共查询到20条相似文献,搜索用时 0 毫秒
1.
AJ Minn CS Kettlun H Liang A Kelekar MG Vander Heiden BS Chang SW Fesik M Fill CB Thompson 《Canadian Metallurgical Quarterly》1999,18(3):632-643
A hydrophobic cleft formed by the BH1, BH2 and BH3 domains of Bcl-xL is responsible for interactions between Bcl-xL and BH3-containing death agonists. Mutants were constructed which did not bind to Bax but retained anti-apoptotic activity. Since Bcl-xL can form an ion channel in synthetic lipid membranes, the possibility that this property has a role in heterodimerization-independent cell survival was tested by replacing amino acids within the predicted channel-forming domain with the corresponding amino acids from Bax. The resulting chimera showed a reduced ability to adopt an open conductance state over a wide range of membrane potentials. Although this construct retained the ability to heterodimerize with Bax and to inhibit apoptosis, when a mutation was introduced that rendered the chimera incapable of heterodimerization, the resulting protein failed to prevent both apoptosis in mammalian cells and Bax-mediated growth defect in yeast. Similar to mammalian cells undergoing apoptosis, yeast cells expressing Bax exhibited changes in mitochondrial properties that were inhibited by Bcl-xL through heterodimerization-dependent and -independent mechanisms. These data suggest that Bcl-xL regulates cell survival by at least two distinct mechanisms; one is associated with heterodimerization and the other with the ability to form a sustained ion channel. 相似文献
2.
Rapid determination of the membrane potential of mitochondria in small biopsy specimens of the liver
Y Yamamoto M Obara W Isselhard J Sturz T Morimoto T Minor S Saad K Ozawa 《Canadian Metallurgical Quarterly》1995,27(3):145-150
In liver transplantation, graft viability is ideally to be determined before implantation. Integrity of mitochondria may be a prerequisite to a viable graft. A new method is presented, which allows for the determination of the membrane potential of mitochondria (MPM; mV) in state 4 respiration within 50 min in 40-mg specimens, employing rhodamine 123 as a probe. Normal control showed a MPM of 239.2 mV. Storage in saline at 37 degrees C yielded an impaired MPM of 153.5 mV within 3 h. The cold storage at 1 degree C could preserve MPM at quasi-normal after 3 h but reduced it significantly after 24 h to 222.2 mV in saline (p < 0.005 vs. control) and 231.0 mV in UW solution (p < 0.05 vs. control): the difference between the 24-hour values was significant (p < 0.05). 相似文献
3.
AE Vercesi SN Moreno CF Bernardes AR Meinicke EC Fernandes R Docampo 《Canadian Metallurgical Quarterly》1993,268(12):8564-8568
Thapsigargin, previously reported to release Ca2+ from non-mitochondrial stores of different cell types, as well as nigericin, were found, when used at high concentrations, to release Ca2+ and collapse the membrane potential of Trypanosoma brucei bloodstream and procyclic trypomastigotes mitochondria in situ. At similarly high concentrations (> 10 microM), thapsigargin was also found to release Ca2+ and collapse the membrane potential of isolated rat liver mitochondria. These results indicate that care should be taken when attributing the effects of thapsigargin in intact cells to the specific inhibition of the sarcoplasmic and endoplasmic reticulum Ca(2+)-ATPase family of calcium pumps. In addition, we have found no evidence for an increase in intracellular Ca2+ by release of the ion from intracellular stores by nigericin, measuring changes in cytosolic Ca2+ by dual wavelength spectrofluorometry in fura-2-loaded T. brucei bloodstream trypomastigotes or measuring Ca2+ transport in digitonin-permeabilized cells. 相似文献
4.
CS Duckett F Li Y Wang KJ Tomaselli CB Thompson RC Armstrong 《Canadian Metallurgical Quarterly》1998,18(1):608-615
The gene encoding human IAP-like protein (hILP) is one of several mammalian genes with sequence homology to the baculovirus inhibitor-of-apoptosis protein (iap) genes. Here we show that hILP can block apoptosis induced by a variety of extracellular stimuli, including UV light, chemotoxic drugs, and activation of the tumor necrosis factor and Fas receptors. hILP also protected against cell death induced by members of the caspase family, cysteine proteases which are thought to be the principal effectors of apoptosis. hILP and Bcl-xL were compared for their ability to affect several steps in the apoptotic pathway. Redistribution of cytochrome c from mitochondria, an early event in apoptosis, was not blocked by overexpression of hILP but was inhibited by Bcl-xL. In contrast, hILP, but not Bcl-xL, inhibited apoptosis induced by microinjection of cytochrome c. These data suggest that while Bcl-xL may control mitochondrial integrity, hILP can function downstream of mitochondrial events to inhibit apoptosis. 相似文献
5.
DM Finucane E Bossy-Wetzel NJ Waterhouse TG Cotter DR Green 《Canadian Metallurgical Quarterly》1999,274(4):2225-2233
A growing body of evidence supports a role for mitochondria and mitochondria-derived factors in the cell death process. In particular, much attention has focused on cytochrome c, a key component of the electron transport chain, that has been reported to translocate from the mitochondria to the cytosol in cells undergoing apoptosis. The mechanism for this release is, as yet, unknown. Here we report that ectopic expression of Bax induces apoptosis with an early release of cytochrome c preceding many apoptosis-associated morphological alterations as well as caspase activation and subsequent substrate proteolysis. A loss of mitochondrial transmembrane potential was detected in vivo, although no mitochondrial swelling or loss of transmembrane potential was observed in isolated mitochondria treated with Bax in vitro. Caspase inhibitors, such as endogenous XIAP and synthetic peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), although capable of altering the kinetics and perhaps mode of cell death, had no influence on this release, suggesting that if cytochrome c plays a role in caspase activation it must precede this step in the apoptotic process. Mitochondrial permeability transition was also shown to be significantly prevented by caspase inhibition, indicating that the translocation of cytochrome c from mitochondria to cytosol is not a consequence of events requiring mitochondrial membrane depolarization. In contrast, Bcl-xL was capable of preventing cytochrome c release while also significantly inhibiting cell death. It would therefore appear that the mitochondrial release of factors such as cytochrome c represents a critical step in committing a cell to death, and this release is independent of permeability transition and caspase activation but is inhibited by Bcl-xL. 相似文献
6.
Employing photometric methods, we have attempted to derive a possible quantitative relationship between volume change and membrane potential for valinomycin-incorporated red blood cells. The cells, collected from a human, rats or bullfrogs, were suspended in test solutions composed of a mixture of NaCl and KCl in varying proportions. The osmolality of the suspending medium was appropriately fixed at different values. After the addition of valinomycin to the suspension, changes in optical density (turbidity) at 620 nm and in fluorescence from a voltage-sensitive permeant dye (diS-C3-[5]) were measured in different concentrations of external potassium ions. The changes in optical density and fluorescence intensity were converted into relative cell volume and membrane potential changes in the test cells, respectively. Cell volume increased with depolarization of the membrane. We derived an empirical equation for the volume change versus membrane potential relation curves obtained experimentally, and have also shown that the observed volume change may be plausibly represented by a hyperbolic function of transmembrane potential involving the medium osmolality as an important parameter. 相似文献
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Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytochemically using two antibodies: a polyclonal antibody raised against the porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptide antibody against the porcine enamelin amino-terminus (EN). Immunochemical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies used. In contrast, the middle and inner enamel layer mainly contained lower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mineralized collagen fibrils of dentin, while EN stained both enamel matrix islands and stippled material. At the matrix formation stage, both antibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneously, while EN primarily stained the prism sheath. The intense immunoreaction over the surface layer of enamel matrix at the matrix formation stage, following staining with 89 E and EN, disappeared by the end of the transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the late differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin continues from late differentiation to the transition stage and the cleavage of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during enamel maturation. 相似文献
10.
RA Schmiedt 《Canadian Metallurgical Quarterly》1996,102(1-2):125-132
In this study we present a strategy for the identification of novel peptide conjugates which may be used to understand the molecular details of the recognition process or to potentially regulate T cell-mediated responses. The approach involves the incorporation of cysteine into a known peptide at a position of interest and subsequent chemical conjugation using thiol-specific agents. Conjugates derived from the nonapeptide QL9 that is recognized by CTL 2C had either enhanced or reduced activity compared to the original cys-peptides. Different classes of thiol-reactive agents (alkyl halides, alkylthiolsulfonates, and disulfides) were tested with increases in activity of over 100-fold. As with standard peptide analogs, the activity depended on the position of the cysteine within the peptide and the nature of the chemically linked functional group. Use of this approach in a cysteine 'scan' of all positions of the original peptide is cost effective and with the availability of many different thiol-specific functional groups will allow the screening of considerably larger libraries of chemically modified peptides than have been used to date. Additionally, these findings may provide insight into the pathogenesis of thiol agents involved in contact sensitivity reactions. 相似文献
11.
Amiodarone affects membrane water permeability properties of human erythrocytes and rat mitochondria
A Federico C Battisti L Manneschi E Gaggelli M Tassini G Valensin A Vivi 《Canadian Metallurgical Quarterly》1996,304(1-3):237-241
Dose-dependent water exchange times and intracellular water contents were measured by NMR (nuclear magnetic resonance) in erythrocytes and mitochondria interacted with the anti-anginal and anti-arrhytmic agent, amiodarone. Addition of the drug up to 26 microM yielded 80% enhancement of the water exchange rate in erythrocytes at 37 degrees C and 41% enhancement at 22 degrees C with 40% and 9%, respectively, increases in the intracellular water content. Similar enhancements were obtained in mitochondria at 22 degrees C. The data suggests a somewhat higher affinity of amiodarone to mitochondrial than to erythrocyte membranes. 相似文献
12.
Ion permeability of internal membrane and a respiration in isolated rat liver mitochondria, further related to as "sodium ones", were studied following replacement of K+ ions for Na+ ones in the mitochondrial matrix. As compared with the control ("potassium mitochondria"), state 4 respiration in the sodium mitochondria, energized by succinate, was shown to be enhanced in KCl or sucrose media. Oxygen consumption rates in the sodium mitochondria, being in state 3 or stimulated by 2,4-dinitrophenol, were lower than rates for the control mitochondria. This effect was much pronounced in the sucrose medium. The coefficients, characterizing the distribution of 137Cs between mitochondria and the medium, were lower for the sodium mitochondria than for the control in the presence of 2.5 mM succinate and 10(-8) M valinomycin. In comparison with the control, a more extensive swelling for the sodium mitochondria was found, first, in the medium containing 25 mM K-acetate and 100 mM sucrose for succinate-energized mitochondria, and second, in the medium containing 125 mM NH4NO3 without mitochondrial energization. Changes disclosed in respiration, swelling and coefficients of 137Cs distribution for the sodium mitochondria are supposed to be caused by non-uniform effects of Na+ and K+ ions on the water structure of mitochondrial matrix, ion permeability of internal membrane, and the activity in oxidative phosphorylation enzymes. 相似文献
13.
N Dekker J Tommassen A Lustig JP Rosenbusch HM Verheij 《Canadian Metallurgical Quarterly》1997,272(6):3179-3184
The outer membrane phospholipase A (OMPLA) of Escherichia coli is present in a dormant state in the cell envelope. The enzyme is activated by various processes, which have in common that they perturb the outer membrane. Kinetic experiments, chemical cross-linking, and analytical ultracentrifugation were carried out with purified, detergent-solubilized OMPLA to understand the underlying mechanism that results in activation. Under conditions in which the enzyme displayed full activity, OMPLA was dimeric. High detergent concentrations or very dilute protein concentrations resulted in low specific activity of the enzyme, and under those conditions the enzyme was monomeric. The cofactor Ca2+ was required for dimerization. Covalent modification of the active site serine with hexadecylsulfonylfluoride resulted in stabilization of the dimeric form and a loss of the absolute calcium requirement for dimerization. The results of these experiments provide evidence for dimerization as the molecular mechanism by which the enzymatic activity of OMPLA is regulated. This dimerization probably plays a role in vivo as well. Data from chemical cross-linking on whole cells indicate that OMPLA is present in the outer membrane as a monomer and that activation of the enzyme induces dimerization concurrent with the appearance of enzymatic activity. 相似文献
14.
The nonmediated inward translocation (flip) of the anionic fluorescent N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- (NBD-)labeled phospholipid phosphatidylmethanol (PM) from the outer to the inner membrane leaflet of human erythrocytes and vice versa depends on membrane potential. Interestingly, inside-positive potentials due to chloride gradients and the native chloride conductance of the cells resulted in an increase of the flip rates. This flip enhancement could be suppressed by addition of gramicidin D, which increases cation conductance, or 4,4'-diisothiocyanatostilbene-2,2'-disufonate (DIDS), which inhibits anion conductance. Conversely, inside negative potentials established by an outward-directed K+ gradient in the presence of gramicidin on DIDS-treated cells resulted in a decrease of flip rate. Flip rate exhibited an exponential dependence on membrane potential. The opposite effects of the positive and negative potentials were obtained for the outward translocation (flop) from the inner to the outer membrane leaflet. Similar potential dependencies were found for the nonmediated flip of anionic NBD-labeled phosphatidic acid (PA) and 2-(N-decyl)aminonaphthalene-6-sulfonic acid (2,6-DENSA) following blockage of the band-3-mediated component of flip. The membrane potential also influences the stationary distribution of the anionic lipids between the inner and outer leaflets. The distribution is shifted to the inner leaflet by increasingly positive potentials and to the outer leaflet by increasingly negative potentials. It is concluded that nonmediated flip-flop of the anionic phospholipids and the long-chain sulfonate represents electrogenic translocation of the unprotonated charged lipids across the hydrophobic barrier. 相似文献
15.
Safranine as a probe of the mitochondrial membrane potential 总被引:1,自引:0,他引:1
16.
S Motoyama M Kitamura S Saito Y Minamiya H Suzuki R Saito K Terada J Ogawa H Inaba 《Canadian Metallurgical Quarterly》1998,249(3):628-636
A modified "Samson" sucrose fading procedure was used to establish voluntary consumption of a 20% ethanol (EtOH) solution in male Sprague-Dawley rats for 18 consecutive months. Intakes were stable over the life span, and corresponded to the moderate to high levels of intake typically observed in human "social" drinkers and alcoholics. The Morris Water Maze (WM), Olton Radial Arm Maze (RM), and a "balance beam" test were used to assess the effects of alcohol and aging on spatial memory and motor function. Aged EtOH-consuming rats (AGED/ALC) demonstrated impaired task acquisition, relative to aged controls (AGED), not reaching criterion performance in either spatial memory task even when given four additional days of training. AGED/ALC rats scored significantly lower on percent correct out of the first eight arm entries, and committed more perseverative errors in the RM. There were no significant performance differences between AGED and AGED/ALC rats on a balance beam test of fine motor coordination and equilibrium, suggesting that deficits observed in the RM and WM were not related to differential motor functioning. These results demonstrated that long-term, moderate, oral self-administration of EtOH, within the range typically consumed by humans, had adverse effects on spatial memory in rats, and that such a pattern of EtOH consumption seemed to exacerbate the decline in cognitive functioning associated with normal aging. 相似文献
17.
Mammalian adenylyl cyclases possess 12 transmembrane-spanning domains and bear a superficial resemblance to certain classes of ion channels. Some evidence suggests that bacterial and sea urchin sperm adenylyl cyclases can be regulated by membrane depolarization. In the present study, we explored the effect of altering membrane potential on the adenylyl cyclase activity of cerebellar granule cells with acute potassium depolarization. A biphasic stimulatory and then inhibitory response is evoked by progressive increases in the extracellular [K]:[Na] ratio in the absence of extracellular Ca2+. This effect does not mimic the linear increase in membrane potential elicited under the same conditions. Instead it appears as though membrane depolarization opens L-type (nimodipine-sensitive) Ca2+ channels, allowing the entry of Na+, which directly stimulates adenylyl cyclase activity. Gramicidin, which generates pores that are permeable to monovalent cations, and concurrently eliminates the membrane potential, permits a similar stimulation by extracellularly applied Na+. Although the results indicate no direct sensitivity of cerebellar granule cell adenylyl cyclase to membrane potential, they do demonstrate that, as a result of membrane depolarization, the influx of Na+, as well as Ca2+, will elevate cAMP levels. 相似文献
18.
The influence of the chlorides of K, Na, Ca, Zn, Cd, Pb, and Ce on the membrane potential of Chlorella fusca (Shihira et Krauss) 211-8b, G?ttingen, has been studied. The potential depended on the concentration of Na und K much less than predicted by the Goldman equation. The dependence on the concentrations of Ca, Zn, and Cd was qualitatively similar to that in the case of the alkali ions. With Pb and Ce, the potential was entirely suppressed already at moderate concentrations, i.e., in the millimolar range. The contrast between the effects of the inessential elements Cd and Pb on the potential corresponds to the difference between their paths of uptake. The dependence of the membrane potential on time has also been studied. With K, Na, and Ca stationary states were reached within about 1 hour. The time course of the depression of the potential by Pb depended on the concentration. At moderate concentration, suppression could be partly reversed by subsequent treatment of the cells with diluted KCl. 相似文献
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Phosphorylation of the vesicle docking protein p115 regulates its association with the Golgi membrane 总被引:1,自引:0,他引:1
The vesicle docking protein p115 was found to be phosphorylated in a cell cycle-specific manner; it was found phosphorylated in interphase but not in mitotic cells. During interphase, however, two forms of p115 were detected in the cells; the phosphorylated form was found exclusively in cytosol, whereas the unphosphorylated form was associated with membranes, mostly of the Golgi complex. The latter form was released from the membranes upon phosphorylation. Mutational analysis revealed that the phosphorylation site of p115 was the Ser942 residue in the C-terminal acidic domain. A mutant with a single substitution of Ser942 --> Ala markedly increased its association with the Golgi membrane. Another mutant with Ser942 --> Asp was able to associate with the membrane, although at a decreased level, indicating that the dissociation of p115 from the membrane is not simply due to the negative charge of phosphorylated Ser942. Taken together, these results suggest that the phosphorylation of Ser942 at the C-terminal acidic domain regulates the interaction of p115 with the Golgi membrane, possibly taking part in the regulatory mechanism of vesicular transport. 相似文献