Development of in vitro reconstituted system for investigating intracellular signal trasduction and cellular function in myocardial cells

开发用于研究心肌细胞内信号转导和细胞功能的体外重建系统

• 批准号: 07557057
• 负责人: HORI Masatsugu
• 金额: $ 9.6万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557057/
• 关键词: intracellular signal transduction; nuclear localization signal; myocardial cell; cellular biology; 心筋; 細胞機能

The aim of this research is to develop an in vitro real-time analyzing system for investigating intracellular signal transduction with CCD camera. In 1995, we succeeded to develop an in vitro reconstituted system for analyzing of intracellular signal transduction, especially nuclear signal transduction. It was found that nuclear protein was transported to the nucleus within 30 minutes, although non-nuclear proteins were not by using homogenates from heart which means that this system reproduces in vivo nuclear protein transport. In 1996, we examined nuclear transport efficiency with extracts from diseased hearts by this in vitro system. The rate of the nuclear transport was more decreased in spontaneous hypertensive rat than of the control. Furthermore, we found the existence of cellular factors that are activated by nuclear localization signal (NLS) and partially purified these cellular factors (FEBS Letters). In the next step, we should improve this system in order to analyze the real-time behavior of molecules quantitatively with CCD camera. The real-time analytical system will provide the precise evaluation of efficiency of nuclear transport that may modulate the cellular function in heart cells.

本研究的目的是开发一种体外实时分析系统，用于CCD相机研究细胞内信号转导。1995年，我们成功地开发了一种体外重建系统，用于分析细胞内信号转导，特别是核信号转导。研究发现，核蛋白在30分钟内被转运到细胞核中，而非核蛋白则不能通过心脏匀浆转运到细胞核中，这意味着该系统可以复制体内核蛋白转运。1996年，我们用这种体外系统检测了患病心脏提取物的核转运效率。自发性高血压大鼠的核转运率明显低于对照组。此外，我们发现了被核定位信号（NLS）激活的细胞因子的存在，并对这些细胞因子进行了部分纯化（FEBS Letters）。下一步，我们需要对该系统进行改进，以便用CCD相机定量分析分子的实时行为。实时分析系统将提供核转运效率的精确评估，可能调节心脏细胞的细胞功能。

项目成果

Yoneda Y. et al.: "Nuclear pore-targetting complex and its role on nuclear protein transport." Arch Histol Cytol. 59. 97-107 (1996)

Yoneda Y. 等人：“核孔靶向复合物及其对核蛋白转运的作用。”

Imamoto N. et al.: "A karyophilic protein forms a stable complex with cytoplasmic components prior to nuclear pore binding." J Biol Chem.270. 8559-85565 (1995)

Imamoto N. 等人：“亲核蛋白在核孔结合之前与细胞质成分形成稳定的复合物。”

Sekimoto T,Yoneda Y.et al.: "Interferon-gamma-dependent nuclear import of Stat 1 is mediated by the GTPase activity of Ran/TC4." J Biol Chem. 271. 31017-31020 (1996)

Sekimoto T、Yoneda Y.等人：“Stat 1 的干扰素γ依赖性核输入是由 Ran/TC4 的 GTP 酶活性介导的。”

Toshinao Kurihara: "Partial purification and characterization of a protein kinase that is activated by nuclear localization signal peptides" FEBS Letters. 380. 241-245 (1996)

Toshinao Kurihara：“核定位信号肽激活的蛋白激酶的部分纯化和表征”FEBS Letters。

Yoneda Y. et al.: "Nuclear export and its significance in retroviral infection." Trends in Microbiology. 4. 1-2 (1996)

Yoneda Y. 等人：“核输出及其在逆转录病毒感染中的意义。”