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CELL BIOLOGICAL RESEARCH WITH THE MOLECULAR MECHANISMS OF LIPID DROPLETS FORMATION AND LIPID METABOLISM, TRANSFORMATION.

脂滴形成和脂质代谢、转化的分子机制的细胞生物学研究。

基本信息

批准号：
10670004
负责人：
AOKI Takeo
金额：
$ 1.22万
依托单位：
Gunma University, School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

TYROSINE PHSPHORYLATION OF CAVEOLIN-1 IN THE ENDOTHELIUM.1) Tyrosine phosphorylation of caveolin-1 occurred in limited locations, including the endothelium of the continuous capillaries and small venules. 2) Cultured endothelial cells were not labeled by PY14 under a standard culture condition, but became positively labeled when exposed to oxidative stresses and/or tyrosine phosphatase inhibitors. 3) The reaction was prohibited by pretreating with herbimycin A or genistein. Vasoactive reagents or physical stimuli did not cause the phosphorylation. 4) The number of invaginated caveolae decreased drastically, and vesicles labeled intensely for caveolin-1 appeared in the cytoplasm ; the average diameter of the vesicles was larger than that of caveolae. 5) The results implies that tyrosine phosphorylation of caveolin-1 occurred at tyrosine-14 in the normal rat endothelium in vivo and may induce caveolar vesiculation and/or fusion.VIMEMTIN-RELATED ANTIGEN AROUND LIPID DROPLETE1) By using a … More monoclonal antibody (mAb A2) raised against the cytoskeleton of goldfish xanthophores, we found that a 190-kDa protein is localized along the rim of lipid droplets in rat pulmonary septal cells. 2) MAb A2 bound to the rim of lipid droplets in the septal cell in vivo. The circular labeling around lipid droplets as well as filamentous labeling in the cytoplasm was observed in cultured septal cells. 3) As the number and size of lipid droplets decreased in cells cultured for several days, the circular labeling became indistinct, whereas the filamentous labeling persisted. 4) In Western blotting, mAb A2 recognized 190-kDa and 58-kDa bands in the cultured septal cell, but the latter turned out to be vimentin. 5) Treatment of septal cells for 4 hr with Colcemid extinguished the labeling around the lipid droplets, and only labeling in a coarse bundle pattern that matched the anti-vimentin labeling pattern remained. 6) After more than 12 hr of Colcemid treatment, the circular labeling reappeared around the lipid droplets. The disappearance of the circular labeling also occurred when septal cells were treated with Taxol or cytochalasin B for more than 4 hr. Less

小窝蛋白-1的酪氨酸磷酸化1)小窝蛋白-1的酪氨酸磷酸化发生在有限的部位，包括连续毛细血管和小静脉的内皮。2)在标准培养条件下，培养的内皮细胞不被PY14标记，但在氧化应激和/或酪氨酸磷酸酶抑制剂作用下，内皮细胞呈阳性标记。3)用去甲二氢青霉素A或金雀异黄素预处理可抑制该反应。血管活性试剂或物理刺激不会引起磷酸化。4)内陷小窝数量急剧减少，胞浆内出现小窝蛋白-1强标记的小泡，小泡的平均直径大于小窝。5)结果提示，在活体大鼠血管内皮细胞中，小窝蛋白-1的酪氨酸磷酸化发生在酪氨酸-14处，并可能诱导小窝泡泡形成和/或融合。更多针对金鱼黄磷细胞骨架的单抗(MAbA2)，我们发现在大鼠肺间隔细胞中有一个190 kDa的蛋白定位于脂滴边缘。2)体内单抗A2与隔细胞内脂滴的边缘结合。在培养的间隔细胞中，可见脂滴周围的圆形标记和胞浆内的丝状标记。3)随着培养数天细胞内脂滴数量和大小的减少，环状标记变得模糊，而丝状标记持续存在。4)在Western blotting中，单抗A2在培养的间隔细胞中识别190 kDa和58 kDa条带，但后者为波形蛋白。5)Colcemid处理隔细胞4h后，脂滴周围的标记消失，仅保留与抗波形蛋白标记模式匹配的粗束状标记。6)Colcemid处理12h以上后，脂滴周围重新出现环形标记。当用紫杉醇或细胞松弛素B处理间隔细胞超过4小时后，环状标记也消失。较少