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1.
Jurkat cells express Fas, and rapidly undergo apoptosis in response to Fas ligand or an agonistic anti-Fas antibody. This apoptotic pathway is mediated by a cascade of caspases. In this report, we show that Fas activation induced the processing of caspase 8 in Jurkat cells with a time frame similar to the activation of caspase 3 and the proteolysis of nuclear proteins. Jurkat cell transformants that overexpress Bcl-2 were partially but not completely resistant to the Fas-induced apoptosis. Little processing of caspase 8 was observed upon Fas activation in these transformants. Furthermore, although caspase 8 was recruited to Fas upon Fas activation in the parental Jurkat cells, the recruitment of caspase 8 to Fas was inhibited in the transformants overexpressing Bcl-2. These results suggest that Bcl-2 inhibits Fas-induced apoptosis by preventing the formation of the death-inducing signaling complex that is composed of Fas, FADD/MORT1, and caspase 8.  相似文献   

2.
Apoptosis requires the activation of caspases (formerly interleukin 1beta-converting enzyme-like proteases), in particular those related to the caspase-3/7/6 subfamily. Recent data, however, revealed that, although caspase-specific inhibitors delay apoptosis, they are often incapable of preventing it. To obtain evidence for caspase-independent steps of apoptosis, we artificially created a high amount of short-lived or aberrant proteins by blocking the ubiquitin degradation pathway. A temperature-sensitive defect in the ubiquitin-activating enzyme E1 induced apoptosis independent of the activation of caspase-3 and -6 and the cleavage of their respective substrates poly(ADP-ribose) polymerase and lamin A. In addition, neither the caspase 3/7-specific inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone nor the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone were capable of blocking this type of cell death. By contrast, Bcl-2 overexpression effectively protected cells from apoptosis induced by a defect in the E1 enzyme at the nonpermissive temperature. Bcl-2 acted downstream of the accumulation of short-lived or aberrant proteins because it did not prevent the overexpression of the short-lived proteins p53, p27(kip1), and cyclins D1 and B1 under conditions of decreased ubiquitination. These results suggest the existence of short-lived proteins that may serve the role of caspase-independent effectors of apoptosis and attractive targets of the death-protective action of Bcl-2.  相似文献   

3.
4.
We have identified and characterized Mtd, a novel regulator of apoptosis. Sequence analysis revealed that Mtd is a member of the Bcl-2 family of proteins containing conserved BH1, BH2, BH3, and BH4 regions and a carboxyl-terminal hydrophobic domain. In adult tissues, Mtd mRNA was predominantly detected in the brain, liver, and lymphoid tissues, while in the embryo Mtd mRNA was detected in the liver, thymus, lung, and intestinal epithelium. Expression of Mtd promoted the death of primary sensory neurons, 293T cells and HeLa cells, indicating that Mtd is a proapoptotic protein. Unlike all other known death agonists of the Bcl-2 family, Mtd did not bind significantly to the survival-promoting proteins Bcl-2 or Bcl-XL. Furthermore, apoptosis induced by Mtd was not inhibited by Bcl-2 or Bcl-XL. A Mtd mutant with glutamine substitutions of highly conserved amino acids in the BH3 domain retained its ability to promote apoptosis, further indicating that Mtd does not promote apoptosis by heterodimerizing with Bcl-2 or Bcl-XL. Mtd-induced apoptosis was not blocked by broad range synthetic caspase inhibitors z-VAD-fmk or a viral protein CrmA. Mtd is the first example of a naturally occurring Bcl-2 family member that can activate apoptosis independently of heterodimerization with survival-promoting Bcl-2 and Bcl-XL.  相似文献   

5.
The aim of this paper is to present a conceptual and practical framework of the case-control design in medical research. To illustrate this method, practical examples directed to clinicians and other health professionals interested in medical research are presented. The case-control method is very versatile and allows for multiple applications. Guidelines for the selection of cases and controls, and some considerations on sample size are presented. In the statistical analysis we use concrete examples of how to estimate odds ratios, confidence intervals, and methods to control for potential confounders, from stratified analysis to logistic regression.  相似文献   

6.
Bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced apoptosis. This might be achieved by preventing virus-induced activation of caspase-3, an IL-1beta-converting enzyme (ICE)-like cysteine protease that has been implicated in the death effector phase of apoptosis. Contrary to this model, we show that three cell types highly overexpressing functional Bcl-2 displayed caspase-3 activation and underwent apoptosis in response to infection with alphaviruses Semliki Forest and Sindbis as efficiently as vector control counterparts. In all three cell types, overexpressed 26 kDa Bcl-2 was cleaved into a 23 kDa protein. Antibody epitope mapping revealed that cleavage occurred at one or two target sites for caspases within the amino acid region YEWD31 (downward arrow) AGD34 (downward arrow) A, removing the N-terminal BH4 region known to be essential for the death-protective activity of Bcl-2. Preincubation of cells with the caspase inhibitor Z-VAD prevented Bcl-2 cleavage and partially restored the protective activity of Bcl-2 against virus-induced apoptosis. Moreover, a murine Bcl-2 mutant having Asp31, Asp34 and Asp36 substituted by Glu was resistant to proteolytic cleavage and abrogated apoptosis following virus infection. These findings indicate that alphaviruses can trigger a caspase-mediated inactivation of Bcl-2 in order to evade the death protection imposed by this survival factor.  相似文献   

7.
Ubiquitination of protein kinase C-alpha and degradation by the proteasome   总被引:1,自引:0,他引:1  
Bryostatins and phorbol esters acutely activate and subsequently down-regulate protein kinase C (PKC) by inducing its proteolysis via an unknown pathway. Here we show that treatment of renal epithelial cells with bryostatin 1 (Bryo) produced novel PKC-alpha species, which were larger than the native protein (80 kDa). The >80 kDa PKC-alpha species contained Ubi as indicated by immunostaining and accumulated in the presence of lactacystin, a selective inhibitor of proteolysis by the proteasome. In vitro experiments with 125I-ubiquitin and membranes from Bryo-treated cells showed that PKC-alpha became ubiquitinated by a reaction that depended on ATP and a cytosolic fraction. Lactacystin or a peptidyl aldehyde, Bz-Gly-Leu-Ala-leucinal, which inhibits certain proteinase activities of the proteasome, inhibited Bryo-evoked disappearance of PKC-alpha protein from the cells. Lacta preserved Bryo-induced 32P-labeled PKC-alpha indicating that the proteasome inhibitor spared activated enzyme from down-regulation in vivo. These findings show that Bryo induces the degradation of PKC-alpha by the ubiquitin-proteasome complex.  相似文献   

8.
Cytosolic degradation of T-cell receptor alpha chains by the proteasome   总被引:1,自引:0,他引:1  
The T-cell antigen receptor (TCR) is an hetero-oligomeric membrane complex composed of at least seven transmembrane polypeptide chains that has served as a model for the assembly and degradation of integral membrane proteins in the endoplasmic reticulum (ER). Unassembled TCRalpha chains fail to mature to the Golgi apparatus and are rapidly degraded by a non-lysosomal "ER degradation" pathway that has been proposed to be autonomous to the ER. In these studies we show that the degradation of core-glycosylated TCRalpha is blocked by N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) and lactacystin, implicating the proteasome in ER degradation. Either acute or chronic treatment of TCRalpha-transfected cells with proteasome inhibitors cause the core-glycosylated TCRalpha chains to progressively shift to an approximately 28-kDa form that lacks N-linked oligosaccharides and the N-terminal signal peptide. The susceptibility of this 28-kDa species to extravesicular protease indicates that it is not protected by the ER membrane and, hence, cytoplasmic. These data suggest a model in which TCRalpha chains that are translocated across the membrane, core-glycosylated, but fail to assemble are dislocated back to the cytoplasm for degradation by cytoplasmic proteasomes. Our data also suggest that covalent modification of TCRalpha with ubiquitin is not required for its degradation.  相似文献   

9.
It has been suggested that overexpression of the Bcl-2 oncoprotein in human cancer cells contributes to their resistance to apoptosis induced by chemotherapy. We report here that a novel dipeptidyl proteasome inhibitor, CEP1612, at low concentrations rapidly induces apoptosis in human Jurkat T cells overexpressing Bcl-2 and also in all human prostate, breast, tongue and brain tumor cell lines we have tested to date, without exception. In contrast, etoposide, a standard anticancer drug, fails to kill these cells when employed under the same conditions. The apoptosis-inducing abilities of CEP1612 and its analogous compounds match precisely their order for inhibition of the proteasome chymotrypsin-like activity. CEP1612-induced apoptosis is p53-independent, inhibitable by a tetrapeptide caspase inhibitor, and associated with accumulation of the cyclin-dependent kinase inhibitors p21 and p27. Furthermore, CEP1612 selectively accumulates p27 and induces apoptosis in simian virus 40-transformed, but not the parental normal, human fibroblasts. Proteasome inhibitors such as those investigated herein might therefore have potential use as novel anticancer drugs.  相似文献   

10.
11.
We have introduced the human bcl-2 gene under the control of the human metallothionein MTIIA promoter into the rat kangaroo PtK2 cell line. Two independent clones were obtained in which the levels of Bcl-2 protein expression can be controlled by the addition of metals in the culture medium. These cell lines were employed to investigate the effects of this protein in UV-induced apoptosis. Overexpression of Bcl-2 in PtK2 cells resulted in a delay in the appearance of apoptosis markers, such as chromatin condensation and internucleosomal DNA fragmentation. However, colony survival after UV was not affected, suggesting that Bcl-2 did not impose a definitive block for cell death. The elimination of cyclobutane pyrimidine dimers through photoreactivation 24 h after irradiation in cells overexpressing Bcl-2 did not affect apoptosis. This indicates that irreversible events in the signaling pathway of apoptosis occur in the period between irradiation and photoreactivation even in the presence of high levels of Bcl-2 protein can delay the onset of UV-induced apoptosis in these marsupial cells, early events triggered by the pyrimidine dimers, upstream from the Bcl-2 action, lead the cell to a state committed to die.  相似文献   

12.
Elucidation of the mechanism and regulation of cell destruction in apoptosis requires knowledge of genome degradation. The cell genome, which encodes the fundamental cell programmes, is most likely to be the main sensitive target in cell apoptosis. Genome breakdown may be achieved by the generation and transduction of apoptogenic signal information to the specific chromatin regions of the nucleus, thus inactivating the basic cell programmes and inducing the endogenous pattern of chromatin degradation, which is determined by the genome organization of the eukaryotic nuclei. Detachment of chromatin from the nuclear matrix attachment regions may be one of the possible mechanisms of switching off the genome function and triggering the multi-step process of endogenous chromatin degradation, thus leading to cell death of terminal differentiation or stress-induced apoptosis.  相似文献   

13.
Mcl-1, a protein increased early in the differentiation of human myeloblastic ML-1 cells, has sequence similarity to Bcl-2. In the present study, we determined whether Mcl-1 has functional similarity to Bcl-2 by testing its ability to inhibit apoptosis induced by c-Myc overexpression. This was carried out using Chinese hamster ovary 5AHSmyc cells which contain the human c-myc proto-oncogene under the control of a heat shock promoter. Heat treatment induces c-Myc overexpression and thus apoptosis as determined by internucleosomal DNA fragmentation. We transfected 5AHSmyc cells with mcl-1 and found that clones expressing the introduced Mcl-1 protein exhibited reduced DNA fragmentation. Mcl-1 was also capable of delaying the onset of cell death as judged by loss of membrane integrity, although it could not provide complete protection from c-Myc overexpression. Thus, Mcl-1 has functional homology to Bcl-2 in that Mcl-1 can enhance cell viability under conditions that otherwise cause apoptosis.  相似文献   

14.
Two pathways have been implicated in the induction of apoptosis by cytotoxic T cells: the granule exocytosis pathway and a pathway using CD95 (Fas/APO-1). To test whether apoptosis induced by either of these pathways could be blocked by Bcl-2, we exposed bcl-2-transfected cells to CTL derived from normal, perforin-deficient, or CD95 ligand mutant (gld) mice. Although the levels of Bcl-2 expression achieved were able to protect FDC-P1 and Yac-1 transfectants from a variety of apoptotic stimuli, the cells were not protected from cytolysis mediated by CTL from any of these sources, by NK cells, or granules isolated from CTL. However, Bcl-2 expression significantly inhibited apoptosis induced by purified granzyme B and perforin. These results suggest that while Bcl-2 is capable of inhibiting the apoptotic pathway utilized by perforin and granzyme B, other granule components can bypass this block. We conclude that CTL harbor potent killing mechanism(s) in addition to those provided by CD95 ligand or perforin and granzyme B that cannot be overcome by Bcl-2.  相似文献   

15.
We report that exposure of aconitase to moderate concentrations of peroxynitrite, 3-morpholinosydnonimine (SIN-1; a superoxide- and nitric oxide-liberating substance), or hydrogen peroxide, inhibits the enzyme and enhances susceptibility to proteolytic digestion by the isolated 20 S proteasome. Exposure to more severe levels of oxidative stress, from these same agents, causes further inhibition of the enzymatic activity of aconitase but actually decreases its proteolytic breakdown by proteasome. It should be noted that the superoxide and nitric oxide liberated by SIN-1 decomposition react to form a steady flux of peroxynitrite. S-Nitroso-N-acetylpenicillamine, a compound that liberates nitric oxide alone, causes only a small loss of aconitase activity (25% or less) and has no effect on the proteolytic susceptibility of the enzyme. Proteasome also seems to be the main protease in cell lysates that can degrade aconitase after it has been oxidatively modified by exposure to peroxynitrite, SIN-1, or hydrogen peroxide. Using cell lysates isolated from K562 cells treated for several days with an antisense oligodeoxynucleotide to the initiation codon region of the C2 subunit of proteasome (a treatment which diminishes proteasome activity by 50-60%), the enhanced degradation of moderately damaged aconitase was essentially abolished. Other model proteins as well as complex mixtures of proteins, such as cell lysates, also exhibit enhanced proteolytic susceptibility after moderate SIN-1 treatment. Therefore we conclude that peroxynitrite reacts readily with proteins and that mild modification by peroxynitrite results in selective recognition and degradation by proteasome.  相似文献   

16.
The aim of the present study was to characterize human CYP2E1 turnover and examine the possible role of the proteasome proteolytic pathway in the rapid degradation of CYP2E1 in a transfected HepG2 cell line expressing human CYP2E1. Microsomes isolated from MVh2E1-9 cells catalyzed a slow degradation of the expressed CYP2E1, which was prevented by the addition of 4-methylpyrazole, a ligand which stabilizes CYP2E1. The addition of the cytosolic fraction of the HepG2 cells to the microsomes produced rapid degradation of CYP2E1. This rapid degradation required MgATP and was completely prevented by 4-methylpyrazole. Pulse-chase experiments after labeling CYP2E1 with [35S]-methionine and immunoprecipitation with anti-human CYP2E1 IgG indicated a biphasic turnover of CYP2E1 with half-lives of 2.5 and 6 hours. The addition of Czb-Ile-Glu(OtBu)-Ala-Leucinal(PSI) as a cell penetrating proteasome inhibitor, at concentrations ranging from 5 to 80 microM resulted in protection against the degradation of CYP2E1. PSI also increased the steady state accumulation of CYP2E1, consistent with its inhibition of CYP2E1 turnover. These results suggest that the proteasome complex plays a major role in the degradation of human CYP2E1 in the transfected HepG2 cells.  相似文献   

17.
bcl-2-/-mice complete embryonic development, but display growth retardation and early mortality postnatally. Hematopoiesis including lymphocyte differentiation is initially normal, but thymus and spleen undergo massive apoptotic involution. Thymocytes require an apoptotic signal to manifest accelerated cell death. Renal failure results from severe polycystic kidney disease characterized by dilated proximal and distal tubular segments and hyperproliferation of epithelium and interstitium. bcl-2-/-mice turn gray with the second hair follicle cycle, implicating a defect in redox-regulated melanin synthesis. The abnormalities in these loss of function mice argue that Bcl-2 is a death repressor molecule functioning in an antioxidant pathway.  相似文献   

18.
Bcl-2-family proteins: the role of the BH3 domain in apoptosis   总被引:1,自引:0,他引:1  
Bcl-2-related proteins have come to occupy a prominent position in the realm of programmed cell death. Members of this fast-growing family are highly related in one or more specific regions, commonly referred to as Bcl-2 homology (BH) domains. BH domains contribute at multiple levels to the function of these proteins in cell death and survival. Particularly intriguing is the emergence of the BH3 domain as a potent 'death domain' and of a growing subclass of pro-apoptotic proteins with no similarity to Bcl-2 beyond their BH3 homology. Here, the authors classify proteins of the Bcl-2 family on the basis of function and domain organization, discuss the importance of the BH3 domain in protein-protein interactions and in cell death and provide possible explanations for the perceived redundancy in the expression of this subclass of death promoters.  相似文献   

19.
Voltage-sensitive dyes NK 2761 and RH 155 were employed (in conjunction with a 12 x 12 photodiode array) to study membrane potential transients in optic lobe neuropils in the eye stalk of the crayfish Procambarus clarkii. By this means we investigated a pathway linking deutocerebral projection neurons, via hemiellipsoid body local interneurons, to an unidentified target (most likely neurons processing visual information) in the medulla terminalis. Rapid (10- to 20-ms duration), transient changes in absorption with the characteristics of action potentials were recorded from the optic nerve and the region occupied by deutocerebral projection neurons after stimulation of the olfactory globular tract in the optic nerve and were blocked by 1 microM tetrodotoxin. Action potentials appeared to propagate to the glomerular layer of the hemiellipsoid body where synaptic responses were recorded from a restricted region of the hemiellipsoid body occupied by dendrites of hemiellipsoid body neurons. Action potentials were also recorded from processes of hemiellipsoid body neurons located in the medulla terminalis. Synaptic responses in the hemiellipsoid body and medulla terminalis were eliminated by addition to the saline of 500 microM Cd2+ or 20 mM Co2+, whereas the action potential attributed to branches of deutocerebral projection neurons in the hemiellipsoid body remained unaffected. Action potentials of hemiellipsoid body neurons in the medulla terminalis evoked postsynaptic potentials (50- to 200-ms duration) with an unidentified target in the medulla terminalis. Transient absorption signals were not detected in either the internal or external medulla nor were they recorded from other parts of the optic lobes in response to electrical stimulation of axons of the deutocerebral projection neurons. Functional maps of optical activity, together with electrophysiological and pharmacological findings, suggest that gamma-aminobutyric acid affects synaptic transmission in glomeruli of the hemiellipsoid body. Synapses of the olfactory pathway located in the medulla terminalis may act as a "filter," modifying visual information processing during olfactory stimulation.  相似文献   

20.
BACKGROUND: Implantable cardioverter-defibrillators have been infrequently used in children as therapy for resuscitated sudden death and syncope due to ventricular arrhythmias unresponsive to antiarrhythmics. METHODS: The medical records of 5 children with implantable cardioverter-defibrillators were retrospectively reviewed. All patients had experienced syncope and 3 (60%) an out-of-hospital cardiac arrest. Underlying pathology included hypertrophic cardiomyopathy in 2, long QT syndrome in 2, and ventricular arrhythmia after remote repair of congenital heart disease in 1. Open thoracotomy with epicardial lead placement and transvenous endocardial approaches were used. RESULTS: There was no early or late mortality in the 5 pediatric patients undergoing implantable cardioverter-defibrillator placement. Postoperative complications occurred more frequently when open thoracotomy was used for placement. At mean follow-up of 34 months, 4 of the 5 (80%) have received shocks. CONCLUSIONS: Implantable cardioverter-defibrillator is a safe and reliable therapy for children with resuscitated sudden death and syncope due to ventricular tachycardia unresponsive to antiarrhythmics. Transvenous lead placement lowers morbidity and hospital length of stay.  相似文献   

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