Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death

Mitochondria play an important role in energy production, Ca2+ homeostasis and cell death. In recent years, the role of the mitochondria in apoptotic and necrotic cell death has attracted much attention. In apoptosis and necrosis, the mitochondrial permeability transition (mPT), which leads to disruption of the mitochondrial membranes and mitochondrial dysfunction, is considered to be one of the key events, although its exact role in cell death remains elusive. We therefore created mice lacking cyclophilin D (CypD), a protein considered to be involved in the mPT, to analyse its role in cell death. CypD-deficient mice were developmentally normal and showed no apparent anomalies, but CypD-deficient mitochondria did not undergo the cyclosporin A-sensitive mPT. CypD-deficient cells died normally in response to various apoptotic stimuli, but showed resistance to necrotic cell death induced by reactive oxygen species and Ca2+ overload. In addition, CypD-deficient mice showed a high level of resistance to ischaemia/reperfusion-induced cardiac injury. Our results indicate that the CypD-dependent mPT regulates some forms of necrotic death, but not apoptotic death.     

Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death

Programmed cell death is a fundamental requirement for embryogenesis, organ metamorphosis and tissue homeostasis. In mammals, release of mitochondrial cytochrome c leads to the cytosolic assembly of the apoptosome-a caspase activation complex involving Apaf1 and caspase-9 that induces hallmarks of apoptosis. There are, however, mitochondrially regulated cell death pathways that are independent of Apaf1/caspase-9. We have previously cloned a molecule associated with programmed cell death called apoptosis-inducing factor (AIF). Like cytochrome c, AIF is localized to mitochondria and released in response to death stimuli. Here we show that genetic inactivation of AIF renders embryonic stem cells resistant to cell death after serum deprivation. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies-the very first wave of cell death indispensable for mouse morphogenesis. AIF-dependent cell death displays structural features of apoptosis, and can be genetically uncoupled from Apaf1 and caspase-9 expression. Our data provide genetic evidence for a caspase-independent pathway of programmed cell death that controls early morphogenesis.     

The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore

A sudden increase in permeability of the inner mitochondrial membrane, the so-called mitochondrial permeability transition, is a common feature of apoptosis and is mediated by the mitochondrial permeability transition pore (mtPTP). It is thought that the mtPTP is a protein complex formed by the voltage-dependent anion channel, members of the pro- and anti-apoptotic BAX-BCL2 protein family, cyclophilin D, and the adenine nucleotide (ADP/ATP) translocators (ANTs). The latter exchange mitochondrial ATP for cytosolic ADP and have been implicated in cell death. To investigate the role of the ANTs in the mtPTP, we genetically inactivated the two isoforms of ANT in mouse liver and analysed mtPTP activation in isolated mitochondria and the induction of cell death in hepatocytes. Mitochondria lacking ANT could still be induced to undergo permeability transition, resulting in release of cytochrome c. However, more Ca2+ than usual was required to activate the mtPTP, and the pore could no longer be regulated by ANT ligands. Moreover, hepatocytes without ANT remained competent to respond to various initiators of cell death. Therefore, ANTs are non-essential structural components of the mtPTP, although they do contribute to its regulation.     

Postconditioning of sevoflurane and propofol is associated with mitochondrial permeability transition pore

Background： Sevoflurane and propofol are effective cardioprotective anaesthetic agents, though the cardioprotection of propofol has not been shown in humans. Their roles and underlying mechanisms in anesthetic postconditioning are unclear. Mitochondrial permeability transition pore （MPTP） opening is a major cause of ischemia-reperfusion injury. Here we investigated sevoflurane- and propofol-induced postconditioning and their relationship with MPTP. Methods： Isolated perfused rat hearts were exposed to 40 min of ischemia followed by 1 h of reperfusion. During the first 15 min of reperfusion, hearts were treated with either control buffer （CTRL group） or buffer containing 20 μmol/L atractyloside （ATR group）, 3% （v/v） sevoflurane （SPC group）, 50 μmol/L propofol （PPC group）, or the combination of atractyloside with respective anesthetics （SPC＋ATR and PPC＋ATR groups）. Infarct size was determined by dividing the total necrotic area of the left ventricle by the total left ventricular slice area （percent necrotic area）. Results： Hearts treated with sevoflurane or propofol showed significantly better recovery of coronary flow, end-diastolic pressures, left ventricular developed pressure and derivatives compared with controls. Sevoflurane resulted in more protective alteration of hemodynamics at most time point of reperfusion than propofol. These improvements were paralleled with the reduction of lactate dehydrogenase release and the decrease of infarct size （SPC vs CTRL： （17.48±2.70）% vs （48.47±6.03）%, P〈0.05; PPC vs CTRL： （35.60±2.10）% vs （48.47±6.03）%,P〈0.05）. SPC group had less infarct size than PPC group （SPC vs PPC： （ 17.48±2.70）% vs （35.60±2.10）%,P〈0.05）. Atractyloside coadministration attenuated or completely blocked the cardioprotective effect of postconditioning of sevoflurane and propofol. Conclusion： Postconditioning of sevoflurane and propofol has cardio-protective effect against ischemia-reperfusion injury of heart     

Identification of a cold receptor reveals a general role for TRP channels in thermosensation

The cellular and molecular mechanisms that enable us to sense cold are not well understood. Insights into this process have come from the use of pharmacological agents, such as menthol, that elicit a cooling sensation. Here we have characterized and cloned a menthol receptor from trigeminal sensory neurons that is also activated by thermal stimuli in the cool to cold range. This cold- and menthol-sensitive receptor, CMR1, is a member of the TRP family of excitatory ion channels, and we propose that it functions as a transducer of cold stimuli in the somatosensory system. These findings, together with our previous identification of the heat-sensitive channels VR1 and VRL-1, demonstrate that TRP channels detect temperatures over a wide range and are the principal sensors of thermal stimuli in the mammalian peripheral nervous system.     

Patch-clamping of the inner mitochondrial membrane reveals a voltage-dependent ion channel

The prime function of mitochondria is to provide the cell with adenosine triphosphate (ATP). ATP synthesis is driven by the protonmotive force (delta p), which is generated and maintained across the inner mitochondrial membrane (IMM) by the activity of the respiratory chain. It is widely believed that the IMM is unlikely to contain ion channels like those present in the plasma membrane, because the high rates of ion transport characteristic of open channels would be expected to dissipate the delta p. Although the small size of the organelle has prevented the use of classical electrophysiological methods, the recent introduction of the patch-clamp technique, which allows currents to be recorded from very small cells, has enabled us to test this hypothesis. By patch-clamping the IMM, we have identified a slightly anion-selective channel, which is voltage-dependent and has a mean conductance of 107 pS in the presence of symmetrical 150 mM KCl.     

Blebbing, free Ca2+ and mitochondrial membrane potential preceding cell death in hepatocytes

Cell surface 'blebbing' is an early consequence of hypoxic and toxic injury to cells. A rise in cytosolic free Ca2+ has been suggested as the stimulus for bleb formation and the final common pathway to irreversible cell injury. Here, using digitized low-light video microscopy, we examine blebbing, cytosolic free Ca2+, mitochondrial membrane potential and loss of cell viability in individual cultured hepatocytes. Unexpectedly, we found that after 'chemical hypoxia' with cyanide and iodoacetate, cytosolic free Ca2+ does not change during bleb formation or before loss of cellular viability. Cell death was precipitated by a sudden breakdown of the plasma membrane permeability barrier, possibly caused by rupture of a cell surface bleb.     

DRP-1-mediated mitochondrial fragmentation during EGL-1-induced cell death in C. elegans

Genetic analyses in Caenorhabditis elegans have been instrumental in the elucidation of the central cell-death machinery, which is conserved from C. elegans to mammals. One possible difference that has emerged is the role of mitochondria. By releasing cytochrome c, mitochondria are involved in the activation of caspases in mammals. However, there has previously been no evidence that mitochondria are involved in caspase activation in C. elegans. Here we show that mitochondria fragment in cells that normally undergo programmed cell death during C. elegans development. Mitochondrial fragmentation is induced by the BH3-only protein EGL-1 and can be blocked by mutations in the bcl-2-like gene ced-9, indicating that members of the Bcl-2 family might function in the regulation of mitochondrial fragmentation in apoptotic cells. Mitochondrial fragmentation is independent of CED-4/Apaf-1 and CED-3/caspase, indicating that it occurs before or simultaneously with their activation. Furthermore, DRP-1/dynamin-related protein, a key component of the mitochondrial fission machinery, is required and sufficient to induce mitochondrial fragmentation and programmed cell death during C. elegans development. These results assign an important role to mitochondria in the cell-death pathway in C. elegans.     

Three key residues form a critical contact network in a protein folding transition state

Determining how a protein folds is a central problem in structural biology. The rate of folding of many proteins is determined by the transition state, so that a knowledge of its structure is essential for understanding the protein folding reaction. Here we use mutation measurements--which determine the role of individual residues in stabilizing the transition state--as restraints in a Monte Carlo sampling procedure to determine the ensemble of structures that make up the transition state. We apply this approach to the experimental data for the 98-residue protein acylphosphatase, and obtain a transition-state ensemble with the native-state topology and an average root-mean-square deviation of 6 A from the native structure. Although about 20 residues with small positional fluctuations form the structural core of this transition state, the native-like contact network of only three of these residues is sufficient to determine the overall fold of the protein. This result reveals how a nucleation mechanism involving a small number of key residues can lead to folding of a polypeptide chain to its unique native-state structure.     

Genomic analysis of metastasis reveals an essential role for RhoC

The most damaging change during cancer progression is the switch from a locally growing tumour to a metastatic killer. This switch is believed to involve numerous alterations that allow tumour cells to complete the complex series of events needed for metastasis. Relatively few genes have been implicated in these events. Here we use an in vivo selection scheme to select highly metastatic melanoma cells. By analysing these cells on DNA arrays, we define a pattern of gene expression that correlates with progression to a metastatic phenotype. In particular, we show enhanced expression of several genes involved in extracellular matrix assembly and of a second set of genes that regulate, either directly or indirectly, the actin-based cytoskeleton. One of these, the small GTPase RhoC, enhances metastasis when overexpressed, whereas a dominant-negative Rho inhibits metastasis. Analysis of the phenotype of cells expressing dominant-negative Rho or RhoC indicates that RhoC is important in tumour cell invasion. The genomic approach allows us to identify families of genes involved in a process, not just single genes, and can indicate which molecular and cellular events might be important in complex biological processes such as metastasis.     

Basal body movements as a mechanism for mitochondrial genome segregation in the trypanosome cell cycle

The mitochondrial genome of Trypanosoma brucei is organized in the form of a complex catenated network of circular DNA molecules. This mass of DNA, known as the kinetoplast, is present at a unique site in the single mitochondrion, and is replicated in a discrete, periodic S phase of the cell cycle. The single-copy nature of the kinetoplast suggests that there is a mechanism ensuring segregation fidelity of replicated copies to each daughter cell. Historically, speculation regarding the nature of this mechanism has often attributed significance to the close association between the kinetoplast and the flagellum basal body. We provide here direct evidence that this mitochondrial DNA complex is indeed linked to the basal body, and segregation of the kinetoplast DNA is dependent on a microtubule-mediated separation of the new and old flagellar basal bodies during the cell cycle. This unique system may represent the remnants of an evolutionarily archaic mechanism for genome segregation.     

异构小蜂窝网中D2D模式选择的联合决策方法

为了在更加贴近实际的环境下研究D2D用户的工作模式选择,提出一种新的异构小蜂窝网络中的D2D模式选择方法。该方法通过分析D2D链路的传输功率和信道开销,给出了D2D链路的传输代价函数表达式,以D2D链路传输代价为标准并使用联合决策算法对D2D链路的模式选择进行控制,使得系统D2D链路的传输总代价达到最小。理论分析和仿真结果表明,所提出的方法能够有效的减小系统传输功耗。     

A role for clonal inactivation in T cell tolerance to Mls-1a

Clonal deletion plays a major part in the maintenance of natural self-tolerance in both normal and transgenic mice. Self antigens that are expressed in the thymus result in the physical elimination of autoreactive thymocytes at a particular stage in their development. For example, the majority V beta 6- and V beta 8.1-bearing T cells that recognize the minor lymphocyte-stimulating antigen, Mls-1a (ref. 10) , are clonally deleted in the thymuses of normal mice and transgenic mice expressing Mls-1a (refs 2, 3, 9). In contrast, a very different mechanism of tolerance involving the functional inactivation, but not elimination, of autoreactive cells, termed clonal inactivation or clonal anergy, has been implicated in some experimentally manipulated systems of tolerance. To test further the mechanisms involved in self-tolerance, we have generated transgenic mice expressing a V beta 8.1 beta chain on greater than 95% of peripheral T cells and have tested tolerance to Mls-1a in these mice. Surprisingly, a significant fraction of the CD4+ peripheral cells that survived deletion were non-responsive in vitro to any stimulus tested. Naturally occurring tolerance to a self antigen expressed in the thymus can thus be mediated by clonal anergy, as well as by clonal deletion.     

大学新生角色转换过程中负性心理调整探析

及时调整大学新生角色转换过程中产生的负性心理,对促进大学新生心理健康和实现角色顺利转换具有重要意义。大学新生负性心理调整是一个抑制、消除负性心理,以及产生、强化正性心理的过程。这一过程既受到新生内部因素的影响,又受到新生外部因素如教育者、校园文化等因素的影响。因此,在新生负性心理调整过程中,要充分发挥新生内部因素和外部因素的综合作用,以期收到好的效果。     

Specific role of mitochondrial electron transport in blood-stage Plasmodium falciparum

The origin of all mitochondria can be traced to the symbiotic arrangement that resulted in the emergence of eukaryotes in a world that was exclusively populated by prokaryotes. This arrangement, however, has been in continuous genetic flux: the varying degrees of gene loss and transfer from the mitochondrial genome in different eukaryotic lineages seem to signify an ongoing 'conflict' between the host and the symbiont. Eukaryotic parasites belonging to the phylum Apicomplexa provide an excellent example to support this view. These organisms contain the smallest mitochondrial genomes known, with an organization that differs among various genera; one genus, Cryptosporidium, seems to have lost the entire mitochondrial genome. Here we show that erythrocytic stages of the human malaria parasite Plasmodium falciparum seem to maintain an active mitochondrial electron transport chain to serve just one metabolic function: regeneration of ubiquinone required as the electron acceptor for dihydroorotate dehydrogenase, an essential enzyme for pyrimidine biosynthesis. Transgenic P. falciparum parasites expressing Saccharomyces cerevisiae dihydroorotate dehydrogenase, which does not require ubiquinone as an electron acceptor, were completely resistant to inhibitors of mitochondrial electron transport. Maintenance of mitochondrial membrane potential, however, was essential in these parasites, as indicated by their hypersensitivity to proguanil, a drug that collapsed the membrane potential in the presence of electron transport inhibitors. Thus, acquisition of just one enzyme can render mitochondrial electron transport nonessential in erythrocytic stages of P. falciparum.