水生动物和大鼠线粒体的呼吸链比较

用陆生哺乳动物线粒体呼吸链与水生动物线粒体呼吸链相比较的研究方法,探讨了呼吸链的功能与环境相适应的关系。研究了淡水中生活的草鱼肝丝线粒体,观察到琥珀酸脱氢酶的活性非常低,而ＮＡＤＨ脱氢酶和泛醌细胞色素Ｃ还原酶的活性较高。但海洋生物海绵的线粒体ＮＡＤＨ脱氢酶和琥垢酸脱氢酶的活性都非常低。     

线粒体蛋白质组学研究进展

线粒体是真核生物中重要的细胞器,其包含的全部蛋白质称为线粒体蛋白质组。人类线粒体大约包含1500多种蛋白质,由核基因和线粒体基因共同编码。线粒体是细胞能量合成和物质代谢的中心,其功能障碍将直接或问接引起许多疾病。目前线粒体蛋白质组学正是系统性地研究线粒体在生理、病理过程中的功能变化以及研究疾病发生机制的重要方法。将线粒体蛋白质组的研究方法、研究进展、线粒体蛋白质组的性质及其在相关疾病研究中的作用进行综述,并对线粒体蛋白质组学在疾病发生机制和诊断治疗中的发展前景进行展望。     

局灶性脑缺血大鼠皮层神经元超微结构及线粒体呼吸功能变化的研究

目的:研究局灶性脑缺血大鼠脑细胞超微结构及脑组织线粒体呼吸链功能的变化。方法:采用改良Zea Longa方法复制大鼠大脑中动脉缺血(MCAO)模型,透射电镜观察缺血后脑组织神经元超微结构的改变;检测呼吸链R3、R4、RCR、OPR等评价呼吸功能的指标。结果:局灶性脑缺血大鼠脑组织神经元细胞结构严重破坏;与对照组相比,脑缺血时大鼠脑线粒体ST3、RCR和OPR降低,ST4升高。结论:脑缺血急性期线粒体结构破坏,功能受损严重,随着时间延长均有所恢复;保护线粒体呼吸链可能对脑缺血损伤有保护作用。     

线粒体呼吸链功能调控机制的研究进展

核基因组与线粒体基因组的相互作用,以及两基因组在调控呼吸链亚基的表达机制方面一直处于探索阶段,线粒体核转录因子的发现,使细胞核调控呼吸链亚基表达的研究得到了很大的发展。本文就近年来对核呼吸因子和细胞核对呼吸链的调控机制研究作一介绍。     

关于呼吸链电子传递与氧化磷酸化机制的教学

近年来,植物生理学中呼吸作用过程及机制研究的新进展和新成果相对较少。在线粒体电子传递系统中,有些传递体的结构和排列、功能尚待进一步确证。目前,对有关呼吸链上伴随着电子传递,线粒体村质中的H~+跨内膜转移到膜间空间、建立质子动力并形成ATP的问题,国内外植物生理学教材有不同提法。在这里,我们试图通过对这些说法的比较分析,以求得到一个比较恰当的说法,这对教学工作可能是有益的。     

冷暴露对中缅树鼩肝脏、膈肌和心肌线粒体呼吸的影响

在4℃急性冷暴露(1 h,4 h,8 h,24 h)和持续冷暴露 (7 d,14 d,28 d)条件下,测定中缅树鼩膈肌、心肌和肝脏的线粒体状态Ⅲ呼吸、状态Ⅳ呼吸、呼吸控制率(RCR)、线粒体蛋白含量以及肝脏线粒体P/O值的变化.结果表明:肝脏线粒体状态Ⅲ、状态Ⅳ呼吸随着低温处理时间的延长,呼吸速率均极显著增加,在28 d后分别增加了132.9%、124.4%(P<0.01),RCR与对照比较,在8 h和7 d组分别显著增加了35.8%和48.4%(P<0.05),线粒体蛋白含量也极显著增加,在28 d后增加了104.7%(P<0.01),P/O值在整个低温处理过程中呈下降趋势,在28 d后降低了40.2%,达到极显著水平(P<0.01);膈肌线粒体状态Ⅲ呼吸在整个低温处理期间没有显著变化,状态Ⅳ呼吸在28 d达到极显著增加(P<0.01,64.9%),RCR在28 d后显著降低(P<0.05, 42.1%),线粒体蛋白只有4 h组有极显著增加(P<0.01,45.2%);心肌的状态Ⅲ呼吸在8 h组有着极显著的增加(P<0.01, 54.7%),状态Ⅳ呼吸随着低温处理时间的增加而显著增加,28 d后增加了94.7%(P<0.01),RCR在28 d后降低37.8%(P<0.01),线粒体蛋白表现出先下降再上升的趋势,8 h组下降37.8%(P<0.01),28 d增加25.2%(P<0.05).说明中缅树鼩在冷胁迫的条件下肝脏线粒体呼吸能力显著增强,主要表现为状态Ⅳ呼吸即质子漏产热的显著增强,膈肌和心肌的线粒体呼吸也具有一定的适应性变化,补偿了冷胁迫条件下中缅树鼩增加的能量需求,是中缅树鼩在冷胁迫中重要的适应对策.     

呼吸末二氧化碳监测在临床中的应用

呼吸末二氧化碳监测是评估患者通气状态的重要指标,也是确保病人安全的重要参数之一。呼吸末二氧化碳分压与动脉血二氧化碳分压之间存在良好的相关关系,可以通过持续监测其动态观察动脉血二氧化碳分压,近年来呼吸末二氧化碳监测已经成为了临床工作中一项常规监测技术手段。在临床的实际工作中呼吸末二氧化碳监测不仅能够确定气管插管的位置,评估心肺复苏的预后,而且能够监测患者的通气功能状态,更好地指导工作中呼吸模式和呼吸机的参数的调整,为撤机提供准确的时机,并能及时发现机械故障和减少不必要的操作。本文重点对呼吸末二氧化碳监测的原理以及其在临床中的应用展开综述。     

烟草愈伤组织中不被KCN加氧肟酸抑制的剩余呼吸研究

烟草愈伤组织中存在有大约占总呼吸20—30%的不被 KCN 加 m-CLAM 抑制的“剩余呼吸”,它的性质和在细胞中的定位都不清楚。本研究发现,乙醇酸和乙醛酸都明显促进不被KCN(或 NaN_3)加 m-CLAM 抑制的剩余呼吸;乙醇酸听引起的氧吸收和剩余呼吸可被α-羟基乙磺酸钠抑制;线粒体呼吸被 KCN 加 m-CLAM 完全抑制;除去线粒体的上清液中发现有乙醇酸和乙醛酸氧化活性的存在。初步确定,烟草愈伤组织中这部分剩余呼吸主要是由非线粒体乙醇酸氧化酶所催化的。     

线粒体呼吸功能与精子活力、核DNA损伤的相关性分析

为探讨线粒体呼吸功能与精子活力、核DNA损伤程度之间的相关性,按WHO标准收集34例不同活力的精液标本,采用蔗糖差速离心法或密度梯度离心法提取精子线粒体,通过铂氧电极-溶氧仪测定线粒体呼吸耗氧率并计算状态III呼吸、状态IV呼吸、呼吸控制率（RCR）、磷氧比（P／0）及氧化磷酸化效率（0PR）;应用精子染色质扩散（sperm chromatin dispersion,SCD）实验检测精子DNA损伤情况。结果表明：不同活力精子线粒体状态Ⅲ呼吸耗氧量之间具有显著差异俨〈0．01）;弱精子症组RcR和OPR与正常对照组比较,分别降低了17．03％（P〈0．05）和40．74％（P〈0。01）;精子DNA损伤程度与精子活力、状态III呼吸及OPR均呈极显著负相关（r值分别是-0．812、-0．788和-0．696）。以上结果提示：精子线粒体呼吸耗氧和氧化磷酸化功能与精子活力之间存在着密切的联系;精子DNA（包括mtDNA）损伤可能影响精子的正常功能。     

线粒体功能的检测方法

线粒体为细胞内的能量工厂,对细胞的增殖、分化、存活以及稳态的维持起着重要的调节作用。线粒体功能障碍与机体生长、发育异常、认知发生障碍以及多种器官病变密切相关。线粒体形态、结构和功能的检测对于了解线粒体的稳态以及功能状态有着重要意义,线粒体的功能状态与线粒体膜电位、线粒体膜通道、线粒体Ca2+浓度、ATP生成、呼吸链复合体活性、活性氧生成以及DNA突变密切相关。本文就线粒体形态、结构和功能的检测方法及其研究进展进行了综述。     

Functional aspects of oxidative phosphorylation and electron transport in cardiac mitochondria of copper-deficient rats

Although dietary copper deficiency causes physiological, morphological, and biochemical abnormalities in cardiac mitochondria, the relationship observed between abnormalities of mitochondrial structure and function have been inconsistent in previous studies. The purpose of the present study was to re-evaluate the respiration rates of cardiac mitochondria from copper-deficient rats and to use several drugs that uncouple and inhibit mitochondrial respiration in order to clarify the mechanisms of mitochondrial dysfunction found in several laboratories. Copper deficiency reduced state 4 and state 3 cardiac mitochondrial respiration rates with all substrates tested. However, neither the ratio of ADP/oxygen consumed nor the acceptor control index was affected by copper deficiency. Cardiac mitochondria of copper-deficient rats showed a resistance to respiratory blockade by oligomycin and an increased ability to hydrolyze ATP in the presence of oligomycin compared with mitochondria of copper-adequate rats. This suggests that copper deficiency affects the function of the cardiac mitochondrial ATP synthase.     

Hyperglycemia Preserves Brain Mitochondrial Respiration During Anoxia

We examined brain mitochondrial function in normo- (5 mM) and hyperglycemic (50 mM) cats after 8 min of anoxia. In anoxic normoglycemic cats, mitochondrial state 3 respiration with NAD-linked substrates glutamate or pyruvate (both plus malate) was inhibited 30-50%. The uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) maximally stimulated respiration, indicating that inhibition of phosphorylation, not impairment of electron transport, substrate transport, or oxidation was present. State 3 respiration with succinate (plus rotenone) was unaffected. Mitochondrial respiratory control ratios trended toward reductions whereas ADP/O ratios remained unchanged. In contrast, brain mitochondria from anoxic hyperglycemic cats showed no such inhibition of state 3 respiration and no differences in function from normo- and hyperglycemic control animals except for trends toward loose coupling. Significantly higher brain tissue glucose concentrations were present in hyperglycemic controls as the only metabolite difference compared to normoglycemic controls. At the end of anoxia, hyperglycemic cats exhibited significantly higher cortical lactate and glucose levels but similarly reduced high-energy phosphate concentrations compared to normoglycemic cats. These results demonstrate that increased availability of glucose to gray matter as a consequence of hyperglycemia maintains normal mitochondrial state 3 respiration during exposure to anoxia. Previous survival studies have shown that lower serum glucose concentrations during anoxia are relatively brain protective. This result indicates that the presently described alterations in mitochondrial respiration must be fully reversible.     

Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria

Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30–35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer''s disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances.     

The Influence of Mitochondrial Concentration and Storage on the Respiratory Control of Isolated Plant Mitochondria

The respiration of mitochondria isolated from various plant tissues was studied over a range of mitochondrial concentrations and at various times after isolation. Respiration at 25 C expressed as nanomoles of O₂ per minute per milligram of protein was constant for mitochondrial concentrations higher than some critical amount, usually 0.25 to 1.0 milligram of protein per reaction. Below this concentration the state 3 respiration rate declined and the mitochondria appeared to lose respiratory control. The respiration of isolated mitochondria stored in ice but measured at 25 C generally declined over long time periods although mitochondria from some tissues showed an initial increase. The results indicate that valid comparisons of the respiratory activity of mitochondria isolated from different tissues or from different parts of the same tissue cannot be made at least until the influence of the above factors has been evaluated.     

Does the respiratory rate in sea urchin embryos increase during early development without proliferation of mitochondria?

During early development of the sea urchin, the respiratory rate, enhanced upon fertilization, is maintained up to hatching (pre-hatching period) and then gradually increases to a maximum at the gastrula stage (post-gastrula period). Except for a short duration after fertilization, respiration in embryos is strongly inhibited by CN⁻ and antimycin A. During the whole span of early development, the amounts of proteins, cytochromes and the specific activities of cytochrome c oxidase and reduced nicotinamide adenine dinucleotide (NADH) cytochrome c reductase in mitochondria are practically the same as in unfertilized eggs. A marked augmentation of mitochondrial respiration after hatching probably occurs without net increase in whole mitochondrial intrinsic capacities. Carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) or tetramethyl p-phenylenediamine (TMPD) enhances the respiratory rate in the pre-hatching period but hardly augments the respiration in the post-gastrula period. In the presence of both FCCP and TMPD, the respiratory rate in the pre-hatching period was as high as in the post-gastrula period. Probably, electron transport in the mitochondrial respiratory chain is regulated by acceptor control and limitation of cytochrome c reduction in the pre-hatching period and released from those regulations in the post-gastrula period. Acceptor control of respiration is experimentally reproduced in isolated mitochondria by making adenine nucleotide levels as those levels in the pre-hatching period.     

Control of mitochondrial respiration by NO*, effects of low oxygen and respiratory state

Nitric oxide (NO.) inhibits mitochondrial respiration by binding to the binuclear heme a3/CuB center in cytochrome c oxidase. However, the significance of this reaction at physiological O2 levels (5-10 microM) and the effects of respiratory state are unknown. In this study mitochondrial respiration, absorption spectra, [O2], and [NO.] were measured simultaneously at physiological O2 levels with constant O2 delivery, to model in vivo respiratory dynamics. Under these conditions NO. inhibited mitochondrial respiration with an IC50 of 0.14 +/- 0.01 microm in state 3 versus 0.31 +/- 0.04 microM in state 4. Spectral data indicate that the higher sensitivity of state 3 respiration to NO. is due to greater control over respiration by an NO.-dependent spectral species in the respiratory chain in this state. These results are discussed in the context of regulation of respiration by NO. in vivo and its implications for the control of vessel-parenchymal O2 gradients.     

Ginkgo biloba Extracts EGb 761 and Bilobalide Increase NADH Dehydrogenase mRNA Level and Mitochondrial Respiratory Control Ratio in PC12 Cells

In the present study, we investigated the effect of Ginkgo biloba extract, EGb 761, and one of its components, bilobalide, on gene expression of subunit 1 of mitochondrial NADH dehydrogenase (ND1) in PC12 cells. By Northern blot analysis we found a 2-fold significant increase in ND1 mRNA level, after 48 and 72 h exposure to 100 g/ml EGb 761 and to 10 g/ml bilobalide. We also evaluated, by oxygraphy measurements, mitochondrial respiration during state 3 and state 4. In cells treated with EGb 761 and bilobalide for 48 and 72 h, state 4 respiration was significantly decreased, and the respiratory control ratio was increased. These results provide evidence that EGb 761 and bilobalide exert their protective effects by up-regulating mitochondrial ND1 gene expression and by decreasing state 4 respiration, whose increase is thought to be responsible for oxidative damage.     

Mitochondrial respiration in heart, liver, and kidney of copper-deficient rats

Morphological observations in some tissues indicate that dietary copper deficiency results in structural damage to mitochondria. The purpose of this study was to determine whether mitochondrial function is impaired as well. Male, weanling Sprague-Dawley rats were fed diets deficient or sufficient in copper for 4 weeks. Mitochondria were isolated from heart, liver, kidney cortex, and kidney medulla. P/O ratio, state 3 and state 4 respiration rates (oxygen consumed in the presence and absence of ADP, respectively), and acceptor control index (ratio of state 3:state 4) were determined using succinate or pyruvate/malate as substrate. State 3 respiration rate in mitochondria from copper-deficient hearts and livers was lower than in mitochondria from copper-sufficient hearts. Copper deficiency reduced the state 4 respiration rate only in cardiac mitochondria. Neither respiration rate was affected by copper deficiency in mitochondria from kidney medulla or cortex. P/O ratio was not significantly affected by copper deficiency in any tissue examined. Acceptor control index was reduced only in liver mitochondria. The observed decreases in respiration rates are consistent with decreased cytochrome c oxidase activity, shown by others to occur in mitochondria isolated from hearts and livers of copper-deficient rats.     

Ischemic preconditioning preserves proton leakage from mitochondrial membranes but not oxidative phosphorylation during heart reperfusion

The aim of this study was to evaluate the role of mitochondria in the recovery of cardiac energetics induced by ischaemic preconditioning at reperfusion. Isolated rat hearts were aerobically perfused (control), subjected to global ischaemia and reperfusion (reperfusion), or subjected to 3 brief cycles of ischaemia/reperfusion and then to the protocol of reperfusion (preconditioning). At the end of the perfusion, antimycin A was delivered to the heart for 25 min, to inhibit mitochondrial respiration and stimulate glycolysis. The increased amount of lactate released in the coronary effluent was correlated with the number of viable cells producing this end-product of glycolysis. Preconditioned hearts released 18% more lactate than reperfused hearts (p < 0.05). This result indicates that preconditioning partially preserved cell viability, as was also evidenced by the MTT assay performed on cardiac biopsies. The difference between antimycin A-stimulated and basal lactate concentration, representing the contribution of mitochondria to the overall energetics of cardiac tissue, was also 18% more elevated in the preconditioned hearts than in the reperfused hearts (p < 0.01). The study of the respiratory function of mitochondria isolated at the end of perfusion, showed that preconditioning did not improve the oxygen-dependent production of ATP (state 3 respiration, ADP/O). On the contrary, state 4 respiration, which is related to proton leakage, was 35.0% lower in the preconditioned group than reperfusion group (p < 0.05). Thus, preconditioning ameliorates cardiac energetics by preserving cell death, but without affecting mitochondrial oxidative phosphorylation. Mitochondria can contribute to cell survival by the attenuation of proton leak from inner membrane.     

Kidney mitochondrial respiration in protein- and energy-malnourished rats

Several lines of evidence show a close association between plasma membrane Na,K-ATPase and mitochondrial respiration. Extending the observation in human erythrocyte membrane (6), Na,K-ATPase activity has been shown to be elevated in kidney microsomal preparations from protein- and energy-malnourished rats (10). Kidney mitochondrial respiration was studied in these rats under various conditions of assay. Sucrose was used as a modifier of mitochondrial morphology and volume to study its effect on these mitochondria. Mitochondrial state 3 respiration was increased by 35% in protein-deficient rats (P less than 0.02). Vmax(ADP) of state 3 respiration was increased by about 47% in protein- as well as energy-restricted rats. Mitochondria from protein- and energy-deficient rats were more tightly coupled as compared to those from control group. Km apparent for (ADP) and (Pi) were elevated in protein- and energy-malnourished rats. The magnitude of increase was much more in energy-deficient rats. Morphological differences between the mitochondria from two dietary manipulations were reflected in differences in the responses of state 3 respiration, Km(ADP), state 4 respiration, and respiratory control ratios to changing sucrose concentrations. This increase in mitochondrial respiration parallels the increased Na,K-ATPase activity in these rats. Increased Km (ADP and Pi) for mitochondrial respiration are perhaps in response to increased availability of these metabolites in the cytosol. The sucrose effect, in addition, distinguishes the morphological differences in mitochondrial membrane due to protein or energy deficiencies. In conclusion, these results, to a great extent, support an association between the activity of Na,K-ATPase and mitochondrial respiration. The study of mechanism(s) which could contribute to the enhancement of mitochondrial respiration will be of general importance to the understanding of regulation of mitochondrial oxidative phosphorylation, and is of particular interest to us.