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Novel factors in the biogenesis of mitochondrial reticulum found from heart of juvenile visceral steatosis mouse

从幼年内脏脂肪变性小鼠心脏发现线粒体网生物发生的新因素

基本信息

批准号：
12480191
负责人：
HIGUTI Tomihiko
金额：
$ 8.19万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12480191/
关键词：
mitochondria JVS mouse carnitin mitochondrial reticulum biogenesis fusogen time-laps deconvolution cytochrome 蛍光蛋白質 HeLa 細胞ディファレンシャルディスプレイ JVS Differential Display 遺伝子発現制御 H^+-ATP合成酵素

项目摘要

Mice with juvenile visceral steatosis (JVS) develop remarkable cardiac hypertrophy and exhibit an increased number of mitochondria in their heart. However, the biochemical characteristics and physiological functions of these mitochondria cardiac are little known. We demonstrated that the respiratory activities at state 3 with glutamate plus malate or succinate in the heart mitochondria of JVS mice were greatly decreased compared with those of control mice. The contents of cytochromes a + a3, b, and c + c1 in the heart mitochondria of these mice were also decreased, to 51 % , 45 % , and 79 % , respectively, of those of the control mice, Oligomycin-sensitive ATPase activity in these mitochondria, however, was increased to about 2 times over that of the control mice. Surprisingly, the ATP-Pi exchange activity of the heart mitochondria of JVS mice was greatly decreased, to 35 % of that of control mice.Since the mitochondrial number in the cardiac cells of JVS mice is increased approximately three-fold compared with that of control mice, factors and their mRNAs which are related with the mitochondrial biogenesis, should dramatically be increased in the cells of JVS mice heart. To find such factors, the fluorescence differential display was performed using the highly purified poly(A) RNAs extracted from hearts of JVS mice and the control mice. We found novel six genes four of which were up-regulated in the hearts of JVS mice and the remain two genes were down-regulated. Furthermore, we established the method to observe mitochondrial reticulum labeled with fluorescence protein by transfecting pDsRed-Mito at high yield of 50-70 % into HeLa cells, and then succeeded to observe the morphological change of mitochondrial reticulum in the cell cycle of the cells by a time-laps deconvolution CCD-fluorescence microscope, which were synchronized at the G1/S boundary by the double thymidine block protocol.

幼年内脏脂肪变性（JVS）小鼠出现显著的心脏肥大，并表现出心脏线粒体数量增加。然而，这些心肌线粒体的生化特性和生理功能却知之甚少。我们证明，在状态3的呼吸活动与谷氨酸加苹果酸或琥珀酸在JVS小鼠的心脏线粒体大大降低与对照小鼠相比。心肌线粒体中细胞色素a + a3、B和c + c1的含量也降低，分别为对照组的51%、45%和79%，但这些线粒体中对寡霉素敏感的ATP酶活性却增加到对照组的2倍左右。令人惊讶的是，JVS小鼠心脏线粒体的ATP-Pi交换活性大大降低，仅为对照小鼠的35%。由于JVS小鼠心脏细胞中的线粒体数量比对照小鼠增加了约3倍，因此与线粒体生物合成相关的因子及其mRNA在JVS小鼠心脏细胞中应显着增加。为了发现这些因子，使用从JVS小鼠和对照小鼠的心脏提取的高度纯化的poly（A）RNA进行荧光差异显示。我们发现了6个新的基因，其中4个在JVS小鼠心脏中表达上调，其余2个基因表达下调。在此基础上，我们建立了荧光蛋白标记的线粒体网状结构的观察方法，将pDsRed-Mito以50- 70%的高收率转染HeLa细胞，并采用双胸苷阻断法将细胞同步化于G1/S期。