Acinus is a caspase-3-activated protein required for apoptotic chromatin condensation.

Apoptosis is defined by several unique morphological nuclear changes, such as chromatin condensation and nuclear fragmentation. These changes are triggered by the activation of a family of cysteine proteases called caspases, and caspase-activated DNase (CAD/DFF40) and lamin protease (caspase-6) have been implicated in some of these changes. CAD/DFF40 induces chromatin condensation in purified nuclei, but distinct caspase-activated factor(s) may be responsible for chromatin condensation. Here we use an in vitro system to identify a new nuclear factor, designated Acinus, which induces apoptotic chromatin condensation after cleavage by caspase-3 without inducing DNA fragmentation. Immunodepletion experiments showed that Acinus is essential for apoptotic chromatin condensation in vitro, and an antisense study revealed that Acinus is also important in the induction of apoptotic chromatin condensation in cells.     

Apoptosis in suspension culture of tobacco cells induced by heat shock

The induction of apoptosis in suspension culture of tobacco cells by heat shock is reported for the first time. Heat treatment (48℃ for 4 h) of tobacco cells led to the appearance of typical hallmarks of apoptosis. It was demonstrated by DNA laddering analysis that the cells treated with heat shock at 48℃ for 4 h had a serious degradation of nuclear DNA into multi-nu-cleosomal sizes, suggesting that heat shock activated endogenous nuclease which led to DNA cleavage at the linkage sites between the nucleosomes, but ladders were very faint for DNA from 2 and 9 h heat-treated cells. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) detection also showed that most of these treated cells (48℃ for 4 h) displayed positive reactions, indicating a serious DNA 3'-OH cleavage in their nuclei. Moreover, some other cytological changes in apoptotic cells, such as cell shrinkage, chromatin aggregation, nucleus collapse, have also been observed by 4', 6'-diamidino-2-phenyl-indole (DAPI) staining.     

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.     

Dynamic changes of apoptosis in duck embryo fibroblasts induced by new type Gosling viral enteritis virus

The monolayer duck embryo fibroblast （DEF） cells were experimentally infected with new type Gosling viral enteritis virus （NGVEV） and the dynamic changes of apoptosis were detected at different time points after NGVEV infection by transmission electron microscopy （TEM）, DNA agarose gel electrophoresis and Annexin V-FITC/PI stained fluorescence-activated cell sorter （FACS）. The result shows that NGVEV can induce infected cells undergoing apoptosis and changing regularly. A series of characteristic apoptotic morphological changes including shrinkage of the cells, chromatin condensation and margination, as well as formation of apoptotic bodies, were observed by TEM. The typical ladder pattern of DNA fragmentation was demonstrated by agarose gel electrophoresis. And using flow cytometry analysis of Annexin V-FITC/PI staining, the dead, viable, apoptotic and necrotic cells could be analyzed quantitatively.     

The changes in telomerase activity and telomere length in HeLa cells undergoing apoptosis induced by sodium butyrate

The changes in telomerase activity and telomere length during apoptosis in HeLa cells as induced by sodium butyrate (SB) have been studied. After a 48 h SB treatment, HeLa cells demonstrated characteristic apoptotic hallmarks including chromatin condensation, formation of apoptotic bodies and DNA Laddering which were caused by the cleavage and degradation of DNA between nucleosomes. There were no significant changes in telomerase activity of apoptotic cells, while the telomere length shortened markedly. In the meanwhile, cells became more susceptible to apoptotic stimuli and telomere became more vulnerable to degradation after telomerase activity was inhibited. All the results suggest that the apoptosis induced by SB is closely related to telomere shortening, while telomerase enhances resistance of HeLa cells to apoptotic stimuli by protecting telomere.     

Dynamic changes of apoptosis in duck embryo fibroblasts induced by new type Gosling viral enteritis virus

The monolayer duck embryo fibroblast (DEF) cells were experimentally infected with new type Gosling viral enteritis virus (NGVEV) and the dynamic changes of apoptosis were detected at different time points after NGVEV infection by using transmission electron microscopy (TEM), DNA agarose gel electrophoresis and Annexin V-FITC/PI stained fluorescence-activated cell sorter (FACS).The result shows that NGVEV can induce infected cells undergo apoptosis and change regularly. A series of characteristic apoptotic morphological changes including shrink of the cells, chromatin condensation and margination, as well as formation of apoptotic bodies were observed by TEM. The typical ladder pattern of DNA fragmentation was demonstrated by agarose gel electrophoresis. And using flow cytometry analysis of Annexin V-FITC/PI staining, the dead, viable, apoptotic and necrotic cells could be analysis quantitatively.     

Apoptosis Induced by High Concentrations of Nicotinamide in Tobacco Suspension Cells

As an inhibitor of poly(ADP-ribose) polymerase (PARP), nicotinamide has a restraining effect on apoptosis at certain low concentrations. In our present study, apoptosis induced by high concentrations of nicotinamide was observed in tobacco suspension cells. When cells were preincubated with 250 mmol/L nicotinamide for 24 h, the hallmarks of apoptosis were detected, including DNA fragments increasing in size by multiples of 180-200 bp，the condensation and peripheral distribution of nuclear chromatin, and a positive reaction to the TUNEL assay. At the same time, the degradation of PARP and the reduction in the potential of the inner membrane of mitochondria appeared in apoptotic cells induced by high concentrations of nicotinamide. This result indicates that apoptosis induced by high concentrations of nicotinamide is associated with caspase-3-1ike activity and with the opening of mitochondrial permeability pores. These results partially support the hypothesis that high concentrations of PARP inhibitor could force cells to enter an apoptotic pathway by delay of DNA repair in replicating cells.     

贝特舒对人角膜上皮细胞凋亡诱导作用的实验研究

为了揭示常用青光眼药物贝特舒(betaxolol)对人角膜上皮(HCEP)细胞的毒性及其作用机理,使用不同质量浓度贝特舒对体外培养HCEP细胞进行了处理,并利用倒置显微镜跟踪观察了细胞的生长和形态,用吖啶橙/溴化乙锭(AO/EB)荧光双染色法检测了质膜的通透性,用琼脂糖凝胶电泳法检测DNA的断片化,用透射电镜检测细胞的超微结构。发现在0.17500~2.80000g/L的质量浓度范围内,贝特舒对HCEP细胞具有显著的毒性,并随着质量浓度的提高和处理时间的延长而逐渐增大,处理24h即可使大部分细胞皱缩死亡;进一步的研究结果发现,质量浓度为0.17500~2.80000g/L的贝特舒能显著提高HCEP细胞的质膜通透性,并使其出现DNA断片化、染色质凝缩和形成凋亡小体等凋亡细胞的结构变化。可见,在贝特舒0.17500~2.80000g/L的质量浓度范围内对HCEP细胞具有显著的毒性,并具有质量浓度和时间依赖性,其毒性作用的发挥是通过诱导细胞凋亡实现的,在眼科临床应用中毒副作用极大。     

Construction of a cell-free system using extracts from apoptotic plant cells

To study the mechanism of plant apoptosis, a cell-free system has been developed using the extracts prepared from tobacco cells that become committed to apoptosis induced by heat shock treatment. Tobacco nuclei have displayed such characteristics of apoptosis as nucleus condensation and formation of apoptotic bodies during incubation in this system. Besides, these morphological changes are accompanied by DNA fragmentation, which is detected by DNA laddering and comet assay. Furthermore, both morphological changes and DNA cleavage are not affected by pepstatin A, leupeptin or EDTA, but are partly inhibited by iodoacetamide. This profile is consistent with Caspases, which are vital to animal apoptosis, suggesting that cysteine-protease like Caspases may be involved in apoptosis in plants.     

利多卡因对人角膜上皮细胞的毒性作用及其机理研究

利用不同浓度利多卡因（ｌｉｄｏｃａｉｎｅ）处理体外培养的人角膜上皮（ＨＣＥＰ）细胞系细胞,利用光镜观察、ＭＴＴ、荧光染色、ＤＮＡ电泳、ＴＵＮＥＬ、流式细胞仪和透射电镜方法研究了利多卡因对 ＨＣＥＰ细胞的毒性作用及其机理。光镜观察和 ＭＴＴ检测结果显示,质量浓度 １２５～１０００ｇ／Ｌ的利多卡因对 ＨＣＥＰ细胞具有显著的毒性作用,并具有浓度和时间依赖性;ＡＯ／ＥＢ荧光双染色结果显示,质量浓度 ０６２５～１００００ｇ／Ｌ的利多卡因可引起 ＨＣＥＰ细胞的质膜通透性显著提高,细胞凋亡率也具有浓度和时间依赖性;ＤＮＡ电泳和 ＴＵＮＥＬ检测结果显示,利多卡因能引起 ＨＣＥＰ细胞发生 ＤＮＡ断片化;ＴＥＭ观察结果显示,利多卡因能引起 ＨＣＥＰ细胞的超微结构出现了凋亡细胞的形态结构特征,如胞质空泡化、染色质浓缩、线粒体膨胀且嵴的结构紊乱、出现凋亡小体等;ＡｎｎｅｘｉｎＶ／ＰＩ染色的流式细胞仪检测结果显示,利多卡因能引起 ＨＣＥＰ细胞质膜中的磷脂酰丝氨酸（ＰＳ）发生外翻变化;ＥＬＩＳＡ检测结果显示,利多卡因还能引起 ＨＣＥＰ细胞中胱冬肽酶3、8、9、10表达量的增加,表明利多卡因确能引起 ＨＣＥＰ细胞发生细胞凋亡,而不是细胞坏死。由此可见,利多卡因在质量浓度大于 0.625ｇ／Ｌ时对 ＨＣＥＰ细胞具有显著的细胞毒性,并具有浓度和时间依赖性,且其毒性作用的发挥是通过诱导细胞凋亡实现的,在眼科临床应用中具有很大的毒副作用,应谨慎使用。     

Menadione-induced apoptosis and its mechanism in plants

Menadione can induce apoptosis in tobacco protoplasts. Typical characteristics are detected including the condensation of chromatin, the formation of apoptotic bodies and the degradation of genomic DNA into “DNA ladder”. Specific DNase is activated during this process. Ca²⁺ and Mg²⁺ are necessary for its activation, while Zn²⁺ and EDTA (Ethylenediaminetetraacetic acid) can inhibit its activation. The fragmentation of DNA and lamin can be inhibited by DEVD (Ac-Asp -Glu- Val- Asp-aldehyde). The fragmentation of lamin can also be inhibited by PMSF (Phenylmethylsulfonyl fluoride) and CH (Cyclohexinide). These results show that activation of specific DNase and proteases is involved in menadion-induced apoptosis in plants.     

荔枝雌花发育过程中花药败育的超微结构观察

应用扫描电镜、透射电镜和DAPI荧光染色方法对荔枝雌花发育过程中花药败育进行了研究,结果发现:败育的花药在减数分裂之前其药壁细胞即开始解体,减数分裂后,药壁组织发生细胞程序性死亡,导致对营养物质竞争力的减弱;发生凋亡的细胞,细胞核染色质固缩,其电子密度高于正常,核仁消失,染色质趋边化的双层核膜降解,有的核变形而凹陷,核膜破裂,在核膜破裂处可见到由核物质组成的凋亡小体.正在凋亡的细胞中,几乎都可观察到细胞核周围有许多线粒体,一些线粒体变形拉长,嵴肿胀,有的线粒体发生缢缩,线粒体膜降解而变得模糊,有的已破裂.表明线粒体与细胞凋亡可能有直接的关系.     

Bcl-2和Caspase-3在HT诱导的HL-60细胞凋亡中的作用

利用常规的HL－60细胞和转染了 bcl－2基因的HL－60/Bcl-2细胞及转染了空载体的对照HL－60／neo细胞为模型,研究Caspase-3及过表达的Bcl-2对凋亡过程中的各种生化,形态学特征的影响,探讨了Caspase-3及Bcl-2在三尖杉酯碱（HT）诱导的HL－60细胞凋亡中的作用,研究表明,Caspase-3的湃化在HT诱导的HL－60细胞凋亡中处于关键地位,导致了质膜出泡,PS翻转,染色质凝集,DNA断裂及凋亡下游的发生,而Bcl-2则通过抑制aspase-3的活化抑制了凋亡的发生,同时还证明,在HT诱导的HL－60细胞凋亡中,通常被认为是凋亡早期特征的磷脂酰丝氨酸PS翻转并不是一个早期特征,它至少晚于Caspase-3的活化,甚至不早于质膜出泡及染色质凝集。     

Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis.

The protein Bid is a participant in the pathway that leads to cell death (apoptosis), mediating the release of cytochrome c from mitochondria in response to signals from 'death' receptors known as TNFR1/Fas on the cell surface. It is a member of the proapoptotic Bcd-2 family and is activated as a result of its cleavage by caspase 8, one of a family of proteolytic cell-death proteins. To investigate the role of Bid in vivo, we have generated mice deficient for Bid. We find that when these mice are injected with an antibody directed against Fas, they nearly all survive, whereas wild-type mice die from hepatocellular apoptosis and haemorrhagic necrosis. About half of the Bid-deficient animals had no apparent liver injury and showed no evidence of activation of the effector caspases 3 and 7, although the initiator caspase 8 had been activated. Other Bid-deficient mice survived with only moderate damage: all three caspases (8 and 37) were activated but their cell nuclei were intact and no mitochondrial cytochrome c was released. We also investigated the effects of Bid deficiency in cultured cells treated with anti-Fas antibody (hepatocytes and thymocytes) or with TNFalpha. (fibroblasts). In these Bid-/- cells, mitochondrial dysfunction was delayed, cytochrome c was not released, effector caspase activity was reduced and the cleavage of apoptosis substrates was altered. This loss-of-function model indicates that Bid is a critical substrate in vivo for signalling by death-receptor agonists, which mediates a mitochondrial amplification loop that is essential for the apoptosis of selected cells.     

Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis.

Phagocyte recognition of cells that have undergone apoptosis (programmed cell death) is an event of broad biological significance. Characterized by endogenous endonuclease activation, which results in chromatin fragmentation and nuclear condensation, apoptosis leads to swift ingestion of intact but 'senescent' or 'unwanted' cells by phagocytes in processes as diverse as the physiological involution of organs, the remodelling of embryonic tissues, and metamorphosis. The cell-surface mechanisms by which macrophages recognize apoptotic cells as 'senescent-self' have remained obscure. Here we report that macrophage recognition of apoptotic cells (both neutrophils and lymphocytes) is mediated by the vitronectin receptor, a heterodimer belonging to the beta 3 or cytoadhesin family of the integrins. Previously, the functions of the vitronectin receptor were believed to be limited to cell anchorage, but our findings indicate that the receptor has a novel and direct role in self-senescent-self intercellular recognition leading to macrophage phagocytosis of cells undergoing apoptosis.     

Apoptotic cell death induced by c-myc is inhibited by bcl-2.

Apoptosis is a form of physiological cell death, characterized by chromatin condensation, cytoplasmic blebbing and DNA fragmentation, which often depends on RNA and protein synthesis by the dying cell. The c-myc proto-oncogene, usually implicated in cell transformation, differentiation and cell-cycle progression also has a central role in some forms of apoptosis. These opposing roles of myc in cell growth and death require that other gene products dictate the outcome of c-Myc expression on a cell. A candidate for such a modifying gene is bcl-2, whose product prolongs cell survival and blocks apoptosis in some systems. Here we demonstrate that Bcl-2 prevents apoptotic death induced by c-Myc, provide a mechanism whereby cells can express c-Myc without undergoing apoptosis, and give a possible explanation for the ability of Bcl-2 to synergize with c-Myc in cell transformation.     

反义HSP90诱导HeLa细胞凋亡的研究

目的 探讨反义ＨＳＰ９０降低ＨｅＬａ细胞恶性度的机制。方法 经荧光染色观察细胞形态,ＤＮＡ Ｌａｄｄｅｒ及流式细胞仪检测凋亡细胞。结果 转染有反义ＨＳＰ９０的ＨｅＬａ细胞经荧光染色细胞呈不均一亮蓝色,荧光染色观察可见细胞变小,染色质浓缩并产生凋亡小体,ＤＮＡ Ｌａｄｄｅｒ可见明显的梯形条带,流式细胞仪检测有凋亡的细胞。结论 反义ＨＳＰ９０可诱导细胞凋亡。     

Apoptosis of carrot nuclei inin vitro system induced by cytochrome c

The apoptosis of carrot nuclei induced in cytosol of carrot cells by cytochrome c is reported. This kind of cell-free apoptosis system of plant, CS-100, is an efficient In vitro inducing system. Several typical characteristics of apoptosis including chromatin condensation, margina-tion and apoptotic bodies could be detected, and the degradation of genome in carrot nuclei into nucleosomal fragments could be detected. TUNEL assay shows the specific breakage of 3'-OH ends of DNA strand. The results indicate that CS-100 is an efficient and easily-operated in vitro system to study the apoptotic mechanism in plant and the apoptosis in plant cells may share similar pathways to apoptosis in animal cells.     

Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome

Apoptosis is an evolutionarily conserved cell suicide process executed by cysteine proteases (caspases) and regulated by the opposing factions of the Bcl-2 protein family. Mammalian caspase-9 and its activator Apaf-1 were thought to be essential, because mice lacking either of them display neuronal hyperplasia and their lymphocytes and fibroblasts seem resistant to certain apoptotic stimuli. Because Apaf-1 requires cytochrome c to activate caspase-9, and Bcl-2 prevents mitochondrial cytochrome c release, Bcl-2 is widely believed to inhibit apoptosis by safeguarding mitochondrial membrane integrity. Our results suggest a different, broader role, because Bcl-2 overexpression increased lymphocyte numbers in mice and inhibited many apoptotic stimuli, but the absence of Apaf-1 or caspase-9 did not. Caspase activity was still discernible in cells lacking Apaf-1 or caspase-9, and a potent caspase antagonist both inhibited apoptosis and retarded cytochrome c release. We conclude that Bcl-2 regulates a caspase activation programme independently of the cytochrome c/Apaf-1/caspase-9 'apoptosome', which seems to amplify rather than initiate the caspase cascade.     

Release of chromatin protein HMGB1 by necrotic cells triggers inflammation

High mobility group 1 (HMGB1) protein is both a nuclear factor and a secreted protein. In the cell nucleus it acts as an architectural chromatin-binding factor that bends DNA and promotes protein assembly on specific DNA targets. Outside the cell, it binds with high affinity to RAGE (the receptor for advanced glycation end products) and is a potent mediator of inflammation. HMGB1 is secreted by activated monocytes and macrophages, and is passively released by necrotic or damaged cells. Here we report that Hmgb1(-/-) necrotic cells have a greatly reduced ability to promote inflammation, which proves that the release of HMGB1 can signal the demise of a cell to its neighbours. Apoptotic cells do not release HMGB1 even after undergoing secondary necrosis and partial autolysis, and thus fail to promote inflammation even if not cleared promptly by phagocytic cells. In apoptotic cells, HMGB1 is bound firmly to chromatin because of generalized underacetylation of histone and is released in the extracellular medium (promoting inflammation) if chromatin deacetylation is prevented. Thus, cells undergoing apoptosis are programmed to withhold the signal that is broadcast by cells that have been damaged or killed by trauma.