NDUFA4通过增强线粒体活化水平促进卵巢癌SKOV3细胞增殖、迁移及侵袭

目的:探讨NDUFA4介导线粒体活化水平对卵巢癌细胞SKOV3增殖、迁移及侵袭的影响。方法:以慢病毒技术建立SKOV3细胞的NDUFA4过表达和沉默模型;CCK-8检测细胞增殖,细胞划痕检测细胞迁移,Transwell检测细胞侵袭,流式细胞术检测线粒体膜电位和凋亡;Real-time PCR检测线粒体DNA(mtDNA)拷贝数,MT-ND1、MT-ND5、MT-CYB、SDHA、PGC-1α、NRF1和mTFA mRNA的表达;电镜观察线粒体形态,分光光度法检测Complex IV活性水平;Western blot检测PGC-1α、NRF1、mTFA蛋白的表达。结果:SKOV3细胞中实现NDUFA4的过表达和沉默;NDUFA4的过表达提高细胞增殖、侵袭和迁移能力(P<0.05),上调mtDNA拷贝数,上调MT-ND1、MT-ND5、MT-CYB、SDHA、PGC-1α、NRF1和mTFA mRNA水平(P<0.05),上调PGC-1α、NRF1和mTFA蛋白水平(P<0.05),上调线粒体膜电位水平以及Complex IV活性水平(P<0.05),线粒体变多形态正常。而NDUFA4的沉默则相反,线粒体出现空泡化。结论:NDUFA4可能通过加强线粒体的活化和氧化磷酸化的水平在SKOV3细胞中发挥促癌功能。

AβOs对大鼠原代海马神经细胞丝裂原活化蛋白激酶信号转导通路的影响

目的:观察β-淀粉样蛋白寡聚体(AβOs)对原代培养大鼠海马神经细胞丝裂原活化蛋白激酶(MAPK)信号转导通路的影响。方法:原代培养大鼠海马神经细胞,用免疫荧光染色鉴定纯度,制备并鉴定AβOs,用不同浓度AβOs(0.25、0.5、1及10μmol/L)处理细胞48 h,采用CCK-8试验检测细胞的存活率,蛋白印迹法(Western blotting)检测细胞中细胞外信号调节蛋白激酶(ERK)、c-Jun氨基末端激酶(JNK)及p38磷酸化及总蛋白表达水平。结果:免疫荧光染色结果显示原代大鼠海马神经细胞的体外培养纯度达85%以上;低于0.5μmol/L AβOs对原代神经细胞无明显毒性作用,但可见phospho-ERK1/2和phospho-JNK蛋白表达升高;1μmol/L及更高浓度的AβOs对原代神经细胞有细胞毒性作用,同时可引起phospho-ERK1/2和phospho-JNK蛋白表达水平均降低,但总ERK1/2及总JNK蛋白表达水平未受明显影响,而phospho-p38及总p38蛋白表达水平与AβOs作用浓度呈负相关。结论:AβOs与MAPK信号通路之间的作用可因AβOs的作用浓度不同而出现差异。

Diurnal Changes of Rubisco and RCA Activities and Their Cellular Localization in Rice

The cellular localization of Rubisco and Rubisco activase (RCA) in rice (Oryza sativa subsp. indica cv. Zhenong 952) leaf was investigated with immunogold-labeled electron microscope techniques on the basis of determining the diurnal changes of photosynthetic rate (Pn), Rubisco and RCA activities, and quantifying two enzyme contents in the leaf with immuno-diffusion method in order to understand why RCA activity decreased in the midday when its contents was high. The results showed that Rubisco mainly was located in chloroplast, and RCA were found both in chloroplast and mitochondria. The lowering of Rubisco in chloroplast as well as Rubisco activity at noon could be one of good reasons to explain the photosynthetic midday depression in leaf. The density of RCA in chloroplast reached the maximum at 14:00 and a valley at 11:00. The result much coincided with the activity of RCA in leaf. In mitochondria, the density of RCA changed abruptly in one day with the highest at 13:00 and it can well elucidate why the activities of Rubisco declined at noon when its amount was increasing. Therefore the cellular localization and/or distribution of Rubisco and RCA during a day is more important for Pn, Rubisco and RCA activities.

Realgar induces differentiation through ROS-dependent mitochondrial pathway in HL-60 cells

Realgar （As 4 S 4 ）, as a mineral drug in traditional Chinese medicine, is currently used as the remedy for acute promyelocytic leukemia and has been proven to have relatively milder side effects as compared to the arsenolite （As 2 O 3 ）-based drugs. We have previously demonstrated that realgar induces differentiation in HL-60 cells, and the differentiation is associated with serine/threonine protein phosphatases, MAPK signaling pathways, and mitochondrial transmembrane potential decrease. In this study, we further explore the roles of mitochondrial permeability transition pore and reactive oxygen species （ROS） in realgar-induced differentiation in HL-60 cells. The differentiation was preceded by marked changes in the cellular level of ROS, and could be enhanced by SB202190, a p38 MAPK inhibitor. In addition, the efficacy of realgar was suppressed by closing the MPTP with an inhibitor. Taken together, these findings indicate that the opening of MPTP and the alteration of ROS generation were involved in realgar-induced differentiation.

Amavadin induced PTP opening not through the promotion of ROS generation in rat kidney mitochondria

Amavadin is a natural vanadium compound that accumulates to high level in poisonous Amanita mushrooms. Recently, amavadin was found to have potential therapeutic effect in cancer treatment. However, its toxicity and the possible mechanism of actions are still not clear. In this study, we investigated the toxic effects of amavadin on rat kidney mitochondria and the possible mechanism. We found that amavadin induced significantly permeability transition pore （PTP） opening in the mitochondria. Amavadin y inhibited the generation of reactive oxygen species （ROS） in succinate buffer, and at high concentration of 200 gM it increased the ROS generation in malate buffer. With the addition of rotenone, the ROS generation in malate buffer was strongly enhanced than that induced by amavadin alone, but remained unchanged in succinate buffer. Results from the present study suggest that amavadin act upon electron transport chain downstream of rotenone, and the ubiquinone binding site in complex I is the most possible binding site.

PGC-1α regulates the cell cycle through ATP and ROS in CH1 cells

Peroxisome proliferator-activated receptor-γ coactivator 1α（PGC-1α） is a transcriptional co-activator involved in mitochondrial biogenesis, respiratory capacity, and oxidative phosphorylation（OXPHOS）. PGC-1α plays an important role in cellular metabolism and is associated with tumorigenesis, suggesting an involvement in cell cycle progression. However, the underlying mechanisms mediating its involvement in these processes remain unclear. To elucidate the signaling pathways involved in PGC-1α function, we established a cell line, CH1 PGC-1α, which stably overexpresses PGC-1α. Using this cell line, we found that over-expression of PGC-1α stimulated extra adenosine triphosphate（ATP） and reduced reactive oxygen species（ROS） production. These effects were accompanied by up-regulation of the cell cycle checkpoint regulators Cyclin D1 and Cyclin B1. We hypothesized that ATP and ROS function as cellular signals to regulate cyclins and control cell cycle progression. Indeed, we found that reduction of ATP levels down-regulated Cyclin D1 but not Cyclin B1, whereas elevation of ROS levels down-regulated Cyclin B1 but not Cyclin D1. Furthermore, both low ATP levels and elevated ROS levels inhibited cell growth, but PGC-1α was maintained at a constant level. Together, these results demonstrate that PGC-1α regulates cell cycle progression through modulation of Cyclin D1 and Cyclin B1 by ATP and ROS. These findings suggest that PGC-1α potentially coordinates energy metabolism together with the cell cycle.

Morphology, mitochondrial development and adipogenic-related genes expression during adipocytes differentiation in grass carp(Ctenopharyngodon idellus)

To investigate the differentiation mechanism of grass carp preadipocytes, a primary adipocytes culture system was established. Confluent preadipocytes were induced to differentiation, and the morphology and gene expression were evaluated at different stages. It was shown that preadipocytes were gradually filled with droplets and the cellular lipid content increased during the differentiation. Ultrastructure observation indicated that the number of mitochondria increased with adipocytes differentiation. Consistently, the mitochondrial protein content was ele- vated in the differentiating adipocytes, qRT-PCR showed that the expression level of lipogenesis-related genes such as peroxisome proliferator activator receptor 7 （PPAR 7）, lipoprotein lipase （LPL）, fatty acid synthase （FAS） and stearoyl-CoA desaturase （SCD） increased during adipocytes differentiation. The mitochondrial relevant gene also elevated when adipocyte differentiation, such as PPAR coactivator-1 （PGC-1 α）, PGC-1β and nuclear respiratory factor （NRF-1）. However, the expression of carnitine palmitoyltransferase 1α （CPT-1 α） gene decreased at the initial stage, but increased at the last stage of cell differ- entiation. These results indicated that the differentiation process of grass carp preadipocytes is similar to that of land animals, but the molecular mechanisms are not exactly the same. The findings revealed in this study provides new information to the study of fish adipocyte differentiation.