Hashimoto,Masayuki Yamamoto,Toru Kawanishi,Takao Hayakawa: "Fluorometric Determination of Aminopolycarboxylate Using Fluo-3"Anal.Biochem.. 270. 329-331 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] H.Tanaka,T.Ichikawa,S.Matsui,K.Okazaki,H.Masumiya,T.Kawanishi and K.Shigenobu: "Calcium channel antagonistic effects of AH-1058,a novel antiarrythmic drug,on guinea-pig myocardium"Res.Commun.Mol.Pathol.Pharmacol.. 104. 13-21 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 川西 徹: "高速共焦点顕微鏡"細胞. 31. 14-18 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Toru Kawanishi,Takehito Kiuchi,Hiroki Asoh,Rie Shibayama,Hiroshi Kawai,et al.: "Effect of Tributyltin Chloride on Release of Calcium Ion from Intracellular Calcium Stores in Rat Hepatocytes"Biochem Pharmacol. (in press).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 川西 徹: "バイオイメージングの最先端(細胞内カルシウムイオンダイナミクスのイメージングを担当)"先端医療技術研究所. 323 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 川西 徹: "基礎生化学実験法第2巻 生体試料(共焦点顕微鏡を担当)"東京化学同人. 245 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hikaru Tanaka, Toshiyuki Sekine, Toru Kawanishi, Ryu Nakamura, and Koki Shigenobu: "Intrasarcomere [Ca^<2+>] Gradients and Their Spatio-Temporal Relation to Ca^<2+> Sparks in Rat Cardiomyocytes."J.Physiol.. 508(1). 145-152 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Isao Kasugai, Toru Kawanishi, Kazushige Morimoto, and Takao Hayakawa: "Establishment of a human leukemia HL-60 cell line that expresses high levels of M-CSF receptors."In Vitro Cell Dev.Biol., -Animals. 34. 93-96 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Masayuki YAMAMOTO, Toru KAWANISHI, Takehito KIUCHI, Miyako OHTA, Isue YAKOTA, Hisayuki OHATA, Kazutaka MOMOSE, Kazuhide INOUE and Takao HAYAKAWA: "Discrepant Intracellular pH Changes Following Intracellular Ca^<2+> Increase Induced by Glutamate and Ca^<2+> Ionophores in Rat Hippocampal Neurons."Life Sci.. 63. 55-63 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Toru KAWANISHI: "Imaging of intracellular Calcium Ion (Japanese)"Tissue, Cellular Engineering. 24. 4-7 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Toru KAWANISHI: "Ion Indicators (Japanese)"Pathology and Clinics. 16. 1141-1147 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Toru KAWANISHI: "Rapid Scannning High Resolution Imaging of Intracellular Calcium Ion Using Confocal Raser Scanning Microscopy (Japanese)"Nihon Yakurigaku Zasshi. 112. 89-95 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Toru KAWANISHI,Hiroki ASOH,Takashi KATO, Chikako UNEYAMA, Kazuhiro TOYODA, Reiko TESHIMA, Hideharu IKEBUCHI, Hisayuki OHATA, Kazutaka MOMOSE, Takao HAYAKAWA, and Michihito TAKAHASHI: "Suppression of Calcium Oscillation by Tributyltin Chloride in Cultured Rat Hepatocytes."Toxicol. Appl. Pharmacol. 155. 54-61 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] M.TAKAHASHI, Y.MISAWA, N.WATANABE, T.KAWANISHI, H.TANAKA, K.SHIGENOBU, and Y.KOBAYASHI: "Role of P-glycoprotein in human natural killer-like cell line-mediated cytotoxicity."Exp. Cell Res. 253. 396-402 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Akiko WATANABE, Teruhide YAMAGUCHI, Toru KAWANISHI, Eriko UCHIDA, Akiko EGUCHI, Hiroyuki MIZUGUCHI, Tadanori MAYUMI, Mahito NAKANISHI and Takao HAYAKAWA: "Target Cell Specificity of Fusogenic Liposomes-Membrane Fusion-mediated Macromolecule Delivery into Human Blood Mononuclear Cells"Biochim. Biophys. Acta. 1416. 339-348 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nana KAWASAKI, Yuan C.LEE, Osamu HASHIMOTO, Masayuki YAMAMOTO, Toru KAWANISHI, Takao HAYAKAWA: "Fluorometric Determination of Aminopolycarboxylate Using Fluo-3"Anal. Biochem.. 270. 329-331 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] H.TANAKA, T.ICHIKAWA, S.MATSUI, K.OKAZAKI, H.MASUMIYA, T.KAWANISHI, and K.SHIGENOBU: "Calcium channel antagonistic effects of AH-1058, a novel antiarrythmic drug, on guinea-pig myocardium"Res.Commun. Mol. Pathol. Pharmacol.. 104. 13-21 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Toru KAWANISHI, Takehito KIUCHI, Hiroki ASOH, Rie SHIBAYAMA, Hiroshi KAWAI, Hisayuki OHATA, Kazutaka, MOMOSE, and Takao HAYAKAWA: "Effect of Tributyltin Chloride on Release of Calcium Ion from Intracellular Calcium Stores in Rat Hepatocytes"Biochem Pharmacol.. (in press).

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2002-03-26  

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