Membrane Deformability and Membrane Tension of Single Isolated Mitochondria

Mitochondria dynamics is crucial to many biological processes such as mitochondria fusion and fission, which is highly correlated to the mechanics of single mitochondria. However, the mechanobiological coupling of mitochondria has been poorly understood. Here membrane deformability and membrane tension of individual mitochondria isolated from MtDsRed labeled human embryonic T-Rex-293 kidney cells were measured using a micropipette aspiration assay. The results demonstrated that membrane deformation of isolated mitochondria exhibited an elastic transition phase followed by an equilibrium phase, and mitochondrial membrane tension was proportional to the area compressibility. It was also indicated that mitochondrial membrane deformability was significantly affected by physical chemical factors such as osmotic pressure or pH value, and was further correlated to mitochondrial functionality in different respiratory states and Ca2+ regulation. These findings provide a new insight into understanding the mechanical regulation of mitochondrial physiology.

Interspecies implantation and mitochondria fate of panda-rabbit cloned embryos

Somatic cell nuclei of giant pandas can dedifferentiate in enucleated rabbit ooplasm, and the reconstructed eggs can develop to blastocysts. In order to observe whether these interspecies cloned embryos can implant in the uterus of an animal other than th

A critical reassessment of the role of mitochondria in tumorigenesis

Background Mitochondrial DNA (mtDNA) is being analyzed by an increasing number of laboratories in order to investigate its potential role as an active marker of tumorigenesis in various types of cancer. Here we question the conclusions drawn in most of these investigations, especially those published in high-rank cancer research journals, under the evidence that a significant number of these medical mtDNA studies are based on obviously flawed sequencing results. Methods and Findings In our analyses, we take a phylogenetic approach and employ thorough database searches, which together have proven successful for detecting erroneous sequences in the fields of human population genetics and forensics. Apart from conceptual problems concerning the interpretation of mtDNA variation in tumorigenesis, in most cases, blocks of seemingly somatic mutations clearly point to contamination or sample mix-up and, therefore, have nothing to do with tumorigenesis. Conclusion The role of mitochondria in tumorigenesis remains unclarified. Our findings of laboratory errors in many contributions would represent only the tip of the iceberg since most published studies do not provide the raw sequence data for inspection, thus hindering a posteriori evaluation of the results. There is no precedent for such a concatenation of errors and misconceptions affecting a whole subfield of medical research.

DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells In vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+](i)) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 mu M) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of ([Ca2+](i)) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

Microcalorimetric studies on the thermogenesis of energy release of mitochondria isolated from rice

After isolation from rice mitochondria still have activity and can live for a long time by using its stored nutrients. The thermogenesis curves of energy release of the mitochondria isolated from variant strains of rice have been determined by using an LKB2277 bioactivity monitor. The differences in shape of the curves and the thermodynamic and kinetic characteristics of the thermogenesis of the mitochondria have been compared. The thermodynamic and kinetic parameters of energy release of the mitochondria in the thermogenesis increasing stage have been calculated, and the experimental thermokinetic equations of the thermogenesis have been established. (C) 2001 Elsevier Science B.V. All rights reserved.

Studies on the energy release of rice mitochondria under different conditions by means of microcalorimetry

The thermodynamic and kinetic behaviors of energy release of mitochondria isolated from rice (Oryza sative L.) were studied by using a LKB 2277 Bioactivity Monitor under different conditions. The thermogenesis curves of energy release of the rice mitochondria (which had been kept at 0-3 degreesC for 15 h and 40 day before the determination) were determined respectively at 25 and 30 degreesC, and the difference in shape of the thermogenesis curves and thermodynamic and kinetic characteristics were compared. The thermodynamic and kinetic parameters of energy release of the mitochondria in the thermogenesis increasing stage have been calculated, and the experimental thermokinetic equations of the thermogenesis have been established. The results indicated that the lower the temperature, the slower the energy release of the rice mitochondria. Both the thermogenesis and the energy release late of the rice mitochondria increased after the mitochondria was kept at lower temperature for 40 days. One can use the methods to characterize the ability of the rice mitochondria to release energy under different conditions. (C) 2001 Published by Elsevier Science B.V.

Species identification in salted products of red snappers by semi-nested PCR-RFLP based on the mitochondria 12S rRNA gene sequence

A molecular approach was developed to distinguish species of red snappers among commercial salted fish products. The specific fragments of the mitocliondrial 12S rRNA gene, which were about 450 bp, were obtained using the semi-nested polymerase chain reaction (semi-nested PCR). Subsequently, PCR amplicons were sequenced, aiming to select restriction endonucleases that generated species-specific restriction fragment length polymorphism (RFLP) profiles. Discrimination of red snappers Lutjanus sanguineus, L. erythopterus from L. argentintaculatus, L. malabarius and other morphologically similar fishes such as Lethrinus leutjanus and Pinjalo pinjalo was feasible by one restriction digestion reaction with three endonucleases Hae III, Sca I and SnaB I, however, for differentiation of L. sattguineus and L. erythopterus, another restriction digestion reaction with single restriction endonuclease Mae II was needed. The seminested PCR-RFLP was demonstrated to be reliable in species identification of salted fish products in this study. (c) 2005 Published by Elsevier Ltd.

烟草和衣藻中ftsZ 基因的克隆及功能分析

作为一种广泛存在于原核细胞中的原始的细胞骨架蛋白，FtsZ在植物中的发现为我们研究植物细胞中质体的分裂机制提供了可能。已有的研究证明了FtsZ与质体的分裂和形态维持有关，但高等植物中FtsZ在质体分裂和形态维持中的作用机制仍不十分清楚，同时高等植物中多个ftsZ成员的存在也使得对FtsZ功能的研究更加复杂。我们从烟草中克隆了两个ftsZ基因，序列和谱系分析表明二者均属于高等植物中的FtsZl基因家族，这也是首次在高等植物中发现多个FtsZl家族的成员。杂交分析表明ftsZ在烟草基因组中是以多拷贝形式存在，并且这两个基因具有相似的表达谱，这些结果暗示着高等植物中FtsZ在质体分裂中的作用更为复杂。GFP标记的原核定位表明二者具有与原核FtsZ类似的功能。此外，利用反义和正义表达的方法研究了二者在烟草质体分裂和形态维持中的作用。反义转化并未对烟草细胞叶绿体的数目和形态造成明显的影响，相反，二者的正义表达均导致细胞中叶绿体数目和形态上的明显变化，这一结果预示着二者在控制质体分裂和形态方面可能具有不同的功能。同时，这些结果也为高等植物中多样化的FtsZ可能具有除质体分裂之外的功能，如质体骨架．提供了证据。 　利用简并引物PCR和RACE从衣藻中扩增得到了一个ftsZ基因的部分cDNA序列，命名为CrFtsZ。序列分析表明该基因编码的蛋白具有FtsZ的典型特点，但同时还有一个与目前已知FtsZ均不同的突出c-末端：分子谱系分析认为CrFtsZ与线粒体进化祖先a -proteobacteria中的FtsZ有着共同起源，因此CrFtsZ可能是一个控制线粒体分裂的FtsZ。此外，CrFtsZ的c-端突出序列还具有目前已知真核生物线粒体分裂相关蛋白dynamin的某些特征，考虑到FtsZ在原核细胞分裂和真核细胞器分裂中的作用，我们推测CrFtsZ可能是FtsZ向dynamin过度的一种中间进化形式。这一发现为线粒体分裂机制的起源和进化提供了新的分子证据，对于认识真核线粒体分裂机制的起源与演化具有重要意义。

DRP蛋白的SUMO化修饰参与线粒体分裂调节的研究

　　SUMO (small ubiquition-like modifier) 是一类小的类泛素修饰物，能在一系列酶的作用下共价结合到目标蛋白上。 这种可逆的SUMO化修饰过程 (SUMOylation) 是调节细胞内蛋白质功能的重要方式之一，参与真核细胞内各种代谢途径。已有的研究发现，植物的SUMO化修饰参与植物花期调控，激素信号转导，抗病防御及逆境应答等生理过程。 　　线粒体是重要的细胞器，它的形态是多样和动态的。在活细胞中，线粒体不断地运动，并频繁地进行分裂与融合，维持着形态的平衡。而这种形态的动态平衡对线粒体功能的行使有着重要的意义。线粒体的分裂需要dynamin相关蛋白DRPs的参与，该类蛋白在进化中是保守的，从植物中已经鉴定出DRP3A和DRP3B两个蛋白。DRP蛋白由胞质招募到线粒体外膜上来参与膜的剪切，但有关DRP向线粒体招募和组装的机制尚不清楚。 　　本文通过对稳定表达GFP-SUMO1的BY-2悬浮细胞观察发现，SUMO1主要定位于细胞核中，在细胞质中也有少量分布；过表达SUMO1能使线粒体的片段化程度增强。而在SAE-RNAi植株及siz1突变体中，线粒体片段化程度减弱，由此提示SUMO化修饰的下调降低了线粒体的分裂水平。经序列分析发现，线粒体分裂蛋白DRP3A和DRP3B有SUMO化修饰的识别序列，而通过表达SUMO-DRP融合蛋白来加强DRP蛋白的SUMO化修饰，则能使线粒体分裂增加，从而表明SUMO化是通过对DRP的调节来影响线粒体的分裂。 我们推测SUMO化修饰的作用是能特异地保护DRP不被降解，最终导致DRP库更为稳定与活跃。总之，本文首次在植物中鉴定出SUMO1在线粒体上的作用，并暗示它可能通过调节DRP蛋白来参与线粒体分裂的新功能。

玉米种子脱水耐性与其线粒体完整性及抗氧化系统的关系

本文以正常性玉米种子‘农大108’(Zea Mays L. ‘Nongda 108’) 种胚为实验材料，研究了玉米种子发育过程中脱水耐性的变化规律，细胞匀浆以及线粒体水平上活性氧清除酶活性与种子脱水耐性/敏感性的关系，以及线粒体结构和功能完整性在发育过程中不同阶段对脱水的应答，以期在亚细胞分区水平上，针对活性氧产生的源头位点 (线粒体) 探明种子细胞脱水耐性/敏感性与抗氧化系统运转的关系。结果表明： 玉米种子在发育过程中先获得萌发能力后获得脱水耐性，并且脱水耐性的获得是一个渐进的过程。人工授粉后26天 (Days after pollination, DAP) 之前的种胚不具有脱水耐性，26 DAP时开始获得脱水耐性，到34 DAP后种胚完全获得脱水耐性。 在发育过程中，种胚线粒体的呼吸速率逐渐降低，并且对脱水的敏感性也逐渐下降。脱水会降低脱水敏感性种胚线粒体的结构完整性；脱水同时会降低线粒体功能的完整性，包括线粒体能量产生的速率和效率，以及三羧酸循环关键酶的活性。但当种胚获得脱水耐性后，脱水将不再影响种胚线粒体结构和功能的完整性。 玉米种胚发育过程中脱水耐性的变化与细胞中的抗氧化系统有关。在细胞匀浆水平上，脱水过程中脂质过氧化产物的积累与细胞脱水耐性的关系不明显；但是在线粒体水平上脱水会明显导致脱水敏感性种胚线粒体膜质过氧化程度的升高。脱水导致脱水敏感种胚细胞中几个重要的抗氧化酶活性的下降，但是与细胞匀浆水平相比，在线粒体水平上抗氧化酶系统对脱水更加敏感。 总之，发育早期玉米胚对脱水之所以敏感有两方面的原因，一方面是发育早期线粒体具有较高的代谢速率因而产生过多的活性氧，另一方面是由于脱水导致各抗氧化酶活性的显著降低，失去了抗氧化保护功能。而在发育晚期，早期本来很活跃的许多代谢随之关闭，呼吸速率降到很低，因而产生的活性氧减少，同时由于抗氧化系统对脱水的耐受性，所以脱水不会对线粒体的结构和功能造成伤害。与细胞匀浆水平相比，线粒体水平上抗氧化系统的运转与种胚在发育过程中脱水耐性的获得的关系更加密切。

低温吸胀对大豆种子呼吸代谢影响的研究

大豆是重要的油料和蛋白植物。在生产实践中，在播种后达到早苗和齐苗是大豆丰收的前提。种子的吸胀冷害是农业生产的严重问题。吸胀冷害发生在种子开始吸水萌动的萌发初始阶段。吸胀冷害不仅发生在高寒地带和低温冷湿地区，尤其在我国东北地区，造成我国乃至全球大豆不同程度的减产。吸胀冷害的原初作用位点在生物膜上，本实验从呼吸代谢的角度研究吸胀冷害对种子活力的影响，探讨吸胀冷害的机制。 本实验选用由黑龙江省黑河农业科学院提供的对低温中度敏感的黑河13 号大豆种子为材料，分别经22°C、10°C 和4°C 恒温培养箱24 h 后，测定其生理指标，通过透射电镜观察细胞超微结构，利用蛋白质组技术研究低温吸胀与种子呼吸代谢的关系，得到的结果如下： 低温吸胀阻碍胚轴膜系统的修复。通过电解质渗漏率测定发现，4°C 到22°C 温度范围内，提高吸胀温度有助于保持细胞膜的完整性，显著降低吸胀后胚轴电解质渗漏。在低温下吸胀，胚轴活性氧清除酶的活性受到抑制，活性氧含量增加，增强了膜脂过氧化作用，进而导致种子活力下降。 通过透射电子显微镜观察，大豆种子在22°C 吸胀24 h 后，胚轴细胞液泡明显变大，在细胞中所占比例很高，并且细胞内膜系统比较发达，能清晰观察到细胞核，线粒体，质膜，内质网整齐有序的形状。胚轴的细胞含有其它结构正常的细胞器，包括细胞壁，胞间连丝，淀粉粒和油体等。线粒体的外膜、内膜、嵴发育较完善。10°C 和4°C 的吸胀严重损伤了胚轴中细胞器的修复，细胞膜不规则，没有发现内质网和胞间连丝，线粒体的体积较小以及膜系统不发达，尤其是4°C 吸胀的胚轴中细胞器的损伤更加严重，细胞膜系统紊乱。 低温吸胀抑制了线粒体从轻线粒体向重线粒体的修复，以及线粒体的耗氧能力。22°C 吸胀的线粒体的总体耗氧能力较高，电子传递主要是利用复合体I 的电子传递途径。10°C 吸胀的线粒体总体耗氧呼吸较低，且其线粒体的电子传递主要以复合体II 的途径。4°C 吸胀的线粒体的耗氧能力则更低。 将分离得到的线粒体进行的蛋白质组分析，共分离400 多个蛋白点，其中有20 个点有表达差异。经ESI-Q-TOF-MS/MS 鉴定，六个下调的蛋白质分别为ATP 合成酶的亚基 (线粒体的氧化磷酸化)，线粒体延长因子Tu（线粒体基因组转录）， 苹果酸脱氢酶（三羧酸循环），精氨酸酶（尿素循环）和2 个线粒体chaperonin-60 （热稳定蛋白）。这些蛋白在低温吸胀时下调表达，影响了线粒体的正常生理代谢，说明它们在维持线粒体正常代谢中起到了重要的作用。 综上所述，低温吸胀影响了线粒体的结构和生理功能的修复，减少了能量和中间物质供应给种子萌发，造成了种子活力的下降。