Functional role and its analysis of cytoslkeletal proteins in stroma cells and in hematopoetic stem cells.

基质细胞和造血干细胞中细胞骨架蛋白的功能作用及其分析。

• 批准号: 05670896
• 负责人: HIGASHIHARA Masaaki
• 金额: $ 1.34万
• 依托单位: 1st Department of Internal Medicine, Faculy of Medicine, University of Tkyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05670896/
• 关键词: Stroma cells; myosin; phosphorylation; phosphatase; monoclonal antibody; モノクロナル抗体; 骨髄幹細胞; 細胞骨格蛋白; サイトカイン; フォスファターゼ; 血小板

We made Monoclonal antibodies (12 clones) specifi for myosin heavy chain of non-muscle cells, although we could not detect the transformation of myosin heavy chain in several stroma cells. We also made DNA probes for these myosin isoforms. However, RT-PCR was unsuccessful using mRNA of HEL cells. Using cDNA of myosin heavy and light chain, we tried the cloning of myosin heavy and light chain of human hematopoietic cells. The problem is that cDNA library of human bone marrow cells we made, was not good. We tried the tranfection of cytokine genes into stroma cell lines (HFL,HUC-Fm) using retrovirus vetors. However, data were variable and non-reprodusible.We Made success in making ten monoclonal antibodies agaist myosin-associated protein phosphatase (MAPP), which was gift of Dr.M.Ikebe, CWRU.However all antibodies had lgM isotype and unfavorable for immunoblotting. Thus we made now clones (lgG) against non-catalytic subunit of phosphatase. We are now working on the characterization of th … More ese antibodies. In the clarifying the role of myosin phosphorylation on chilling -induced platelet shape change, four types of shape change were observed and these changed in a time-dependent fashion. The atractive finding is that these shape change were correlated with the extent of phosphorylation of LC20, and these changes were reversible. Contribution of myosin on the shape changes was further confirmed by two findings. First, association of myosin into the triton X-insoluble fractions were proceded in a time-ddependent fashion. Secondly, electron microscopic observation shwed that myosins were associated in the bundles of fibers in the filopodias. We previously reported the purifiation of myosin-associated protein phosphatase and Q10 of this phosphatase is very high.i.e.about 5.0 in contrast to MLCK 2.0. The suppression of this phosphatase activity was much more than that of MLCK during the change in temperature from 37C to 0-4ﾟC.This unbalace can induced the phosphorylation of LC20 even if [Ca^<2+>] i does not increase by chilling. Less

我们制备了12株非肌细胞肌球蛋白重链特异性单克隆抗体，但在几种间质细胞中未检测到肌球蛋白重链的转化。我们还为这些肌球蛋白亚型做了DNA探针。然而，使用HEL细胞的mRNA的RT-PCR是不成功的。利用肌球蛋白重链和轻链的cDNA，尝试克隆人造血细胞的肌球蛋白重链和轻链。问题是我们制作的人骨髓细胞cDNA文库并不好。我们尝试用逆转录病毒载体将细胞因子基因转染到基质细胞系（HFL、HUC-Fm）中。我们成功地制备了10株抗肌球蛋白相关蛋白磷酸酶（MAPP）的单克隆抗体，这些抗体是由M.Ikebe，CWRU赠送的，但所有抗体都是IgM型，不利于免疫印迹。因此，我们现在针对磷酸酶的非催化亚基制备克隆（IgG）。我们现在正在研究 ...更多信息 这些抗体。在阐明肌球蛋白磷酸化对低温诱导的血小板形状变化的作用时，观察到四种类型的形状变化，并且这些变化以时间依赖性的方式变化。引人注目的发现是，这些形状变化与LC 20的磷酸化程度相关，并且这些变化是可逆的。肌球蛋白对形状变化的贡献被两个发现进一步证实。首先，协会肌球蛋白成triton X-不溶性馏分进行了时间依赖性的方式。电镜观察显示肌球蛋白与丝状伪足内的纤维束相连。我们以前报道过肌球蛋白相关蛋白磷酸酶的纯化，该磷酸酶的Q_（10）很高，约为5.0，而MLCK为2.0。从37 ℃到0-4 ℃的温度变化过程中，这种磷酸酶活性的抑制作用比MLCK的要大得多。即使[Ca^<2+>] i不因低温而增加，这种不平衡也能诱导LC 20的磷酸化。少

项目成果

Higashihara M., Ikebe M.: "Inhibition of20-kDa myosin light chain exchangeby monoclonal antibodies against 17-kDa light chain" Biochem.Biophys.Res.Commun. 363. 57-60 (1995)

Higashihara M.，Ikebe M.：“针对 17-kDa 轻链的单克隆抗体抑制 20-kDa 肌球蛋白轻链交换”Biochem.Biophys.Res.Commun。

Kawakami, H. et al: "Okadaic acid induces marked shape change of human platelets" J. Smooth Muscle Reseach. 30. 57-64 (1994)

Kawakami, H. 等人：“Okadaic 酸诱导人类血小板的显着形状变化”J. Smooth Muscle Reseach。

Kawakami H et al: "Okadaic acid induces marked shape change of human platelets" J.Smooth Muscle Res. 30. 57-64 (1994)

Kawakami H 等人：“冈田酸诱导人血小板形状发生显着变化”J.Smooth Muscle Res。

Higashihara M.et al: "Inhibition of 20-KDa myosin light clain exchange by monocloral antibodies against 17-KDa myosin light clain" FEBS Letters. (in press). (1995)

Higashihara M.et al：“针对 17-KDa 肌球蛋白轻克莱因的单克隆抗体抑制 20-KDa 肌球蛋白轻克莱因交换”FEBS Letters。

Tange T., Hasegawa Y., Oka T., Sunaga S., Higashihara M., Matuo K., Miyazaki H,, Shimosaka A., Pkano A., TodokoroK., Ishikawa T., Machinami R.: "Establishiment and characterization of a new human mesothelioma cell line (T-85) from malignant peritoneal mes

Tange T.、Hasekawa Y.、Oka T.、Sunaga S.、Higashihara M.、Matuo K.、Miyazaki H、、Shimosaka A.、Pkano A.、TodokoroK.、Ishikawa T.、Machinami R.：“建立和