①目的探讨不同提取方案对心肌细胞线粒体质量的影响。②方法在体外用Langendorff法灌注雄性Wistar大鼠心脏,取左心室组织并分离线粒体; Western blot法检测确定分离线粒体的纯度,透射电子显微镜(scanning electron microscopy,SEM)检...①目的探讨不同提取方案对心肌细胞线粒体质量的影响。②方法在体外用Langendorff法灌注雄性Wistar大鼠心脏,取左心室组织并分离线粒体; Western blot法检测确定分离线粒体的纯度,透射电子显微镜(scanning electron microscopy,SEM)检测分离线粒体的结构形态,线粒体呼吸控制率(respiratory control ratio,RCR)测定分离线粒体的功能。③结果当分离转速为3500g时,与12000g相比,分离得到的线粒体Tubulin蛋白和COXⅣ蛋白表达均较低,表明低转速能够提高分离线粒体的纯度。与此结果一致,电镜结果显示与3500g组相比,12000g组可见较多微丝微管结构及线粒体内外膜分离。与12000g组相比,3500g组Ⅲ态呼吸及RCR值明显增高,表明低转速分离获得的线粒体呼吸功能增强。④结论低转速不仅可以提高分离线粒体的纯度,而且有利于维持分离线粒体的正常结构和功能。展开更多
We isolated and purified mitochondria from mouse livers and spinach leaves. When added into egg extracts of Xenopus laevis, they caused nuclei of mouse liver to undergo apoptotic changes. Chromatin condensation, margi...We isolated and purified mitochondria from mouse livers and spinach leaves. When added into egg extracts of Xenopus laevis, they caused nuclei of mouse liver to undergo apoptotic changes. Chromatin condensation, margination and DNA ladder were observed. After incubating isolated mitochondria in some hypotonic solutions, and centrifuging these mixtures at high speed, we got mitochondrial supernatants. It was found that in the absence of cytosolic factor, the supernatant alone was able to induce apoptotic changes in nuclei. The effective components were partly of protein. DNA fragmentation was partly inhibited by caspase inhibitors AC-DEVD-CHO and AC- YVADCHO. Meanwhile, caspase inhibitors fully blocked chromatin condensation. Primary characterization of the nuclear endonuclease(s) induced by mitochondrial supernatants was also conducted. It was found that this endonuclease is different from endonuclease G, cytochrome c-induced nuclease, or Ca2+-activated endonuclease.展开更多
Present study describes the results of an efficient protocol for the isolation of good quality DNA from human saliva. The protocol includes collection of saliva in sterile specimen tubes, followed by the cell lysis. A...Present study describes the results of an efficient protocol for the isolation of good quality DNA from human saliva. The protocol includes collection of saliva in sterile specimen tubes, followed by the cell lysis. After formation of cell lysate, proteins wereextracted by phenol chloroform treatment for purification of DNA. The purified DNA was precipitated by adding equal volume of isopropanol to the treated supernatant. After isolation DNA pellet was washed with 70% ethanol, air-dried and was suspended in 30 pL of double distilled water. Best quality of DNA was extracted from the saliva samples and the PCR product was amplified for hyper-variable regions （HVI＆ HV2） of the mitochondrial DNA. The genes were cleaned with GeneAll gel elution kit （Gel SV） （Cat. No. 102-10） and sequenced accordingly. The DNA isolation protocol presented here is recommended for the isolation, best quality and yield of DNA from the human saliva.展开更多
文摘①目的探讨不同提取方案对心肌细胞线粒体质量的影响。②方法在体外用Langendorff法灌注雄性Wistar大鼠心脏,取左心室组织并分离线粒体; Western blot法检测确定分离线粒体的纯度,透射电子显微镜(scanning electron microscopy,SEM)检测分离线粒体的结构形态,线粒体呼吸控制率(respiratory control ratio,RCR)测定分离线粒体的功能。③结果当分离转速为3500g时,与12000g相比,分离得到的线粒体Tubulin蛋白和COXⅣ蛋白表达均较低,表明低转速能够提高分离线粒体的纯度。与此结果一致,电镜结果显示与3500g组相比,12000g组可见较多微丝微管结构及线粒体内外膜分离。与12000g组相比,3500g组Ⅲ态呼吸及RCR值明显增高,表明低转速分离获得的线粒体呼吸功能增强。④结论低转速不仅可以提高分离线粒体的纯度,而且有利于维持分离线粒体的正常结构和功能。
基金the National Natural Science Fundation of China(Grant No. 39800075 and 19890380) and the special Funds for Major StateBasic
文摘We isolated and purified mitochondria from mouse livers and spinach leaves. When added into egg extracts of Xenopus laevis, they caused nuclei of mouse liver to undergo apoptotic changes. Chromatin condensation, margination and DNA ladder were observed. After incubating isolated mitochondria in some hypotonic solutions, and centrifuging these mixtures at high speed, we got mitochondrial supernatants. It was found that in the absence of cytosolic factor, the supernatant alone was able to induce apoptotic changes in nuclei. The effective components were partly of protein. DNA fragmentation was partly inhibited by caspase inhibitors AC-DEVD-CHO and AC- YVADCHO. Meanwhile, caspase inhibitors fully blocked chromatin condensation. Primary characterization of the nuclear endonuclease(s) induced by mitochondrial supernatants was also conducted. It was found that this endonuclease is different from endonuclease G, cytochrome c-induced nuclease, or Ca2+-activated endonuclease.
文摘Present study describes the results of an efficient protocol for the isolation of good quality DNA from human saliva. The protocol includes collection of saliva in sterile specimen tubes, followed by the cell lysis. After formation of cell lysate, proteins wereextracted by phenol chloroform treatment for purification of DNA. The purified DNA was precipitated by adding equal volume of isopropanol to the treated supernatant. After isolation DNA pellet was washed with 70% ethanol, air-dried and was suspended in 30 pL of double distilled water. Best quality of DNA was extracted from the saliva samples and the PCR product was amplified for hyper-variable regions （HVI＆ HV2） of the mitochondrial DNA. The genes were cleaned with GeneAll gel elution kit （Gel SV） （Cat. No. 102-10） and sequenced accordingly. The DNA isolation protocol presented here is recommended for the isolation, best quality and yield of DNA from the human saliva.
