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Permeabilization
Kinds of Permeabilization Selected AbstractsAn antifungal compound produced by Bacillus subtilis YM 10,20 inhibits germination of Penicillium roqueforti conidiosporesJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2003G.S. Chitarra Abstract Aims: To identify and characterize an antifungal compound produced by Bacillus subtilis YM 10-20 which prevents spore germination of Penicillium roqueforti. Methods and Results: The antifungal compound was isolated by acid precipitation with HCl. This compound inhibited fungal germination and growth. Identification by HPLC and mass spectrometry analysis showed high similarity to iturin A. Permeabilization and morphological changes in P. roqueforti conidia in the presence of the inhibitor were revealed by fluorescence staining and SEM, respectively. Conclusions: The iturin-like compound produced by B. subtilis YM 10-20 permeabilizes fungal spores and blocks germination. Significance and Impact of the Study: Fluorescence staining in combination with flow cytometry and scanning electron microscopy are efficient tools for assessing the action of antifungal compounds against spores. Iturin-like compounds may permeabilize fungal spores and inhibit their germination. [source] Intracellular uptake and trafficking of Pluronic micelles in drug-sensitive and MDR cells: Effect on the intracellular drug localizationJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 1 2002Natalya Rapoport Abstract The intracellular uptake and localization of a fluorescently labeled Pluronic P-105 in HL-60 leukemia cells and in A2780 drug-sensitive and A2780/ADR MDR ovarian carcinoma cells were characterized by flow cytometry and fluorescence microscopy. Pluronic P-105 molecules were labeled with a pH-sensitive fluorescent label, 5-(and 6-)carboxy-2,7,-dichlorofluorescein. The fluorescence intensity of labeled Pluronic was about twofold higher at pH 7.4 than at pH 5.5. At Pluronic concentrations exceeding the critical micelle concentration (cmc), flow cytometry histograms manifested bimodal distribution of cell fluorescence for all types of cells. Cell population characterized by higher fluorescence intensity presumably resulted from Pluronic transfer from the acidic environment of cytoplasmic vesicles (endosomes or lysosomes) into the neutral environment of the cytoplasm and cell nuclei, which suggested the permeabilization of the membranes of acidic vesicle by Pluronic molecules. For the MDR cells, the bimodal distribution of cell fluorescence was already observed at very low Pluronic concentrations in the incubation medium (i.e., below the cmc). The data suggest that the membranes of acidic vesicles of MDR cells are more susceptible to the action of polymeric surfactants than those of drug-sensitive cells. Permeabilization of acidic vesicles had a dramatic effect on the intracellular trafficking of drugs: when delivered in PBS, the anthracyclin drug ruboxyl (Rb) sequestered in cytoplasmic vesicles and was excluded from cell nuclei; however, when delivered in Pluronic micelles, drug accumulated in cell nuclei. Drug uptake from/with Pluronic micelles was substantially enhanced by ultrasound. These findings suggest that the nuclear accumulation of drugs internalized via fluid-phase endocytosis can be enhanced by the application of Pluronic micelles and can be further augmented by ultrasonic irradiation. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:157,170, 2002 [source] Anthocyanins Protect Against A2E Photooxidation and Membrane Permeabilization in Retinal Pigment Epithelial Cells,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2005Young P. Jang ABSTRACT The pyridinium bisretinoid A2E, an autofluorescent pigment that accumulates in retinal pigment epithelial cells with age and in some retinal disorders, can mediate a detergent-like perturbation of cell membranes and light-induced damage to the cell. The photodynamic events initiated by the sensitization of A2E include the generation of singlet oxygen and the oxidation of A2E at carbon-carbon double bonds. To assess the ability of plant-derived anthocyanins to modulate adverse effects of A2E accumulation on retinal pigment epithelium (RPE) cells, these flavylium salts were isolated from extracts of bilberry. Nine anthocyanin fractions reflecting monoglycosides of delphinidin, cyanidin, petunidin and malvidin were obtained and all were shown to suppress the photooxidation of A2E at least in part by quenching singlet oxygen. The anthocyanins tested exhibited antioxidant activity of variable efficiency. The structural characteristics relevant to this variability likely included the ability to form a stable quinonoidal anhydro base at neutral pH, a conjugated diene structure in the C (pyrane) ring, the presence of hydroxyl groups on the B (benzene) ring and the relative hydrophobicity conferred by the arrangement of substituents on the B ring. Cells that had taken up anthocyanins also exhibited a resistance to the membrane permeabilization that occurs as a result of the detergent-like action of A2E. [source] Ca2+ -independent hypoxic vasorelaxation in porcine coronary arteryTHE JOURNAL OF PHYSIOLOGY, Issue 3 2005Min Gu To demonstrate a Ca2+ -independent component of hypoxic vasorelaxation and to investigate its mechanism, we utilized permeabilized porcine coronary arteries, in which [Ca2+] could be clamped. Arteries permeabilized with ,-escin developed maximum force in response to free Ca2+ (6.6 ,m), concomitant with a parallel increase in myosin regulatory light chain phosphorylation (MRLC-Pi), from 0.183 ± 0.023 to 0.353 ± 0.019 MRLC-Pi (total light chain),1. Hypoxia resulted in a significant decrease in both force (,31.9 ± 4.1% prior developed force) and MRLC-Pi (from 0.353 to 0.280 ± 0.023), despite constant [Ca2+] buffered by EGTA (4 mm). Forces developed in response to Ca2+ (6.6 ,m), Ca2+ (0.2 ,m) + GTP,S (1 mm), or in the absence of Ca2+ after treatment with ATP,S (1 mm), were of similar magnitude. Hypoxia also relaxed GTP,S contractures but importantly, arteries could not be relaxed after treatment with ATP,S. Permeabilization with Triton X-100 for 60 min also abolished hypoxic relaxation. The blocking of hypoxic relaxation after ATP,S suggests that this Ca2+ -independent mechanism(s) may operate through alteration of MRLC-Pi or of phosphorylation of the myosin binding subunit of myosin light chain phosphatase. Treatment with the Rho kinase inhibitor Y27632 (1 ,m) relaxed GTP,S and Ca2+ contractures; but the latter required a higher concentration (10 ,m) for consistent relaxation. Relaxations to N2 and/or Y27632 averaged 35% and were not additive or dependent on order. Our data suggest that the GTP-mediated, Rho kinase-coupled pathway merits further investigation as a potential site of this novel, Ca2+ -independent O2 -sensing mechanism. Importantly, these results unambiguously show that hypoxia-induced vasorelaxation can occur in permeabilized arteries where the Ca2+ is clamped at a constant value. [source] P2X7 receptors in rat parotid acinar cells: formation of large poresAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 4 2001Simon J. Gibbons 1 Permeabilization of cells mediated by P2X7 receptors occurs to varied degrees in native and heterologous expression systems. Previous studies on P2X7 receptors in parotid acinar cells suggested that ATP does not permeabilize these cells. 2 Modification of the assay conditions showed that ATP permeabilizes freshly dissociated rat parotid acinar cells to the fluorescent dye YOPRO-1. 3 The pharmacological and physiological properties of this effect indicate that permeabilization is mediated by the P2X7 receptor. Adenosine 5,-triphosphate (ATP) and 3,- O -(4-benzoyl)benzoyl adenosine 5,-triphosphate (BzBzATP) were effective agonists with EC50 values of 49.3 and 0.6 ,M, respectively. 4 Permeabilization was best observed in low divalent cation concentrations and at physiological temperatures. Previous studies failed to detect permeabilization because of the sensitivity of this effect to temperature and divalent cations. 5 An important consideration in understanding the effect of divalent cations is that the fluorescence of YOPRO-1/nucleic acid complexes is directly quenched by addition of divalent cations. This must be considered if quantitative study of the interaction of divalent cations with P2X7 receptors is carried out using fluorescent DNA-binding dyes. 6 In summary, our data show that P2X7 receptors in parotid acinar cells can form large pores in the plasma membrane. This property likely contributes to signalling and may be cytotoxic and have particular significance in damaged or inflamed salivary glands. [source] Role of mitochondrial ion channels in cell deathBIOFACTORS, Issue 4 2010Shin-Young Ryu Abstract Ion channels located in the outer and inner mitochondrial membranes are key regulators of cellular signaling for life and death. Permeabilization of mitochondrial membranes is one of the most critical steps in the progression of several cell death pathways. The mitochondrial apoptosis-induced channel (MAC) and the mitochondrial permeability transition pore (mPTP) play major roles in these processes. Here, the most recent progress and current perspectives about the roles of MAC and mPTP in mitochondrial membrane permeabilization during cell death are presented. The crosstalk signaling of MAC and mPTP formation/activation mediated by cytosolic Ca2+ signaling, Bcl-2 family proteins, and other mitochondrial ion channels is also discussed. Understanding the mechanisms that regulate opening and closing of MAC and mPTP has revealed new therapeutic targets that potentially could control cell death in pathologies such as cancer, ischemia/reperfusion injuries, and neurodegenerative diseases. [source] Purification and Concentration of Alkaline Phosphatase by Selective Permeabilization of Escherichia coli Using Reverse Micellar SolutionsBIOTECHNOLOGY PROGRESS, Issue 6 2003Ritu Bansal-Mutalik Recovery of alkaline phosphatase (AP) from the periplasm of Escherichia coli using reverse micellar solutions (RMSs) of sodium dioctyl sulfosuccinate (AOT) in aliphatic hydrocarbons has been attempted. A variety of surface-active agents, solvents, and reverse micellar conditions were screened, and an excellent recovery of the enzyme in a concentrated form, with a high purification factor, was obtained in a single-step process. The permeabilization process strongly depended on the water content of the RMS as well as on the amount of water coating the microbial cell surface. The product was almost free from nucleic acids. In addition, because of the low affinity of AOT and the organic solvent for the aqueous phase, contamination by the permeabilizing agents would also be negligible. [source] Membrane Permeabilization of a Mammalian Neuroendocrine Cell Type (PC12) by the Channel-Forming Peptides Zervamicin, Alamethicin, and GramicidinCHEMISTRY & BIODIVERSITY, Issue 6 2007Abstract Zervamicin IIB (ZER) is a 16-mer peptaibol that produces voltage-dependent conductances in artificial membranes, a property considered responsible for its antimicrobial activity to mainly Gram -positive microorganisms. In addition, ZER appears to inhibit the locomotor activity of the mouse (see elsewhere in this Issue), probably by affecting the brain. To examine whether the electrophysiological properties of the neuronal cells of the central neural system might be possibly influenced by the pore forming ZER, the present study was undertaken as a first attempt to unravel the molecular mechanism of this biological activity. To this end, membrane permeabilization of the neuron-like rat pheochromocytoma cell (PC12) by the channel-forming ZER was studied with the whole-cell patch-clamp technique, and compared with the permeabilizations of the well-known voltage-gated peptaibol alamethicin F50/5 (ALA) and the cation channel-forming peptide-antibiotic gramicidin D (GRAM). While 1,,M GRAM addition to PC12 cells kept at a membrane potential Vm=0,mV causes an undelayed gradual increase of a leak conductance with a negative reversal potential of ca. ,24,mV, ZER and ALA are ineffective at that concentration and potential. However, if ZER and ALA are added in 5,10,,M concentrations while Vm is kept at ,60,mV, they cause a sudden and strong permeabilization of the PC12 cell membrane after a delay of 1,2,min, usually leading to disintegrating morphology changes of the patched cell but not of the surrounding cells of the culture at that time scale. The zero reversal potential of the established conductance is consistent with the known aselectivity of the channels formed. This sudden permeabilization does not occur within 10,20,min at Vm=0,mV, in accordance with the known voltage dependency of ZER and ALA channel formation in artificial lipid membranes. The permeabilizing action of these peptaibols on the culture as a whole is further supported by K+ -release measurements from a PC12 suspension with a K+ -selective electrode. Further analysis suggested that the permeabilizing action is associated with extra- or intracellular calcium effects, because barium inhibited the permeabilizing effects of ZER and ALA. We conclude, for the membrane of the mammalian neuron-like PC12 cell, that the permeabilizing effects of the peptides ZER and ALA are different from those of GRAM, consistent with earlier studies of these peptides in other (artificial) membrane systems. They are increased by cis -positive membrane potentials in the physiological range and may include calcium entry into the PC12 cell. [source] Measurement of barbed ends, actin polymerization, and motility in live carcinoma cells after growth factor stimulation,CYTOSKELETON, Issue 4 2004Mike Lorenz Abstract Motility is associated with the ability to extend F-actin-rich protrusions and depends on free barbed ends as new actin polymerization sites. To understand the function and regulation of different proteins involved in the process of generating barbed ends, e.g., cofilin and Arp2/3, fixed cell approaches have been used to determine the relative barbed end concentration in cells. The major disadvantages of these approaches are permeabilization and fixation of cells. In this work, we describe a new live-cell time-lapse microscopy assay to determine the increase of barbed ends after cell stimulation that does not use permeabilization and provides a better time resolution. We established a metastatic carcinoma cell line (MTLn3) stably expressing GFP-,-actin at physiological levels. Stimulation of MTLn3 cells with epidermal growth factor (EGF) causes rapid and transient lamellipod protrusion along with an increase in actin polymerization at the leading edge, which can be followed in live cell experiments. By measuring the increase of F-actin at the leading edge vs. time, we were able to determine the relative increase of barbed ends after stimulation with a high temporal resolution. The F-actin as well as the barbed end concentration agrees well with published data for this cell line. Using this newly developed assay, a decrease in lamellipod extension and a large reduction of barbed ends was documented after microinjecting an anti-cofilin function blocking antibody. This assay has a high potential for applications where rapid changes in the dynamic filament population are to be measured. Cell Motil. Cytoskeleton 57:207,217, 2004. © 2004 Wiley-Liss, Inc. [source] CXCL10-induced cell death in neurons: role of calcium dysregulationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2006Yongjun Sui Abstract Chemokines play a key role in the regulation of central nervous system disease. CXCL10 over-expression has been observed in several neurodegenerative diseases, including multiple sclerosis, Alzheimer's disease and HIV-associated dementia. More recent studies by others and us have shown that CXCL10 elicits apoptosis in fetal neurons. The mechanism of CXCL10-mediated neurotoxicity, however, remains unclear. In this study, we provide evidence for the direct role of Ca2+ dysregulation in CXCL10-mediated apoptosis. We demonstrate that treatment of fetal neuronal cultures with exogenous CXCL10 produced elevations in intracellular Ca2+ and that this effect was modulated via the binding of CXCL10 to its cognate receptor, CXCR3. We further explored the association of intracellular Ca2+ elevations with the caspases that are involved in CXC10-induced neuronal apoptosis. Our data showed that increased Ca2+, which is available for uptake by the mitochondria, is associated with membrane permeabilization and cytochrome c release from this compartment. The released cytochrome c then activates the initiator active caspase-9. This initiator caspase sequentially activates the effector caspase-3, ultimately leading to apoptosis. This study identifies the temporal signaling cascade involved in CXCL10-mediated neuronal apoptosis and provides putative targets for pharmaceutical intervention of neurological disorders associated with CXCL10 up-regulation. [source] Involvement of caspase 1 and its activator Ipaf upstream of mitochondrial events in apoptosisFEBS JOURNAL, Issue 12 2006Subhash Thalappilly PTP-S2/TC45 is a nuclear protein tyrosine phosphatase that activates p53 and induces caspase 1-dependent apoptosis. We analyzed the role of ICE protease-activating factor (Ipaf), an activator of caspase 1 in p53-dependent apoptosis. We also determined the sequence of events that lead to apoptosis upon caspase 1 activation by Ipaf. PTP-S2 expression induced Ipaf mRNA in MCF-7 cells which was dependent on p53. PTP-S2-induced apoptosis was inhibited by a dominant-negative mutant of Ipaf and also by an Ipaf-directed short-hairpin RNA. Doxorubicin-induced apoptosis was potentiated by the expression of caspase 1 (but not by a catalytic mutant of caspase 1) and required endogenous Ipaf. Doxorubicin treatment of MCF-7 cells resulted in activation of exogenous caspase 1, which was partly dependent on endogenous Ipaf. An activated form of Ipaf induced caspase 1-dependent apoptosis that was inhibited by Bcl2 and also by a dominant inhibitor of caspase 9 (caspase 9s). Caspase 1-dependent apoptosis induced by doxorubicin was also inhibited by Bcl2 and caspase 9s, but caspase 1 activation by activated Ipaf was not inhibited by Bcl2. Mitochondrial membrane permeabilization was induced by caspase 1 and activated Ipaf, which was inhibited by Bcl2, but not by caspase 9s. Expression of caspase 1 with activated Ipaf resulted in the activation of Bax at mitochondria. Our results suggest that Ipaf is involved in PTP-S2-induced apoptosis and that caspase 1, when activated by Ipaf, causes release of mitochondrial proteins (cytochrome c and Omi) through Bax activation, thereby functioning as an initiator caspase. [source] Detergent-resistant membranes are platforms for actinoporin pore-forming activity on intact cellsFEBS JOURNAL, Issue 4 2006Jorge Alegre-Cebollada Sticholysin II is a pore-forming toxin produced by the sea anemone Stichodactyla helianthus. We studied its cytolytic activity on COS-7 cells. Fluorescence spectroscopy and flow cytometry revealed that the toxin permeabilizes cells to propidium cations in a dose-dependent and time-dependent manner. This permeabilization is impaired by preincubation of cells with cyclodextrin. Isolation of detergent-resistant cellular membranes showed that sticholysin II colocalizes with caveolin-1 in fractions corresponding to raft-like domains. The interaction of sticholysin II with such domains is only lipid dependent as it also occurs in the absence of any other membrane-associated protein. Toxin binding to raft-like lipid vesicles inhibited cell permeabilization. The results suggest that sticholysin II promotes pore formation in COS-7 cells through interaction with membrane domains which behave like cellular rafts. [source] Interaction of ostreolysin, a cytolytic protein from the edible mushroom Pleurotus ostreatus, with lipid membranes and modulation by lysophospholipidsFEBS JOURNAL, Issue 6 2003Kristina Sep Ostreolysin is a 16-kDa cytolytic protein specifically expressed in primordia and fruiting bodies of the edible mushroom Pleurotus ostreatus. To understand its interaction with lipid membranes, we compared its effects on mammalian cells, on vesicles prepared with either pure lipids or total lipid extracts, and on dispersions of lysophospholipids or fatty acids. At nanomolar concentrations, the protein lysed human, bovine and sheep erythrocytes by a colloid-osmotic mechanism, compatible with the formation of pores of 4 nm diameter, and was cytotoxic to mammalian tumor cells. A search for lipid inhibitors of hemolysis revealed a strong effect of lysophospholipids and fatty acids, occurring below their critical micellar concentration. This effect was distinct from the capacity of ostreolysin to bind to and permeabilize lipid membranes. In fact, permeabilization of vesicles occurred only when they were prepared with lipids extracted from erythrocytes, and not with lipids extracted from P. ostreatus or pure lipid mixtures, even if lysophospholipids or fatty acids were included. Interaction with lipid vesicles, and their permeabilization, correlated with an increase in the intrinsic fluorescence and ,-helical content of the protein, and with aggregation, which were not detected with lysophospholipids. It appears that either an unknown lipid acceptor or a specific lipid complex is required for binding, aggregation and pore formation. The inhibitory effect of lysophospholipids may reflect a regulatory role for these components on the physiological action of ostreolysin and related proteins during fruiting. [source] Achieving efficient delivery of morpholino oligos in cultured cellsGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 3 2001Paul A. Morcos Abstract Summary: One of the many features that make morpholino oligos unique among the antisense structural types is an uncharged backbone. While this feature eliminates the nonspecific interactions of traditional S-oligos, it also renders the morpholino undeliverable via the traditional lipid-based delivery systems. This article describes a highly efficient method of delivering morpholino oligos into adherent and nonadherent cultured cells. In this system, a nonionic morpholino oligo is paired to a complementary DNA "carrier." The DNA is then bound electrostatically to a partially ionized, weakly-basic ethoxylated polyethylenimine (EPEI). This morpholino/DNA/EPEI complex is efficiently endocytosed, and when the pH drops within the endosome, the EPEI more fully ionizes, resulting in permeabilization of the endosomal membrane and release of the morpholino into the cytosol. This article describes optimization of delivery in HeLa cells and provides the basis for delivery in any cultured endocytic cell type. genesis 30:94,102, 2001. © 2001 Wiley-Liss, Inc. [source] Involvement of adenylate cyclase and tyrosine kinase signaling pathways in response of crayfish stretch receptor neuron and satellite glia cell to photodynamic treatmentGLIA, Issue 3 2005Anatoly Uzdensky Abstract Neuroglial interactions are most profound during development or damage of nerve tissue. We studied the responses of crayfish stretch receptor neurons (SRN) and satellite glial cells to photosensitization with sulfonated aluminum phthalocyanine Photosens. Although Photosens was localized mainly in the glial envelope, neurons were very sensitive to photodynamic treatment. Photosensitization gradually inhibited and then abolished neuron activity. Neuronal and glial nuclei shrank. Some neurons and glial cells lost the integrity of the plasma membrane and died through necrosis after the treatment. The nuclei of other glial cells but not neurons become fragmented, indicating apoptosis. The number of glial nuclei around neuron soma increased, probably indicating proliferation for enhanced neuron protection. Adenylate cyclase (AC) inhibition by MDL-12330A, or tyrosine kinase (TK) inhibition by genistein, shortened neuron lifetime, whereas AC activation by forskolin or protein tyrosine phosphatases (PTP) inhibition by sodium orthovanadate prolonged neuronal activity. Therefore, cAMP and phosphotyrosines produced by AC and TK, respectively, protected SRN against photoinactivation. AC inhibition reduced photodamage of the plasma membrane and subsequent necrosis in neuronal and glial cells. AC activation prevented apoptosis in photosensitized glial cells and stimulated glial proliferation. TK inhibition protected neurons but not glia against photoinduced membrane permeabilization and subsequent necrosis whereas PTP inhibition more strongly protected glial cells. Therefore, both signaling pathways involving cAMP and phosphotyrosines might contribute to the maintenance of neuronal activity and the integrity of the neuronal and glial plasma membranes. Adenylate cyclase but not phosphotyrosine signaling pathways modulated glial apoptosis and proliferation under photooxidative stress. © 2004 Wiley-Liss, Inc. [source] Mitochondrial protection by the JNK inhibitor leflunomide rescues mice from acetaminophen-induced liver injury,HEPATOLOGY, Issue 2 2007Calivarathan Latchoumycandane Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses but which can precipitate liver injury at high doses. We have previously found that the antirheumatic drug leflunomide is a potent inhibitor of APAP toxicity in cultured human hepatocytes, protecting them from mitochondria-mediated cell death by inhibiting the mitochondrial permeability transition. The purpose of this study was to explore whether leflunomide protects against APAP hepatotoxicity in vivo and to define the molecular pathways of cytoprotection. Male C57BL/6 mice were treated with a hepatotoxic dose of APAP (750 mg/kg, ip) followed by a single injection of leflunomide (30 mg/kg, ip). Leflunomide (4 hours after APAP dose) afforded significant protection from liver necrosis as assessed by serum ALT activity and histopathology after 8 and 24 hours. The mechanism of protection by leflunomide was not through inhibition of cytochrome P450 (CYP),catalyzed APAP bioactivation or an apparent suppression of the innate immune system. Instead, leflunomide inhibited APAP-induced activation (phosphorylation) of c-jun NH2 -terminal protein kinase (JNK), thus preventing downstream Bcl-2 and Bcl-XL inactivation and protecting from mitochondrial permeabilization and cytochrome c release. Furthermore, leflunomide inhibited the APAP-mediated increased expression of inducible nitric oxide synthase and prevented the formation of peroxynitrite, as judged from the absence of hepatic nitrotyrosine adducts. Even when given 8 hours after APAP dose, leflunomide still protected from massive liver necrosis. Conclusion: Leflunomide afforded protection against APAP-induced hepatotoxicity in mice through inhibition of JNK-mediated activation of mitochondrial permeabilization. (HEPATOLOGY 2007.) [source] Involvement of Ca2+ and ROS in ,-tocopheryl succinate-induced mitochondrial permeabilizationINTERNATIONAL JOURNAL OF CANCER, Issue 8 2010Vladimir Gogvadze Abstract Release of mitochondrial proteins such as cytochrome c, AIF, Smac/Diablo etc., plays a crucial role in apoptosis induction. A redox-silent analog of vitamin E, ,-tocopheryl succinate (,-TOS), was shown to stimulate cytochrome c release via production of reactive oxygen species (ROS) and Bax-mediated permeabilization of the outer mitochondrial membrane. Here we show that ,-TOS facilitates mitochondrial permeability transition (MPT) in isolated rat liver mitochondria, Tet21N neuroblastoma cells and Jurkat T-lymphocytes. In particular, in addition to ROS production, ,-TOS stimulates rapid Ca2+ entry into the cells with subsequent accumulation of Ca2+ in mitochondria,a prerequisite step for MPT induction. Alteration of mitochondrial Ca2+ buffering capacity was observed as early as 8 hr after incubation with ,-TOS, when no activation of Bax was yet detected. Ca2+ accumulation in mitochondria was important for apoptosis progression, since inhibition of mitochondrial Ca2+ uptake significantly mitigated the apoptotic response. Importantly, Ca2+ -induced mitochondrial destabilization might cooperate with Bax-mediated mitochondrial outer membrane permeabilization to induce cytochrome c release from mitochondria. [source] Flow cytometric method for simultaneous assay of foetal haemoglobin containing red cells, reticulocytes and foetal haemoglobin containing reticulocytesINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 3 2001Y. Mundee Level of foetal haemoglobin (HbF) containing red cells (F cells) is a parameter for monitoring sickle cell anaemia (SS) patients undergoing treatment with HbF modulating drugs (including hydroxyurea (HU)). One convenient technique for F cell assay is flow cytometry. A flow cytometric method for the simultaneous assay of F cells, reticulocytes and HbF-containing reticulocytes (F reticulocytes) is described in this paper. These three parameters can be obtained within 2 h using double colour staining flow cytometry. Glutaraldehyde fixation, Triton X-100 permeabilization, monoclonal antibody to HbF Tri-colour® conjugate (MoAb-HbF-TC; deep-red fluorescence) immuno-staining and thiazole orange (TO; green fluorescence) are employed. The red cell gate was set on forward scatter (FSC) and logarithmic side scatter (logSSC) for 50 000 cells on the flow cytometer. Fluorescent signals were acquired from fluorescent channel 1 (FL1; green) and (FL4; deep-red). Coefficient of variation percent (%CVs) of intra- and inter-assay were less than 9% and 15%, respectively. EDTA, citrate, heparin and CTAD anticoagulants are all suitable; the samples can be stored at 4°C for up to 6 days. The method is a simple, rapid, convenient, reproducible and useful way of determining F cell, reticulocyte and F reticulocyte levels in sickle cell and thalassaemic patients. [source] Nitrosative stress induced cytotoxicity in Giardia intestinalisJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2003D. Lloyd Abstract Aims: To investigate the antigiardial properties of the nitrosating agents: sodium nitrite, sodium nitroprusside and Roussin's black salt. Methods and Results: Use of confocal laser scanning microscopy and flow cytometry indicated permeabilization of the plasma membrane to the anionic fluorophore, DiBAC4(3) [bis(1,3-dibutylbarbituric acid) trimethine oxonol]. Loss of plasma membrane electrochemical potential was accompanied by loss of regulated cellular volume control. Changes in ultrastructure revealed by electron microscopy and capacity for oxygen consumption, were also consequences of nitrosative stress. Roussin's black salt (RBS), active at micromolar concentrations was the most potent of the three agents tested. Conclusions: These multitargeted cytotoxic agents affected plasma membrane functions, inhibited cellular functions in Giardia intestinalis and led to loss of viability. Significance and Impact of the Study: Nitrosative damage, as an antigiardial strategy, may have implications for development of chemotherapy along with suggesting natural host defence mechanisms. [source] Mitochondria, the killer organelles and their weaponsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2002Luigi Ravagnan Apoptosis is a cell-autonomous mode of death that is activated to eradicate superfluous, damaged, mutated, or aged cells. In addition to their role as the cell's powerhouse, mitochondria play a central role in the control of apoptosis. Thus, numerous pro-apoptotic molecules act on mitochondria and provoke the permeabilization of mitochondrial membranes. Soluble proteins contained in the mitochondrial intermembrane space are released through the outer membrane and participate in the organized destruction of the cell. Several among these lethal proteins can activate caspases, a class of cysteine proteases specifically activated in apoptosis, whereas others act in a caspase-independent fashion, by acting as nucleases (e.g., endonuclease G), nuclease activators (e.g., apoptosis-inducing factor), or serine proteases (e.g., Omi/HtrA2). In addition, mitochondria can generate reactive oxygen species, following uncoupling and/or inhibition of the respiratory chain. The diversity of mitochondrial factors participating in apoptosis emphasizes the central role of these organelles in apoptosis control and unravels novel mechanisms of cell death execution. © 2002 Wiley-Liss, Inc. [source] OPTIMIZATION OF PERMEABILIZATION PROCESS FOR LACTOSE HYDROLYSIS IN WHEY USING RESPONSE SURFACE METHODOLOGYJOURNAL OF FOOD PROCESS ENGINEERING, Issue 3 2009GURPREET KAUR ABSTRACT To overcome the permeability barrier and prepare whole cell biocatalysts with high activities, permeabilization of Kluyveromyces marxianus var. lactis NCIM 3566 in relation to, -galactosidase activity was optimized using cetyltrimethylammonium bromide (CTAB) as permeabilizing agent. Permeabilized whole cells can be advantageous over pure enzyme preparations in terms of cost-effectiveness and increased stability maintained by the intracellular environment. Response surface methodology (RSM) was applied to optimize concentration of CTAB, temperature and the treatment time for maximum permeabilization of yeast cells. The optimum operating conditions for permeabilization process to achieve maximum enzyme activity obtained by RSM were 0.06% (w/v) CTAB concentration, 28C temperature and process duration of 14 min. At these conditions of process variables, the maximum value of enzyme activity was found to be 1,334 IU/g. The permeabilized yeast cells were highly effective and resulted in 90.5% lactose hydrolysis in whey. PRACTICAL APPLICATION , -Galactosidase is one of the most promising enzymes, which has several applications in the food, fermentation and dairy industry. However, the industrial applications of , -galactosidase have been hampered by the costs involved in downstream processing. The present investigation was focused on developing the low-cost technology for lactose hydrolysis based on permeabilization process. Disposal of lactose in whey and whey permeates is one of the most significant problems with regard to economics and environmental impact faced by the dairy industries. Keeping this in view, lactose hydrolysis in whey has been successfully performed using permeabilized Kluyveromyces marxianus cells. Hydrolysis of lactose using , -galactosidase converts whey into a potentially very useful food ingredient, which has immense applications in food industries. Its use has increased significantly in recent years, mainly in the dairy products and in digestive preparations. Lactose hydrolysis causes several potential changes in the manufacture and marketing of dairy products, including increased solubility, sweetness and broader fermentation possibilities. [source] Onion Cells After High Pressure and Thermal Processing: Comparison of Membrane Integrity Changes Using Different Analytical Methods and Impact on Tissue TextureJOURNAL OF FOOD SCIENCE, Issue 7 2010Maria E. Gonzalez Abstract:, Two different analytical methods were evaluated for their capacity to provide quantitative information on onion cell membrane permeability and integrity after high pressure and thermal processing and to study the impact of these processing treatments on cell compartmentalization and texture quality. To determine changes in cell membrane permeability and/or integrity the methodologies utilized were: (1) measurement of a biochemical product, pyruvate, formed as a result of membrane permeabilization followed by enzymatic activity and (2) leakage of electrolytes into solution. These results were compared to previously determined methods that quantified cell viability and 1H-NMR T2 of onions. These methods allowed for the monitoring of changes in the plasma and tonoplast membranes after high pressure or thermal processing. High pressure treatments consisted of 5 min holding times at 50, 100, 200, 300, or 600 MPa. Thermal treatments consisted of 30 min water bath exposure to 40, 50, 60, 70, or 90 °C. There was strong agreement between the methods in the determination of the ranges of high pressure and temperature that induce changes in the integrity of the plasma and tonoplast membranes. Membrane rupture could clearly be identified at 300 MPa and above in high pressure treatments and at 60 °C and above in the thermal treatments. Membrane destabilization effects could already be visualized following the 200 MPa and 50 °C treatments. The texture of onions was influenced by the state of the membranes and was abruptly modified once membrane integrity was lost. Practical Application:, In this study, we used chemical, biochemical, and histological techniques to obtain information on cell membrane permeability and onion tissue integrity after high pressure and thermal processing. Because there was strong agreement between the various methods used, it is possible to implement something relatively simple, such as ion leakage, into routine quality assurance measurements to determine the severity of preservation methods and the shelf life of processed vegetables. [source] Ca2+ -induced permeabilization promotes free radical release from rat brain mitochondria with partially inhibited complex IJOURNAL OF NEUROCHEMISTRY, Issue 3 2005Tatyana V. Votyakova Abstract Mitochondrial complex I dysfunction has been implicated in a number of brain pathologies, putatively owing to an increased rate of reactive oxygen species (ROS) release. However, the mechanisms regulating the ROS burden are poorly understood. In this study we investigated the effect of Ca2+ loads on ROS release from rat brain mitochondria with complex I partially inhibited by rotenone. The addition of 20 nm rotenone to brain mitochondria increased ROS release. Ca2+ (100 µm) alone had no effect on ROS release, but greatly potentiated the effects of rotenone. The effect of Ca2+ was decreased by ruthenium red. Ca2+ -challenged mitochondria lose about 88% of their glutathione and 46% of their cytochrome c under these conditions, although this depends only on Ca2+ loading and not complex I inhibition. ADP in combination with oligomycin decreased the loss of glutathione and cytochrome c and free radical generation. Cyclosporin A alone was ineffective in preventing these effects, but augmented the protection provided by ADP and oligomycin. Non-specific permeabilization of mitochondria with alamethicin also increased the ROS signal, but only when combined with partial inhibition of complex I. These results demonstrate that Ca2+ can greatly increase ROS release by brain mitochondria when complex I is impaired. [source] Mitogenic effects of phospholipase D and phosphatidic acid in transiently permeabilized astrocytes: effects of ethanolJOURNAL OF NEUROCHEMISTRY, Issue 1 2003Beate Schatter Abstract Investigations of lipid-mediated signalling pathways are often limited by a lack of methods for the intracellular delivery of lipid messengers. We established a procedure for the transient permeabilization of astrocytes by an oxygen-insensitive mutant of streptolysin-O (SLO) to investigate the participation of the phospholipase D (PLD) signalling pathway in astroglial cell proliferation. Exogenous PLD, when incubated in the presence of SLO, caused an increase in DNA synthesis (measured by thymidine incorporation) which was completely suppressed by ethanol (0.3%, v/v). In parallel experiments, phosphatidic acid also induced a dose-dependent mitogenic response which, however, was not affected by the presence of ethanol. Phosphatidic acid was more effective in this assay than diacylglycerol but its effect was sensitive to the protein kinase inhibitor Ro 31-8220. Our findings provide direct evidence that disruption of the PLD signalling pathway by ethanol is sufficient to suppress astroglial proliferation, an effect that might contribute to the inhibition of brain growth in alcoholic embryopathy. [source] Antimicrobial peptide interactions with silica bead supported bilayers and E. coli: buforin II, magainin II, and arenicin,JOURNAL OF PEPTIDE SCIENCE, Issue 8 2009Ryan W. Davis Abstract Using the unique quantitative capabilities of hyperspectral confocal microscopy combined with multivariate curve resolution, a comparative approach was employed to gain a deeper understanding of the different types of interactions of antimicrobial peptides (AMPs) with biological membranes and cellular compartments. This approach allowed direct comparison of the dynamics and local effects of buforin II, magainin II, and arenicin with nanoporous silica bead supported bilayers and living E. coli. Correlating between experiments and comparing these responses have yielded several important discoveries for pursuing the underlying biophysics of bacteriocidal specificity and the connection between structure and function in various cellular environments. First, a novel fluorescence method for direct comparison of a model and living system is demonstrated by utilizing the membrane partitioning and environmental sensitivity of propidium iodide. Second, measurements are presented comparing the temporal dynamics and local equilibrium concentrations of the different antimicrobial agents in the membrane and internal matrix of the described systems. Finally, we discuss how the data lead to a deeper understanding of the roles of membrane penetration and permeabilization in the action of these AMPs. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. [source] The role of surfactants in the reversal of active transport mediated by multidrug resistance proteinsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 6 2003Katrijn Bogman Abstract A variety of seven nonionic, one amphoteric and, one anionic surfactant that are applied or investigated as surfactants in drug formulation, were analyzed for their capacity to modulate carrier-mediated transport by efflux pumps. Two cell lines, murine monocytic leukemia cells overexpressing P-glycoprotein (P-gp) and Madin-Darby canine kidney cells stably overexpresssing human multidrug resistance-associated protein 2 (MRP2), were used as test systems. The modulation of P-gp and of MRP2 function was studied by the reversal of rhodamine 123 and of methylfluorescein-glutathione conjugate transport, respectively. Mechanisms that were not transporter related and could lead to misinterpretations were identified, such as probe quenching, probe encapsulation by micelles, and membrane damage. P-gp-mediated rhodamine 123 transport was inhibited by five nonionic surfactants in a concentration-dependent manner and in the order TPGS,>,Pluronic PE8100,>,Cremophor EL,>,Pluronic PE6100,,,Tween 80. In contrast, none of the surfactants showed a significant inhibition of MRP2-mediated efflux in Madin-Darby canine kidney/MRP2 cells. In conclusion, the results indicate that surfactants demonstrate a transporter-specific interaction, rather than unspecific membrane permeabilization. The present analysis offers insight in the possible mechanisms of surfactant interactions with biological membranes and could help to identify specific drug formulations. © 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:1250,1261, 2003 [source] Intracellular uptake and trafficking of Pluronic micelles in drug-sensitive and MDR cells: Effect on the intracellular drug localizationJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 1 2002Natalya Rapoport Abstract The intracellular uptake and localization of a fluorescently labeled Pluronic P-105 in HL-60 leukemia cells and in A2780 drug-sensitive and A2780/ADR MDR ovarian carcinoma cells were characterized by flow cytometry and fluorescence microscopy. Pluronic P-105 molecules were labeled with a pH-sensitive fluorescent label, 5-(and 6-)carboxy-2,7,-dichlorofluorescein. The fluorescence intensity of labeled Pluronic was about twofold higher at pH 7.4 than at pH 5.5. At Pluronic concentrations exceeding the critical micelle concentration (cmc), flow cytometry histograms manifested bimodal distribution of cell fluorescence for all types of cells. Cell population characterized by higher fluorescence intensity presumably resulted from Pluronic transfer from the acidic environment of cytoplasmic vesicles (endosomes or lysosomes) into the neutral environment of the cytoplasm and cell nuclei, which suggested the permeabilization of the membranes of acidic vesicle by Pluronic molecules. For the MDR cells, the bimodal distribution of cell fluorescence was already observed at very low Pluronic concentrations in the incubation medium (i.e., below the cmc). The data suggest that the membranes of acidic vesicles of MDR cells are more susceptible to the action of polymeric surfactants than those of drug-sensitive cells. Permeabilization of acidic vesicles had a dramatic effect on the intracellular trafficking of drugs: when delivered in PBS, the anthracyclin drug ruboxyl (Rb) sequestered in cytoplasmic vesicles and was excluded from cell nuclei; however, when delivered in Pluronic micelles, drug accumulated in cell nuclei. Drug uptake from/with Pluronic micelles was substantially enhanced by ultrasound. These findings suggest that the nuclear accumulation of drugs internalized via fluid-phase endocytosis can be enhanced by the application of Pluronic micelles and can be further augmented by ultrasonic irradiation. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:157,170, 2002 [source] Alcohol and Mitochondria in Cardiac Apoptosis: Mechanisms and VisualizationALCOHOLISM, Issue 5 2005György Hajnóczky Apoptosis of myocytes is likely to contribute to a variety of heart conditions and could also be important in the development of alcoholic heart disease. A fundamental pathway to apoptosis is through mitochondrial membrane permeabilization and release of proapoptotic factors from the mitochondrial intermembrane space to the cytosol. The authors' results show that prolonged exposure of cultured cardiac cells to ethanol (35 mM for 48 hr) promotes Ca2+ -induced activation of the mitochondrial permeability transition pore (PTP). PTP-dependent mitochondrial membrane permeabilization is followed by release of cytochrome c and execution of apoptosis. The authors propose that chronic ethanol exposure, in combination with other stress signals, may allow for activation of the PTP by physiological calcium oscillations, providing a trigger for cardiac apoptosis during chronic alcohol abuse. Coincidence of apoptosis promoting factors occurs in only a small fraction of myocytes, but because of the absence of regeneration, even a modest increase in the rate of cell death may contribute to a decrease in cardiac contractility. Detection of apoptotic changes that are present in only a few myocytes at a certain time in the heart is not feasible with most of the apoptotic assays. Fluorescence imaging is a powerful technology to visualize changes that are confined to a minor fraction of cells in a tissue, and the use of multiphoton excitation permits imaging in situ deep in the wall of the intact heart. This article discusses potential mechanisms of the effect of alcohol on mitochondrial membrane permeabilization and visualization of mitochondria-dependent apoptosis in cardiac muscle. [source] A novel lysis system in PM2, a lipid-containing marine double-stranded DNA bacteriophageMOLECULAR MICROBIOLOGY, Issue 6 2007Mart Krupovi Summary In this study we investigated the lysis system of the lipid-containing double-stranded DNA bacteriophage PM2 infecting Gram-negative marine Pseudoalteromonas species. We analysed wt and lysis-deficient phage-induced changes in the host physiology and ascribed functions to two PM2 gene products (gp) involved in lysis. We show that bacteriophage PM2 uses a novel system to disrupt the infected cell. The novelty is based on the following findings: (i) gp k is needed for the permeabilization of the cytoplasmic membrane and appears to play the role of a typical holin. However, its unique primary structure [53 aa, 1 transmembrane domain (TMD)] places it into a new class of holins. (ii) We have proposed that, unlike other bacteriophages studied, PM2 relies on lytic factors of the cellular origin for digestion of the peptidoglycan. (iii) gp l (51 aa, no TMDs) is needed for disruption of the outer membrane, which is highly rigidified by the divalent cations abundant in the marine environment. The gp l has no precedent in other phage lytic systems studied so far. However, the presence of open reading frame l-like genes in genomes of other bacterial viruses suggests that the same system might be used by other phages and is not unique to PM2. [source] Anthocyanins Protect Against A2E Photooxidation and Membrane Permeabilization in Retinal Pigment Epithelial Cells,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2005Young P. Jang ABSTRACT The pyridinium bisretinoid A2E, an autofluorescent pigment that accumulates in retinal pigment epithelial cells with age and in some retinal disorders, can mediate a detergent-like perturbation of cell membranes and light-induced damage to the cell. The photodynamic events initiated by the sensitization of A2E include the generation of singlet oxygen and the oxidation of A2E at carbon-carbon double bonds. To assess the ability of plant-derived anthocyanins to modulate adverse effects of A2E accumulation on retinal pigment epithelium (RPE) cells, these flavylium salts were isolated from extracts of bilberry. Nine anthocyanin fractions reflecting monoglycosides of delphinidin, cyanidin, petunidin and malvidin were obtained and all were shown to suppress the photooxidation of A2E at least in part by quenching singlet oxygen. The anthocyanins tested exhibited antioxidant activity of variable efficiency. The structural characteristics relevant to this variability likely included the ability to form a stable quinonoidal anhydro base at neutral pH, a conjugated diene structure in the C (pyrane) ring, the presence of hydroxyl groups on the B (benzene) ring and the relative hydrophobicity conferred by the arrangement of substituents on the B ring. Cells that had taken up anthocyanins also exhibited a resistance to the membrane permeabilization that occurs as a result of the detergent-like action of A2E. [source] | |||||||||||||||||||||||||||||||