| Project/Area Number |
13558078
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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 | 展開研究 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | The University of Tokyo |
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Principal Investigator |
KIKUSHI Kazuya The University of Tokyo, Graduate School of Pharmaceutical Sciences, Associate Professor, 大学院・薬学系研究科, 助教授 (70292951)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥13,600,000 (Direct Cost: ¥13,600,000)
Fiscal Year 2003: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2002: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2001: ¥5,600,000 (Direct Cost: ¥5,600,000)
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| Keywords | FRET / ratio imaging / fluorescent probe / PTP / ehergy fransfer / overlap integral / arachidonic acid / fluorescein / 蛍光共鳴エネルギー移動 / チロシンフォスファターゼ / レシオ測定法 / バイオイメージング / フルオレッセイン / クマリン / 可視化解析 / マクロCALI / クロモフォア / レーザー分子不活化 / IP_3受容体 / レーザー照射 / 平滑筋組織 / 細胞内Ca^<2+> / 亜鉛イオン / CA1 / 海馬 / 虚血 / 可視化 / 歯状回 |
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| Research Abstract |
A novel rationietric fluorescent probe 1 for protein tyrosine phosphatase (PTP) activity based on spectral overlap integral switching of FRET was designed and was synthesized. The fluorescence spectra of 1 exhibited a large shift in their emission wavelength after reaction with PTPs in vitro. Within the 1 labeled cells, the increase in the emission ratio was observed, and it was suppressed by the addition of a PTP inhibitor orthovanadate and hydrogen peroxide. So it was shown that 1 could detect PTP activity in living cells. Next, we applied this imaging method to biological studies on the regulation of PTP activity during contact growth inhibition of normal cells. In normal cells, PTP activity of dense cells was markedly enhanced compared to that of sparse or medium-density cells. The increased PTP activity was also observed in sparse cells treated with a reducing agent, lipoxygenase inhibitor; and PLA_2 inhibitor, and resulted in cell growth inhibition. These observations suggest that PTP activity could be regulated by ROS produced in lipoxygenase-mediated arachidonate metabolism and play a key role in density-dependent growth inhibition.
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