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101.
Micol Silic-Benussi Enrica Cannizzaro Andrea Venerando Valeria Petronilli Oriano Marin Fabio Di Lisa Paolo Bernardi 《BBA》2009,1787(7):947-516
Human T-cell leukemia virus type-1 (HTLV-1) expresses an 87-amino acid protein named p13 that is targeted to the inner mitochondrial membrane. Previous studies showed that a synthetic peptide spanning an alpha helical domain of p13 alters mitochondrial membrane permeability to cations, resulting in swelling. The present study examined the effects of full-length p13 on isolated, energized mitochondria. Results demonstrated that p13 triggers an inward K+ current that leads to mitochondrial swelling and confers a crescent-like morphology distinct from that caused by opening of the permeability transition pore. p13 also induces depolarization, with a matching increase in respiratory chain activity, and augments production of reactive oxygen species (ROS). These effects require an intact alpha helical domain and strictly depend on the presence of K+ in the assay medium. The effects of p13 on ROS are mimicked by the K+ ionophore valinomycin, while the protonophore FCCP decreases ROS, indicating that depolarization induced by K+ vs. H+ currents has different effects on mitochondrial ROS production, possibly because of their opposite effects on matrix pH (alkalinization and acidification, respectively). The downstream consequences of p13-induced mitochondrial K+ permeability are likely to have an important influence on the redox state and turnover of HTLV-1-infected cells. 相似文献
102.
Tamara Azarashvili Irina Odinokova Anush Bakunts Vadim Ternovsky Olga Krestinina Jaana Tyynelä Nils-Erik Leo Saris 《Cell calcium》2014
Phosphorylated and non-phosphorylated forms of the F0F1-ATPase subunit c from rat liver mitochondria (RLM) were purified and their effect on the opening of the permeability transition pore (mPTP) was investigated. Addition of dephosphorylated subunit c to RLM induced mitochondrial swelling, decreased the membrane potential and reduced the Ca2+ uptake capacity, which was prevented by cyclosporin A. The same effect was observed in the presence of storage subunit c purified from livers of sheep affected with ceroid lipofuscinosis. In black-lipid bilayer membranes subunit c increased the conductance due to formation of single channels with fast and slow kinetics. The dephosphorylated subunit c formed channels with slow kinetics, i.e. the open state being of significantly longer duration than in the case of channels formed by the phosphorylated form that had short life spans and fast kinetics. The channels formed were cation-selective more so with the phosphorylated form. Subunit c of rat liver mitochondria was able to bind Ca2+. Collectively, the data allowed us to suppose that subunit c F0F1-ATPase might be a structural/regulatory component of mPTP exerting its role in dependence on phosphorylation status. 相似文献
103.
zlem Alptekin S. Seyhan Tükel Deniz Yldrm Dilek Alagz 《Journal of Molecular Catalysis .B, Enzymatic》2009,58(1-4):124-131
Bovine liver catalase was covalently immobilized onto controlled pore glass (CPG) beads modified with 3-aminopropyltriethoxysilane (3-APTES) followed by treatment with glutaraldehyde. Coupling of catalase onto CPG was optimized to improve the efficiency of the overall immobilization procedure. The optimum coupling conditions: pore diameter of CPG, pH, buffer concentration, temperature, coupling time and initial catalase amount per grams of carrier were determined as 70 nm, 6.0, 75 mM, 5 °C, 7 h and 6 mg catalase, respectively. Catalytic efficiencies (kcat/Km) and thermal inactivation rate constants (ki) of ICPG1 were determined and compared with that of free catalase. Suitability of ICPG1 was also investigated by using it in batch and plug-flow type reactors. When the remaining activity of ICPG1 retained was about 50% of its initial activity the highest total productivity of ICPG1 was determined as 7.6 × 106 U g immobilized catalase−1 in plug-flow type reactor. However, the highest total productivity of ICPG1 was 6.2 × 105 U g immobilized catalase−1 in batch type reactor. ICPG1 may have great potentials as biocatalyst for the application in decomposition of hydrogen peroxide in plug-flow type reactor. 相似文献
104.
目的:探讨谷氨酰胺(Gln)对过度训练状态下心肌线粒体膜通透性转换孔(PTP)开放的干预作用及其可能机制。方法:30只SD大鼠随机分为3组(n=10):对照组(CG组)、过度训练组(OG组)和补充Gln+过度训练组(GOG组)。采用分光光度法检测大鼠心肌线粒体PTP开放程度,电化学法检测心肌丙二醛(MDA)、还原型谷胱苷肽(GSH)含量和磷脂酶A2(PLA2)活性。结果:OG组与GOG组比较,吸光度(A0)显著下降(P<0.05),吸光度变化(△A)值显著降低(P<0.05);荧光剂罗丹明123(Rh123)的荧光强度(F0)显著增强(P<0.05),Rh123荧光强度变化(△F)值明显降低(P<0.05)。与GOG组比较,线粒体GSH含量显著降低(P<0.05),PLA2活性显著增加(P<0.05);MDA含量显著升高(P<0.05)。结论:过度训练可导致心肌细胞线粒体PTP开放增加,过度训练状态下线粒体活性氧生成增多,PLA2活性增加及GSH的含量下降,补充外源性的Gln对这些变化有显著的干预作用。 相似文献
105.
A cohesion/tension model for the gating of aquaporins allows estimation of water channel pore volumes in Chara 总被引:2,自引:2,他引:0
The water permeability (hydraulic conductivity; Lp) of turgid, intact internodes of Chara corallina decreased exponentially as the concentration of osmolytes applied in the medium increased. Membranes were permeable to osmolytes and therefore they could be applied on both sides of the plasma membrane at concentrations of up to 2.0 m (5.0 MPa of osmotic pressure). Organic solutes of different molecular size (molecular weight, MW) and reflection coefficients (σs) were used [heavy water HDO, MW: 19, σs: 0.004; acetone, MW: 58, σs: 0.15; dimethyl formamide (DMF), MW: 73, σs: 0.76; ethylene glycol monomethyl ether (EGMME), MW: 76, σs: 0.59; diethylene glycol monomethyl ether (DEGMME), MW: 120, σs: 0.78 and triethylene glycol monoethyl ether (TEGMEE), MW: 178, σs: 0.80]. The larger the molecular size of the osmolyte, the more efficient it was in reducing cell Lp at a given concentration. The residual cell Lp decreased with increasing size of osmolytes. The findings are in agreement with a cohesion/tension model of the osmotic dehydration of water channels (aquaporins; AQPs), which predicts both reversible exponential dehydration curves and the dependence on the size of osmolytes which are more or less excluded from AQPs (Ye, Wiera & Steudle, Journal of Experimental Botany 55, 449–461, 2004). In the presence of big osmolytes, dehydration curves were best described by the sum of two exponentials (as predicted from the theory in the presence of two different types of AQPs with differing pore diameters and volumes). AQPs with big diameters could not be closed in the presence of osmolytes of small molecular size, even at very high concentrations. The cohesion/tension theory allowed pore volumes of AQPs to be evaluated, which was 2.3 ± 0.2 nm3 for the narrow pore and between 5.5 ± 0.8 and 6.1 ± 0.8 nm3 for the wider pores. The existence of different types of pores was also evident from differences in the residual Lp. Alternatively, pore volumes were estimated from ratios between osmotic (Pf) and diffusional (Pd) water flow, yielding the number of water molecules (N) in the pores. N-values ranged between 35 and 60, which referred to volumes of 0.51 and 0.88 nm3/pore. Values of pore volumes obtained by either method were bigger than those reported in the literature for other AQPs. Absolute values of pore volumes and differences obtained by the two methods are discussed in terms of an inclusion of mouth parts of AQPs during osmotic dehydration. It is concluded that the mouth part contributed to the absolute values of pore volumes depending on the size of osmolytes. However, this can not explain the finding of the existence of two different types or groups of AQPs in the plasma membrane of Chara. 相似文献
106.
The access resistance (AR) of a channel is an important component of the conductance of ion channels, particularly in wide and short channels, where it accounts for a substantial fraction of the total resistance to the movement of ions. The AR is usually calculated by using a classical and simple expression derived by Hall from electrostatics (J.E. Hall 1975 J. Gen. Phys. 66:531-532), though other expressions, both analytical and numerical, have been proposed. Here we report some numerical results for the AR of a channel obtained by solving the Poisson-Nernst-Planck equations at the entrance of a circular pore. Agreement is found between numerical calculations and analytical results from Hall's equation for uncharged pores in neutral membranes. However, for channels embedded in charged membranes, Hall's expression overestimates the AR, which is much lower and can even be neglected in some cases. The weak dependence of AR on the pore radius for charged membranes at low salt concentration can be exploited to separate the channel and the access contributions to the measured conductance. 相似文献
107.
Chinopoulos C Starkov AA Grigoriev S Dejean LM Kinnally KW Liu X Ambudkar IS Fiskum G 《Journal of bioenergetics and biomembranes》2005,37(4):237-247
Mitochondria contribute to cytosolic Ca2+ homeostasis through several uptake and release pathways. Here we report that 1,2-sn-diacylglycerols (DAGs) induce Ca2+ release from Ca2+-loaded mammalian mitochondria. Release is not mediated by the uniporter or the Na+/Ca2+ exchanger, nor is it attributed to putative catabolites. DAGs-induced Ca2+ efflux is biphasic. Initial release is rapid and transient, insensitive to permeability transition inhibitors, and not accompanied by mitochondrial swelling. Following initial rapid release of Ca2+ and relatively slow reuptake, a secondary progressive release of Ca2+ occurs, associated with swelling, and mitigated by permeability transition inhibitors. The initial peak of DAGs-induced Ca2+ efflux is abolished by La3+ (1 mM) and potentiated by protein kinase C inhibitors. Phorbol esters, 1,3-diacylglycerols and 1-monoacylglycerols do not induce mitochondrial Ca2+ efflux. Ca2+-loaded mitoplasts devoid of outer mitochondrial membrane also exhibit DAGs-induced Ca2+ release, indicating that this mechanism resides at the inner mitochondrial membrane. Patch clamping brain mitoplasts reveal DAGs-induced slightly cation-selective channel activity that is insensitive to bongkrekic acid and abolished by La3+. The presence of a second messenger-sensitive Ca2+ release mechanism in mitochondria could have an important impact on intracellular Ca2+ homeostasis. 相似文献
108.
109.
BACKGROUND: One major barrier limiting the transfection efficiency of polyplexes is poor endosomal release, especially when small particles are applied. In an approach to overcome this barrier, covalent attachment of the membrane-active peptide all-(L)-melittin to polyethylenimine (PEI) polyplexes was found to enhance gene transfer efficiency. METHODS: The N-terminus of natural all-(L)- or non-immunogenic all-(D)-melittin was covalently coupled to PEI. In addition, two different all-(D)-melittin conjugates were synthesized, with PEI covalently attached to either the C-terminus (C-mel-PEI) or the N-terminus of melittin (N-mel-PEI). Melittin-PEI polyplexes with particle sizes < 150 nm were generated in HEPES-buffered glucose and tested in transfection experiments. The membrane lytic activities of conjugates and polyplexes were analyzed at neutral and endosomal pH. RESULTS: All-(D)-melittin conjugates mediated enhanced gene expression similar to the natural all-(L)-stereoisomer, with up to 160-fold higher luciferase activity than unmodified PEI. The site of melittin linkage strongly influenced the membrane-destabilizing activities of both conjugates and polyplexes. C-mel-PEI was highly lytic at neutral pH and therefore elevated doses of C-mel-PEI polyplexes induced high toxicity. In contrast, N-mel-PEI was less lytic at neutral pH but retained higher lytic activity than C-mel-PEI at endosomal pH. This apparently promoted better endosomal release of N-mel-PEI polyplexes resulting in efficient gene delivery in different cell lines. CONCLUSIONS: The high potency of C-mel-PEI to destabilize membranes at neutral pH is presumably due to a reported destabilization mechanism proceeding through membrane insertion of the peptide. In contrast, N-mel-PEI is supposed to induce lysis by insertion-independent pore formation according to the toroidal pore model. 相似文献
110.
Yoshimoto M Wang S Fukunaga K Fournier D Walde P Kuboi R Nakao K 《Biotechnology and bioengineering》2005,90(2):231-238
The reactivity of immobilized glucose oxidase-containing liposomes (IGOL) prepared in our previous work (Wang et al. [2003] Biotechnol Bioeng 83:444-453) was considerably improved here by incorporating the channel protein OmpF from Escherichia coli into the liposome membrane as well as by entrapping inside the liposome's aqueous interior not only glucose oxidase (GO), but also catalase (CA), both from Aspergillus niger. CA was used for decomposing the hydrogen peroxide produced in the glucose oxidation reaction inside the liposomes. The presence of OmpF enhanced the transport of glucose molecules from the exterior of the liposomes to the interior. In a first step of the work, liposomes containing GO and CA (GOCAL) were prepared and characterized. A remarkable protection effect of the liposome membrane on CA inside the liposomes at 40 degrees C was found; the remaining CA activity at 72 h incubation was more than 60% for GOCAL, while less than 20% for free CA. In a second step, OmpF was incorporated into GOCAL membranes, leading to the formation of OmpF-embedded GOCAL (abbreviated GOCAL-OmpF). The activity of GO inside GOCAL-OmpF increased up to 17 times in comparison with that inside GOCAL due to an increased glucose permeation across the liposome bilayer, without any leakage of GO or CA from the liposomes. The optimal system was estimated to contain on average five OmpF molecules per liposome. Finally, GOCAL-OmpF were covalently immobilized into chitosan gel beads. The performance of this novel biocatalyst (IGOCAL-OmpF) was examined by following the change in glucose conversion, as well as by following the remaining GO activity in successive 15-h air oxidations for repeated use at 40 degrees C in an airlift bioreactor. IGOCAL-OmpF showed higher reactivity and reusability than IGOL, as well as IGOL containing OmpF (IGOL-OmpF). The IGOCAL-OmpF gave about 80% of glucose conversion even when the catalyst was used repeatedly four times, while the corresponding conversions were about 60% and 20% for the IGOL and IGOL-OmpF, respectively. Due to the absence of CA, IGOL-OmpF was less stable and resulted in drastically inhibited GO. 相似文献