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ABSTRACT: The connection between the action of microbial RNases and Ca2+-activated K+ (KCa) channels was investigated in human embryo kidney cells HEKhSK4 artificially expressing the channels. These channels protected HEKhSK4 cells from apoptosis induced by binase and 5K charge reversal mutant of RNase Sa. After the first 24h, potassium current increased without increase in intracellular Ca2+, and mitochondrial potential remained high. After 72 h, the concentration of calcium increased and mitochondria lost their potential. Whole-cell recordings of membrane currents through KCa channels in RNase-treated cells demonstrated a biphasic pattern: initially their activity in cell population increased, peaked at 24h, and then gradually decreased. In each individual cell we observed either an increase of the amplitude of KCa current, or a complete shutdown of the channels. The activity of KCa channels could be restored by removing RNases from the media. Based on this pattern and especially its timing, we hypothesize that toxic RNases downregulate KCa channels at the level of transcription or translation. Our results indicate that new anticancer agents could be created on the basis of microbial RNases targeting KCa channels.
Biochimie 06/2008; 90(5):717-25. · 3.02 Impact Factor
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ABSTRACT: Escherichia coli alpha-hemolysin (HlyA), archetype of a bacterial pore-forming toxin, has been reported to deregulate physiological Ca2+ channels, thus inducing periodic low-frequency Ca2+ oscillations that trigger transcriptional processes in mammalian cells. The present study was undertaken to delineate the mechanisms underlying the Ca2+ oscillations. Patch-clamp experiments were combined with single cell measurements of intracellular Ca2+ and with flowcytometric analyses. Application of HlyA at subcytocidal concentrations provoked Ca2+ oscillations in human renal and endothelial cells. However, contrary to the previous report, the phenomenon could not be inhibited by the Ca2+ channel blocker nifedipine and Ca2+ oscillations showed no constant periodicity at all. Ca2+ oscillations were dependent on the pore-forming activity of HlyA: application of a nonhemolytic but bindable toxin had no effect. Washout experiments revealed that Ca2+ oscillations could not be maintained in the absence of toxin in the medium. Analogously, propidium iodide flux into cells occurred in the presence of HlyA, but cells rapidly became impermeable toward the dye after toxin washout, indicating resealing or removal of the membrane lesions. Finally, patch-clamp experiments revealed temporal congruence between pore formation and Ca2+ influx. We conclude that the nonperiodic Ca2+ oscillations induced by HlyA are not due to deregulation of physiological Ca2+ channels but derive from pulsed influxes of Ca2+ as a consequence of formation and rapid closure of HlyA pores in mammalian cell membranes.
The FASEB Journal 06/2006; 20(7):973-5. · 5.71 Impact Factor
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ABSTRACT: Recently, class II fusion proteins have been identified on the surface of alpha- and flaviviruses. These proteins have two functions besides membrane fusion: they generate an isometric lattice on the viral surface and they form ion-permeable pores at low pH. An attempt was made to identify inhibitors for the ion pores generated by the fusion proteins of the alphaviruses Semliki Forest virus and Sindbis virus. These pores can be detected and analysed in three situations: (i) in the target membrane during virus entry, by performing patch-clamp measurements of membrane currents; (ii) in the virus particle, by studying the entry of propidium iodide; and (iii) in the plasma membrane of infected cells, by Fura-2 fluorescence imaging of Ca2+ entry into infected cells. It is shown here that, at a concentration of 0.1 mM, rare earth ions block the ion permeability of alphavirus ion pores in all three situations. Even at a concentration of 0.5 mM, these ions do not block formation of the viral fusion pore, as they do not inhibit entry or multiplication of alphaviruses. The data indicate that ions flow through the ion pores into the virus particle in the endosome and from the endosome into the cytoplasm after fusion of the viral envelope with the endosomal membrane. These ion flows, however, are not necessary for productive infection. The possibility that the ability of class II fusion proteins to form ion-permeable pores reflects their origin from protein toxins that form ion-permeable pores, and that entry via class II fusion proteins may resemble the entry of non-enveloped viruses, is discussed.
Journal of General Virology 01/2006; 86(Pt 12):3311-20. · 3.36 Impact Factor
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ABSTRACT: Cystic fibrosis (CF) is one of the most common monogenic disorders in the caucasian population. Gene therapy for CF is principally feasible and bacterial transfer systems might provide novel possibilities for therapy. However, transfection efficiencies are low and need to be improved. Thus, more detailed understanding of the DNA transfer mechanism is necessary to systematically eliminate these restrictions.
Functional transfer of GFP-CFTR (cystic fibrosis transmembrane conductance regulator) to eukaryotic cells using attenuated Listeria monocytogenes mediated gene transfer (bacteriofection) was shown by fluorescent microscopy, flow cytometry, immunoblotting and whole cell patch clamping. The characteristics of plasmid transfer were studied by use of electron and fluorescence microscopy, flow cytometry and Southern blotting. Polymerase chain reaction (PCR) was used to screen the genome of bacteriofected cells for cotransfer of chromosomal bacterial DNA.
Correct intracellular localization and functionality of the GFP-CFTR fusion protein after bacteriofection was shown. Efficient bacterial lysis and release of bacterial content was demonstrated using antibiotics to kill intracellular bacteria. Although only low transfection rates were observed, high numbers of transferred plasmids were detected in host cells under these conditions. However, they were associated with high molecular weight entities and not available to cytosolic transcription. Cotransfer of bacterial genomic DNA was observed in transfectants but occurred at low frequencies.
In this work we demonstrate that low rates of bacteria-mediated transfection are not due to poor invasion of bacteria, insufficient bacterial lysis, or plasmid DNA degradation. Our data suggest that the transferred plasmid DNA is associated with higher macromolecular structures inhibiting nuclear transport and transgene transcription.
The Journal of Gene Medicine 09/2005; 7(8):1097-112. · 2.48 Impact Factor
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ABSTRACT: Studies using the alphavirus Semliki Forest virus have indicated that the viral E1 fusion protein forms two types of pore: fusion pores and ion-permeable pores. The formation of ion-permeable pores has not been generally accepted, partly because it was not evident how the protein might form these different pores. Here it is proposed that the choice of the target membrane determines whether a fusion pore or ion-permeable pores are formed. The fusion protein is activated in the endosome and for steric reasons only a fraction of the activated molecules can interact with the endosomal membrane. This target membrane reaction forms the fusion pore. It is proposed that the rest of the activated molecules interact with the membrane in which the protein is anchored and that this self-membrane reaction leads to formation of ion-permeable pores, which can be detected in the target membrane after fusion of the viral membrane into the target membrane.
Journal of General Virology 07/2004; 85(Pt 6):1695-701. · 3.36 Impact Factor
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ABSTRACT: K(Ca) channels are involved in control of cell proliferation and differentiation. Here we have revealed their role in overcoming the RNase-induced cytotoxicity. Toxic effects of Streptomyces aureofaciens RNases Sa, Sa2, Sa3, and of RNase Sa charge reversal mutants on the human embryonic kidney cell lines differing only by the presence of K(Ca) channels were characterized. In contrast to other RNases, a basic variant of RNase Sa and RNase Sa3 exhibit significant cytotoxic activity of the same order of magnitude as onconase. Our data indicate the absence of a correlation between catalytic activity and stability of RNases and cytotoxicity. On the other hand, cationization enhances toxic effect of an RNase indicating the major role of a positive charge. Essentially lower sensitivity to cytotoxic microbial RNases of cells expressing K(Ca) channels was found. These results suggest that cells without the K(Ca) channel activity cannot counteract toxic effect of RNases.
Biochemical and Biophysical Research Communications 03/2004; 314(2):550-4. · 2.48 Impact Factor
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ABSTRACT: Recently, we presented evidence that the E1 fusion protein of the alphavirus Semliki Forest virus forms ion-permeable pores in the target membrane after fusion. We proposed that the homologous fusion proteins of flaviviruses and hepatitis C virus form similar pores. To test this hypothesis for the E fusion protein of flaviviruses, the release of [(3)H]choline from liposomes by the flavivirus West Nile (WN) virus was determined. [(3)H]Choline was released at mildly acid pH. The pH threshold depended on the lipid composition. Release from certain liposomes was activated even at neutral pH. To identify the generation of individual pores, single cells were investigated with the patch-clamp technique. The formation of individual pores during low pH-induced WN virus entry at the plasma membrane occurred within seconds. These experiments were performed in parallel with Semliki Forest virus. The results indicated that, similar to alphavirus infection, infection with flaviviruses via endosomes leads to the formation of ion-permeable pores in the endosome after fusion, which allows the flow of protons from the endosome into the cytoplasm during virus entry. However, in vitro translation experiments of viral cores showed that, in contrast to alphaviruses, which probably need this proton flow for core disassembly, the genome RNA of WN virus present in the viral core is directly accessible for translation. For entry of flaviviruses, therefore, a second pathway for productive infection may exist, in which fusion of the viral membrane is activated at neutral pH by contact with a plasma membrane of appropriate lipid composition.
Journal of General Virology 08/2003; 84(Pt 7):1711-21. · 3.36 Impact Factor
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ABSTRACT: Listeriolysin (LLO) is a major virulence factor of Listeria monocytogenes, a Gram-positive bacterium that can cause life-threatening diseases. Various signalling events and cellular effects, including modulation of gene expression, are triggered by LLO through unknown mechanisms. Here, we demonstrate that LLO applied extracellularly at sublytic concentrations causes long-lasting oscillations of the intracellular Ca2+ level of human embryonic kidney cells; resulting from a pulsed influx of extracellular Ca2+ through pores that are formed by LLO in the plasma membrane. Calcium influx does not require the activity of endogenous Ca2+ channels. LLO-formed pores are transient and oscillate between open and closed states. Pore formation and Ca2+ oscillations were also observed after exposure of cells to native Listeria monocytogenes. Our data identify LLO as a tool used by Listeria monocytogenes to manipulate the intracellular Ca2+ level without direct contact of the bacterium with the target cell. As Ca2+ oscillations modulate cellular signalling and gene expression, our findings provide a potential molecular basis for the broad spectrum of Ca2+-dependent cellular responses induced by LLO during Listeria infection.
Cellular Microbiology 09/2002; 4(8):483-91. · 5.46 Impact Factor
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ABSTRACT: KCa channels are involved in control of cell proliferation and differentiation. Here we have revealed their role in overcoming the RNase-induced cytotoxicity. Toxic effects of Streptomyces aureofaciens RNases Sa, Sa2, Sa3, and of RNase Sa charge reversal mutants on the human embryonic kidney cell lines differing only by the presence of KCa channels were characterized. In contrast to other RNases, a basic variant of RNase Sa and RNase Sa3 exhibit significant cytotoxic activity of the same order of magnitude as onconase. Our data indicate the absence of a correlation between catalytic activity and stability of RNases and cytotoxicity. On the other hand, cationization enhances toxic effect of an RNase indicating the major role of a positive charge. Essentially lower sensitivity to cytotoxic microbial RNases of cells expressing KCa channels was found. These results suggest that cells without the KCa channel activity cannot counteract toxic effect of RNases.
Biochemical and Biophysical Research Communications.
