Carbonyl Cyanide 3-chlorophenylhydrazone Research Articles

Determination of imipenem efflux-mediated resistance in Acinetobacter spp., using an efflux pump inhibitor

In recent years, reports of Acinetobacter strains resistant to all known antibiotics have caused a great concern in medical communities. Overexpression of efflux pumps is one of the major causes of resistance in bacteria. The aim of this study was to investigate the role of efflux pumps in conferring resistance to imipenem in clinically important Acinetobacter spp; Acinetobacter baumannii and Acinetobacter lwoffii. A total number of 46 clinical Acinetobacter isolates, including 33 A. baumannii and 13 A. lwoffii isolates, previously collected from Shahid Kamyab and Ghaem hospitals of Mashhad, Iran were used in this study. Imipenem susceptibility testing was carried out by the disc diffusion method. Imipenem minimum inhibitory concentration (MIC) for resistant Acinetobacter isolates were determined both in the presence and absence of the efflux pumps inhibitor, carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Resistance to imipenem was observed in 38 isolates including 30 A. baumannii and 8 A. lwoffii isolates. Experiments in the presence of CCCP showed a 2 to 16384 fold reduction in imipenem MICs in 14 A. baumannii and 2 A. lwoffii isolates. The results obtained showed high levels of resistance to imipenem and contribution of efflux pumps in conferring resistance in both Acinetobacter species in this study. Moreover, imipenem efflux mediated resistance highlights the importance of this mechanism not only in A. baumannii but also in non-baumannii Acinetobacter Spp. which have been neglected in antibiotic resistance studies.

Molecular characterization of the orphan transporter SLC16A9, an extracellular pH- and Na+-sensitive creatine transporter

Human monocarboxylate transporters (hMCTs) mediate the transport of monocarboxylates across plasma membranes. One such transporter, hMCT9, has been shown to be related to serum uric acid levels and the risk of renal overload gout. However, the functional characteristics of hMCT9 remain unknown. The aim of this study was to investigate the expression and localization of hMCT9 using a Xenopus laevis oocyte heterologous expression system and characterize its transport properties. Kinetic analysis of hMCT9-mediated creatine uptake revealed that uptake consisted of two components, with apparent Km values of 237 mm (low-affinity) and 23.7 mm (high-affinity), respectively. The transport activity of hMCT9 was dependent on the extracellular pH and activity sharply increased with increasing pH. Under Na+-free conditions, hMCT9-mediated creatine uptake was reduced by one-half, indicating that hMCT9 is a Na+-sensitive transporter. Moreover, carbonyl cyanide 3-chlorophenylhydrazone (a protonophore) inhibited hMCT9 activity, whereas valinomycin (a K+-ionophore) did not inhibit the transporter. These results suggest that hMCT9 is susceptible to changes in H+ gradients. A cis-inhibition assay of hMCT9-and hMCT12-mediated creatine transport revealed that cyclocreatine, creatine, guanidineacetate, and 3-guanidinopropionate are recognized by the transporter, and 4-guanidinobutyrate and guanidinoethyl sulfonate selectively inhibited hMCT9 activity. These findings demonstrate that hMCT9 is an extracellular pH- and Na+-sensitive creatine transporter.

EFFLUX MEDIATED MULTIDRUG RESISTANT PSEUDOMONAS AERUGINOSA ISOLATED FROM DIFFERENT ENVIRONMENTAL SOURCES

Pseudomonas aeruginosa is an important opportunistic pathogen and one of the leading causes of multi-drug resistant nosocomial infections. This study was therefore carried out to determine the resistance nature, and the role of efflux pump in multidrug resistance of Pseudomonas aeruginosa isolated from different environmental sources using the efflux pump inhibitor, Carbonyl Cyanide 3-Chlorophenylhydrazone (CCCP). A total of 220 environmental samples were collected and processed following standard techniques. Susceptibility to antibiotics was performed using disc diffusion methods as described by the Clinical and Laboratory Standards Institute. Activity of the efflux pump system was carried out using the efflux pump inhibitor, CCCP. Results obtained identified 100 (45.5%) Pseudomonas aeruginosa and 72 (32.7%) other strains of Pseudomonas spp. The susceptibility testing revealed that all the identified strains of Pseudomonas aeruginosa that were subjected to susceptibility test were significantly resistant to ampicillin and cefotaxime, But the resistance profile of isolates to tetracycline, chloramphenicol, ceftriaxone, cefuroxime and perfloxacin were 93%, 72.1%, 79.1%, 58.1% and 51.2% respectively. However, imipenem was the most sensitive (100%), followed by cefepime (65%) and gentamicin (44%). Carbonyl Cyanide 3-Chlorophenylhydrazone decreased the minimum inhibitory concentration (MIC) of the isolates by 2 folds. Results obtained have shown the ubiquitous presence of multi-drug resistant P. aeruginosa from the environmental samples examined. Furthermore, it indicated the role of efflux pump in antibiotics resistance in P. aeruginosa isolates which indicate that P. aeruginosa strains from environmental sources could resist antibiotics by the efflux mechanism. 
 

Hydrogen peroxide depolarizes mitochondria and inhibits IP3-evoked Ca2+ release in the endothelium of intact arteries

Hydrogen peroxide (H2O2) is a mitochondrial-derived reactive oxygen species (ROS) that regulates vascular signalling transduction, vasocontraction and vasodilation. Although the physiological role of ROS in endothelial cells is acknowledged, the mechanisms underlying H2O2 regulation of signalling in native, fully-differentiated endothelial cells is unresolved. In the present study, the effects of H2O2 on Ca2+ signalling were investigated in the endothelium of intact rat mesenteric arteries. Spontaneous local Ca2+ signals and acetylcholine evoked Ca2+ increases were inhibited by H2O2. H2O2 inhibition of acetylcholine-evoked Ca2+ signals was reversed by catalase. H2O2 exerts its inhibition on the IP3 receptor as Ca2+ release evoked by photolysis of caged IP3 was supressed by H2O2. H2O2 suppression of IP3-evoked Ca2+ signalling may be mediated by mitochondria. H2O2 depolarized mitochondria membrane potential. Acetylcholine-evoked Ca2+ release was inhibited by depolarisation of the mitochondrial membrane potential by the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or complex 1 inhibitor, rotenone. We propose that the suppression of IP3-evoked Ca2+ release by H2O2 arises from the decrease in mitochondrial membrane potential. These results suggest that mitochondria may protect themselves against Ca2+ overload during IP3-linked Ca2+ signals by a H2O2 mediated negative feedback depolarization of the organelle and inhibition of IP3-evoked Ca2+ release.

Cadmium absorption and translocation of amaranth (Amaranthus mangostanus L.) affected by iron deficiency

Amaranth (Amaranthus mangostanus L.) has superior capability for accumulating cadmium (Cd) and has the potential to be used for phytoremediation of Cd contaminated soils. Iron (Fe) is chemically similar to Cd and may mediate Cd-induced physiological or metabolic impacts in plants. The purpose was to investigate the model of time-dependent and concentration-dependent kinetics of Cd absorption under Fe deficiency, understanding the physiological mechanism of Cd absorption in amaranth roots. The kinetic characteristics of Cd uptake by amaranth grown in Cd enriched nutritional solution with or without Fe addition and with methanol-chloroform, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and lanthanum chloride (LaCl3) were compared using 109Cd2+ isotope labeling technique. The results showed that Cd uptake was time-dependent and about 90–93% of uptake occurred during the first 150 min. The kinetics of Cd uptake showed that two stages were involved. The saturation stage fitted the Michaelis-Menten model when concentrations of Cd were lower than 12.71 μmol/L and then the absorption of Cd by roots was increased linearly during the second stage. Only linear absorption was observed with methanol-chloroform treatment while the metabolic inhibitor CCCP inhibited only the saturation absorption process, and the Ca channel inhibitor LaCl3 partially inhibited the two stages of absorption. These results indicated that the root absorption of 109Cd2+ was enhanced under Fe deficiency which induced more Fe transporters in the root cell membrane, and the Ca channel, apoplastic and symplastic pathways enhanced the Cd absorption in roots.

A rapid, low pH, nutrient stress, assay to determine the bactericidal activity of compounds against non-replicating Mycobacterium tuberculosis.

There is an urgent need for new anti-tubercular agents which can lead to a shortened treatment time by targeting persistent or non-replicating bacilli. In order to assess compound activity against non-replicating Mycobacterium tuberculosis, we developed a method to detect the bactericidal activity of novel compounds within 7 days. Our method uses incubation at low pH in order to induce a non-replicating state. We used a strain of M. tuberculosis expressing luciferase; we first confirmed the linear relationship between luminescence and viable bacteria (determined by colony forming units) under our assay conditions. We optimized the assay parameters in 96-well plates in order to achieve a reproducible assay. Our final assay used M. tuberculosis in phosphate-citrate buffer, pH 4.5 exposed to compounds for 7 days; viable bacteria were determined by luminescence. We recorded the minimum bactericidal concentration at pH 4.5 (MBC4.5) representing >2 logs of kill. We confirmed the utility of the assay with control compounds. The ionophores monensin, niclosamide, and carbonyl cyanide 3-chlorophenylhydrazone and the anti-tubercular drugs pretomanid and rifampicin were active, while several other drugs such as isoniazid, ethambutol, and linezolid were not.

Protective effects of Platycodon grandiflorus polysaccharides against apoptosis induced by carbonyl cyanide 3-chlorophenylhydrazone in 3D4/21 cells

This study aimed to investigate the potential protective effects of Platycodon grandiflorus polysaccharide (PGPS) on carbonyl cyanide 3-chlorophenylhydrazone (CCCP)-induced mitochondrial apoptosis in 3D4/21 cells. Apoptosis-related indicators such as cell viability, apoptosis rate, mitochondrial membrane potential (MMP), and apoptosis-related protein were examined. Results indicated that PGPSt can inhibit CCCP-induced cell damage, with cell-survival rate reaching 81% and apoptotic rate decreasing to 23%. Nuclear deformation was also significantly reduced in the PGPSt group, and changes in MMP were inhibited by PGPSt. Further analyses showed that the protein expression of Caspase-9 and Bcl-2 increased and the expression of cleaved Caspase-3 decreased, indicating that PGPSt significantly inhibited the CCCP-induced change in apoptotic protein expression. All these results suggested that PGPSt can antagonize 3D4/21 cell apoptosis by restoring MMP, protecting the integrity of nuclear morphology, and increasing Bcl-2 expression.

Glaucoma-Associated Mutations in the Optineurin Gene Have Limited Impact on Parkin-Dependent Mitophagy

Glaucoma results in progressive degeneration of the optic nerve and irreversible vision loss. Several mutations in the gene encoding optineurin (OPTN), the receptor for Parkin-dependent mitochondrial autophagy (mitophagy), are associated with glaucoma and amyotrophic lateral sclerosis (ALS). ALS mutations in the ubiquitin-binding domain of OPTN impair Parkin-dependent mitophagy. However, the effects of glaucoma mutations in this region remain unknown. We examined the impact of glaucoma-associated OPTN mutations on Parkin-dependent mitophagy. The mitochondria-localized, pH-sensitive fluorescent protein mito-Keima was used to monitor mitophagy. HeLa cells expressing Parkin were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or oligomycin/antimycin A (O/A) to induce Parkin-dependent mitophagy. Two complementary mitophagy receptors, OPTN and NDP52, were deleted in HeLa cells expressing mito-Keima and Parkin (DKO_HeLa). The mutant OPTN genes were re-introduced into DKO_HeLa cells using retroviruses or through transfection. Mitophagy activity and OPTN localization were evaluated via microscopic analyses. OPTN binding to ubiquitin was examined using an immunoprecipitation assay. Parkin-dependent mitophagy was inhibited in DKO_HeLa cells. Introduction of two glaucoma mutations in the ubiquitin-interacting region of OPTN restored mitophagy in CCCP-treated DKO_HeLa cells, whereas the two ALS mutations failed to replicate this effect. Under treatment with CCCP, the two glaucoma-mutant OPTN proteins normally translocated to mitochondria and bound to ubiquitinated proteins. Furthermore, five additional glaucoma-mutant OPTN proteins restored CCCP-induced mitophagy. Moreover, treatment with O/A exhibited similar results. Glaucoma-mutant OPTN proteins retain their normal properties as mitophagy receptors, suggesting that mutations in the OPTN gene cause glaucoma through a mechanism independent of mitophagy defects.

USP33 deubiquitinates PRKN/parkin and antagonizes its role in mitophagy

ABSTRACTPRKN/parkin activation through phosphorylation of its ubiquitin and ubiquitin-like domain by PINK1 is critical in mitophagy induction for eliminating the damaged mitochondria. Deubiquitinating enzymes (DUBs) functionally reversing PRKN ubiquitination are critical in controlling the magnitude of PRKN-mediated mitophagy process. However, potential DUBs that directly target PRKN and antagonize its pro-mitophagy effect remains to be identified and characterized. Here, we demonstrated that USP33/VDU1 is localized at the outer membrane of mitochondria and serves as a PRKN DUB through their interaction. Cellular and in vitro assays illustrated that USP33 deubiquitinates PRKN in a DUB activity-dependent manner. USP33 prefers to remove K6, K11, K48 and K63-linked ubiquitin conjugates from PRKN, and deubiquitinates PRKN mainly at Lys435. Mutation of this site leads to a significantly decreased level of K63-, but not K48-linked PRKN ubiquitination. USP33 deficiency enhanced both K48- and K63-linked PRKN ubiquitination, but only K63-linked PRKN ubiquitination was significantly increased under mitochondrial depolarization. Further, USP33 knockdown increased both PRKN protein stabilization and its translocation to depolarized mitochondria leading to the enhancement of mitophagy. Moreover, USP33 silencing protects SH-SY5Y human neuroblastoma cells from the neurotoxin MPTP-induced apoptotic cell death. Our findings convincingly demonstrate that USP33 is a novel PRKN deubiquitinase antagonizing its regulatory roles in mitophagy and SH-SY5Y neuron-like cell survival. Thus, USP33 inhibition may represents an attractive new therapeutic strategy for PD patients.Abbreviations: CCCP: carbonyl cyanide 3-chlorophenylhydrazone; DUB: deubiquitinating enzymes; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; OMM: outer mitochondrial membrane; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; TM: transmembrane; Ub: ubiquitin; UBA1: ubiquitin like modifier activating enzyme 1; UBE2L3/UbcH7: ubiquitin conjugating enzyme E2 L3; USP33: ubiquitin specific peptidase 33; WT: wild type.

Abstract 160: Atg7-Dependent Activation of Mitochondrial Autophagy in Cardiomyocytes

Introduction: Autophagy-related protein 7 (Atg7) has been studied for its role in cellular macroautophagy induction and regulation in different cell systems. We have previously reported that Atg7 regulates cardiac macroautophagy. However, whether Atg7 plays any role in the initiation and regulation of mitochondrial autophagy (mitophagy) in cardiomyocytes remains elusive. Objective: We investigated subcellular localization and molecular function of Atg7 in the regulation of mitophagy through a series of in vitro and in vivo experiments. Methods and Results: We isolated mitochondria from mouse hearts by subcellular fractionation and conducted biochemical experiments which revealed localization of Atg7 on the mitochondrial membrane. Next, we co-stained for Atg7 in isolated heart mitochondria stained with Mitotracker dye and found Atg7 localizes to mitochondrial membrane. To elucidate molecular function of Atg7 on mitophagy, adenovirus-mediated Atg7 overexpressed cultured neonatal rat cardiomyocytes (NRCs) were treated with mitophagy inducer carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in vitro and stained with organelle-specific mitochondria and lysosome dyes to count mitophagy fluorescent dots by confocal microscopy. Atg7 overexpressed NRCs exhibited an increase in mitophagy dots than β-gal treated NRCs following CCCP induction. Further, we have treated cardiac specific Atg7 overexpressed (Atg7 Tg) mice and littermate non-transgenic (Ntg) mice with CCCP to assess mitophagy induction in vivo . Immunoblotting confirmed enhanced accumulation of mature autophagolysosomal marker LC3-II protein in mitochondrial fraction from Atg7 Tg hearts. Conclusions: Our in vitro and in vivo findings indicate that Atg7 localizes to the mitochondrial membrane, regulates recruitment and initiation of mitophagy machinery, and facilitates mitophagy in cardiomyocytes.

Interaction of Potent Mitochondrial Uncouplers with Thiol-Containing Antioxidants.

It is generally considered that reactive oxygen species (ROS) are involved in the development of numerous pathologies. The level of ROS can be altered via the uncoupling of oxidative phosphorylation by using protonophores causing mitochondrial membrane depolarization. Here, we report that the uncoupling activity of potent protonophores, such as carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and fluazinam, can be abrogated by the addition of thiol-containing antioxidants to isolated mitochondria. In particular, N-acetylcysteine, glutathione, cysteine, and dithiothreitol removed both a decrease in the mitochondrial membrane potential and an increase in the respiration rate that is caused by FCCP. The thiols also reduced the electrical current that is induced by FCCP and CCCP across planar bilayer lipid membranes. Thus, when speculating on the mechanistic roles of ROS level modulation by mitochondrial uncoupling based on the antioxidant reversing certain FCCP and CCCP effects on cellular processes, one should take into account the ability of these protonophoric uncouplers to directly interact with the thiol-containing antioxidants.

Clearance of damaged mitochondria via mitophagy is important to the protective effect of ischemic preconditioning in kidneys

ABSTRACTIschemic preconditioning (IPC) affords tissue protection in organs including kidneys; however, the underlying mechanism remains unclear. Here we demonstrate an important role of macroautophagy/autophagy (especially mitophagy) in the protective effect of IPC in kidneys. IPC induced autophagy in renal tubular cells in mice and suppressed subsequent renal ischemia-reperfusion injury (IRI). The protective effect of IPC was abolished by pharmacological inhibitors of autophagy and by the ablation of Atg7 from kidney proximal tubules. Pretreatment with BECN1/Beclin1 peptide induced autophagy and protected against IRI. These results suggest the dependence of IPC protection on renal autophagy. During IPC, the mitophagy regulator PINK1 (PTEN induced putative kinase 1) was activated. Both IPC and BECN1 peptide enhanced mitolysosome formation during renal IRI in mitophagy reporter mice, suggesting that IPC may protect kidneys by activating mitophagy. We further established an in vitro model of IPC by inducing ‘chemical ischemia’ in kidney proximal tubular cells with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Brief treatment with CCCP protected against subsequent injury in these cells and the protective effect was abrogated by autophagy inhibition. In vitro IPC increased mitophagosome formation, enhanced the delivery of mitophagosomes to lysosomes, and promoted the clearance of damaged mitochondria during subsequent CCCP treatment. IPC also suppressed mitochondrial depolarization, improved ATP production, and inhibited the generation of reactive oxygen species. Knockdown of Pink1 suppressed mitophagy and reduced the cytoprotective effect of IPC. Together, these results suggest that autophagy, especially mitophagy, plays an important role in the protective effect of IPC.Abbreviations: ACTB: actin, beta; ATG: autophagy related; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; BUN: blood urea nitrogen; CASP3: caspase 3; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; COX4I1: cytochrome c oxidase subunit 4I1; COX8: cytochrome c oxidase subunit 8; DAPI: 4ʹ,6-diamidino-2-phenylindole; DNM1L: dynamin 1 like; EGFP: enhanced green fluorescent protein; EM: electron microscopy; ER: endoplasmic reticulum; FC: floxed control; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; H-E: hematoxylin-eosin; HIF1A: hypoxia inducible factor 1 subunit alpha; HSPD1: heat shock protein family D (Hsp60) member 1; IMMT/MIC60: inner membrane mitochondrial protein; IPC: ischemic preconditioning; I-R: ischemia-reperfusion; IRI: ischemia-reperfusion injury; JC-1: 5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethylbenzimidazolylcarbocyanine iodide; KO: knockout; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; mito-QC: mito-quality control; mRFP: monomeric red fluorescent protein; NAC: N-acetylcysteine; PINK1: PTEN induced putative kinase 1; PPIB: peptidylprolyl isomerase B; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; RPTC: rat proximal tubular cells; SD: standard deviation; sIPC: simulated IPC; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling

Mitochondrial membrane potential played crucial roles in the accumulation of berberine in HepG2 cells.

Berberine is a natural alkaloid that has antineoplastic effects. However, in hepatoma cells like HepG2, the expressions of uptake transporters are minimal but efflux transporters are relatively high. Hence, how berberine enters and reaches a cytocidal concentration remains to be elucidated. In the present study, we revealed the accumulation mechanism of berberine in HepG2 cells. Cell organelles were isolated based on differential centrifugation; berberine concentration was measured using a liquid chromatography-tandem mass chromatography method or flow cytometry. Subcellular distribution of berberine was observed using a laser scanning confocal microscopy. The results showed that berberine was concentration-, temperature-, and time-dependently taken up and accumulated in HepG2 cells. Membrane drug transporters and cell membrane potential had limited effects in berberine uptake. However, qualitative and quantitative studies showed that berberine was enriched in the mitochondria; inhibition of mitochondrial membrane potential (MMP) by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) significantly decreased the intracellular berberine by up to 70%. More importantly, MMP not only significantly enhanced berberine uptake driven by cell membrane potential (P<0.01) but also inhibited p-glycoprotein (P-gp)-mediated berberine efflux (P<0.01). In brief, our results for the first time showed that MMP played crucial roles in berberine accumulation in HepG2 cells.

High-throughput identification of the sensitivities of an Escherichia coli ΔrecA mutant strain to various chemical compounds.

Antibiotic resistance is considered a global threat to public health. Adaptive resistance mutations and the acquisition of resistance genes by horizontal gene transfer are known to be facilitated by the RecA-dependent SOS response during antibiotic treatment, making RecA inhibitors promising agents for the prevention of antibiotic resistance. However, the impact of RecA inactivation on antibiotic sensitivities remains unclear. Therefore, in this study, we performed high-throughput screening to determine the minimum inhibitory concentrations (MICs) of 217 chemicals, including both antibiotics and toxic chemicals of unknown drug action, in the wild-type MDS42 and the ΔrecA mutant strains of Escherichia coli. The ΔrecA mutant showed increased sensitivity to DNA-damaging agents, DNA replication inhibitors, and chromate stress, as well as to other chemicals, such as S-(2-aminoethyl)-L-cysteine, L-histidine, ruthenium red, D-penicillamine, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), cerulenin, and L-cysteine. Microarray analysis showed further that the ΔrecA mutant had lower expressions of glnK, nac, and glnLG, which encode nitrogen assimilation regulators, as well as amtB, which encodes an ammonium transporter, compared with the wild type. These findings suggest that the ΔrecA mutation affects not only the SOS response but also amino acid metabolism.

Inactivation of the oprD porin gene by a novel insertion sequence ISPa195 associated with large deletion in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate.

Alteration of the porin-encoding gene oprD by insertion sequences (ISs) is one mechanism conferring carbapenem resistance in Pseudomonas aeruginosa. Here we describe a carbapenem-resistant clinical P. aeruginosa isolate 36-989 harbouring a novel IS (ISPa195) in oprD. Minimum inhibitory concentrations (MICs) of antimicrobial agents were determined by the broth microdilution method. Carbapenemase activity was assessed using a MALDI-TOF/MS-based assay of meropenem hydrolysis. Efflux-dependent carbapenem resistance was evaluated using an assay with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The oprD gene and IS sequence were analysed by the Sanger method. Whole-genome sequencing was performed on an Illumina HiSeq 2500 platform. Antimicrobial susceptibility testing demonstrated that P. aeruginosa 36-989 was resistant to imipenem (MIC=32mg/L) and meropenem (MIC=16mg/L). No carbapenemase activity was detected, however an efflux-mediated component of carbapenem resistance was revealed. A new IS element (ISPa195) was found in the oprD gene of P. aeruginosa 36-989. ISPa195 was 1190bp in length, belonging to the IS3 family, and contained two open reading frames that overlapped through a ribosomal slippage to translate the full-size transposase enzyme. There was an IS-associated 284-bp deletion in the oprD gene; no direct repeats at flanking regions of the IS were detected. The absence of direct repeats at flanking regions in combination with the IS-associated deletion distinguished ISPa195 from other ISs previously detected in oprD. Carbapenem resistance in P. aeruginosa 36-989 was conferred by a combination of oprD alteration and carbapenem efflux.

Expression analysis of 10 efflux pump genes in multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis clinical isolates.

Active extrusion of antituberculosis drugs via efflux pumps (EPs) has been suggested as contributing to drug resistance in Mycobacterium tuberculosis. This study was conducted to determine the role of 10 drug efflux transporters in the development of drug resistance in a series of clinical M. tuberculosis isolates. A total of 31 clinical M. tuberculosis isolates without drug exposure [21 multi/extensively drug-resistant (M/XDR-TB) and 10 drug-susceptible isolates] were studied. The expression profile of 10 EP genes, including efpA, mmr, stp, drrA, drrB, mmpL7, Rv1250, Rv1634, Rv2994 and Rv1258c, was investigated against the H37Rv standard strain by quantitative reverse transcription PCR (RT-qPCR). Among the 21M/XDR-TB isolates, 10 showed significantly increased levels of gene expression (>4-fold) for at least one of the studied EPs. Moreover, of the isolates with overexpressed genes, three and seven lacked genetic alterations in the surveyed regions of the rpoB+katG+inhA and katG+inhA genes, respectively. Whilst no elevation was observed in the expression of mmr, Rv1250, Rv1634 and Rv1258c genes in any of the isolates, drrA, stp and drrB were found to be the most commonly overexpressed, being overexpressed in seven, five and three isolates, respectively. Decreased minimum inhibitory concentrations (MICs) of rifampicin, but not isoniazid, were observed in the presence of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Overexpression of EP genes can contribute to the emergence of a MDR phenotype in M. tuberculosis. Inhibition of EPs may provide a promising strategy for improving tuberculosis treatment outcomes in patients infected with M/XDR-TB isolates.

NO-synthase activity in mitochondria of uterus smooth muscle: identification and biochemical properties.

Information about the catalytic and kinetic properties of mitochondria NO-synthase from uterus smooth muscle is missing currently. According to the data on MitoTracker Orange CM-H2TMRos and 4-аmino-5-methylamino-2',7'-difluorescein, diaminofluorescein-FM (DAF-FM) dye co-localization in uterine smooth muscle cells, presented in this paper, NO can be synthesized in their mitochondria. High activity of NO synthase requires the presence of substrates of respiration, L-arginine, Ca2+ and NADPH. It is established that the dependence of NO production on the concentration of L-arginine has a bell-shaped character with a maximum of 75 μM, and the apparent affinity constant for L-arginine is 28.9 ± 9.1 μM. The dependence of NO production on Ca2+ concentration has a maximum at 100-250 μM; the activation constant for Ca2+ is 44.4 ± 14.5 μM. The inhibitor of Ca2+ transport in mitochondria ruthenium red (RuR), as well as the inhibitor of NO-synthase NG-nitro-L-arginine (NA), reduces NO production. The biosynthesis of NO by mitochondria depends on its energized level: it is stimulated by the addition of respiration substrates, suppressed with specific inhibitors of the electron transport chain (rotenone and antimycin A) and carbonyl-cyanide 3-chlorophenylhydrazone (CCCP) protonophore.

Mitochondrial functionality modifies human sperm acrosin activity, acrosome reaction capability and chromatin integrity.

In addition to sperm motility, which major biological characteristics of sperm fertility potential are associated with mitochondrial functionality? Sperm fertilization capacities, including acrosin activity, acrosome reaction (AR) capability and chromatin integrity, are related to the mitochondria functionality as evaluated by the mitochondrial membrane potential (MMP). Correlative studies suggest a potential role of sperm MMP in predicting sperm fertilization ability and ensuring sperm motility. However, researches characterizing other determinants of sperm fertility potential according to MMP are lacking. The sperm MMP was examined in 627 young college students in the Male Reproductive Health in Chongqing College Students (MARHCS) cohort study in 2014. Among these participants, acrosin activity and chromatin integrity were measured in 378 and 604 subjects, respectively. These two determinants of sperm fertility potential were first compared among high-, moderate- and low-MMP groups in the college population. The effects of MMP collapse caused by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on acrosin activity, AR, DNA fragmentation, reactive oxygen species (ROS) production, and ATP content in human spermatozoa were evaluated in vitro. The sperm MMP was evaluated by using JC-1 staining, acrosin activity was measured using a N-α-benzoyl-dl-arginine-para-nitroanilide HCl (BAPNA) substrate method, the integrity of chromatin represented by DNA fragmentation index (DFI) was measured by sperm chromatin structure assay (SCSA), AR was evaluated with chlortetracycline staining, and intracellular ROS production was evaluated with dihydroethidium. ATP concentration was determined with luciferase. Measurements were performed by spectrophotometry or flow cytometry. Nonparametric analysis revealed significantly higher acrosin activity and a lower DFI in subjects with moderate or high MMP compared to those with low MMP. After adjustment for potential confounders, increases of 7.9 and 44.4% in sperm acrosin activity and deceases of 12.0 and 25.2% in the sperm DFI were found in the moderate- and high-MMP groups, respectively. The MMP dissipation induced by CCCP caused significant declines in acrosin activity and AR capacity and increased DFI in human spermatozoa. Moreover, sperm MMP dissipation induced ROS overproduction and decreased ATP content. We cannot exclude a contribution of leukocytes to ROS production and no size gating was used to exclude these cells from the FACS measurements. No simultaneous live-dead staining was done and a contribution of dead sperm to the MMP and acrosome assays cannot be excluded. Mitochondrial functionality might be necessary to maintain sperm acrosin activity, AR and chromatin integrity. Tests of mitochondrial functionality should be developed and used independently of or in addition to conventional semen parameters in infertility diagnosis or risk-assessment processes. This study was supported by the Key Program of the National Natural Science Foundation of China (No. 81630087) and the National Natural Science Foundation of China (No. 81703254). None of the authors have any competing interests to declare.

First detection of efrAB, an ABC multidrug efflux pump in Enterococcus faecalis in Tehran, Iran.

Enterococcus faecalis is one of the most significant pathogen in both nosocomial and community-acquired infections. Reduced susceptibility to antibiotics is in part due to efflux pumps. This study was conducted on 80 isolates of E. faecalis isolated from outpatients with urinary tract infection during a period of 1year from April 2014 to April 2015. The antibiotic susceptibility patterns of isolates were determined by the disk diffusion method and presence of efrA and efrB genes was detected by PCR and sequencing. Minimum inhibitory concentrations (MICs) to ciprofloxacin (CIP) were measured with and without carbonyl cyanide 3-chlorophenylhydrazone (CCCP) by broth microdilution. The highest resistance rate was observed to erythromycin (83.3%) and the prevalence of efrA and efrB genes in all E. faecalis isolates was 100%. This study showed that 9 out of 13 (69.2%) ciprofloxacin-resistant isolates became less resistant at least fourfolds to CIP in the presence of efflux pump inhibitor. Our result showed that CCCP as an efflux inhibitor can increase effect of CIP as an efficient antibiotic and it is suggested that efrAB efflux pumps are involved in resistance to fluoroquinolone.

Phenotypic Detection of Efflux Mechanism in Panaminoglycoside Resistant Acinetobacter baumannii from Egyptian Clinical Isolates

Background: Over the last several years, Acinetobacter has emerged as a leading cause of hospital-acquired infections. Aminoglycosides are frequently used in the treatment of invasive infections. Factors associated with the resistance to aminoglycosides include the reduction of drug uptake, modification of aminoglycosides, and aminoglycoside efflux. Objectives: The aim of our study was to phenotypically detect the presence of efflux mechanism using carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in aminoglycoside resistant Acinetobacter baumannii strains isolated from different hospital wards. Methods: In total, 57 A. baumannii isolates were collected from two Egyptian hospitals. The antimicrobial susceptibility pattern was determined. The activity of the efflux system was evaluated using CCCP. Results: Among 57 A. baumannii isolates, most resistance was observed against tobramycin, amikacin, kanamycin, neomycin, and gentamicin. The minimum inhibitory concentrations (MIC) range of A. baumannii was between 2 and 1024 µg/mL based on the tested antibiotics. The phenotypic detection of efflux pumps displaying a reduction of at least two folds in the MICs of antibiotics after addition of the efflux pump inhibitor showed that 19.4% of the isolates became less resistant to kanamycin, 44% to tobramycin, and 46% to amikacin but lower rates were recorded against gentamicin (12.2%) and neomycin (9.4 %). Conclusions: Our study suggests that the efflux mechanism is getting widespread in clinical settings to play an important role in aminoglycoside resistance. Acinetobacter baumannii has the ability to gain resistance to different antibiotic classes through these active efflux pumps. Hence, the application of strict infection control measures together with novel approaches to eradicate those efflux transporters should be applied in hospital settings.