μM Carbonyl Cyanide M-chlorophenylhydrazone Research Articles

Protective effect of mitophagy against aluminum-induced MC3T3-E1 cells dysfunction

Aluminum (Al) is a ubiquitous environmental metal toxicant that causes osteoblast (OB) damage which leads to Al-related bone diseases. Mitochondrial damage plays a key role in Al-related bone diseases, and while mitophagy can clear damaged mitochondria and improve OB function, the relationship between mitophagy and Al-induced OB dysfunction is unknown. To explore the role of mitophagy in Al-induced OB dysfunction in vitro, we used 2 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 0.4 μM Cyclosporin A (CsA) to activate and inhibit mitophagy, respectively. MC3T3-E1 cells were treated with 0 mM AlCl3 (control group); 2 mM AlCl3 (Al group); 2 μM CCCP (CCCP group); 2 μM CCCP and 2 mM AlCl3 (CCCP + Al group); 0.4 μM CsA (CsA group); 0.4 μM CsA and 2 mM AlCl3 (CsA + Al group). The results showed that Al induced ultrastructural and functional impairment of MC3T3-E1 cells. Compared to the Al group, mitophagy activation caused mitochondrial membrane potentials to collapse, up-regulated PINK1, Parkin, and LC3 expression, down-regulated p62 expression, and increased mitophagosome numbers. Mitophagy activation also reduced Al-induced oxidative stress and MC3T3-E1 cell functional damage, as seen in improvement in cell viability, cellular calcium and phosphorus contents, and collagen I, osteocalcin, and bone alkaline phosphatase gene expression. Mitophagy inhibition had the opposite effects on activation. Overall, these results show that mitophagy can protect against Al-induced OB dysfunction.

Regulation of biohydrogen production by protonophores in novel green microalgae Parachlorella kessleri

The green microalgae Parachlorella kessleri RA-002 isolated in Armenia can produce biohydrogen (H2) during oxygenic photosynthesis. Addition of protonophores, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNF) enhances H2 yield in P. kessleri. The maximal H2 yield of ~2.20 and 2.08 mmol L−1 was obtained in the presence of 15 μM CCCP and 50 μM DNF, respectively. During dark conditions H2 production by P. kessleri was not observed even in the presence of protonophores, indicating that H2 formation in these algae was mediated by light conditions. The enhancing effect of protonophores can be coupled with dissipation of proton motive force across thylakoid membrane in P. kessleri, facilitating the availability of protons and electrons to [Fe-Fe]-hydrogenase, which led to formation of H2. At the same time H2 production was not observed in the presence of diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), a specific inhibitor of PS II. Moreover, diuron inhibits H2 yield in P. kessleri in the presence of protonophores. The inhibitory effect of diuron coupled with suppression of electron transfer from PS II. The results showed that in these algae operates PS II-dependent pathway of H2 generation. This study is important for understanding of the mechanisms of H2 production by green microalgae P. kessleri and developing of its biotechnology.

167 Basal and maximal oxygen consumption of oocytes from young and old mares

Mitochondria play a critical role in oocyte developmental competence by providing energy to the oocyte through oxidative phosphorylation. We hypothesised that oxygen consumption, a measure of mitochondrial function, would be lower in the oocytes of old versus young mares as a potential mechanism of reduced developmental competence associated with equine aging. To test this hypothesis, we used a novel electrochemical sensor device to compare basal and maximal oxygen consumption rates of oocytes obtained from mares aged 6 to 12 years (Young, n=8 from 7 mares) and=20 years (Old, n=12 from 8 mares). Cumulus-oocyte complexes (COC) were collected by transvaginal, ultrasound-guided follicular aspirations of dominant follicles (35mm) during oestrus and at 16±2h after induction of follicular maturation (2000IU of hCG and 0.75mg of deslorelin acetate). Recovered COC were incubated in medium [TCM-199; Life Technologies, Grand Island, NY, USA) with 10% fetal calf serum, 25mg mL−1 gentamicin, and 0.2mM pyruvate] at 38.5°C in 5% CO2 and air for 26±2h. After maturation, COC were stripped of cumulus cells and checked for the presence of an extruded polar body before electrochemical measurements using amperometry. A microchamber with a 3-electrode system (working, counter, and reference electrodes) was filled with 120µL of medium (G-MOPS™, Vitrolife, Englewood, CO, USA) and overlaid with 120µL of paraffin oil (OVOIL™, Vitrolife). The baseline current was measured before adding the oocyte through the oil layer and over the centre of the working electrode. Oxygen consumption was monitored as the linear decrease in the oxygen reduction current over time, which was converted to femtomoles of O2 consumed per second (fmol s−1) using a calibration curve for the sensor. Basal (nonstimulated) oxygen consumption was measured for 5min, followed by assessment of maximal noncoupled respiratory capacity obtained by titrating 1 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 8-min intervals. The highest value observed during CCCP titrations was considered the maximal oxygen consumption. Two-tailed Student’s t-tests were used to analyse data. Oocytes from young mares had higher basal oxygen consumption compared with old mares, respectively (mean±s.e.m., 1.74±0.18 and 1.27±0.09 fmol s−1; P=0.04) as well as higher maximal oxygen consumption (2.67±0.19 and 2.05±0.12 fmol s−1; P=0.016). Mitochondrial efficiency (maximal/basal oxygen consumption) was not different for Young and Old, respectively (1.62±0.13 and 1.65±0.07; P=0.9). In summary, we used a novel electrochemical sensor device to measure basal and maximal oxygen consumption of oocytes and to confirm our hypothesis that aging impairs mitochondrial respiratory capacity in equine oocytes, which may contribute to age-associated changes in equine fertility.

Secretion of d-alanine by Escherichia coli.

Escherichia coli has an l-alanine export system that protects the cells from toxic accumulation of intracellular l-alanine in the presence of l-alanyl-l-alanine (l-Ala-l-Ala). When a DadA-deficient strain was incubated with 6.0 mM l-Ala-l-Ala, we detected l-alanine and d-alanine using high-performance liquid chromatography (HPLC) analysis at a level of 7.0 mM and 3.0 mM, respectively, after 48 h incubation. Treatment of the culture supernatant with d-amino acid oxidase resulted in the disappearance of a signal corresponding to d-alanine. Additionally, the culture supernatant enabled a d-alanine auxotroph to grow without d-alanine supplementation, confirming that the signal detected by HPLC was authentic d-alanine. Upon introduction of an expression vector harbouring the alanine racemase genes, alr or dadX, the extracellular level of d-alanine increased to 11.5 mM and 8.5 mM, respectively, under similar conditions, suggesting that increased metabolic flow from l-alanine to d-alanine enhanced d-alanine secretion. When high-density DadA-deficient cells preloaded with l-Ala-l-Ala were treated with 20 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), secretion of both l-alanine and d-alanine was enhanced ~twofold compared with that in cells without CCCP treatment. In contrast, the ATPase inhibitor dicyclohexylcarbodiimide did not exert such an effect on the l-alanine and d-alanine secretion. Furthermore, inverted membrane vesicles prepared from DadA-deficient cells lacking the l-alanine exporter AlaE accumulated [3H]D-alanine in an energy-dependent manner. This energy-dependent accumulation of [3H]D-alanine was strongly inhibited by CCCP. These results indicate that E. coli has a transport system(s) that exports d-alanine and that this function is most likely modulated by proton electrochemical potential.

Structural and Functional Studies of a Newly Grouped Haloquadratum walsbyi Bacteriorhodopsin Reveal the Acid-resistant Light-driven Proton Pumping Activity

Retinal bound light-driven proton pumps are widespread in eukaryotic and prokaryotic organisms. Among these pumps, bacteriorhodopsin (BR) proteins cooperate with ATP synthase to convert captured solar energy into a biologically consumable form, ATP. In an acidic environment or when pumped-out protons accumulate in the extracellular region, the maximum absorbance of BR proteins shifts markedly to the longer wavelengths. These conditions affect the light-driven proton pumping functional exertion as well. In this study, wild-type crystal structure of a BR with optical stability under wide pH range from a square halophilic archaeon, Haloquadratum walsbyi (HwBR), was solved in two crystal forms. One crystal form, refined to 1.85 Å resolution, contains a trimer in the asymmetric unit, whereas another contains an antiparallel dimer was refined at 2.58 Å. HwBR could not be classified into any existing subgroup of archaeal BR proteins based on the protein sequence phylogenetic tree, and it showed unique absorption spectral stability when exposed to low pH values. All structures showed a unique hydrogen-bonding network between Arg(82) and Thr(201), linking the BC and FG loops to shield the retinal-binding pocket in the interior from the extracellular environment. This result was supported by R82E mutation that attenuated the optical stability. The negatively charged cytoplasmic side and the Arg(82)-Thr(201) hydrogen bond may play an important role in the proton translocation trend in HwBR under acidic conditions. Our findings have unveiled a strategy adopted by BR proteins to solidify their defenses against unfavorable environments and maintain their optical properties associated with proton pumping.

Active Plasma Membrane P-type H+-ATPase Reconstituted into Nanodiscs Is a Monomer

Plasma membrane H(+)-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H(+)-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins.

Characterization of H 2 photoproduction by a new marine green alga, Platymonas helgolandica var. tsingtaoensis

A significant improvement in H 2 photoproduction was observed in the marine green alga Platymonas helgolandica var. tsingtaoensis in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The addition of 15 μM CCCP to algal cultures led to a 260-fold increase in H 2 production compared with a negligible volume of H 2 gas produced without CCCP. The photosystem (PS) II photochemical activity was shown to be completely inhibited in the presence of CCCP under illumination. The added herbicide 3-(3,4-dichlorophenyl)-1, 1-dimethylurea further resulted in a twofold increase in H 2 photoproduction in the CCCP-treated cultures. These results demonstrated the absence of PSII-dependent pathway of H 2 photoproduction. H 2 production essentially depends on electrons donated to the hydrogenase by the endogenous substrate catabolism in CCCP-treated P. helgolandica var. tsingtaoensis. An analysis of the O 2-tolerance of the hydrogenase of P. helgolandica var. tsingtaoensis suggested that this enzyme may belong to the class [FeFe]-hydrogenase.

Mitochondrial Uncoupling Agents Trigger Ventricular Fibrillation in Isolated Rat Hearts

Sudden cardiac death resulting from ventricular fibrillation (VF) remains a major cause of mortality. The purpose of this study was to investigate the roles of loss of oxidative phosphorylation and activation of the mitochondrial ATP-sensitive K+ channel and permeability transition pore in VF development during myocardial ischemia by using mitochondrial uncoupling agents (carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol) and channel blockers (5-hydroxydecanoate and cyclosporine A) at concentrations that have been demonstrated to block the intended targets selectively. Isolated rat hearts (n = 8 per group) were perfused with 0.3 μM carbonyl cyanide m-chlorophenylhydrazone, 100 μM 2,4-dinitrophenol, 0.2 μM cyclosporine A, 100 μM 5-hydroxydecanoate, or vehicle solution and regional ischemia induced after 10 minutes. Carbonyl cyanide m-chlorophenylhydrazone and 2,4 dinitrophenol caused profound QT shortening and triggered VF in 100% of hearts before ischemia. During ischemia, neither cyclosporine A (88%) nor 5-hydroxydecanoate (100%) reduced VF incidence compared with control (100% VF). In separate hearts, carbonyl cyanide m-chlorophenylhydrazone decreased tissue ATP content, and glibenclamide or glimepiride delayed the QT shortening and onset of VF triggered by carbonyl cyanide m-chlorophenylhydrazone. In conclusion, mitochondrial uncoupling agents trigger VF, likely as a result of ATP depletion with subsequent activation of sarcolemmal ATP-sensitive K+ currents. The mechanism of VF in ischemia does not involve activation of the mitochondrial ATP-sensitive K+ channel or permeability transition pore.

Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium

The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na(+), Li(+), or H(+). In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na(+) or Li(+) and stimulated by equimolar concentrations of internal and external Na(+) or Li(+). Melibiose exchange is faster than efflux in the presence of H(+) or Na(+) and stimulated by an inwardly directed Na(+) gradient. Thus, sugar is released from MelB-ST externally prior to the release of cation in agreement with current models proposed for MelB of Escherichia coli (MelB-EC) and LacY. Although Li(+) stimulates efflux, and an outwardly directed Li(+) gradient increases exchange, it is striking that internal and external Li(+) with no gradient inhibits exchange. Furthermore, Trp → dansyl FRET measurements with a fluorescent sugar (2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside) demonstrate that MelB-ST, in the presence of Na(+) or Li(+), exhibits (app)K(d) values of ∼1 mM for melibiose. Na(+) and Li(+) compete for a common binding pocket with activation constants for FRET of ∼1 mM, whereas Rb(+) or Cs(+) exhibits little or no effect. Taken together, the findings indicate that MelB-ST utilizes H(+) in addition to Na(+) and Li(+). FRET studies also show symmetrical emission maximum at ∼500 nm with MelB-ST in the presence of 2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside and Na(+), Li(+), or H(+), which implies a relatively homogeneous distribution of conformers of MelB-ST ternary complexes in the membrane.

Temporary Sequestration of Potassium by Mitochondria in Astrocytes

Increases in extracellular potassium concentration ([K(+)](o)), which can occur during neuronal activity and under pathological conditions such as ischemia, lead to a variety of potentially detrimental effects on neuronal function. Although astrocytes are known to contribute to the clearance of excess K(+)(o), the mechanisms are not fully understood. We examined the potential role of mitochondria in sequestering K(+) in astrocytes. Astrocytes were loaded with the fluorescent K(+) indicator PBFI and release of K(+) from mitochondria into the cytoplasm was examined after uncoupling the mitochondrial membrane potential with carbonyl cyanide m-chlorophenylhydrazone (CCCP). Under the experimental conditions employed, transient applications of elevated [K(+)](o) led to increases in K(+) within mitochondria, as assessed by increases in the magnitudes of cytoplasmic [K(+)] ([K(+)](i)) transients evoked by brief exposures to CCCP. When mitochondrial K(+) sequestration was impaired by prolonged application of CCCP, there was a robust increase in [K(+)](i) upon exposure to elevated [K(+)](o). Blockade of plasmalemmal K(+) uptake routes by ouabain, Ba(2+), or a mixture of voltage-activated K(+) channel inhibitors reduced K(+) uptake into mitochondria. Also, reductions in mitochondrial K(+) uptake occurred in the presence of mito-K(ATP) channel inhibitors. Rises in [K(+)](i) evoked by brief applications of CCCP following exposure to high [K(+)](o) were also reduced by gap junction blockers and in astrocytes isolated from connexin43-null mice, suggesting that connexins also play a role in K(+) uptake into astrocyte mitochondria. We conclude that mitochondria play a key role in K(+)(o) handling by astrocytes.

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha1 (residues Gly-9 to Arg-21), alpha2 (residues Glu-27 to Asn-40), alpha3 (residues Arg-44 to Thr-54), alpha4 (residues Leu-57 to Tyr-64), and alpha5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

Cystic Fibrosis Transmembrane Conductance Regulator-independent Phagosomal Acidification in Macrophages

It was reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) is required for acidification of phagosomes in alveolar macrophages (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Here we determined whether the CFTR chloride channel is a generalized pathway for chloride entry into phagosomes in macrophages and whether mutations in CFTR could contribute to alveolar macrophage dysfunction. The pH of mature phagolysosomes in macrophages was measured by fluorescence ratio imaging using a zymosan conjugate containing Oregon Green(R) 488 and tetramethylrhodamine. Acidification of phagolysosomes in J774A.1 macrophages (pH approximately 5.1 at 45 min), murine alveolar macrophages (pH approximately 5.3), and human alveolar macrophages (pH approximately 5.3) was insensitive to CFTR inhibition by the thiazolidinone CFTR(inh)-172. Acidification of phagolysosomes in alveolar macrophages isolated from mice homozygous for DeltaF508-CFTR, the most common mutation in cystic fibrosis, was not different compared with that in alveolar macrophages isolated from wild-type mice. We also measured the kinetics of phagosomal acidification in J774A.1 and murine alveolar macrophages using a zymosan conjugate containing fluorescein and tetramethylrhodamine. Phagosomal acidification began within 3 min of zymosan binding and was complete within approximately 15 min of internalization. The rate of phagosomal acidification in J774A.1 cells was not slowed by CFTR(inh)-172 and was not different in alveolar macrophages from wild-type versus DeltaF508-CFTR mice. Our data indicate that phagolysosomal acidification in macrophages is not dependent on CFTR channel activity and do not support a proposed mechanism for cystic fibrosis lung disease involving defective phagosomal acidification and bacterial killing in alveolar macrophages.

Saccharomyces cerevisiae Lacking Btn1p Modulate Vacuolar ATPase Activity to Regulate pH Imbalance in the Vacuole

The vacuolar H(+)-ATPase (V-ATPase) along with ion channels and transporters maintains vacuolar pH. V-ATPase ATP hydrolysis is coupled with proton transport and establishes an electrochemical gradient between the cytosol and vacuolar lumen for coupled transport of metabolites. Btn1p, the yeast homolog to human CLN3 that is defective in Batten disease, localizes to the vacuole. We previously reported that Btn1p is required for vacuolar pH maintenance and ATP-dependent vacuolar arginine transport. We report that extracellular pH alters both V-ATPase activity and proton transport into the vacuole of wild-type Saccharomyces cerevisiae. V-ATPase activity is modulated through the assembly and disassembly of the V(0) and V(1) V-ATPase subunits located in the vacuolar membrane and on the cytosolic side of the vacuolar membrane, respectively. V-ATPase assembly is increased in yeast cells grown in high extracellular pH. In addition, at elevated extracellular pH, S. cerevisiae lacking BTN1 (btn1-Delta), have decreased V-ATPase activity while proton transport into the vacuole remains similar to that for wild type. Thus, coupling of V-ATPase activity and proton transport in btn1-Delta is altered. We show that down-regulation of V-ATPase activity compensates the vacuolar pH imbalance for btn1-Delta at early growth phases. We therefore propose that Btn1p is required for tight regulation of vacuolar pH to maintain the vacuolar luminal content and optimal activity of this organelle and that disruption in Btn1p function leads to a modulation of V-ATPase activity to maintain cellular pH homeostasis and vacuolar luminal content.

Hypertonic Induction of COX-2 in Collecting Duct Cells by Reactive Oxygen Species of Mitochondrial Origin

Our previous studies have documented MAPK mediation of the hypertonicity-induced stimulation of COX-2 expression in cultured renal medullary epithelial cells. The present study extends this observation by examining the role of reactive oxygen species (ROSs). ROS levels, determined using dichlorodihydrofluorescence diacetate and cytochrome c, were rapidly and significantly increased following exposure of mIMCD-K2 cells to media made hypertonic by adding NaCl. Hypertonic treatment (550 mosmol/kg) for 16 h induced a 5.6-fold increase in COX-2 protein levels and comparable increases in prostaglandin E(2) release, both of which were completely abolished by the NADPH oxidase inhibitor diphenyleneiodonium (25-50 microM). The general antioxidant N-acetyl-l-cysteine (6 mM), and the superoxide dismutase mimetic TEMPO (2.0 mm) reduced COX-2 levels by 75.6 and 79.8%, respectively. Exposure of mIMCD-K2 cells to exogenous O(2)(-.) generated by the xanthine/xanthine oxidase system mimicked the effect of hypertonicity on COX-2 expression and prostaglandin E(2) release. The increases in phosphorylation of ERK1/2 and p38 were detected 20 min following the hypertonic treatment and were both prevented by N-acetyl-l-cysteine. The increases in ROSs in response to hypertonic treatment were completely blocked by any one of the mitochondrial inhibitors tested, such as rotenone, thenoyltrifluoroacetone, or carbonyl cyanide m-chlorophenylhydrazone, associated with remarkable inhibition of COX-2 expression. In contrast, the increases in ROSs were not significantly altered in IMCD cells deficient in either gp91(phox) or p47(phox), nor were the increases in COX-2 expression. We conclude that ROSs derived from mitochondria, but not NADPH oxidase, mediate the hypertonicity-induced phosphorylation of MAPK and the stimulation of COX-2 expression.

Calcium buffering activity of mitochondria controls basal growth hormone secretion and modulates specific neuropeptide signaling

Goldfish somatotropes contain multiple functionally distinct classes of non-mitochondrial intracellular Ca 2+ stores. In this study, we investigated the role of mitochondrial Ca 2+ handling in the control of hormone secretion. Inhibition of mitochondrial Ca 2+ uptake with 10 μM ruthenium red (RR) and 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) caused a small and reversible increase in cytosolic [Ca 2+]. Despite relatively modest global Ca 2+ signals, RR and CCCP stimulated robust GH secretion under basal culture conditions. CCCP-stimulated hormone release was abolished in cells pre-incubated with 50 μM BAPTA-AM, suggesting that elevations in cytosolic [Ca 2+] mediate this release of GH. Both caffeine-sensitive intracellular Ca 2+ stores and l-type Ca 2+ channels can be the source of the Ca 2+ buffered by mitochondria in somatotropes. The stimulatory effect of RR on caffeine-stimulated GH release was enhanced dramatically in the presence of ryanodine, pointing to a complex interaction between these three Ca 2+ stores. Inhibition of mitochondrial Ca 2+ uptake with RR augmented GH release evoked by only one of the two endogenous gonadotropin-releasing hormones. Thus, we provide the first evidence that mitochondrial Ca 2+ buffering is differentially involved in specific agonist Ca 2+ signaling pathways and plays an important role in the control of basal GH release.

Mutants of Mycobacterium smegmatis unable to grow at acidic pH in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone

Mycobacterium smegmatis is able to grow and survive at acidic pH, and exhibits intracellular pH homeostasis under these conditions. In this study, the authors have identified low proton permeability of the cytoplasmic membrane, and high cytoplasmic buffering capacity, as determinants of intrinsic acid resistance of M. smegmatis. To identify genes encoding proteins involved in protecting cells from acid stress, a screening method was developed using the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). CCCP was used to suppress intrinsic acid resistance of M. smegmatis. The screen involved exposing cells to pH 5.0 in the presence of CCCP, and survivors were rescued at various time intervals on solid medium at pH 7.5. Cells capable of responding to intracellular acidification (due to CCCP-induced proton equilibration) will survive longer under these conditions than acid-sensitive cells. From a total pool of 5000 transposon (Tn611) insertion mutants screened, eight acid-sensitive M. smegmatis mutants were isolated. These acid-sensitive mutants were unable to grow at pH 5.0 in the presence of 1-5 microM CCCP, a concentration not lethal to the wild-type strain mc2155. The DNA flanking the site of Tn611 was identified using marker rescue in Escherichia coli, and DNA sequencing to identify the disrupted locus. Acid-sensitive mutants of M. smegmatis were disrupted in genes involved in phosphonate/phosphite assimilation, methionine biosynthesis, the PPE multigene family, xenobiotic-response regulation and lipid biosynthesis. Several of the acid-sensitive mutants were also defective in stationary-phase survival, suggesting that overlapping stress protection systems exist in M. smegmatis.

The Carboxyl-terminal Third of the Dicarboxylate Carrier Is Crucial for Productive Association with the Inner Membrane Twin-pore Translocase

The carrier proteins of the mitochondrial inner membrane consist of three structurally related tandem repeats (modules). Several different, and in some cases contradictory, views exist on the role individual modules play in carrier transport across the mitochondrial membranes and how they promote protein insertion into the inner membrane. Thus, by use of specific translocation intermediates, we performed a detailed analysis of carrier biogenesis and assessed the physical association of carrier modules with the inner membrane translocation machinery. Here we have reported that each module of the dicarboxylate carrier contains sufficient targeting information for its transport across the outer mitochondrial membrane. The carboxyl-terminal module possesses major targeting information to facilitate the direct binding of the carrier protein to the inner membrane twin-pore translocase and subsequent insertion into the inner membrane in a membrane potential-dependent manner. We concluded that, in this case, a single structural repeat can drive inner membrane insertion, whereas all three related units contribute targeting information for outer membrane translocation.

Effect of uncouplers on endogenous respiration and ferrous iron oxidation in a chemolithoautotrophic bacterium Acidithiobacillus ( Thiobacillus) ferrooxidans

Oxidation of ferrous iron (Fe 2+) to ferric iron (Fe 3+) with oxygen (O 2) by Acidithiobacillus ( Thiobacillus) ferrooxidans is considered to be inhibited by uncouplers. Oxidation of the endogenous substrates (presumably NADH) with O 2 or Fe 3+, on the other hand, was stimulated by uncouplers, 2,4-dinitrophenol (DNP) and carbonylcyanide- m-chlorophenyl-hydrazone (CCCP), as expected in respiratorily controlled mitochondria or heterotrophic bacteria. Amytal and rotenone were inhibitory. Fe 3+ reduction by endogenous substrates was studied extensively and was found to be stimulated by a permeable anion, SCN − and weak acids, as well as the above uncouplers. Proton translocating properties of some of these stimulators were shown by following a pH change in the cell suspension. It was concluded that any compounds that destroy proton electrochemical gradient, Δ p, stimulated endogenous respiration. Stimulation of Fe 2+ or ascorbate oxidation by lower concentrations of uncouplers was successfully demonstrated by shortening the reaction time, but only to a small extent. Uncouplers at concentrations stimulatory to endogenous respiration inhibited Fe 2+ oxidation if present before Fe 2+ addition. The inhibition by 10 μM CCCP was reversed by washing the cells in a buffer. Complex I inhibitors, atabrine, rotenone and amytal inhibited Fe 2+ oxidation, more strongly in the presence of 0.1 mM DNP. It is proposed that Fe 2+ oxidation required Δ p perhaps to climb an energetically uphill reaction or to reduce NAD + to NADH by reversed electron flow for CO 2 fixation. The latter interpretation implies some obligatory coupling between Fe 2+ oxidation and NAD + reduction.

A Specific Role of the Yeast Mitochondrial Carriers Mrs3/4p in Mitochondrial Iron Acquisition under Iron-limiting Conditions

The yeast genes MRS3 and MRS4 encode two members of the mitochondrial carrier family with high sequence similarity. To elucidate their function we utilized genome-wide expression profiling and found that both deletion and overexpression of MRS3/4 lead to up-regulation of several genes of the "iron regulon." We therefore analyzed the two major iron-utilizing processes, heme formation and Fe/S protein biosynthesis in vivo, in organello (intact mitochondria), and in vitro (mitochondrial extracts). Radiolabeling of yeast cells with 55Fe revealed a clear correlation between MRS3/4 expression levels and the efficiency of these biosynthetic reactions indicating a role of the carriers in utilization and/or transport of iron in vivo. Similar effects on both heme formation and Fe/S protein biosynthesis were seen in organello using mitochondria isolated from cells grown under iron-limiting conditions. The correlation between MRS3/4 expression levels and the efficiency of the two iron-utilizing processes was lost upon detergent lysis of mitochondria. As no significant changes in the mitochondrial membrane potential were observed upon overexpression or deletion of MRS3/4, our results suggest that Mrs3/4p carriers are directly involved in mitochondrial iron uptake. Mrs3/4p function in mitochondrial iron transport becomes evident under iron-limiting conditions only, indicating that the two carriers do not represent the sole system for mitochondrial iron acquisition.

Thiol oxidation by 2,2′-dithiodipyridine induced calcium mobilization in MG63 human osteosarcoma cells

2,2′-dithiodipyridine (2,2′-DTDP), a reactive disulphide that mobilizes Ca 2+ in muscle, induced an increase in cytoplasmic free Ca 2+concentrations ([Ca 2+] i) in MG63 human osteosarcoma cells loaded with the Ca 2+-sensitive dye fura-2. 2,2′-DTDP acted in a concentration-independent manner with an EC 50 of 50 μM. The Ca 2+ signal comprised an initial spike and a prolonged increase. Removing extracellular Ca 2+ did not alter the Ca 2+ signal, suggesting that the Ca 2+ signal was due to store Ca 2+ release. In Ca 2+-free medium, the 2,2′-DTDP-induced [Ca 2+] i increase was not changed by depleting store Ca 2+ with 50 μM bredfeldin A (a Golgi apparatus permeabilizer), 2 μM carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler), 1 μM thapsigargin (an endoplasmic reticulum Ca 2+pump inhibitor) or 5 μM ryanodine. Conversely, 2,2′-DTDP pretreatment abolished CCCP and thapsigargin-induced [Ca 2+] i increases. 2,2′-DTDP-induced Ca 2+ signals in Ca 2+-containing medium were not affected by modulation of protein kinase C activity or suppression of phospholipase C activity. However, 2,2′-DTDP-induced Ca 2+ release was inhibited by a thiol-selective reducing reagent, dithiothreitol (5-25 μM) in a concentration-dependent manner. Collectively, this study shows that 2,2′-DTDP induced [Ca 2+] i increases in human osteosarcoma cells via releasing store Ca 2+from multiple stores in a manner independent of protein kinase C or phospholipase C activity. The 2,2′-DTDP-induced store Ca 2+ release appeared to be dependent on oxidation of membranes.