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1.
Proteins of the Bcl-2 protein family, including pro-apoptotic Bax and anti-apoptotic Bcl-xL, are critical for mitochondrial-mediated apoptosis regulation. Since yeast lacks obvious orthologs of Bcl-2 family members, heterologous expression of these proteins has been used to investigate their molecular and functional aspects. Active Bax is involved in the formation of mitochondrial outer membrane pores, through which cytochrome c (cyt c) is released, triggering a cascade of downstream apoptotic events. However, when in its inactive form, Bax is largely cytosolic or weakly bound to mitochondria. Given the central role of Bax in apoptosis, studies aiming to understand its regulation are of paramount importance towards its exploitation as a therapeutic target. So far, studies taking advantage of heterologous expression of human Bax in yeast to unveil regulation of Bax activation have relied on the use of artificial mutated or mitochondrial tagged Bax for its activation, rather than the wild type Bax (Bax α). Here, we found that cell death could be triggered in yeast cells heterologoulsy expressing Bax α with concentrations of acetic acid that are not lethal to wild type cells. This was associated with Bax mitochondrial translocation and cyt c release, closely resembling the natural Bax function in the cellular context. This regulated cell death process was reverted by co-expression with Bcl-xL, but not with Bcl-xLΔC, and in the absence of Rim11p, the yeast ortholog of mammalian GSK3β. This novel system mimics human Bax α regulation by GSK3β and can therefore be used as a platform to uncover novel Bax regulators and explore its therapeutic modulation. 相似文献
2.
Delftia acidovorans P4a (DSMZ 10474) was grown in mineral medium on acetic acid at pH 8.0 without an additional supply of nutrients like yeast extract or polypeptone. Using acetic acid and γ‐butyrolactone (GBL), copolymers with a 4HB content from 2–90 mol % were detected in batch experiments, depending on the ratio of the both carbon substrates. Due to the different consumption rates of the individual carbon substrates a multitude of different target mole fractions were difficult to produce by fed‐batch fermentation. Therefore, the two‐stage continuous cultivation technique was applied with two fermenters connected in series. At stage 2, the optimum PHA productivity of the bioreactor and a target 4HB content of the polymer could be precisely adjusted by the composition of the two substrates. This cultivation strategy was especially convenient when toxic substrates like acetic acid and GBL were employed. Using mixtures of acetic acid and GBL (3.5–23.5 mol % GBL), copolymers with a target mole fraction of 2.7–19 % 4HB could be produced. The PHA content was in the range of 52–60 %. The dilution rates ( D) of the first and second fermenter were 0.2 h –1 and 0.06 h –1, respectively. 相似文献
3.
KD115 (ol1), an unsaturated fatty acid auxotroph of S. cerevisiae, was grown in a semi-synthetic medium supplemented with 3.3 x 10(-4) M palmitoleic (cis 16:1) or palmitelaidic (trans 16:1) acids. The parent strain S288C was studied as a control. The lipid composition (fatty acids, neutral lipids, and phospholipids), respiratory activity (O2 consumption), and ultrastructure were compared in mutant yeast grown with each unsaturated fatty acid supplement. The fatty acid supplement represented 70-80% of the yeast fatty acids. Yeast grown in trans 16:1 contained more squalene, a higher ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC), and had 10-20% of the respiratory activity compared to the same yeast grown in cis 16:1. The mitochondrial morphology of yeast in each growth supplement was notably different. The use of mixtures of cis and trans 16:1 in different proportions revealed that the PE/PC ratio, the squalene content, the respiratory defect, and the mitochondrial morphology were all similarly dependent on the fraction of trans 16:1 in the mixtures. As little as 10-20% of cis 16:1 in the mixture was sufficient to abrogate the physiological effects of trans 16:1 on each of the parameters noted above. The combined effects of high content of trans unsaturated fatty acid and the altered phospholipid composition seem to account for the decrease in lipid fluidity, the defective structure and function of the mitochondrial membrane. 相似文献
4.
The Bcl‐2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro‐apoptotic proteins. Yet the mechanistic details of the Bax‐induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring‐like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super‐resolution data provide direct evidence in support of large Bax‐delineated pores in the mitochondrial outer membrane as being crucial for Bax‐mediated MOMP in cells. 相似文献
5.
The regulation of both mitochondrial dynamics and apoptosis is key for maintaining the health of a cell. Bcl-2 family proteins, central in apoptosis regulation, also have roles in the maintenance of the mitochondrial network. Here we report that Bax and Bak participate in the regulation of mitochondrial fusion in mouse embryonic fibroblasts, primary mouse neurons and human colon carcinoma cells. To assess how Bcl-2 family members may regulate mitochondrial morphogenesis, we determined the binding of a series of chimeras between Bcl-xL and Bax to the mitofusins, mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2). One chimera (containing helix 5 (H5) of Bax replacing H5 of Bcl-xL (Bcl-xL/Bax H5)) co-immunoprecipitated with Mfn1 and Mfn2 significantly better than either wild-type Bax or Bcl-xL. Expression of Bcl-xL/Bax H5 in cells reduced the mobility of Mfn1 and Mfn2 and colocalized with ectopic Mfn1 and Mfn2, as well as endogenous Mfn2 to a greater extent than wild-type Bax. Ultimately, Bcl-xL/Bax H5 induced substantial mitochondrial fragmentation in healthy cells. Therefore, we propose that Bcl-xL/Bax H5 disturbs mitochondrial morphology by binding and inhibiting Mfn1 and Mfn2 activity, supporting the hypothesis that Bcl-2 family members have the capacity to regulate mitochondrial morphology through binding to the mitofusins in healthy cells. 相似文献
6.
By employing two electron microscopy techniques, postembedding double- and triple immunocytochemical gold-labelling combined with embedment-free electron microscopy (EF-EM), we have detected previously unreported nuclear and cytoplasmic complexes between different proapoptatic proteins in a human cancer cell line COLO 205 stimulated to apoptosis by nimesulide, a specific cyclooxygenase-2 inhibitor. Experiments with the use of double- and triple immunolabelling visualized the colocalization of proapoptotic proteins such as Bax with Bid, Bax with Bid and voltage-dependent anion channel protein (VDAC-1), and Bax with Bid and caspase-8, on organellar membranes and within the nucleus. Application of this technique in combination with EF-EM technique augments our knowledge on the precise identification and relationship of subcellular structures containing Bax, Bid, VDAC-1 and caspase-8. 相似文献
7.
To identify genes related to plant mitochondrial morphology and dynamics, novel mutants with respect to mitochondrial morphology were isolated from an ethyl methane sulphonate (EMS)-mutated population of Arabidopsis thaliana. Mitochondria were visualized by transforming Arabidopsis with a gene for a fusion protein consisting of GFP and a mitochondria-targeting pre-sequence. From 19,000 M2 populations, 17 mutants were isolated by fluorescent microscopic observations. All mitochondria in these mutants were longer and/or larger than wild-type mitochondria. The approximate chromosomal loci of the mutations of seven mutants that grew well were determined. The mitochondrial phenotypes of six of the mutants were recessive but the mitochondrial phenotype of the seventh mutant was dominant. Chromosomal rough mapping of the seven mutants showed that the mutations occurred at four different loci. At least one of these loci was novel, i.e., it was different from loci of other known mitochondrial morphology mutants of Arabidopsis and different from loci of Arabidopsis homologues of yeast genes related to mitochondrial morphology. 相似文献
8.
The objective of this study was to evaluate mitochondrial alterations in a cell-based model of myocardial ischemia/reperfusion (I/R) injury. Using GFP-biosensors and fluorescence deconvolution microscopy, we investigated mitochondrial morphology in relation to Bax and Bid activation in the HL-1 cardiac cell line. Mitochondria underwent extensive fragmentation during ischemia. Bax translocation from cytosol to mitochondria was initiated during ischemia and proceeded during reperfusion. However, Bax translocation was not sufficient to induce cell death or mitochondrial dysfunction. Bid processing was caspase-8 dependent, and Bid translocation to mitochondria occurred after Bax translocation and clustering, and minutes before cell death. Clustering of Bax into distinct regions on mitochondria could be prevented by CsA, an inhibitor of the mitochondrial permeability transition pore, and also by SB203580, an inhibitor of p38 MAPK. Surprisingly, mitochondrial fragmentation which occurred during ischemia and before Bax translocation could be reversed by the addition of the p38 inhibitor SB203580 at reperfusion. Taken together, these results implicate p38 MAPK in the mitochondrial remodeling response to I/R that facilitates Bax recruitment to mitochondria. 相似文献
9.
Mitochondria exist as a network of interconnected organelles undergoing constant fission and fusion. Current approaches to study mitochondrial morphology are limited by low data sampling coupled with manual identification and classification of complex morphological phenotypes. Here we propose an integrated mechanistic and data-driven modeling approach to analyze heterogeneous, quantified datasets and infer relations between mitochondrial morphology and apoptotic events. We initially performed high-content, multi-parametric measurements of mitochondrial morphological, apoptotic, and energetic states by high-resolution imaging of human breast carcinoma MCF-7 cells. Subsequently, decision tree-based analysis was used to automatically classify networked, fragmented, and swollen mitochondrial subpopulations, at the single-cell level and within cell populations. Our results revealed subtle but significant differences in morphology class distributions in response to various apoptotic stimuli. Furthermore, key mitochondrial functional parameters including mitochondrial membrane potential and Bax activation, were measured under matched conditions. Data-driven fuzzy logic modeling was used to explore the non-linear relationships between mitochondrial morphology and apoptotic signaling, combining morphological and functional data as a single model. Modeling results are in accordance with previous studies, where Bax regulates mitochondrial fragmentation, and mitochondrial morphology influences mitochondrial membrane potential. In summary, we established and validated a platform for mitochondrial morphological and functional analysis that can be readily extended with additional datasets. We further discuss the benefits of a flexible systematic approach for elucidating specific and general relationships between mitochondrial morphology and apoptosis. 相似文献
10.
The initiating events that lead to the induction of apoptosis mediated by the chemopreventative agent beta-phenyethyl isothiocyanate (PEITC) have yet to be elucidated. In the present investigation, we examined the effects of PEITC on mitochondrial function and apoptotic signaling in hepatoma HepG2 cells and isolated rat hepatocyte mitochondria. PEITC induced a conformational change in Bax leading to its translocation to mitochondria in HepG2 cells. Bax accumulation was associated with a rapid loss of mitochondrial membrane potential (Deltapsim), impaired respiratory chain enzymatic activity, release of mitochondrial cytochrome c and the activation of caspase-dependent cell death. Caspase inhibition did not prevent Bax translocation, the release of cytochrome c or the loss of Deltapsim, but blocked caspase-mediated DNA fragmentation and cell death. To determine whether PEITC dependent Bax translocation caused loss of Deltapsim by the activation of the mitochondrial permeability transition (MPT), we examined the effects of PEITC in isolated rat hepatocyte mitochondria. Interestingly, PEITC did not induce MPT in isolated rat mitochondria. Accordingly, using pharmacological inhibitors of MPT namely cyclosporine A, trifluoperazine and Bongkrekic acid we were unable to block PEITC mediated apoptosis in HepG2 cells, this suggesting that mitochondrial permeablisation is a likely consequence of Bax dependent pore formation. Taken together, our data suggest that mitochondria are a key target in PEITC induced apoptosis in HepG2 cells via the pore forming ability of pro-apoptotic Bax. 相似文献
11.
In the present study, we evaluated proapoptotic protein Bax on mitochondria and Ca2+ homeostasis in primary cultured astrocytes. We found that recombinant Bax (rBax, 10 and 100 ng/ml) induces a loss in mitochondrial membrane potential (Delta Psi m). This effect might be related to the inhibition of respiratory rates and a partial release of cytochrome c, which may change mitochondrial morphology. The loss of Delta Psi m and a selective permeabilization of mitochondrial membranes contribute to the release of Ca2+ from the mitochondria. This was inhibited by cyclosporin A (5 microM) and Ruthenium Red (1 microg/ml), indicating the involvement of mitochondrial Ca2+ transport mechanisms. Bax-induced mitochondrial Ca2+ release evokes Ca2+ waves and wave propagation between cells. Our results show that Bax induces mitochondrial alteration that affects Ca2+ homeostasis and signaling. These changes show that Ca2+ signals might be correlated with the proapoptotic activities of Bax. 相似文献
12.
The Bcl-2 proapoptotic proteins Bax and Bak mediate the permeabilization of the mitochondrial outer membrane during apoptosis. Current models consider that Bax and Bak form pores at the mitochondrial outer membrane that are responsible for the release of cytochrome c and other larger mitochondrial apoptotic factors ( i.e. Smac/DIABLO, AIF, and endoglycosidase G). However, the properties and nature of Bax/Bak apoptotic pores remain enigmatic. Here, we performed a detailed analysis of the membrane permeabilizing activity of Bax and Bak at the single vesicle level. We directly visualized that cBid-activated Bax and BakΔC21 can form membrane pores large enough to release not only cytochrome c, but also allophycocyanine, a protein of 104 kDa. Interestingly, the size of Bax and BakΔC21 pores is not constant, as typically observed in purely proteinaceous channels, but evolves with time and depends on protein concentration. We found that Bax and BakΔC21 formed long-lived pores, whose areas changed with the amount of Bax/BakΔC21 but not with cardiolipin concentration. Altogether, our results demonstrate that Bax and BakΔC21 follow similar mechanisms of membrane permeabilization characterized by the formation of protein-permeable pores of dynamic size, in agreement with the proteolipidic nature of these apoptotic pores. 相似文献
13.
A yeast mutant was isolated that was resistant to Bax-induced cell death. It supports a mutation leading to decreased amounts of the protein Uth1p. A strain in which the UTH1 gene is disrupted also exhibits resistance to Bax expression. The absence of Uth1p does not change the mitochondrial localization of Bax, its insertion in the mitochondrial outer membrane or its cytochrome c-releasing activity. On the other hand, the absence of Uth1p does prevent the appearance of other hallmarks related to Bax expression in yeast, such as oxidation of mitochondrial lipid, production of reactive oxygen species and maintenance of plasma membrane properties after ethanol stress. The absence of Uth1p was also found to induce resistance to rapamycin, a specific inducer of autophagy. This resistance only appears when cells are grown under respiratory conditions, but not under fermentative conditions, suggesting that Uth1p acts in an autophagic pathway involving mitochondria, in accordance with its main localization in the outer mitochondrial membrane. Taken together, these data show that Bax is able to activate a death pathway related to autophagy in yeast, which also exhibits typical hallmarks of apoptosis, revealing a possible dual function of Bax in both types of death. This hypothesis is discussed in the light of observations suggesting a co-regulation of apoptosis and autophagy in mammalian cells. 相似文献
14.
A 8.7 kDa lectin with high agglutin activity was isolated by affinity chromatography and cloned from Allium chinense in this study. For the MTT assay, approximately 60 µg/ml A. chinense lectin (ACL) inhibited 50% of the human hepatoma Hep-3B cells grown after 48 h. In addition, no antiproliferative effect was observed on normal human umbilical vein endothelial cells (HUVEC) even at 100 µg/ml concentration. After treatments with ACL on Hep-3B cells, morphologic changes in the nucleus and cytoskeleton were observed under laser scanning confocal microscopy with 4′,6-diamidino-2-phenylindole and tubulin Alexa Fluor 488 staining; whereas, the mitochondrial membrane potential was observed through Mito Tracker Red CMXRos staining. The results showed that ACL led to cell morphology and structure change (e.g., round cell shrinkage). Moreover, ACL resulted in significant change in the shape of the nucleus, damaged the cytoskeleton when tubulin was degraded, and reduced the mitochondrial transmembrane potential. By contrast, no changes were observed on HUVEC cells under the same treatment conditions. DNA fragmentation analysis was used to detect DNA damage. Western blot showed that ACL upregulated caspase-3 and Bax expression during apoptosis and cloned the structural gene of ACL with an open reading frame of 456 bp encoding 151 amino acid residues. The results showed that ACL is a potential anticancer drug. 相似文献
15.
The morphology and replication of Mycoplasma felis in relation to growth phase in culture were studied by electron microscopy. The organisms showed 1.0 to 1.45-hr doubling times with typical bacterial-type growth curves when grown in dialysate broth supplemented with horse serum. Organisms were fixed for electron microscopy by using Veronal acetate-buffered 0.8% OsO(4) (pH 6.1) in 20% sucrose. The morphology of exponential-phase organisms differed markedly from that of stationary or death-phase organisms, which were essentially large round forms with either dispersed or abnormally aggregated cytoplasm. Plasticine models prepared from serial sections of organisms in exponential phase showed the organisms to be either disc-shaped, triangular, horseshoe-shaped, or multilobular. A central "hole" was frequently present in these structures and could be visualized in the lobular forms as an interconnecting circular membrane. The inner surface of this membrane often showed contact with a small membranous body about 0.12 mum in diameter. The significance of this body is unknown. The morphology of the various shapes was confirmed by using the phosphotungstic acid and critical point methods. When the ratios of the various forms in exponential-phase cultures were determined, it was found that a replication sequence could be proposed which accounted for not only the volume increase required to accommodate deoxyribonucleic acid (DNA) replication but also the distribution of that DNA. Although it is likely that DNA replication in M. felis is a binary process, it appears that the mechanism for production of new cells need not be a binary process. 相似文献
16.
The combined effects of lactic acid and acetic acid on ethanol production by S. cerevisiae in corn mash, as influenced by temperature, were examined. Duplicate full factorial experiments (three lactic acid concentrations
× three acetic acid concentrations) were performed to evaluate the interaction between lactic and acetic acids on the ethanol
production of yeast at each of the three temperatures, 30, 34, and 37°C. Corn mash at 30% dry solids adjusted to pH 4 after
lactic and acetic acid addition was used as the substrate. Ethanol production rates and final ethanol concentrations decreased
( P<0.001) progressively as the concentration of combined lactic and acetic acids in the corn mash increased and the temperature
was raised from 30 to 37°C. At 30°C, essentially no ethanol was produced after 96 h when 0.5% w/v acetic acid was present
in the mash (with 0.5, 2, and 4% w/v lactic acid). At 34 and 37°C, the final concentrations of ethanol produced by the yeast
were noticeably reduced by the presence of 0.3% w/v acetic acid and ≥2% w/v lactic acid. It can be concluded that, as in previous
studies with defined media, lactic acid and acetic acid act synergistically to reduce ethanol production by yeast in corn
mash. In addition, the inhibitory effects of combined lactic and acetic acid in corn mash were more apparent at elevated temperatures. 相似文献
17.
The influence of different alkanes on spore morphology, glyoxlate pathway enzyme activities, total lipid contents and fatty acid composition of Isaria fumosorosea were investigated under laboratory conditions. Fungal spores grown on different alkanes showed higher germination and mycelial growth when compared to control. A strong induction of glyoxlate cycle enzymes in cell-free extracts was observed for cells grown on different alkanes when compared to glucose and control. Higher activities of glyoxlate cycle enzymes were observed for cells grown on alkanes when compared to other treatments. Even numbered fatty acids accounted for the majority of fatty acid production with a significant increase in relative amounts of linoleic acid and palmatic acid observed for conidia grown on alkanes. These results indicate that addition of alkanes to culture media can be a tool to pre-induce metabolic adaptations that can facilitate successful infection of insect host by entomopathogenic fungi. 相似文献
18.
The effect of acetic acid and formic acid on acetone-butanol-ethanol (ABE) production by solventogenic Clostridia was investigated.
The ABE concentration in Clostridium acetobutylicum was found to have increased slightly on addition of 3.7 ∼ 9.7 g/L acetic acid, but was found to have drastically reduced
in the presence of 11.7 g/L acetic acid. However, the solvent production of C. beijerinckii was not affected by addition of acetic acid in the range of 3.7 ∼ 11.7 g/L. C. acetobutylicum was more vulnerable to formic acid than C. beijerinckii. In C. acetobutylicum, the total ABE production decreased to 77% on addition of 0.4 g/L formic acid and 25% with 1.0 g/L formic acid. The total
ABE production by C. acetobutylicum was also noted to have decreased from 15.1 to 8.6 g/L when 8.7 g/L acetic acid and 0.4 g/L formic acid co-existed. The solvent
production by C. beijerinckii was not affected at all under the tested concentration range of formic acid (0.0 ∼ 1.0 g/L) and co-presence of acetic acid
and formic acid. Therefore, C. beijerinckii is more favorable than C. acetobutylicum when the ABE is produced using lignocellulosic hydrolysate containing acetic and formic acid. 相似文献
19.
Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase‐activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N‐terminus, generating a potent proapoptotic 18‐kDa fragment (Bax/p18). Both the calpain‐mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane‐enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase‐3, cleavage of poly(ADP‐ribose) polymerase, and fragmentation of DNA. Unlike the full‐length Bax, Bax/p18 did not interact with the antiapoptotic Bcl‐2 protein in the mitochondrial fraction of drug‐treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide‐induced calpain activation, Bax cleavage, cytochrome c release, and caspase‐3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase‐3‐mediated apoptosis that was not blocked by overexpression of Bcl‐2 protein. Therefore, Bax/p18 has a cytochrome c–releasing activity that promotes cell death independent of Bcl‐2. Finally, Bcl‐2 overexpression inhibited etoposide‐induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution. J. Cell. Biochem. 80:53–72, 2000. © 2000 Wiley‐Liss, Inc. 相似文献
20.
The morphology and cell wall composition of Bacillus coagulans, a facultative thermophile, were examined as a function of growth temperature. The morphology of the organism varied when it was grown at different temperatures; at 37 C the organism grew as individual cells which increased in length with increasing growth temperature. At 55 C it grew in long chains of cells. Cell wall prepared from cells grown at 37 C contained 44% teichoic acid by weight, whereas cells grown at 55 C contained 29% teichoic acid. Teichoic acid from these cells was a polymer of glycerol phosphate containing galactose and ester alanine. The ratio of ester alanine to phosphate was significantly higher in cell walls and teichoic acid from 37 C-grown cells compared with those from 55 C-grown cells. Other differences observed were that cells grown at 55 C contained a lower level of autolytic ability, produced cell walls which bound more Mg(2+), and contained less peptide cross-bridging in its peptidoglycan layer than cells grown at 37 C. 相似文献
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