Effect Of Carbonyl Cyanide M-chlorophenylhydrazone Research Articles

Effect of Carbonyl Cyanide M-chlorophenylhydrazone on Ciprofloxacin Resistance and Biofilm Formation in Hospital-acquired Uropathogenic Escherichia coli and Klebsiella pneumoniae

Antibiotic resistant and biofilm forming uropathogenic Enterobacteriaceae are rising. This study was conducted to evaluate the efflux pump and plasmid mediated efflux genes in ciprofloxacin (CIP) resistant hospital acquired uropathogenic Escherichia coli and Klebsiella pneumoniae. Also, to assess the anti-biofilm action of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Uropathogenic E. coli and K. pneumoniae isolates were collected from Mansoura University Hospitals in Mansoura, Egypt. The effect of Sub- minimum inhibitory concentration (MIC) of CCCP on CIP MIC was evaluated and the MIC decrease factor (MDF) was calculated. The presence of oqxAB and qepA genes was detected by PCR. The effect CCCP on biofilm was detected in strong biofilm formers. 56 and 47 CIP-resistant uropathogenic E. coli and K. pneumoniae isolates respectively were detected. Significant MDF by CCCP was observed in 55.3% of these isolates. The qepA gene was only present in E. coli. However, oqxAB genes were found only in K. pneumoniae. Biofilm formation was detected in 58.9% and 72.3% of CIP-resistant E. coli and K. pneumoniae isolates, respectively. Biofilm formation was significantly decreased by CCCP. According to these findings, CIP resistance and plasmid-mediated efflux pumps in uropathogenic E. coli and K. pneumoniae are of rising concern. Efflux pump inhibitor CCCP represents a possible option to decrease the biofilm formation in these resistant urinary pathogens.

Effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on microbial activity and polycyclic aromatic hydrocarbons (PAH) degradation in contaminated river sediments

Polycyclic aromatic hydrocarbons (PAHs) are of continued interest because of their carcinogenic nature and persistence in the environment. Among volatilization, sorption, and chemical oxidation, microbial degradation is the main path of PAHs disappearance. The majority of degradation studies have used pure cultures, were spiked with added PAHs, or have been performed using single pollutants. Because contaminated aquatic ecosystems retain mixtures of xenobiotics, experiments using environmental samples that incorporate the role of microbes provide more realistic conditions to study degradation. We tested the inhibitory effects of [carbonyl cyanide m-chlorophenylhydrazone (CCCP)] on microbial respiration, which in turn altered PAH degradation. In this study, 3.5 mM of CCCP (two orders of magnitude higher to what was needed to inhibit growth of E. coli in pure culture) was shown to inhibit respiration of indigenous microbes in complex sediment samples by 79 % at 25 °C and 64 % at 37 °C. After 15 days of incubation sediment slurries without CCCP showed higher PAH degradation rates (between 60 and 90 %) compared to sediment slurries with CCCP, which showed much lower degradation rates (<40 %). This study also reported the highest recorded rates of INT reduction (29 nkat/g) due to both short incubation times and the potential to stimulate high microbial activity in the sediments. These data indicated the effectiveness of CCCP as a microbial respiratory inhibitor in sediments, and that indigenous microbes in long-term PAH contaminated anaerobic sediments can be stimulated to degrade PAHs present in their environment.

Mitochondrial Transmembrane Potential and Reactive Oxygen Species Generation Regulate the Enhanced Effect of CCCP on TRAIL-Induced SNU-638 Cell Apoptosis

TRAIL is a member of the tumor necrosis factor family and engages apoptosis via recruitment and rapid activation of caspase-8. This study investigated the effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a classic uncoupler of oxidative phosphorylation, on TRAIL-induced apoptosis in SNU-638 cells derived from human gastric cancer cells. It was found that treatment with CCCP followed by incubation with TRAIL markedly enhanced apoptosis by 2 fold compared with treatment with TRAIL alone. This effect was accompanied by reduction in mitochondrial transmembrane potential and generation of reactive oxygen species. This sensitization was inhibited by N-acetyl-l-cysteine, which restored the mitochondrial transmembrane potential and reduced reactive oxygen species generation. Treatment with N-acetyl-L-cysteine also inhibited expression of apoptotic proteins such as Bax and Smac and abrogated caspase-8 activation. Moreover, treatment with N-acetyl-L-cysteine prior to induction with TRAIL increased expression of the anti-apoptotic Bcl-2 protein. These data indicate that CCCP enhanced TRAIL-induced apoptosis by dissipation of mitochondrial transmembrane potential and reactive oxygen species, suggesting that treatment with CCCP combined with that with TRAIL can be an efficient method to induce death of tumor cells, particularly cells that are resistant to TRAIL-induced apoptosis.

New Plasmid-Mediated Fluoroquinolone Efflux Pump, QepA, Found in an Escherichia coli Clinical Isolate

Plasmid-mediated Qnr and AAC(6')-Ib-cr have been recognized as new molecular mechanisms affecting fluoroquinolone (FQ) resistance. C316, an Escherichia coli strain demonstrating resistance to various FQs, was isolated in Japan. Resistance to FQs was augmented in an E. coli CSH2 transconjugant, but PCR failed to detect qnr genes, suggesting the presence of novel plasmid-mediated FQ resistance mechanisms. Susceptibility tests, DNA manipulation, and analyses of the gene and its product were performed to characterize the genetic determinant. A novel FQ-resistant gene, qepA, was identified in a plasmid, pHPA, of E. coli C316, and both qepA and rmtB genes were mediated by a probable transposable element flanked by two copies of IS26. Levels of resistance to norfloxacin, ciprofloxacin, and enrofloxacin were significantly elevated in E. coli transformants harboring qepA under AcrB-TolC-deficient conditions. QepA showed considerable similarities to transporters belonging to the 14-transmembrane-segment family of environmental actinomycetes. The effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on accumulation of norfloxacin was assayed in a qepA-harboring E. coli transformant. The intracellular accumulation of norfloxacin was decreased in a qepA-expressing E. coli transformant, but this phenomenon was canceled by CCCP. The augmented FQ resistance level acquired by the probable intergeneric transfer of a gene encoding a major facilitator superfamily-type efflux pump from some environmental microbes to E. coli was first identified. Surveillance of the qepA-harboring clinical isolates should be encouraged to minimize further dissemination of the kind of plasmid-dependent FQ resistance determinants among pathogenic microbes.

CCCP enhances catecholamine release from the perfused rat adrenal medulla

The present study was designed to investigate the effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, on secretion of catecholamines from the isolated perfused model of the rat adrenal gland and to establish the mechanism of its adrenomedullary secretion. The perfusion of CCCP (3 × 10 − 5 M) into an adrenal vein of for 90 min caused a great increase in catecholamine secretion. Tachyphylaxis to catecholamine-releasing effect of CCCP was not observed by repeated perfusion of it. The net catecholamine-releasing effects of CCCP were depressed by pretreament with pirenzepine (a selective muscarinic M 1-receptor antagonist), chlorisondamine (a selective neuronal nicotinic receptor antagonist), nicardipine (an L-type Ca 2+-channel antagonist), TMB-8 (an intracellular Ca 2+-antagonist), and the perfusion of EGTA plus Ca 2+-free medium, respectively. In the presence of CCCP (3 × 10 − 5 M), catecholamine secretory responses induced by ACh (5.32 × 10 − 3 M), high K + (5.6 × 10 − 2 M, a direct membrane depolarizer), DMPP (10 − 4 M, (a selective neuronal nicotinic receptor agonist), and McN-A-343 (10 − 4 M, (a selective muscarinic M 1-receptor agonist) were significantly enhanced. CCCP also significantly enhanced the catecholamine secretory responses evoked by Bay-K-8644 (10 − 5 M), L-type Ca 2+ channel activator, and cyclopiazonic acid (10 − 5 M), an inhibitor of Ca 2+-ATPase. Furthermore, the perfusion of FCCP (3 × 10 − 5 M), a similar mitochondrial uncoupler, into an adrenal vein of for 90 min also caused a great increase in catecholamine secretion in a similar pattern with CCCP. Taken together, the results demonstrate that CCCP causes the catecholamine secretion from the perfused rat adrenal medulla in a calcium-dependent fashion. It is thought that this catecholamine secretory enhancement of CCCP may be mediated by both cholinergic receptor stimulation and membrane depolarization, which are relevant to the cytoplasmic Ca 2+ increase by stimulation of the Ca 2+ influx as well as by the inhibition of Ca 2+ uptake into the cytoplasmic Ca 2+ stores (both endoplasmic reticulum and mitochondria in chromaffin cells). It also seems that protonophores, such as CCCP, suppress mitochondrial Ca 2+ uptake and increase the stimulated secretion of catecholamine by the secretagogues. These results indicate that mitochondria modulate catecholamine secretion by regulating the Ca 2+ mobilization for exocytosis.

Pregnancy-Specific Modulatory Role of Mitochondria on Adenosine 5′-Triphosphate-Induced Cytosolic [Ca2+] Signaling in Uterine Artery Endothelial Cells

Vascular endothelial cells respond to extracellular ATP by inositol 1,4,5-trisphosphate-mediated Ca2+ release from the endoplasmic reticulum followed by Ca2+ influx and subsequent synthesis of vasodilators. In this study, the contribution of mitochondria in shaping the ATP-induced Ca2+ increase was examined in ovine uterine artery endothelial cells from nonpregnant and pregnant (late gestation) ewes (NP- and P-UAEC, passage 4). The mitochondrial protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) induced a rapid mitochondrial depolarization. CCCP also slowly increased cytosolic [Ca2+] ([Ca2+]c), which then gradually declined to 10-20 nM above resting level. Pretreatment with CCCP for 30 min significantly inhibited both ATP and thapsigargin-induced [Ca2+]c, with inhibition in NP-UAEC more effective than in P-UAEC. Pretreatment of mitochondrial permeability transition pore inhibitor cyclosporine A did not affect CCCP-induced mitochondrial depolarization, but delayed CCCP-induced [Ca2+]c for about 12-15 min (we termed this the "window of time"). During the cyclosporine A-delayed window of time of CCCP-induced [Ca2+]c, ATP induced a normal Ca2+ response, but after this window of time, ATP-induced [Ca2+]c was significantly inhibited. Pretreatment of oligomycin B to prevent intracellular ATP depletion by F0F1-ATPase did not reduce the inhibition of ATP-induced [Ca2+]c by CCCP. Ruthenium red, a mitochondrial Ca2+ uptake blocker, did not mimic the inhibition of Ca2+ signaling by CCCP. In conclusion, our data show that mitochondrial Ca2+ depletion after dissipation of mitochondrial membrane potential with CCCP inhibits ATP-induced [Ca2+]c, mediated at the level of Ca2+ release from the endoplasmic reticulum. Moreover, our data revealed that P-UAEC is more resistant to the inhibitory effect of CCCP on [Ca2+]c than NP-UAEC.

Flagellar motors of marine bacteria Halomonas are driven by both protons and sodium ions.

Bacterial cells in aquatic environments are able to reach or stay near nutrient patches by using motility. Motility is usually attained by rotating flagellar motors that are energized by electrochemical potential of H+ or Na+. In this paper, the ion specificity for flagellar rotation of two marine isolates Halomonas spp. strains US172 and US201 was investigated. Both isolates require sodium for growth and possess a respiratory-driven primary sodium pump. They are motile because of lateral flagella regardless of the presence of sodium ions. Their swimming speed under various concentrations of sodium ions with and without carbonylcyanide m-chlorophenylhydrazone, a proton conductor, and with and without phenamil, a specific inhibitor for the sodium-driven flagellar motors, was examined. The effect of carbonylcyanide m-chlorophenylhydrazone on the transmembrane proton gradient was also determined. Our results showed that the flagellar motors of the Halomonas strains were energized by both H+ and Na+ in one cell. The bimodal nature of Halomonas spp. motility with respect to the driving energy source may reflect ecophysiological versatility to adapt to a wide range of salt conditions of the marine environment.

Lack of effect of carbonyl cyanide m-chlorophenylhydrazone on KB-5246 accumulation by Staphylococcus aureus.

The accumulation of KB-5246 in a quinolone-susceptible strain of Staphylococcus aureus was about 70 times that of norfloxacin. Carbonyl cyanide m-chlorophenylhydrazone increased the accumulation of norfloxacin about eightfold, but it did not influence that of KB-5246. The low efflux of KB-5246 from S. aureus may contribute to its potent antibacterial activity.

Effect of carbonyl cyanide m-chlorophenylhydrazone on Escherichia coli halotolerance.

The growth-inhibitory effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) was less on members of the family Enterobacteriaceae (halotolerant organisms) than it was on species of Vibrio (moderately halophilic organisms). When sodium chloride concentration increased from 0.5 to 0.85 M, this effect was more pronounced for Escherichia coli; it remained relatively stable for Vibrio spp. The effect of carbonyl cyanide m-chlorophenylhydrazone was antagonized by the addition of glycine betaine or proline or by growth in a rich medium.

O2 Uptake in the Light in Chlamydomonas

The nature of the process responsible for the stationary O(2) uptake occurring in the light under saturating CO(2) concentration in Chlamydomonas reinhardii has been investigated. For this purpose, a mass spectrometer with a membrane inlet system was used to measure O(2) uptake and evolution in the algal suspension. First, we observed that the O(2) uptake rate was constant (about 0.5 micromoles of O(2) per milligram chlorophyll per minute) during a light to dark transition and was not affected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Salicylhydroxamic acid had no effect on O(2) uptake in the dark or in the light, but was found to have the same inhibitory effect either in the dark or in the light when added to cyanide-treated algae. The stimulation of the O(2) uptake rate due to the uncoupling effect of carbonyl cyanide m-chlorophenylhydrazone was about the same in the dark or in the light. From these results, we conclude that mitochondrial respiration is maintained during illumination and therefore is not inhibited by high ATP levels. Another conclusion is that in conditions where photorespiration is absent, no other light-dependent O(2) uptake process occurs. If Mehler reactions are involved, in Chlamydomonas, under conditions where both photosynthetic carbon oxidation and reduction cycles cannot operate (as in cyanide-treated algae), their occurrence in photosynthesizing algae either under saturating CO(2) concentration or at the CO(2) compensation point appears very unlikely. The comparison with the situation previously reported in Scenedesmus (R. J. Radmer and B. Kok 1976 Plant Physiol 58: 336-340) suggests that different O(2) uptake processes might be present in these two algal species.

Ca2+ transport and Ca2+-dependent ATP hydrolysis by Golgi vesicles from lactating rat mammary glands.

Ca2+ transport across mammary-gland Golgi membranes was measured after centrifugation of the membrane vesicles through silicone oil. In the presence of 2.3 microM free Ca2+ the vesicles accumulated 5.8 nmol of Ca2+/mg of protein without added ATP, and this uptake was complete within 0.5 min. In the presence of 1 mM-ATP, Ca2+ was accumulated at a linear rate for 10 min after the precipitation of intravesicular Ca2+ with 10 mM-potassium oxalate. ATP-dependent Ca2+ uptake exhibited a Km of 0.14 microM for Ca2+ and a Vmax. of 3.1 nmol of Ca2+/min per mg of protein. Ca2+-dependent ATP hydrolysis exhibited a Km of 0.16 microM for Ca2+ and a Vmax. of 10.1 nmol of Pi/min per mg of protein. The stoichiometry between ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase varied between 0.3 and 0.7 over the range 0.03-8.6 microM-Ca2+. Both Ca2+ uptake and Ca2+-stimulated ATPase were strongly inhibited by orthovanadate, which suggests that the major mechanism by which Golgi vesicles accumulate Ca2+ is through the action of the Ca2+-stimulated ATPase. However, Ca2+ uptake was also decreased by the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone), indicating that it may occur by other mechanisms too. The effect of CCCP may be related to the existence of transmembrane pH gradients (delta pH) in these vesicles: the addition of 30 microM-CCCP reduced delta pH from a control value of 1.06 to 0.73 pH unit. Golgi vesicles also possess a Ca2+-efflux pathway which operated at an initial rate of 0.5-0.57 nmol/min per mg of protein.

Metabolic changes in Crithidia fasciculata accompanying physiological adaptation to growth in the presence of carbonyl cyanide [formula omitted

1. 1. Crithidia fasciculata adapted to growth in the presence of 10 −5 M carbonyl cyanide m- chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, maintained adenosine phosphate pools and an adenylate energy charge comparable to those of control cells. 2. 2. CCCP-adapted cells in the presence of the uncoupler respire endogenous substrate at a greater rate than control cells and this effect of CCCP appears readily reversible. 3. 3. CCCP-treated, adapted cells, supporting high endogenous respiration rates, were not responsive to added substrates which significantly stimulated the oxygen utilization of normal C. fasciculata. 4. 4. CCCP-adapted cells, provided with [U- 14C]-labeled proline, utilize this substrate at 67% the rate of control cells, but divide the isotopic label between CO 2 and protein in a ratio identical to that of normal cells. 5. 5. The transport of alamine and proline by adapted C. fasciculata was severely impaired, while the transport of tyrosine and leucine was unaffected.

Effect of carbonylcyanide m-chlorophenylhydrazone on the calcium-stimulated ATPase activity of erythrocyte ghosts

Incubation of erythrocyte ghosts with carbonylcyanide m-chlorophenylhydrazone (CCCP) plus Ca 2+ resulted in inactivation of the Ca 2+-stimulated ATPase activity. Omission of Ca 2+ or lowering of the temperature below 25 °C eliminated the inhibitory effect, as also did the presence of ATP during the incubation. On the other hand, the addition of β-mercaptoethanol did not influence the Ca 2+-dependent inhibition by CCCP. Compared with the level of CCCP which uncouples oxidative phosphorylation, a rather high level (0.5 mM) of CCCP was required to inhibit the ATPase activity in ghosts. However, once the inhibition had been accomplished, almost all of the CCCP could be removed from the ghost membrane by washing with a Ca 2+-containing solution, without affecting the inhibition of ATPase. If ethylene-glycol-bis( β-aminoethyl acid was included in the washing medium, the inhibition of ATPase was nearly completely reversed by washing. The results indicate that only the Ca 2+-stimulated, Mg 2+-ATPase was inhibited by 0.5 mM CCCP, while the remaining components of the ATPase activity were slightly inhibited by higher levels of the uncoupler. Low levels of CCCP (0.1 mM) stimulated the Mg 2+-ATPase slightly. CCCP was much more specific for the Ca 2+-stimulated ATPases than N-(1-naphthyl)maleimide, an unusually effective sulfhydryl reagent, and the requirement of Ca 2+ for inactivation was also quite specific.

Inhibitory site of carbonyl cyanide m-chlorophenylhydrazone in the electron transfer system of the chloroplasts

The inhibitory effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the electron transport system of chloroplasts was investigated. At concentrations higher than 1 · 10 −7 M, CCCP acted as an inhibitor of the Hill reaction. Two different processes of CCCP inhibition were discernible; the one which was accomplished very rapidly and independently of light, and the other which developed slowly and only in light. Both processes of inhibition were not reversed by washing the inhibited chloroplasts. The rapid type of inhibition in the dark was observed not only in the Hill reaction in normal (untreated) chloroplasts, but also in ascorbate plus hydroquinonesupported NADP + photoreduction and manganese-supported 2,6-dichlorophenol-indophenol (DCIP) photoreduction in heated chloroplasts, whereas NADP + photoreduction supported by reduced DCIP was not inhibited. CCCP inactivated the Hill reaction by affecting the quantum efficiency of the reaction without influencing the rate constant of the dark reaction. There was no loss in the manganese content in chloroplasts caused by incubation with CCCP in the dark. The variable fluorescence in chloroplasts was decreased by CCCP at concentrations higher than 1 · 10 −7 M, with concomitant elimination of the induction phenomenon. However, on addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to the CCCP-inhibited chloroplasts there occurred a recovery of the induction phase of fluorescence. The pool size of the electron acceptor of photosystem II decreased with increasing concentrations of CCCP, but the pool size of the primary electron acceptor determined in the presence of DCMU was not affected by the poison. It is postulated that the site of CCCP inhibition is located between the endogenous redox substance (Y 1) which receives electrons from artificial electron donors such as ascorbate, and the primary electron donor of photosystem II (Y 2).

Effect of carbonylcyanide m-chlorophenylhydrazone on the photochemical reactions of isolated chloroplasts.

Abstract Carbonylcyanide m -chlorophenylhydrazone is a powerful inhibitor of both photosynthetic phosphorylation and photoreduction of O 2 in isolated spinach chloroplasts. The inhibition of phosphorylation was observed in three cyclic systems (PMS, vitamin K 3 and FMN) and in three non-cyclic systems (ferricyanide-water, TPN + -water and TPN + -ascorbate). Inhibition of O 2 evolution was also obtained in the ferricyanide-water and TPN + -water systems but only at higher concentrations of CCCP. The only photochemical activity of chloroplasts which was found to be resistant to CCCP inhibition was the photoreduction of TPN + with ascorbate-dichlorophenol-indophenol as the electron donor (TPN + -ascorbate system). In the cyclic system (PMS) but not in the non-cyclic system (ferricyanide-water) CCCP inhibition of photophosphorylation varied with light intensity. Thus, in the PMS system, at 1000 lux, 0.4 μM CCCP gave 50% inhibition of photophosphorylation, whereas at 50 000 lux, 8 μM CCCP was required to give 50% inhibition. A model is proposed to account for the differences between cyclic and non-cyclic phosphorylation. The inhibition of photophosphorylation by CCCP was completely reversed by the addition of cysteine or by washing.