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(In Japanese)内分泌かく乱化学物質の細胞内標的分子の同定と新しいバイオモニタリング

Research report code R000000334
Posted date Sep 30, 2002
  • (In Japanese)梅澤 喜夫
  • (In Japanese)東京大学大学院理学系研究科
Research organization
  • (In Japanese)東京大学大学院理学系研究科
  • (In Japanese)東京大学環境安全研究センター
  • (In Japanese)東京大学大学院医学系研究科
  • (In Japanese)金沢大学大学院医学研究科
  • (In Japanese)北里大学薬学部
Report name (In Japanese)内分泌かく乱化学物質の細胞内標的分子の同定と新しいバイオモニタリング
Technology summary (In Japanese)内分泌撹乱化学物質(EDC)のリスクアセスメントのための化学物質の新しい高速前試験法,(High Throughput Pre-Screening)として,化学物質による細胞内シグナル伝達の撹乱を可視化検出する新規プローブ分子を開発した。まず蛍光共鳴エネルギー(FRET)に基づく蛍光プローブ分子として第二次情報伝達物質(cGMP,およびPIP3, DAG などの脂質メッセンジャー),Ca2+情報伝達分子(CaMK),蛋白質のリン酸化に対するプローブを開発した。また,細胞内蛋白質?蛋白質相互作用を検出する最も一般的方法として,プロテインスプライシングを用いたスプリットGFPおよびスプリットルシフェラーゼシステムを開発した。これらのプローブ分子が細胞内シグナル伝達を撹乱する化学物質の新しい分析法として用いうることをEDCを含む化学物質を例に検証した。更にヒト乳ガン細胞株(MCF-7),およびヒト神経芽細胞腫NB-1細胞株のEDCにより発現が変化する遺伝子の系統的解析を行った。またヒト肝臓組織内の遺伝子発現の系統的解析を行った。
Research field
  • Influence on animals
  • Physiology and anatomy of endocrine system
  • Toxicity of organic compounds
  • Health damage
  • Pollutants
Published papers related (In Japanese)(1)Fluorescent Indicators for Imaging Protein Phosphorylation in Single Living Cells. M. Sato, T. Ozawa, K. Inukai, T. Asano, and Y. Umezawa, Nature Biotech., submitted.
(2)Protein Splicing-Based Reconsition of Split Green Fluorescent Protein for Monitoring Protein-Protein Interactions in Bacteria: Improved Sensitivity and Reduced Screening Time. T. Ozawa, M. Takeuchi, A. Kaihara, M. Sato and Y. Umezawa, Anal. Chem., in press.
(3)A Screening Method for Antagonists that Inhibit the Binding of Calmodulin to a Target Peptide Using Surface Plasmon Resonance. K. Sasaki, T. Ozawa and Y. Umezawa, Anal. Chim. Acta, in press.
(4)Detection of Protein?Protein Interactions in Vivo Based on Protein Splicing. T. Ozawa and Y. Umezawa, Current Opinion in Chemical Biology; Section Analytical techniques, 5, 578-583 (2001).
(5)Assay and Screening Methods for Bioactive Substances Based on Cellular Signaling Pathways, Y. Umezawa, Reviews in Molecular Biology, special issue on “Biomolecular Sensors and Bioelectronics”, 5, 578-583, 2001.
(6)Imaging of Conformational Changes of Proteins with a New Environment-Sensitive Fluorescent Probe Designed for Site-Specific Labeling of Recombinant Proteins in Live Cells. J. Nakanishi, T. Nakajima, M, Sato, T, Ozawa, K. Tohda and Y. Umezawa, Anal. Chem., 73, No. 13, 2920-2928 (2001).
(7)Split Luciferase as an Optical Probe for Detecting Protein?Protein Interactions in Mammalian Cells Based on Protein Splicing. T. Ozawa, , A. Kaihara, M. Sato, K. Tachihara and Y. Umezawa, Anal. Chem., 73, No. 11, 2516-2521 (2001).
(8)How Can Ca2+ Selectively Activate Recoverin in the Presence of Mg2+? Surface Plasmon Resonance and FT-IR Spectroscopic Studies. T. Ozawa, M. Fukuda, M. Nara, A. Nakamura, K. Kohama and Y. Umezawa, Biochemistry, 39, No. 47, 14495-14503 (2000).
(9)Fluorescent Indicators for Cyclic GMP Based on Cyclic GMP-Dependent Protein Kinase I? and Green Fluorescent Proteins. M. Sato, N. Hida, T. Ozawa and Y. Umezawa, Anal. Chem., 72, No. 24, 5918-5924 (2000).
(10)A Fluorescent Indicator for Detecting Protein??Protein Interactions in Vivo Based on Protein Splicing. T. Ozawa, S. Nogami, M. Sato, Y. Ohya and Y. Umezawa, Anal. Chem., 72, No. 21, 5151-5157 (2000).
(11)Electrochemical Detection of a One-Base Mismatch in an Oligonucleotide Using Ion-Channel Sensors with Self-Assembled PNA Monolayers. H. Aoki, P. Buhlmann and Y. Umezawa, Electroanalysis, 12, No. 16, 1272-1276 (2000).
(12)A Screening Method for Substrates of Multidrug Resistance-associated Protein (MRP). Z. Quan, T. Ozawa, M. Sato and Y. Umezawa, Anal. Chim. Acta, 423, 197-203 (2000).
(13)A Screening Method for Antigen-Specific IgE Using Mast Cells Based on Intracellular Calcium Signaling. S. Aketani, R. Teshima, J. Sawada and Y. Umezawa, Anal. Chem., 72, No. 11, 2653-2658 (2001).
(14)An SPR-based Screening Method for Agonist Selectivity for Insulin Signaling Pathways Based on the Binding of Phosphotyrosine to its Specific Binding Protein. T. Yoshida, M. Sato, T. Ozawa and Y. Umezawa, Anal. Chem., 72, No. 1, 6-11 (2000).
(15)Metal Ion Selectivity for Formation of the Calmodulin-Metal-Target Peptide Ternary Complex Studied by Surface Plasmon Resonance Spectroscopy. T. Ozawa, K. Sasaki and Y. Umezawa, Biochim. Biophys. Acta , 1434, 211-220 (1999).
(16)WISP-2 as a Novel Estrogen-Responsive Gene in Human Breast Cancer Cells. H. Inadera, S. Hashimoto, H-Y. Dong, T. Suzuki, S. Nagai, T. Yamashita, N. Toyoda and K. Matsushima, Biochem. Biophys. Res. Commun. 275, 108-114 (2000).
(17)Comprehensive Gene Expression Profile of a Normal Human Liver. T. Yamashita, S. Hashimoto, S. Kaneko, S. Nagai, N. Toyoda, T. Suzuki, K. Kobayashi and K. Matsushima, Biochem. Biophys. Res. Commun., 269, 110-116 (2000).
(18)Identification of Differentially Expressed Genes in Hepatocellular Carcinoma With cDNA Microarrays. Y. Shirota, S Kaneko, M Honda, H F Kawai, and K Kobayashi, Hepatology, in press.
(19)Comprehensive Gene Expression Profile of Hepatocellular Carcinoma Complicated in Chronic Hepatitis C. T. Yamashita, S. Kaneko, S. Hashimoto, T. Sato, S. Nagai, N. Toyoda, T. Suzuki, K. Kobayashi and K. Matsushima. Biochem. Biophys. Res. Comm., in press.
(20)Differential Gene Expression Between Chronic Hepatitis B and C Hepatic Lesion. M. Honda, S. Kaneko, H. F. Kawai, Y. Shirota and K. Kobayashi, Gastroenterology, 120, 955-966 (2001).
(21)Alpha-Fetoprotein-Producing Hepatoma Cell Lines Share Common Expression Profiles of Genes in Various Categories Demonstrated by cDNA Microarray Analysis. H. F. Kawai, S. Kaneko, M. Honda, Y. Shirota and K. Kobayashi, Hepatology, 33, 676-691 (2001).
(22)Effects of Cadmium on Neurite Outgrowth and Gene Eexpression in Human Neuroblastoma NB-1 Cells. R. Pramanik, M. Ishido and M. Kunimoto, J. Health Sci., 47, 478-482 (2001).
Research project
  • Core Research for Evolutional Science and Technology;Endocrine Disrupters
Information research report
  • (In Japanese)梅澤 喜夫. 内分泌かく乱物質 内分泌かく乱化学物質の細胞内標的分子の同定と新しいバイオモニタリング. 戦略的基礎研究推進事業 平成11年度 研究年報.科学技術振興事業団, 2000. p.1205 - 1210.