Rotenone-sensitive mitochondrial potential in Phytomonas serpens: electrophoretic Ca(2+) accumulation.
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Phytomonas sp. are flagellated trypanosomatid plant parasites that cause diseases of economic importance in plantations of coffee, oil palm, cassava and coconuts. Here we investigated Ca(2+) uptake by the vanadate-insensitive compartments using permeabilized Phytomonas serpens promastigotes. This uptake occurs at a rate of 1.13+/-0.23 nmol Ca(2+) mg x protein(-1) min(-1). It is completely abolished by the H(+) ionophore FCCP and by valinomycin and nigericin. It is also inhibited by 2 microM ruthenium red, which, at this low concentration, is known to inhibit the mitochondrial calcium uniport. Furthermore, salicylhydroxamic acid (SHAM) and propylgallate, specific inhibitors of the alternative oxidase in plant and parasite mitochondria, are also effective as inhibitors of the Ca(2+) transport. These compounds abolish the membrane potential that is monitored with safranine O. Rotenone, an inhibitor of NADH-CoQ oxidoreductase, can also dissipate 100% of the membrane potential. It is suggested that the mitochondria of P. serpens can be energized via oxidation of NADH in a pathway involving the NADH-CoQ oxidoreductase and the alternative oxidase to regenerate the ubiquinone. The electrochemical H(+) gradient can be used to promote Ca(2+) uptake by the mitochondria. (+info)
A novel mechanism of regulation of cardiac contractility by mitochondrial functional state.
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It is generally considered that mitochondria regulate cardiac cell contractility by providing ATP for cellular ATPases and by participating in Ca2+ homeostasis. However, other possible mechanisms by which mitochondria can influence contractility have been largely overlooked. Here, we demonstrate that inhibition of the mitochondrial electron transport chain strongly increases Ca2+-dependent and independent isometric force development in rat ventricular fibers with selectively permeabilized sarcolemma. This effect is unrelated to the ATP-generating activity of mitochondria or Ca2+ homeostasis. Furthermore, various conditions that increase K+ accumulation in the mitochondrial matrix (activation of ATP- or Ca2+-dependent K+ channels as well as inhibition of the K+ efflux pathway via the K+/H+ exchanger) induce a similar mechanical response. Modulators of mitochondrial function that augment isometric force also cause swelling of mitochondria in the vicinity of myofibrils in situ, as shown by confocal microscopy. Osmotic compression of intracellular structures abolishes the effect of mitochondria-induced force modulation, suggesting a mechanical basis for the interaction between the organelles. These findings suggest a novel mechanism for cellular regulation of myofibrillar function, whereby increases in mitochondrial volume can impose mechanical constraints inside the cell, leading to an increase in force developed by myofibrils. (+info)
Phenotypic and genotypic changes in Salmonella enterica subsp. enterica serotype typhimurium during passage in intestines of broiler chickens fed on diets that included ionophore anticoccidial supplements.
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The effect of continuous in-feed administration of anticoccidial agents on antimicrobial sensitivity and the level of bacterial shedding in poultry experimentally infected with Salmonella enterica subsp. enterica serotype Typhimurium definitive type 104 (DT104) were investigated. On day 0, 1,200 1-day-old Salmonella-free broiler chicks were placed into 50 pens, and the pens were randomly allocated to one of five treatments: nonsupplemented (negative control; T1), monensin at 120 mg/kg of diet (T2), salinomycin at 60 mg/kg of diet (T3), semduramicin at 20 mg/kg of diet (T4), or semduramicin at 25 mg/kg of diet (T5). Each bird was inoculated with a well-characterized strain of serotype Typhimurium DT104 on day 10. On day 49, the birds were euthanatized humanely. Bulk fecal samples were collected on days 13, 43, and 48 and were examined for organisms which had acquired resistance. The genetic basis of acquired resistance was determined from representative samples of isolates. Of 784 Salmonella-selective plates supplemented with antimicrobial agents, only 33 showed growth. These isolates came from all treatment regimens, including the nonsupplemented control. A number of phenotypic changes were observed; these included changes in motility, phage type, and agglutination properties. Supplementation of the diet with an anticoccidial drug does not appear to affect antimicrobial resistance or the level of excretion of salmonellae. Most of the changes observed do not seem to be related to the presence of a supplement in feed. Salmonellae appear to be capable of acquiring antimicrobial resistance and phenotypic changes independently of specific antimicrobial selection pressures. (+info)
ATP-dependent cadmium transport by the cadA cadmium resistance determinant in everted membrane vesicles of Bacillus subtilis.
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Resistance to cadmium conferred by the staphylococcal plasmid pI258 occurs by means of energy-dependent efflux, resulting in decreased intracellular accumulation of cadmium. Recent sequence information suggested that efflux is mediated by a P-type ATPase. The cadA gene was previously expressed in Bacillus subtilis, conferring resistance to cadmium. Everted membrane vesicles were prepared from B. subtilis cells harboring either a plasmid containing the cadA system or the vector plasmid alone. 109Cd2+ transport into the everted membranes was measured in the presence of various energy sources. Cadmium transport was detected only in the presence of ATP as an energy source. The production of an electrochemical proton gradient (delta mu H+) by using NADH or phenazine methosulfate plus ascorbate was not able to drive transport. Reagents which dissipate delta pH abolished calcium transport due to the Ca2+/H+ antiporter but only partially inhibited cadmium transport. Inhibition of transport by the antibiotic bafilomycin A1 occurred at concentrations comparable to those which inhibit P-type ATPases. A band corresponding to the cadA gene product was identified on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and antibodies to the protein were prepared. (+info)
Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes.
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Inorganic polyphosphate (polyP) has been identified and measured in human platelets. Millimolar levels (in terms of Pi residues) of short chain polyP were found. The presence of polyP of approximately 70-75 phosphate units was identified by 31P NMR and by urea-polyacrylamide gel electrophoresis of platelet extracts. An analysis of human platelet dense granules, purified using metrizamide gradient centrifugation, indicated that polyP was preferentially located in these organelles. This was confirmed by visualization of polyP in the dense granules using 4',6-diamidino-2-phenylindole and by its release together with pyrophosphate and serotonin upon thrombin stimulation of intact platelets. Dense granules were also shown to contain large amounts of calcium and potassium and both bafilomycin A1-sensitive ATPase and pyrophosphatase activities. In agreement with these results, when human platelets were loaded with the fluorescent calcium indicator Fura-2 acetoxymethyl ester to measure their intracellular Ca2+ concentration ([Ca2+]i), they were shown to possess a significant amount of Ca2+ stored in an acidic compartment. This was indicated by the following: 1) the increase in [Ca2+]i induced by nigericin, monensin, or the weak base, NH4Cl, in the nominal absence of extracellular Ca2 and 2) the effect of ionomycin, which could not take Ca2+ out of acidic organelles and was more effective after alkalinization of this compartment by the previous addition of nigericin, monensin, or NH4Cl. All of these characteristics of the platelet dense granules, together with their known acidity and high density (both by weight and by electron microscopy), are similar to those of acidocalcisomes (volutin granules, polyP bodies) of bacteria and unicellular eukaryotes. The results suggest that acidocalcisomes have been conserved during evolution from bacteria to humans. (+info)
Implicating the glutathione-gated potassium efflux system as a cause of electrophile-induced activated sludge deflocculation.
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The glutathione-gated K(+) efflux (GGKE) system represents a protective microbial stress response that is activated by electrophilic or thiol-reactive stressors. It was hypothesized that efflux of cytoplasmic K(+) occurs in activated sludge communities in response to shock loads of industrially relevant electrophilic chemicals and results in significant deflocculation. Novosphingobium capsulatum, a bacterium consistent with others found in activated sludge treatment systems, responded to electrophilic thiol reactants with rapid efflux of up to 80% of its cytoplasmic K(+) pool. Furthermore, N. capsulatum and activated sludge cultures exhibited dynamic efflux-uptake-efflux responses very similar to those observed by others in Escherichia coli K-12 exposed to the electrophilic stressors N-ethylmaleimide and 1-chloro-2,4-dinitrobenzene and the reducing agent dithiothreitol. Fluorescent LIVE/DEAD stains were used to show that cell lysis was not the cause of electrophile-induced K(+) efflux. Nigericin was used to artificially stimulate K(+) efflux from N. capsulatum and activated sludge cultures as a comparison to electrophile-induced K(+) efflux and showed that cytoplasmic K(+) efflux by both means corresponded with activated sludge deflocculation. These results parallel those of previous studies with pure cultures in which GGKE was shown to cause cytoplasmic K(+) efflux and implicate the GGKE system as a probable causal mechanism for electrophile-induced, activated sludge deflocculation. Calculations support the notion that shock loads of electrophilic chemicals result in very high K(+) concentrations within the activated sludge floc structure, and these K(+) levels are comparable to that which caused deflocculation by external (nonphysiological) KCl addition. (+info)
Mitochondrial defects in cis-diamminedichloroplatinum(II)-resistant human ovarian carcinoma cells.
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We have been studying the membranes of cisplatin (DDP)-resistant 2008 human ovarian carcinoma cells (C13* cells) for alterations that may account for their decreased DDP accumulation. We now report that C13* cells have significant changes in their mitochondrial and plasma membrane potentials and in their mitochondrial morphology. C13* cells accumulated 2.0 +/- 0.1-fold more of the membrane potential marker [3H]tetraphenylphosphonium cation (TPP+) than sensitive cells. In high K+ medium, which depolarizes the plasma membrane but not the mitochondrial membrane, [3H]TPP+ accumulation was still 2.3 +/- 0.1- fold greater in resistant cells, indicating that the mitochondrial membrane potential was higher. In the presence of carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, which depolarizes the mitochondrial membrane but not the plasma membrane, [3H]TPP+ accumulation demonstrated that the plasma membrane potential in C13* cells was elevated as well. These elevations were also present in C8 cells with low-level DDP resistance. After ouabain treatment, exposure to nigericin stimulated [3H]TPP+ accumulation 3-fold in sensitive cells but had no effect in C13* cells, indicating either that: (a) the mitochondrial pH gradient was minimal; or (b) the mitochondrial electric potential was already at a maximal level in C13* cells. Fluorescence microscopy of living cells stained with the mitochondria-specific dye rhodamine 123 revealed that resistant cells had significant changes in their mitochondrial morphology. Electron microscopy also revealed major alterations in the cristae structure. The C13* cells, which were approximately 15-fold resistant to DDP, were 5-fold hypersensitive to the mitochondrial poison rhodamine 123. We conclude that these DDP-resistant 2008 cells have an elevated plasma membrane potential and alterations in their mitochondria as indicated by their membrane potential, morphology, and sensitivity to mitochondrial poisons. These results imply that mitochondria play an important role in the cellular pharmacology of DDP. (+info)
Role of H+-ATPase-mediated acidification in sorting and release of the regulated secretory protein chromogranin A: evidence for a vesiculogenic function.
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The constitutive and regulated secretory pathways represent the classical routes for secretion of proteins from neuroendocrine cells. Selective aggregation of secretory granule constituents in an acidic, bivalent cation-rich environment is considered to be a prerequisite for sorting to the regulated secretory pathway. The effect of selective vacuolar H+-ATPase (V-ATPase) inhibitor bafilomycin A1 on the pH gradient along the secretory pathway was used here to study the role of acidification on the trafficking of the regulated secretory protein chromogranin A (CgA) in PC12 cells. Sorting of CgA was assessed by three-dimensional deconvolution microscopy, subcellular fractionation, and secretagogue-stimulated release, examining a series of full-length or truncated domains of human CgA (CgA-(1-115), CgA-(233-439)) fused to either green fluorescent protein or to a novel form of secreted embryonic alkaline phosphatase (EAP). We show that a full-length CgA/EAP chimera is sorted to chromaffin granules for exocytosis. Inhibition of V-ATPase by bafilomycin A1 markedly reduced the secretagogue-stimulated release of CgA-EAP by perturbing sorting of the chimera (at the trans-Golgi network or immature secretory granule) rather than the late steps of exocytosis. The effect of bafilomycin A1 on CgA secretion depends on a sorting determinant located within the amino terminus (CgA-(1-115)) but not the C-terminal region of the granin. Moreover, examination of chromaffin granule abundance in PC12 cells exposed to bafilomycin A1 reveals a substantial decrease in the number of dense-core vesicles. We propose that a V-ATPase-mediated pH gradient in the secretory pathway is an important factor for the formation of dense-core granules by regulating the ability of CgA to form aggregates, a crucial step that may underlie the granulogenic function of the protein. (+info)