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Sample records for active efflux system

  1. [The role of cell wall organization and active efflux pump systems in multidrug resistance of bacteria].

    PubMed

    Hasdemir, Ufuk

    2007-04-01

    Multiple antibiotic resistance of clinically important bacteria are of major concern worldwide. Alterations of drug targets or enzymatic inactivation of antimicrobial agents are the well known mechanisms of antimicrobial drug resistance. Besides these well known mechanisms, recent studies have shown that a further resistance mechanism, active drug efflux, has become increasingly important in the current threat of multidrug resistance. It involves certain bacterial transport proteins which pump out toxic antimicrobial compounds from the cell. Drug efflux pump proteins in bacteria fall into five distinct protein super families [ATP binding cassette super family (ABC), Major facilitator super family (MFS), Small multidrug resistance super family (SMR), Multidrug and toxic compound extrusion (MATE) super family, Resistance-nodulation-cell division (RND) super family] and are mostly encoded by chromosomal genes. Among them, the members of RND protein super family are widely distrubuted in Gram negative bacteria and play siginificant role in both, intrinsic and acquired multidrug resistance of these bacteria with very wide substrate specificity. RND type multidrug efflux proteins usually function together with an outer membrane canal protein (OMP) and a membrane fusion protein (MFP) to pump out drugs. AcrAB-TolC of Escherichia coli and MexAB-OprM of Pseudomonas aeruginosa are the typical examples of these tripartite systems. They are constitutively expressed in wild type cells and play significant role in intrinsic resistance of these bacteria. However, multidrug resistance which is of major clinical significance, rises as a result of overexpression of these pump systems due to mutations and elevated levels of resistance are recorded to structurally unrelated antimicrobial drugs such as fluoroquinolones, beta-lactams, tetracyclines, chloramphenicol, trimethoprim, aminoglycosides and toxic compunds. Synthesis of RND type pump proteins are regulated by complex genetic

  2. KefF, the regulatory subunit of the potassium efflux system KefC, shows quinone oxidoreductase activity.

    PubMed

    Lyngberg, Lisbeth; Healy, Jessica; Bartlett, Wendy; Miller, Samantha; Conway, Stuart J; Booth, Ian R; Rasmussen, Tim

    2011-09-01

    Escherichia coli and many other Gram-negative pathogenic bacteria protect themselves from the toxic effects of electrophilic compounds by using a potassium efflux system (Kef). Potassium efflux is coupled to the influx of protons, which lowers the internal pH and results in immediate protection. The activity of the Kef system is subject to complex regulation by glutathione and its S conjugates. Full activation of KefC requires a soluble ancillary protein, KefF. This protein has structural similarities to oxidoreductases, including human quinone reductases 1 and 2. Here, we show that KefF has enzymatic activity as an oxidoreductase, in addition to its role as the KefC activator. It accepts NADH and NADPH as electron donors and quinones and ferricyanide (in addition to other compounds) as acceptors. However, typical electrophilic activators of the Kef system, e.g., N-ethyl maleimide, are not substrates. If the enzymatic activity is disrupted by site-directed mutagenesis while retaining structural integrity, KefF is still able to activate the Kef system, showing that the role as an activator is independent of the enzyme activity. Potassium efflux assays show that electrophilic quinones are able to activate the Kef system by forming S conjugates with glutathione. Therefore, it appears that the enzymatic activity of KefF diminishes the redox toxicity of quinones, in parallel with the protection afforded by activation of the Kef system.

  3. Comparative study of the active cadmium efflux systems operating at the plasma membrane and tonoplast of cucumber root cells.

    PubMed

    Migocka, Magdalena; Papierniak, Anna; Kosatka, Ewelina; Klobus, Grazyna

    2011-10-01

    The strategies developed by plants to avoid the toxicity of cadmium (Cd) and other heavy metals involve active sequestration of metals into the apoplast and vacuoles. The protein systems excluding heavy metals from the cell cytosol localize to the plasma membrane and tonoplast and are energized either by ATP or by the electrochemical gradient generated by H(+)-ATPase or by V-ATPase and pyrophosphatase (PPase), respectively. In this work, a comparative study on the contribution of both the plasma membrane and tonoplast in the active detoxification of plant cells after treatment with Cd was performed. The studies using plants treated and untreated with Cd reveal that both, H(+)-coupled and MgATP-driven efflux of Cd across plasma membranes and tonoplast is markedly stimulated in the presence of Cd in the environment. Previous studies on plasma-membrane localized H(+)-coupled Cd efflux together with the present data demonstrating tonoplast H(+)/Cd(2+) antiport activity suggest that H(+)-coupled secondary transport of Cd displays a lower affinity for Cd when compared with Cd primary pumps driven by MgATP. In addition, it is shown that MgATP-energized Cd efflux across both membranes is significantly enhanced by cysteine, dithiothreitol, and glutathione. These results suggest that Cd is excluded from the cytosol through an energy-dependent system as a free ion as well as a complexed form. Although both membranes contribute in the active exclusion of ionized and complexed Cd from the cytosol, the overall calculation of Cd accumulation in the everted plasma membranes and vacuolar vesicles suggests that the tonoplast and vacuole have a major function in Cd efflux from the cytosol in the roots of cucumber subjected to Cd stress.

  4. Proton-dependent multidrug efflux systems.

    PubMed Central

    Paulsen, I T; Brown, M H; Skurray, R A

    1996-01-01

    Multidrug efflux systems display the ability to transport a variety of structurally unrelated drugs from a cell and consequently are capable of conferring resistance to a diverse range of chemotherapeutic agents. This review examines multidrug efflux systems which use the proton motive force to drive drug transport. These proteins are likely to operate as multidrug/proton antiporters and have been identified in both prokaryotes and eukaryotes. Such proton-dependent multidrug efflux proteins belong to three distinct families or superfamilies of transport proteins: the major facilitator superfamily (MFS), the small multidrug resistance (SMR) family, and the resistance/ nodulation/cell division (RND) family. The MFS consists of symporters, antiporters, and uniporters with either 12 or 14 transmembrane-spanning segments (TMS), and we show that within the MFS, three separate families include various multidrug/proton antiport proteins. The SMR family consists of proteins with four TMS, and the multidrug efflux proteins within this family are the smallest known secondary transporters. The RND family consists of 12-TMS transport proteins and includes a number of multidrug efflux proteins with particularly broad substrate specificity. In gram-negative bacteria, some multidrug efflux systems require two auxiliary constituents, which might enable drug transport to occur across both membranes of the cell envelope. These auxiliary constituents belong to the membrane fusion protein and the outer membrane factor families, respectively. This review examines in detail each of the characterized proton-linked multidrug efflux systems. The molecular basis of the broad substrate specificity of these transporters is discussed. The surprisingly wide distribution of multidrug efflux systems and their multiplicity in single organisms, with Escherichia coli, for instance, possessing at least nine proton-dependent multidrug efflux systems with overlapping specificities, is examined. We also

  5. Enhanced Efflux Activity Facilitates Drug Tolerance in Dormant Bacterial Cells

    PubMed Central

    Pu, Yingying; Zhao, Zhilun; Li, Yingxing; Zou, Jin; Ma, Qi; Zhao, Yanna; Ke, Yuehua; Zhu, Yun; Chen, Huiyi; Baker, Matthew A.B.; Ge, Hao; Sun, Yujie; Xie, Xiaoliang Sunney; Bai, Fan

    2016-01-01

    Summary Natural variations in gene expression provide a mechanism for multiple phenotypes to arise in an isogenic bacterial population. In particular, a sub-group termed persisters show high tolerance to antibiotics. Previously, their formation has been attributed to cell dormancy. Here we demonstrate that bacterial persisters, under β-lactam antibiotic treatment, show less cytoplasmic drug accumulation as a result of enhanced efflux activity. Consistently, a number of multi-drug efflux genes, particularly the central component TolC, show higher expression in persisters. Time-lapse imaging and mutagenesis studies further establish a positive correlation between tolC expression and bacterial persistence. The key role of efflux systems, among multiple biological pathways involved in persister formation, indicates that persisters implement a positive defense against antibiotics prior to a passive defense via dormancy. Finally, efflux inhibitors and antibiotics together effectively attenuate persister formation, suggesting a combination strategy to target drug tolerance. PMID:27105118

  6. A combined long-term recording system for single-unit activity and neurotransmitter efflux of a brain slice

    NASA Astrophysics Data System (ADS)

    Sheu, Y. H.; Young, M. S.

    1998-04-01

    A combined long-term measurement and recording system for neurotransmission research of brain slices is presented in this study. This system, based on the IBM PC or compatible computer, is capable of simultaneously measuring and recording both single-unit neural electropotential signals and the electrochemical signals of neurotransmitter efflux from the same neuron in a brain slice for long periods of time (time limited largely by hard disk capacity, 100 h or more not being unreasonable with contemporary hardware) using a single carbon microelectrode for both measurements. The combined long-term recording system uses a simple switching circuit to switch periodically the single microelectrode between two data acquisition subsystems, one for electrochemical data and one for electrophysiological data. The simple switching circuit separates the electrophysiological signals and electrochemical signals, overcoming the traditional interference problem caused by the two different measuring techniques. Software designed for the proposed system allows easy reconstruction of the full time course of the compressed measured data and easy, simultaneous display of both types of signals on the same time scale. On-line and recorded displays are available. Test results of a practical implementation of the proposed system verify that the combined long-term recording system meets actual requirements for electrophysiological and neurochemical research.

  7. Calcium Efflux Systems in Stress Signaling and Adaptation in Plants

    PubMed Central

    Bose, Jayakumar; Pottosin, Igor I.; Shabala, Stanislav S.; Palmgren, Michael G.; Shabala, Sergey

    2011-01-01

    Transient cytosolic calcium ([Ca2+]cyt) elevation is an ubiquitous denominator of the signaling network when plants are exposed to literally every known abiotic and biotic stress. These stress-induced [Ca2+]cyt elevations vary in magnitude, frequency, and shape, depending on the severity of the stress as well the type of stress experienced. This creates a unique stress-specific calcium “signature” that is then decoded by signal transduction networks. While most published papers have been focused predominantly on the role of Ca2+ influx mechanisms to shaping [Ca2+]cyt signatures, restoration of the basal [Ca2+]cyt levels is impossible without both cytosolic Ca2+ buffering and efficient Ca2+ efflux mechanisms removing excess Ca2+ from cytosol, to reload Ca2+ stores and to terminate Ca2+ signaling. This is the topic of the current review. The molecular identity of two major types of Ca2+ efflux systems, Ca2+-ATPase pumps and Ca2+/H+ exchangers, is described, and their regulatory modes are analyzed in detail. The spatial and temporal organization of calcium signaling networks is described, and the importance of existence of intracellular calcium microdomains is discussed. Experimental evidence for the role of Ca2+ efflux systems in plant responses to a range of abiotic and biotic factors is summarized. Contribution of Ca2+-ATPase pumps and Ca2+/H+ exchangers in shaping [Ca2+]cyt signatures is then modeled by using a four-component model (plasma- and endo-membrane-based Ca2+-permeable channels and efflux systems) taking into account the cytosolic Ca2+ buffering. It is concluded that physiologically relevant variations in the activity of Ca2+-ATPase pumps and Ca2+/H+ exchangers are sufficient to fully describe all the reported experimental evidence and determine the shape of [Ca2+]cyt signatures in response to environmental stimuli, emphasizing the crucial role these active efflux systems play in plant adaptive responses to environment. PMID:22639615

  8. Contribution of efflux activity to isoniazid resistance in the Mycobacterium tuberculosis complex.

    PubMed

    Rodrigues, Liliana; Machado, Diana; Couto, Isabel; Amaral, Leonard; Viveiros, Miguel

    2012-06-01

    Resistance to isoniazid (INH), one of the main drugs used in tuberculosis (TB) therapy, is mostly due to chromosomal mutations in target genes. However, approximately 20-30% of INH resistant Mycobacterium tuberculosis isolates do not have mutations in any of the genes associated with INH resistance. This suggests that other mechanism(s) may be involved, namely efflux pump systems capable of extruding the drug to the exterior of the cell. In a previous work, we have induced clinical INH susceptible M. tuberculosis isolates and the H37Rv reference strain to high-level resistance to INH, by gradual exposure to increasing concentrations of this drug. In the present study, we have characterized these strains and Mycobacterium bovis BCG induced to INH resistance with respect to their efflux activity and its contribution to INH resistance using the following approach: determination of the susceptibility to INH in the presence and absence of the efflux inhibitors (EIs) chlorpromazine, thioridazine and verapamil; evaluation of efflux activity by a semi-automated fluorometric method; and quantification of the expression level of genes coding for efflux pumps by real-time RT-qPCR. The EIs decreased INH resistance in the INH induced strains, in particular verapamil promoted a reversal of resistance in some of the strains tested. The induced strains presented an increased efflux activity that was inhibited by the EIs and showed overexpression of the efflux pump genes efpA, mmpL7, mmr, p55 and the Tap-like gene Rv1258c. Altogether, these results correlate efflux activity with INH resistance and demonstrate that efflux pumps play an important role in acquired INH resistance in M. tuberculosis complex. The development of EIs that can restore the antimicrobial activity of the antibiotic subject to efflux is an approach that can be useful in order to prevent the emergence of this resistance and guide the development of new effective anti-TB therapeutical approaches.

  9. RND efflux pump and its interrelationship with quorum sensing system.

    PubMed

    Zhibin, Liang; Yumei, Chen; Yufan, Chen; Yingying, Cheng; Lianhui, Zhang

    2016-10-20

    Antibiotic resistance has become a serious concern in treatment of bacterial infections. Overexpression of efflux pump is one of the important mechanisms in antibiotic resistance. In Gram negative bacteria, RND (Resistance-nodulation-cell division) superfamily efflux pump plays a vital important role in antibiotics resistance. Recent research progress unveils an intriguing interrelationship between RND efflux pump and the bacterial quorum sensing system, whose regulation is dependent on small signal molecules. This article reviews the latest findings on the structure and transport mechanism of RND efflux pump, as well as the general features and regulatory mechanisms of quorum sensing, with a special focus on the role and mechanism of quorum sensing system in regulation of RND efflux pump, and the influence of efflux pump on quorum sensing signal transportation. Further investigation of the interrelationship between RND efflux pumps and the bacterial quorum sensing systems is critical for elucidation of the regulatory mechanisms that govern the expression of the RND efflux pumps genes, and may also provide useful clues to overcome the efflux pump mediated antibiotic resistance.

  10. Efflux systems in bacteria and their metabolic engineering applications.

    PubMed

    Jones, Christopher M; Hernández Lozada, Néstor J; Pfleger, Brian F

    2015-11-01

    The production of valuable chemicals from metabolically engineered microbes can be limited by excretion from the cell. Efflux is often overlooked as a bottleneck in metabolic pathways, despite its impact on alleviating feedback inhibition and product toxicity. In the past, it has been assumed that endogenous efflux pumps and membrane porins can accommodate product efflux rates; however, there are an increasing number of examples wherein overexpressing efflux systems is required to improve metabolite production. In this review, we highlight specific examples from the literature where metabolite export has been studied to identify unknown transporters, increase tolerance to metabolites, and improve the production capabilities of engineered bacteria. The review focuses on the export of a broad spectrum of valuable chemicals including amino acids, sugars, flavins, biofuels, and solvents. The combined set of examples supports the hypothesis that efflux systems can be identified and engineered to confer export capabilities on industrially relevant microbes.

  11. Multidrug Efflux Systems in Microaerobic and Anaerobic Bacteria

    PubMed Central

    Xu, Zeling; Yan, Aixin

    2015-01-01

    Active drug efflux constitutes an important mechanism of antibiotic and multidrug resistance in bacteria. Understanding the distribution, expression, and physiological functions of multidrug efflux pumps, especially under physiologically and clinically relevant conditions of the pathogens, is the key to combat drug resistance. In animal hosts, most wounded, infected and inflamed tissues display low oxygen tensions. In this article, we summarize research development on multidrug efflux pumps in the medicinally relevant microaerobic and anaerobic pathogens and their implications in the effort to combat drug-resistant infections. PMID:27025630

  12. Neuroinflammation activates efflux transport by NFκB

    PubMed Central

    Yu, Chuanhui; Argyropoulos, George; Zhang, Yan; Kastin, Abba J.; Hsuchou, Hung; Pan, Weihong

    2009-01-01

    Background/aims Although it is known that drug delivery across the blood-brain barrier (BBB) may be hampered by efflux transport activity of the multidrug resistance (mdr) gene product P-glycoprotein, it is not clear how inflammation regulates efflux transporters. In rat brain endothelial (RBE4) cells of BBB origin, the proinflammatory cytokine TNF mainly induces transcriptional upregulation of mdr1b, and to a lesser extent mdr1a, resulting in greater efflux of the substrates (Yu C et al., Cell Physiol Biochem, 2007). This study further determined the mechanisms by which TNF activates mdr1b promoter activity. Methods/Results Luciferase reporter assays and DNA binding studies show that (a) maximal basal promoter activity was conferred by a 476 bp sequence upstream to the mdr1b transcriptional initiation site; (2) TNF induced upregulation of promoter activity by NFkB nuclear translocation; and (3) the NFκB binding site of the mdr1b promoter was solely responsible for basal and TNF-activated gene transcription, whereas the p53 binding site was not involved. Binding of the p65 subunit of NFκB to nuclear DNA from RBE4 cells was shown by electrophoretic mobility shift assay and chromatin immunoprecipitation assays. Conclusion Thus, NFκB mediated TNF-induced upregulation of mdr1b promoter activity, illustrating how inflammation activates BBB efflux transport. PMID:19088456

  13. Volume-activated amino acid efflux from term human placental tissue: stimulation of efflux via a pathway sensitive to anion transport inhibitors.

    PubMed

    Shennan, D B; McNeillie, S A

    1995-04-01

    The effect of a hyposmotic challenge and hence cell-swelling upon the efflux of a variety of solutes from isolated human placental tissue has been examined. A hyposmotic shock increased the fractional release of taurine, the most abundant free amino acid in placental tissue, via a pathway sensitive to niflumic acid, DIDS (4,4'-Diisothiocyanatostilbene-2',2'-disulphonic acid,) NPPB (5-Nitro-2(3-phenylpropylamino)benzoic acid) and DIOA (R(+)[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden -5-y) oxy] acetic acid). In contrast, tamoxifen was without effect. The cell-swelling induced efflux of taurine was attenuated (40 per cent) by replacing external Cl- with NO3-. The efflux of glutamic acid was also markedly increased by a hyposmotic challenge. Niflumic acid inhibited both basal and volume-activated glutamic acid efflux. A hyposmotic shock also increased alpha-aminoisobutyric acid efflux but not that of 3-O-methylglucose and SO4(2)-. The results suggest that the human placenta can respond to cell-swelling by releasing organic osmolytes such as amino acids via a pathway which is sensitive to anion transport inhibitors. However, it appears that the volume-activated amino acid transport system is independent from the placental anion-exchange pathways. The efflux of these compounds may act with K+ and Cl- efflux to effect a regulatory volume decrease in placental tissue. In addition, volume-activated transport may play a role in transplacental amino acid transfer.

  14. Microbial Efflux Systems and Inhibitors: Approaches to Drug Discovery and the Challenge of Clinical Implementation

    PubMed Central

    Kourtesi, Christina; Ball, Anthony R; Huang, Ying-Ying; Jachak, Sanjay M; Vera, D Mariano A; Khondkar, Proma; Gibbons, Simon; Hamblin, Michael R; Tegos, George P

    2013-01-01

    Conventional antimicrobials are increasingly ineffective due to the emergence of multidrug-resistance among pathogenic microorganisms. The need to overcome these deficiencies has triggered exploration for novel and unconventional approaches to controlling microbial infections. Multidrug efflux systems (MES) have been a profound obstacle in the successful deployment of antimicrobials. The discovery of small molecule efflux system blockers has been an active and rapidly expanding research discipline. A major theme in this platform involves efflux pump inhibitors (EPIs) from natural sources. The discovery methodologies and the available number of natural EPI-chemotypes are increasing. Advances in our understanding of microbial physiology have shed light on a series of pathways and phenotypes where the role of efflux systems is pivotal. Complementing existing antimicrobial discovery platforms such as photodynamic therapy (PDT) with efflux inhibition is a subject under investigation. This core information is a stepping stone in the challenge of highlighting an effective drug development path for EPIs since the puzzle of clinical implementation remains unsolved. This review summarizes advances in the path of EPI discovery, discusses potential avenues of EPI implementation and development, and underlines the need for highly informative and comprehensive translational approaches. PMID:23569468

  15. Efflux pumps of Mycobacterium tuberculosis play a significant role in antituberculosis activity of potential drug candidates.

    PubMed

    Balganesh, Meenakshi; Dinesh, Neela; Sharma, Sreevalli; Kuruppath, Sanjana; Nair, Anju V; Sharma, Umender

    2012-05-01

    Active efflux of drugs mediated by efflux pumps that confer drug resistance is one of the mechanisms developed by bacteria to counter the adverse effects of antibiotics and chemicals. To understand these efflux mechanisms in Mycobacterium tuberculosis, we generated knockout (KO) mutants of four efflux pumps of the pathogen belonging to different classes. We measured the MICs and kill values of two different compound classes on the wild type (WT) and the efflux pump (EP) KO mutants in the presence and absence of the efflux inhibitors verapamil and l-phenylalanyl-l-arginyl-β-naphthylamide (PAβN). Among the pumps studied, the efflux pumps belonging to the ABC (ATP-binding cassette) class, encoded by Rv1218c, and the SMR (small multidrug resistance) class, encoded by Rv3065, appear to play important roles in mediating the efflux of different chemical classes and antibiotics. Efflux pumps encoded by Rv0849 and Rv1258c also mediate the efflux of these compounds, but to a lesser extent. Increased killing is observed in WT M. tuberculosis cells by these compounds in the presence of either verapamil or PAβN. The efflux pump KO mutants were more susceptible to these compounds in the presence of efflux inhibitors. We have shown that these four efflux pumps of M. tuberculosis play a vital role in mediating efflux of different chemical scaffolds. Inhibitors of one or several of these efflux pumps could have a significant impact in the treatment of tuberculosis. The identification and characterization of Rv0849, a new efflux pump belonging to the MFS (major facilitator superfamily) class, are reported.

  16. Predicting Efflux Ratios and Blood-Brain Barrier Penetration from Chemical Structure: Combining Passive Permeability with Active Efflux by P-Glycoprotein

    PubMed Central

    2012-01-01

    In order to reach their pharmacologic targets, successful central nervous system (CNS) drug candidates have to cross a complex protective barrier separating brain from the blood. Being able to predict a priori which molecules can successfully penetrate this barrier could be of significant value in CNS drug discovery. Herein we report a new computational approach that combines two mechanism-based models, for passive permeation and for active efflux by P-glycoprotein, to provide insight into the multiparameter optimization problem of designing small molecules able to access the CNS. Our results indicate that this approach is capable of distinguishing compounds with high/low efflux ratios as well as CNS+/CNS– compounds and provides advantage over estimating P-glycoprotein efflux or passive permeability alone when trying to predict these emergent properties. We also demonstrate that this method could be useful for rank-ordering chemically similar compounds and that it can provide detailed mechanistic insight into the relationship between chemical structure and efflux ratios and/or CNS penetration, offering guidance as to how compounds could be modified to improve their access into the brain. PMID:23421687

  17. Homologs of the Acinetobacter baumannii AceI Transporter Represent a New Family of Bacterial Multidrug Efflux Systems

    PubMed Central

    Liu, Qi; Henderson, Peter J. F.

    2015-01-01

    ABSTRACT Multidrug efflux systems are a major cause of resistance to antimicrobials in bacteria, including those pathogenic to humans, animals, and plants. These proteins are ubiquitous in these pathogens, and five families of bacterial multidrug efflux systems have been identified to date. By using transcriptomic and biochemical analyses, we recently identified the novel AceI (Acinetobacter chlorhexidine efflux) protein from Acinetobacter baumannii that conferred resistance to the biocide chlorhexidine, via an active efflux mechanism. Proteins homologous to AceI are encoded in the genomes of many other bacterial species and are particularly prominent within proteobacterial lineages. In this study, we expressed 23 homologs of AceI and examined their resistance and/or transport profiles. MIC analyses demonstrated that, like AceI, many of the homologs conferred resistance to chlorhexidine. Many of the AceI homologs conferred resistance to additional biocides, including benzalkonium, dequalinium, proflavine, and acriflavine. We conducted fluorimetric transport assays using the AceI homolog from Vibrio parahaemolyticus and confirmed that resistance to both proflavine and acriflavine was mediated by an active efflux mechanism. These results show that this group of AceI homologs represent a new family of bacterial multidrug efflux pumps, which we have designated the proteobacterial antimicrobial compound efflux (PACE) family of transport proteins. PMID:25670776

  18. Physiological characterisation of the efflux pump system of antibiotic-susceptible and multidrug-resistant Enterobacter aerogenes.

    PubMed

    Martins, A; Spengler, G; Martins, M; Rodrigues, L; Viveiros, M; Davin-Regli, A; Chevalier, J; Couto, I; Pagès, J M; Amaral, L

    2010-10-01

    Enterobacter aerogenes predominates amongst Enterobacteriaceae species that are increasingly reported as producers of extended-spectrum beta-lactamases. Although this mechanism of resistance to beta-lactams is important, other mechanisms bestowing a multidrug-resistant (MDR) phenotype in this species are now well documented. Amongst these mechanisms is the overexpression of efflux pumps that extrude structurally unrelated antibiotics prior to their reaching their targets. Interestingly, although knowledge of the genetic background behind efflux pumps is rapidly advancing, few studies assess the physiological nature of the overall efflux pump system of this, or for that matter any other, bacterium. The study reported here evaluates physiologically the efflux pump system of an E. aerogenes ATCC reference as well as two strains whose MDR phenotypes are mediated by overexpressed efflux pumps. The activities of the efflux pumps in these strains are modulated by pH and glucose, although the effects of the latter are essentially restricted to pH 8, suggesting the presence of two general efflux pump systems, i.e. proton-motive force-dependent and ABC transporter types, respectively.

  19. Anti-androgens inhibit ABCB1 efflux and ATPase activity and reverse docetaxel resistance in advanced prostate cancer

    PubMed Central

    Zhu, Yezi; Liu, Chengfei; Armstrong, Cameron; Lou, Wei; Sandher, Amandeep; Gao, Allen C.

    2015-01-01

    Purpose Previous studies show that inhibition of ABCB1 expression overcomes acquired docetaxel resistance in C4-2B-TaxR cells. In this study, we examined if anti-androgens, such as bicalutamide and enzalutamide, could inhibit ABCB1 activity and overcome resistance to docetaxel. Experimental Design ABCB1 efflux activity was determined using a rhodamine efflux assay. ABCB1 ATPase activity was determined by Pgp-Glo™ assay systems. The effects of the anti-androgens bicalutamide and enzalutamide on docetaxel sensitivity were determined by cell growth assays and tumor growth in vivo. Results We found that bicalutamide and enzalutamide inhibit ABCB1 ATP-binding cassette transporter activity through blocking ABCB1 efflux activity. Bicalutamide inhibited ABCB1 efflux activity by 40%, while enzalutamide inhibited ABCB1 efflux activity by ~60%. Both bicalutamide and enzalutamide inhibit ABCB1 ATPase activity. In addition, bicalutamide and enzalutamide inhibit ABCB1 efflux activity and desensitize docetaxel resistant and androgen receptor (AR)-negative DU145 cells. Combination of bicalutamide with docetaxel had a significant anti-tumor effect in both AR-positive and AR-negative docetaxel resistant xenograft models, suggesting that bicalutamide desensitizes docetaxel resistant cells to docetaxel treatment independent of AR status. Conclusions We identified a novel mechanism of action for anti-androgens such as bicalutamide and enzalutamide as inhibitors of ABCB1 efflux and ATPase activity. Bicalutamide and enzalutamide desensitize docetaxel resistant prostate cancer cells to docetaxel treatment independent of AR status. These studies may lead to the development of combinational therapies with bicalutamide/enzalutamide and docetaxel as an effective regiment to treat advanced castration resistant prostate cancer (CRPC) independent of AR status. PMID:25995342

  20. Swelling-activated taurine and creatine effluxes from rat cortical astrocytes are pharmacologically distinct.

    PubMed

    Bothwell, J H; Styles, P; Bhakoo, K K

    2002-01-15

    Primary cultures of rat cortical astrocytes undergo a swelling-activated loss of taurine and creatine. In this study, the pharmacological characteristics of the taurine and creatine efflux pathways were compared, and significant differences were shown to exist between the two. Both taurine and creatine effluxes were rapidly activated upon exposure of astrocytes to hypo-osmotic media, and rapidly inactivated upon their return to iso-osmotic media. The relative rates of taurine and creatine efflux depended upon the magnitude of the hypo-osmotic shock. Anion-transport inhibitors strongly inhibited taurine efflux, with the order of potency being NPPB > DIDS > niflumic acid. DIDS and NPPB had less of an inhibitory effect on creatine efflux, whereas tamoxifen and niflumic acid actually stimulated creatine efflux. These data are consistent with separate pathways for taurine and creatine loss during astrocyte swelling.

  1. Enhancement of antibiotic activity by efflux inhibitors against multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil.

    PubMed

    Coelho, Tatiane; Machado, Diana; Couto, Isabel; Maschmann, Raquel; Ramos, Daniela; von Groll, Andrea; Rossetti, Maria L; Silva, Pedro A; Viveiros, Miguel

    2015-01-01

    Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA) to study single combinations between antituberculosis drugs and efflux inhibitors (EIs) against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC) indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates.

  2. The Cus efflux system removes toxic ions via a methionine shuttle.

    PubMed

    Su, Chih-Chia; Long, Feng; Yu, Edward W

    2011-01-01

    Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel diverse toxic compounds from the cell. These efflux systems span the entire cell envelope to mediate the phenomenon of bacterial multidrug resistance. The three parts of the efflux complexes are: (1) a membrane fusion protein (MFP) connecting (2) a substrate-binding inner membrane transporter to (3) an outer membrane-anchored channel in the periplasmic space. One such efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. We recently determined the crystal structures of both the inner membrane transporter CusA and MFP CusB of the CusCBA tripartite efflux system from E. coli. These are the first structures of the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here, we summarize the structural information of these two efflux proteins and present the accumulated evidence that this efflux system utilizes methionine residues to bind and export Cu(I)/Ag(I). Genetic and structural analyses suggest that the CusA pump is capable of picking up the metal ions from both the periplasm and cytoplasm. We propose a stepwise shuttle mechanism for this pump to extrude metal ions from the cell.

  3. Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in Acinetobacter baumannii

    PubMed Central

    Cain, Amy K.; Huang, TaoTao; Liu, Qi; Elbourne, Liam D. H.; Boinett, Christine J.; Brzoska, Anthony J.; Li, Liping; Ostrowski, Martin; Nhu, Nguyen Thi Khanh; Nhu, Tran Do Hoang; Baker, Stephen; Paulsen, Ian T.

    2016-01-01

    ABSTRACT Multidrug efflux pumps provide clinically significant levels of drug resistance in a number of Gram-negative hospital-acquired pathogens. These pathogens frequently carry dozens of genes encoding putative multidrug efflux pumps. However, it can be difficult to determine how many of these pumps actually mediate antimicrobial efflux, and it can be even more challenging to identify the regulatory proteins that control expression of these pumps. In this study, we developed an innovative high-throughput screening method, combining transposon insertion sequencing and cell sorting methods (TraDISort), to identify the genes encoding major multidrug efflux pumps, regulators, and other factors that may affect the permeation of antimicrobials, using the nosocomial pathogen Acinetobacter baumannii. A dense library of more than 100,000 unique transposon insertion mutants was treated with ethidium bromide, a common substrate of multidrug efflux pumps that is differentially fluorescent inside and outside the bacterial cytoplasm. Populations of cells displaying aberrant accumulations of ethidium were physically enriched using fluorescence-activated cell sorting, and the genomic locations of transposon insertions within these strains were determined using transposon-directed insertion sequencing. The relative abundance of mutants in the input pool compared to the selected mutant pools indicated that the AdeABC, AdeIJK, and AmvA efflux pumps are the major ethidium efflux systems in A. baumannii. Furthermore, the method identified a new transcriptional regulator that controls expression of amvA. In addition to the identification of efflux pumps and their regulators, TraDISort identified genes that are likely to control cell division, cell morphology, or aggregation in A. baumannii. PMID:27601573

  4. Bacillus subtilis MntR coordinates the transcriptional regulation of manganese uptake and efflux systems.

    PubMed

    Huang, Xiaojuan; Shin, Jung-Ho; Pinochet-Barros, Azul; Su, Tina T; Helmann, John D

    2017-01-01

    The Bacillus subtilis MntR metalloregulatory protein senses manganese, an essential element required for central metabolism, oxidative stress resistance and replication. An mntR null mutant is highly sensitive to Mn(II) intoxication, which is attributed in part to the constitutive expression of two importers: the proton-dependent NRAMP family transporter MntH and the ABC transporter MntABCD. Here, we show that an mntR null mutant is still sensitive to Mn(II) intoxication even if both of the import systems are absent. This Mn(II) sensitivity results from the requirement for MntR to activate the transcription of two genes encoding cation diffusion facilitator (CDF) family efflux pumps. Physiological studies indicate that MneP (formerly YdfM) serves as the primary Mn(II) efflux pump with MneS (formerly YeaB) playing a secondary role. Mutant strains lacking mneP are Mn(II) sensitive and accumulate elevated levels of Mn(II), and these effects are exacerbated in a mneP mneS double mutant. DNA-binding and in vitro transcription studies demonstrate that MntR binds to both the mneP and mneS regulatory regions and directly activates transcription in response to levels of Mn(II) several-fold higher than required for repression of import genes. These results highlight the delicate balance of Mn(II) uptake and efflux systems controlled by MntR.

  5. Interspecies signalling: Pseudomonas putida efflux pump TtgGHI is activated by indole to increase antibiotic resistance.

    PubMed

    Molina-Santiago, Carlos; Daddaoua, Abdelali; Fillet, Sandy; Duque, Estrella; Ramos, Juan-Luis

    2014-05-01

    In Gram-negative bacteria, multidrug efflux pumps are responsible for the extrusion of chemicals that are deleterious for growth. Some of these efflux pumps are induced by endogenously produced effectors, while abiotic or biotic signals induce the expression of other efflux pumps. In Pseudomonas putida, the TtgABC efflux pump is the main antibiotic extrusion system that respond to exogenous antibiotics through the modulation of the expression of this operon mediated by TtgR. The plasmid-encoded TtgGHI efflux pump in P. putida plays a minor role in antibiotic resistance in the parental strain; however, its role is critical in isogenic backgrounds deficient in TtgABC. Expression of ttgGHI is repressed by the TtgV regulator that recognizes indole as an effector, although P. putida does not produce indole itself. Because indole is not produced by Pseudomonas, the indole-dependent antibiotic resistance seems to be part of an antibiotic resistance programme at the community level. Pseudomonas putida recognizes indole added to the medium or produced by Escherichia coli in mixed microbial communities. Transcriptomic analyses revealed that the indole-specific response involves activation of 43 genes and repression of 23 genes. Indole enhances not only the expression of the TtgGHI pump but also a set of genes involved in iron homeostasis, as well as genes for amino acid catabolism. In a ttgABC-deficient P. putida, background ampicillin and other bactericidal compounds lead to cell death. Co-culture of E. coli and P. putida ΔttgABC allowed growth of the P. putida mutant in the presence of ampicillin because of induction of the indole-dependent efflux pump.

  6. Phytochemicals increase the antibacterial activity of antibiotics by acting on a drug efflux pump

    PubMed Central

    Ohene-Agyei, Thelma; Mowla, Rumana; Rahman, Taufiq; Venter, Henrietta

    2014-01-01

    Drug efflux pumps confer resistance upon bacteria to a wide range of antibiotics from various classes. The expression of efflux pumps are also implicated in virulence and biofilm formation. Moreover, organisms can only acquire resistance in the presence of active drug efflux pumps. Therefore, efflux pump inhibitors (EPIs) are attractive compounds to reverse multidrug resistance and to prevent the development of resistance in clinically relevant bacterial pathogens. We investigated the potential of pure compounds isolated from plants to act as EPIs. In silico screening was used to predict the bioactivity of plant compounds and to compare that with the known EPI, phe-arg-β-naphthylamide (PAβN). Subsequently, promising products have been tested for their ability to inhibit efflux. Plumbagin nordihydroguaretic acid (NDGA) and to a lesser degree shikonin, acted as sensitizers of drug-resistant bacteria to currently used antibiotics and were able to inhibit the efflux pump-mediated removal of substrate from cells. We demonstrated the feasibility of in silico screening to identify compounds that potentiate the action of antibiotics against drug-resistant strains and which might be potentially useful lead compounds for an EPI discovery program. PMID:25224951

  7. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID.

    PubMed

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inês C R; Willige, Björn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

    2014-06-19

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the--in many cells--asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  8. Antimicrobial and Efflux Pump Inhibitory Activity of Caffeoylquinic Acids from Artemisia absinthium against Gram-Positive Pathogenic Bacteria

    PubMed Central

    Fiamegos, Yiannis C.; Kastritis, Panagiotis L.; Exarchou, Vassiliki; Han, Haley; Bonvin, Alexandre M. J. J.; Vervoort, Jacques; Lewis, Kim; Hamblin, Michael R.; Tegos, George P.

    2011-01-01

    Background Traditional antibiotics are increasingly suffering from the emergence of multidrug resistance amongst pathogenic bacteria leading to a range of novel approaches to control microbial infections being investigated as potential alternative treatments. One plausible antimicrobial alternative could be the combination of conventional antimicrobial agents/antibiotics with small molecules which block multidrug efflux systems known as efflux pump inhibitors. Bioassay-driven purification and structural determination of compounds from plant sources have yielded a number of pump inhibitors which acted against gram positive bacteria. Methodology/Principal Findings In this study we report the identification and characterization of 4′,5′-O-dicaffeoylquinic acid (4′,5′-ODCQA) from Artemisia absinthium as a pump inhibitor with a potential of targeting efflux systems in a wide panel of Gram-positive human pathogenic bacteria. Separation and identification of phenolic compounds (chlorogenic acid, 3′,5′-ODCQA, 4′,5′-ODCQA) was based on hyphenated chromatographic techniques such as liquid chromatography with post column solid-phase extraction coupled with nuclear magnetic resonance spectroscopy and mass spectroscopy. Microbial susceptibility testing and potentiation of well know pump substrates revealed at least two active compounds; chlorogenic acid with weak antimicrobial activity and 4′,5′-ODCQA with pump inhibitory activity whereas 3′,5′-ODCQA was ineffective. These intitial findings were further validated with checkerboard, berberine accumulation efflux assays using efflux-related phenotypes and clinical isolates as well as molecular modeling methodology. Conclusions/Significance These techniques facilitated the direct analysis of the active components from plant extracts, as well as dramatically reduced the time needed to analyze the compounds, without the need for prior isolation. The calculated energetics of the docking poses supported the

  9. Heavy metal transport by the CusCFBA efflux system

    PubMed Central

    Delmar, Jared A; Su, Chih-Chia; Yu, Edward W

    2015-01-01

    It is widely accepted that the increased use of antibiotics has resulted in bacteria with developed resistance to such treatments. These organisms are capable of forming multi-protein structures that bridge both the inner and outer membrane to expel diverse toxic compounds directly from the cell. Proteins of the resistance nodulation cell division (RND) superfamily typically assemble as tripartite efflux pumps, composed of an inner membrane transporter, a periplasmic membrane fusion protein, and an outer membrane factor channel protein. These machines are the most powerful antimicrobial efflux machinery available to bacteria. In Escherichia coli, the CusCFBA complex is the only known RND transporter with a specificity for heavy metals, detoxifying both Cu+ and Ag+ ions. In this review, we discuss the known structural information for the CusCFBA proteins, with an emphasis on their assembly, interaction, and the relationship between structure and function. PMID:26258953

  10. Low levels of graphene and graphene oxide inhibit cellular xenobiotic defense system mediated by efflux transporters.

    PubMed

    Liu, Su; Jiang, Wei; Wu, Bing; Yu, Jing; Yu, Haiyan; Zhang, Xu-Xiang; Torres-Duarte, Cristina; Cherr, Gary N

    2016-01-01

    Low levels of graphene and graphene oxide (GO) are considered to be environmentally safe. In this study, we analyzed the potential effects of graphene and GO at relatively low concentrations on cellular xenobiotic defense system mediated by efflux transporters. The results showed that graphene (<0.5 μg/mL) and GO (<20 μg/mL) did not decrease cell viability, generate reactive oxygen species, or disrupt mitochondrial function. However, graphene and GO at the nontoxic concentrations could increase calcein-AM (CAM, an indicator of membrane ATP-binding cassette (ABC) transporter) activity) accumulation, indicating inhibition of ABC transporters' efflux capabilities. This inhibition was observed even at 0.005 μg/mL graphene and 0.05 μg/mL GO, which are 100 times and 400 times lower than their lowest toxic concentration from cytotoxicity experiments, respectively. The inhibition of ABC transporters significantly increased the toxicity of paraquat and arsenic, known substrates of ABC transporters. The inhibition of ABC transporters was found to be based on graphene and GO damaging the plasma membrane structure and fluidity, thus altering functions of transmembrane ABC transporters. This study demonstrates that low levels of graphene and GO are not environmentally safe since they can significantly make cell more susceptible to other xenobiotics, and this chemosensitizing activity should be considered in the risk assessment of graphene and GO.

  11. Antibiotic resistance, efflux pump genes and virulence determinants in Enterococcus spp. from surface water systems.

    PubMed

    Molale, L G; Bezuidenhout, Cornelius Carlos

    2016-11-01

    The aim of this study was to report on antibiotic susceptibility patterns as well as highlight the presence of efflux pump genes and virulence genetic determinants in Enterococcus spp. isolated from South African surface water systems. One hundred and twenty-four Enterococcus isolates consisting of seven species were identified. Antimicrobial susceptibility testing revealed a high percentage of isolates was resistant to β-lactams and vancomycin. Many were also resistant to other antibiotic groups. These isolates were screened by PCR, for the presence of four efflux pump genes (mefA, tetK, tetL and msrC). Efflux genes mefA and tetK were not detected in any of the Enterococcus spp. However, tetL and msrC were detected in 17 % of the Enterococcus spp. The presence of virulence factors in the Enterococcus spp. harbouring efflux pump genes was determined. Virulence determinants were detected in 86 % of the Enterococcus spp. harbouring efflux pump genes. Four (asa1, cylA, gel and hyl) of the five virulence factors were detected. The findings of this study have demonstrated that Enterococcus from South African surface water systems are resistant to multiple antibiotics, some of which are frequently used for therapy. Furthermore, these isolates harbour efflux pump genes coding for resistance to antibiotics and virulence factors which enhance their pathogenic potential.

  12. Effects of pharmaceuticals and personal care products (PPCPs) on multixenobiotic resistance (MXR) related efflux transporter activity in zebrafish (Danio rerio) embryos.

    PubMed

    Cunha, V; Burkhardt-Medicke, K; Wellner, P; Santos, M M; Moradas-Ferreira, P; Luckenbach, T; Ferreira, M

    2017-02-01

    Certain ATP binding cassette (ABC) transporter proteins, such as zebrafish Abcb4, are efflux pumps acting as a cellular defence against a wide range of different, potentially toxic chemical compounds thus mediating so called multixenobiotic resistance (MXR). Certain chemicals target MXR proteins and, as so called chemosensitisers, inhibit the activity of these proteins thus increasing the toxicity of other chemicals that would normally be effluxed. In this study 14 pharmaceuticals and personal care products (PPCPs) that are being increasingly detected in aquatic systems, were assessed for interference with the MXR system of zebrafish (Danio rerio). Concentration dependent effects of test compounds were recorded with the dye accumulation assay using zebrafish embryos and in ATPase assays with recombinant zebrafish Abcb4. In the dye accumulation assay embryos at 24h post fertilisation (hpf) were exposed to 8µm rhodamine 123 along with test compounds for 2h. The rhodamine 123 tissue levels upon the exposure served as a measure for MXR transporter efflux activity of the embryo (low rhodamine levels - high activity; high levels - low activity). The known ABC protein inhibitors MK571, vinblastine and verapamil served as positive controls. All tested PPCPs affected rhodamine 123 accumulation in embryos. For seven compounds rhodamine tissue levels were either both decreased and increased depending on the compound concentration indicating both stimulation and inhibition of rhodamine 123 efflux by those compounds, only increased (inhibition, six compounds) or only decreased (stimulation, one compound). Recombinant zebrafish Abcb4 was obtained with the baculovirus expression system and PPCPs were tested for stimulation/inhibition of basal transporter ATPase activity and for inhibition of the transporter ATPase activity stimulated with verapamil. Eight of the tested PPCPs showed effects on Abcb4 ATPase activity indicating that their effects in the dye accumulation assay may

  13. ACTIVE EFFLUX OF ORGANIC SOLVENTS BY PSEUDOMONAS PUTIDA S12 IS INDUCED BY SOLVENTS

    EPA Science Inventory

    Induction of the membrane-associated organic solvent efflux system SrpABC of Pseudomonas putida S12 was examined by cloning a 312-bp DNA fragment, containing the srp promoter, in the broad-host-range reporter vector pKRZ-1. Compounds that are capable of inducing expression of the...

  14. Properties of AdeABC and AdeIJK Efflux Systems of Acinetobacter baumannii Compared with Those of the AcrAB-TolC System of Escherichia coli

    PubMed Central

    Sugawara, Etsuko

    2014-01-01

    Acinetobacter baumannii contains RND-family efflux systems AdeABC and AdeIJK, which pump out a wide range of antimicrobial compounds, as judged from the MIC changes occurring upon deletion of the responsible genes. However, these studies may miss changes because of the high backgrounds generated by the remaining pumps and by β-lactamases, and it is unclear how the activities of these pumps compare quantitatively with those of the well-studied AcrAB-TolC system of Escherichia coli. We expressed adeABC and adeIJK of A. baumannii, as well as E. coli acrAB, in an E. coli host from which acrAB was deleted. The A. baumannii pumps were functional in E. coli, and the MIC changes that were observed largely confirmed the substrate range already reported, with important differences. Thus, the AdeABC system pumped out all β-lactams, an activity that was often missed in deletion studies. When the expression level of the pump genes was adjusted to a similar level for a comparison with AcrAB-TolC, we found that both A. baumannii efflux systems pumped out a wide range of compounds, but AdeABC was less effective than AcrAB-TolC in the extrusion of lipophilic β-lactams, novobiocin, and ethidium bromide, although it was more effective at tetracycline efflux. AdeIJK was remarkably more effective than a similar level of AcrAB-TolC in the efflux of β-lactams, novobiocin, and ethidium bromide, although it was less so in the efflux of erythromycin. These results thus allow us to compare these efflux systems on a quantitative basis, if we can assume that the heterologous systems are fully functional in the E. coli host. PMID:25246403

  15. The role of multidrug resistance protein (MRP-1) as an active efflux transporter on blood-brain barrier (BBB) permeability.

    PubMed

    Lingineni, Karthik; Belekar, Vilas; Tangadpalliwar, Sujit R; Garg, Prabha

    2017-01-03

    Drugs acting on central nervous system (CNS) may take longer duration to reach the market as these compounds have a higher attrition rate in clinical trials due to the complexity of the brain, side effects, and poor blood-brain barrier (BBB) permeability compared to non-CNS-acting compounds. The roles of active efflux transporters with BBB are still unclear. The aim of the present work was to develop a predictive model for BBB permeability that includes the MRP-1 transporter, which is considered as an active efflux transporter. A support vector machine model was developed for the classification of MRP-1 substrates and non-substrates, which was validated with an external data set and Y-randomization method. An artificial neural network model has been developed to evaluate the role of MRP-1 on BBB permeation. A total of nine descriptors were selected, which included molecular weight, topological polar surface area, ClogP, number of hydrogen bond donors, number of hydrogen bond acceptors, number of rotatable bonds, P-gp, BCRP, and MRP-1 substrate probabilities for model development. We identified 5 molecules that fulfilled all criteria required for passive permeation of BBB, but they all have a low logBB value, which suggested that the molecules were effluxed by the MRP-1 transporter.

  16. Distribution of AdeABC efflux system genes in genotypically diverse strains of clinical Acinetobacter baumannii.

    PubMed

    Wieczorek, Piotr; Sacha, Paweł; Czaban, Sławomir; Hauschild, Tomasz; Ojdana, Dominika; Kowalczuk, Oksana; Milewski, Robert; Poniatowski, Bogusław; Nikliński, Jacek; Tryniszewska, Elżbieta

    2013-10-01

    Acinetobacter baumannii has emerged as a highly problematic hospital-associated pathogen. Different mechanisms contribute to the formation of multidrug resistance in A. baumannii, including the AdeABC efflux system. Distribution of the structural and regulatory genes encoding the AdeABC efflux system among genetically diverse clinical A. baumannii strains was achieved by using PCR and pulsed-field gel electrophoresis techniques. The distribution of adeABRS genes is extremely high among our A. baumannii strains, except the adeC gene. We have observed a large proportion of strains presenting multidrug-resistance phenotype for several years. The efflux pump could be an important mechanism in these strains in resistance to antibiotics.

  17. NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux.

    PubMed

    He, Yuan; Zeng, Melody Y; Yang, Dahai; Motro, Benny; Núñez, Gabriel

    2016-02-18

    Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases. So far, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor protein ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-18 proteins. The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases as well as cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations. Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by many stimuli. Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. Here we report the identification of NEK7, a member of the family of mammalian NIMA-related kinases (NEK proteins), as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of NEK7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasomes. NLRP3-activating stimuli promoted the NLRP3-NEK7 interaction in a process that was dependent on potassium efflux. NLRP3 associated with the catalytic domain of NEK7, but the catalytic activity of NEK7 was shown to be dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3-NEK7 complex, which, along with ASC oligomerization and ASC speck formation, was abrogated in the absence of NEK7. NEK7 was required for macrophages containing the CAPS-associated NLRP3(R258W) activating mutation to activate caspase-1. Mouse chimaeras reconstituted with wild-type, Nek7(-/-) or Nlrp3(-/-) haematopoietic cells showed that NEK7 was required for NLRP3 inflammasome activation in vivo. These studies demonstrate that NEK7 is an essential protein that acts downstream of potassium efflux to

  18. The Contribution of Antibiotic Resistance Mechanisms in Clinical Burkholderia cepacia Complex Isolates: An Emphasis on Efflux Pump Activity

    PubMed Central

    Tseng, Sung-Pin; Tsai, Wan-Chi; Liang, Chih-Yuan; Lin, Yin-Shiou; Huang, Jun-Wei; Chang, Chung-Yu; Tyan, Yu-Chang; Lu, Po-Liang

    2014-01-01

    Due to the limited information of the contribution of various antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates, Antibiotic resistance mechanisms, including integron analysis, identification of quinolone resistance-determining region mutations, measurement of efflux pump activity, and sequence analysis of efflux pump regulators, were investigated in 66 clinical B. cepacia complex isolates. Species were identified via recA-RFLP and MALDI-TOF. Four genomovars were identified by recA-RFLP. B. cenocepacia (genomovar III) was the most prevalent genomovar (90.1%). Most isolates (60/66, 90.9%) were correctly identified by MALDI-TOF analysis. Clonal relatedness determined by PFGE analysis revealed 30 pulsotypes, including two major pulsotypes that comprised 22.7% and 18.2% of the isolates, respectively. Seventeen (25.8%) isolates harboured class 1 integron with various combinations of resistance genes. Among six levofloxacin-resistant isolates, five had single-base substitutions in the gyrA gene and three demonstrated efflux pump activities. Among the 42 isolates exhibiting resistance to at least one antimicrobial agent, 94.4% ceftazidime-resistant isolates (17/18) and 72.7% chloramphenicol-resistant isolates (16/22) demonstrated efflux pump activity. Quantitation of efflux pump RNA level and sequence analysis revealed that over-expression of the RND-3 efflux pump was attributable to specific mutations in the RND-3 efflux pump regulator gene. In conclusion, high-level expression of efflux pumps is prevalent in B. cepacia complex isolates. Mutations in the RND-3 efflux pump regulator gene are the major cause of efflux pump activity, resulting in the resistance to antibiotics in clinical B. cepacia complex isolates. PMID:25153194

  19. The contribution of antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates: an emphasis on efflux pump activity.

    PubMed

    Tseng, Sung-Pin; Tsai, Wan-Chi; Liang, Chih-Yuan; Lin, Yin-Shiou; Huang, Jun-Wei; Chang, Chung-Yu; Tyan, Yu-Chang; Lu, Po-Liang

    2014-01-01

    Due to the limited information of the contribution of various antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates, Antibiotic resistance mechanisms, including integron analysis, identification of quinolone resistance-determining region mutations, measurement of efflux pump activity, and sequence analysis of efflux pump regulators, were investigated in 66 clinical B. cepacia complex isolates. Species were identified via recA-RFLP and MALDI-TOF. Four genomovars were identified by recA-RFLP. B. cenocepacia (genomovar III) was the most prevalent genomovar (90.1%). Most isolates (60/66, 90.9%) were correctly identified by MALDI-TOF analysis. Clonal relatedness determined by PFGE analysis revealed 30 pulsotypes, including two major pulsotypes that comprised 22.7% and 18.2% of the isolates, respectively. Seventeen (25.8%) isolates harboured class 1 integron with various combinations of resistance genes. Among six levofloxacin-resistant isolates, five had single-base substitutions in the gyrA gene and three demonstrated efflux pump activities. Among the 42 isolates exhibiting resistance to at least one antimicrobial agent, 94.4% ceftazidime-resistant isolates (17/18) and 72.7% chloramphenicol-resistant isolates (16/22) demonstrated efflux pump activity. Quantitation of efflux pump RNA level and sequence analysis revealed that over-expression of the RND-3 efflux pump was attributable to specific mutations in the RND-3 efflux pump regulator gene. In conclusion, high-level expression of efflux pumps is prevalent in B. cepacia complex isolates. Mutations in the RND-3 efflux pump regulator gene are the major cause of efflux pump activity, resulting in the resistance to antibiotics in clinical B. cepacia complex isolates.

  20. Multixenobiotic resistance efflux activity in Daphnia magna and Lumbriculus variegatus.

    PubMed

    Vehniäinen, Eeva-Riikka; Kukkonen, Jussi V K

    2015-04-01

    Multixenobiotic resistance is a phenomenon in which ATP-binding cassette (ABC) family proteins transfer harmful compounds out of cells. Daphnia magna and Lumbriculus variegatus are model species in aquatic ecotoxicology, but the presence and activity of ABC proteins have not been well described in these species. The aim of this work was to study the presence, activity, and inhibition of ABC transport proteins in D. magna and L. variegatus. The presence of abcb1 and abcc transcripts in 8-9-day-old D. magna was investigated by qRT-PCR. The activity of MXR in D. magna and L. variegatus was explored by influx of the fluorescent ABC protein substrates rhodamine B and calcein-AM, with and without the model inhibitors verapamil (unspecific ABC inhibitor), reversin 205 (ABCB1 inhibitor) and MK571 (ABCC inhibitor). Juvenile D. magna possessed all examined abcb and abcc transcripts, but only reversin 205 inhibited MXR activity. The MXR activity in L. variegatus was inhibited by MK571, and to a lesser extent by verapamil, whereas reversin 205 seemed to stimulate the transport activity. Whereas calcein-AM worked better as an MXR substrate in D. magna, rhodamine B was a better substrate for L. variegatus MXR activity measurements. This is the first report on MXR activity in the order Lumbriculida, subclass Oligochaeta, and class Clitellata.

  1. Substrate-Linked Conformational Change in the Periplasmic Component of a Cu(I)/Ag(I) Efflux System

    SciTech Connect

    Bagai, I.; Liu, W.; Rensing, C.; Blackburn, N.J.; McEvoy, M.M.

    2009-06-02

    Gram-negative bacteria utilize dual membrane resistance nodulation division-type efflux systems to export a variety of substrates. These systems contain an essential periplasmic component that is important for assembly of the protein complex. We show here that the periplasmic protein CusB from the Cus copper/silver efflux system has a critical role in Cu(I) and Ag(I) binding. Isothermal titration calorimetry experiments demonstrate that one Ag(I) ion is bound per CusB molecule with high affinity. X-ray absorption spectroscopy data indicate that the metal environment is an all-sulfur 3-coordinate environment. Candidates for the metal-coordinating residues were identified from sequence analysis, which showed four conserved methionine residues. Mutations of three of these methionine residues to isoleucine resulted in significant effects on CusB metal binding in vitro. Cells containing these CusB variants also show a decrease in their ability to grow on copper-containing plates, indicating an important functional role for metal binding by CusB. Gel filtration chromatography demonstrates that upon binding metal, CusB undergoes a conformational change to a more compact structure. Based on these structural and functional effects of metal binding, we propose that the periplasmic component of resistance nodulation division-type efflux systems plays an active role in export through substrate-linked conformational changes.

  2. Distribution and expression of the Ade multidrug efflux systems in Acinetobacter baumannii clinical isolates.

    PubMed

    Pagdepanichkit, Sirawit; Tribuddharat, Chanwit; Chuanchuen, Rungtip

    2016-09-01

    One hundred Acinetobacter baumannii clinical isolates were examined for inhibitory effect of reserpine and carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the antimicrobial susceptibility and expression of 4 resistant-nodulation-cell division (RND)-type multidrug efflux systems, including AdeABC, AdeDE, AdeIJK, and AdeFGH, using RT-PCR. Ten A. baumannii isolates expressing AdeABC, AdeIJK, or AdeFGH were randomly selected for determination of transcription level and regulatory mutations. While all the isolates were resistant to multiple drugs, the reserpine and CCCP experiment showed that the multidrug resistance phenotype in most A. baumannii isolates was associated with efflux pumps. Most isolates expressed at least one of the RND-type efflux pumps tested (97%). AdeIJK expression was most common (97%), but none of the isolates produced AdeDE. Fifty-two percent of the A. baumannii isolates simultaneously produced up to 3 RND-type efflux systems (i.e., AdeABC, AdeFGH, and AdeIJK). No good correlation between the expression of RND-type efflux pumps and the type of antimicrobial resistance was observed. Overexpression of AdeABC, AdeIJK, and AdeFGH was not always related to the presence of mutations in their corresponding regulatory genes. This study highlights (i) the universal presence of the RND-type efflux pumps with variable levels of expression level among the A. baumannii in this collection and (ii) the complexity of their regulation of expression.

  3. Brain Distribution of Cediranib Is Limited by Active Efflux at the Blood-Brain Barrier

    PubMed Central

    Wang, Tianli; Agarwal, Sagar

    2012-01-01

    Cediranib is an orally active tyrosine kinase inhibitor that targets the vascular endothelial growth factor receptor family. Because of its potent antiangiogenic and antitumor activities, cediranib has been evaluated for therapy in glioma, a primary brain tumor. This study investigated the influence of two important efflux transporters at the blood-brain barrier, P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), on the delivery of cediranib to the central nervous system. In vitro studies indicated that cediranib is a dual substrate for both P-gp and Bcrp. It is noteworthy that in spite of the in vitro data the in vivo mouse disposition studies conclusively showed that P-gp was the dominant transporter restricting the brain distribution of cediranib. The brain-to-plasma partitioning (AUCbrain/AUCplasma, where AUC is area under the curve) and the steady-state brain-to-plasma concentration ratio of cediranib were approximately 20-fold higher in Mdr1a/b(−/−) and Mdr1a/b(−/−)Bcrp1(−/−) mice compared with wild-type and Bcrp1(−/−) mice. Moreover, there was no significant difference in brain distribution of cediranib between wild-type and Bcrp1(−/−) mice and between Mdr1a/b(−/−) and Mdr1a/b(−/−)Bcrp1(−/−) mice. These results show that, unlike other tyrosine kinase inhibitors that are dual substrates for P-gp and Bcrp, Bcrp does not restrict the distribution of cediranib across the blood-brain barrier. We also show that inhibition of P-gp using specific or nonspecific inhibitors resulted in significantly enhanced delivery of cediranib to the brain. Concurrent administration of cediranib with chemical modulators of efflux transporters can be used as a strategy to enhance delivery and thus efficacy of cediranib in the brain. These findings are clinically relevant to the efficacy of cediranib chemotherapy in glioma. PMID:22323823

  4. Modulation of P-glycoprotein efflux pump: induction and activation as a therapeutic strategy.

    PubMed

    Silva, Renata; Vilas-Boas, Vânia; Carmo, Helena; Dinis-Oliveira, Ricardo Jorge; Carvalho, Félix; de Lourdes Bastos, Maria; Remião, Fernando

    2015-05-01

    P-glycoprotein (P-gp) is an ATP-dependent efflux pump encoded by the MDR1 gene in humans, known to mediate multidrug resistance of neoplastic cells to cancer therapy. For several decades, P-gp inhibition has drawn many significant research efforts in an attempt to overcome this phenomenon. However, P-gp is also constitutively expressed in normal human epithelial tissues and, due to its broad substrate specificity, to its cellular polarized expression in many excretory and barrier tissues, and to its great efflux capacity, it can play a crucial role in limiting the absorption and distribution of harmful xenobiotics, by decreasing their intracellular accumulation. Such a defense mechanism can be of particular relevance at the intestinal level, by significantly reducing the intestinal absorption of the xenobiotic and, consequently, avoiding its access to the target organs. In this review, the current knowledge on this important efflux pump is summarized, and a new focus is brought on the therapeutic interest of inducing and/or activating P-gp for limiting the toxicity caused by its substrates. Several in vivo and in vitro studies validating the use of such a therapeutic strategy are discussed. An extensive literature search for reported P-gp inducers/activators and for the experimental models used in their characterization was conducted. Those studies demonstrate that effective antidotal pathways can be achieved by efficiently promoting the P-gp-mediated efflux of deleterious xenobiotics, resulting in a significant reduction in their intracellular levels and, consequently, in a significant reduction of their toxicity.

  5. Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux

    PubMed Central

    Vrins, Carlos L. J.; van der Velde, Astrid E.; van den Oever, Karin; Levels, Johannes H. M.; Huet, Stephane; Oude Elferink, Ronald P. J.; Kuipers, Folkert; Groen, Albert K.

    2009-01-01

    Peroxisome proliferator-activated receptor delta (PPARδ) is involved in regulation of energy homeostasis. Activation of PPARδ markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased hepatobiliary cholesterol secretion, nor by reduced cholesterol absorption. To test the hypothesis that PPARδ activation leads to stimulation of transintestinal cholesterol efflux (TICE), we quantified it by intestine perfusions in FVB mice treated with PPARδ agonist GW610742. To exclude the effects on cholesterol absorption, mice were also treated with cholesterol absorption inhibitor ezetimibe or ezetimibe/GW610742. GW601742 treatment had little effect on plasma lipid levels but stimulated both fecal neutral sterol excretion (∼200%) and TICE (∼100%). GW610742 decreased intestinal Npc1l1 expression but had no effect on Abcg5/Abcg8. Interestingly, expression of Rab9 and LIMPII, encoding proteins involved in intracellular cholesterol trafficking, was increased upon PPARδ activation. Although treatment with ezetimibe alone had no effect on TICE, it reduced the effect of GW610742 on TICE. These data show that activation of PPARδ stimulates fecal cholesterol excretion in mice, primarily by the two-fold increase in TICE, indicating that this pathway provides an interesting target for the development of drugs aiming at the prevention of atherosclerosis. PMID:19439761

  6. K+ efflux agonists induce NLRP3 inflammasome activation independently of Ca2+ signaling1

    PubMed Central

    Katsnelson, Michael A.; Rucker, L. Graham; Russo, Hana M.; Dubyak, George R.

    2015-01-01

    Perturbation of intracellular ion homeostasis is a major cellular stress signal for activation of NLRP3 inflammasome signaling that results in caspase-1 mediated production of IL-1β and pyroptosis. However, the relative contributions of decreased cytosolic [K+] versus increased cytosolic [Ca2+] remain disputed and incompletely defined. We investigated roles for elevated cytosolic [Ca2+] in NLRP3 activation and downstream inflammasome signaling responses in primary murine dendritic cells and macrophages in response to two canonical NLRP3 agonists (ATP and nigericin) that facilitate primary K+ efflux by mechanistically distinct pathways or the lysosome-destabilizing agonist Leu-Leu-O-methyl ester (LLME). The study provides three major findings relevant to this unresolved area of NLRP3 regulation. First, increased cytosolic [Ca2+] was neither a necessary nor sufficient signal for the NLRP3 inflammasome cascade during activation by endogenous ATP-gated P2X7 receptor channels, the exogenous bacterial ionophore nigericin, or the lysosomotropic agent LLME. Second, agonists for three Ca2+-mobilizing G protein-coupled receptors (formyl peptide receptor/FPR; P2Y2 purinergic receptor/P2Y2R; calcium-sensing receptor/CaSR) expressed in murine dendritic cells were ineffective as activators of rapidly induced NLRP3 signaling when directly compared to the K+ efflux agonists. Third, the intracellular Ca2+ buffer, BAPTA, and the channel blocker, 2-aminoethoxydiphenyl borate (2-APB), widely used reagents for disruption of Ca2+-dependent signaling pathways, strongly suppressed nigericin-induced NLRP3 inflammasome signaling via mechanisms dissociated from their canonical or expected effects on Ca2+ homeostasis. The results indicate that the ability of K+ efflux agonists to activate NLRP3 inflammasome signaling can be dissociated from changes in cytosolic [Ca2+] as a necessary or sufficient signal. PMID:25762778

  7. Soil surface carbon dioxide efflux of bioenergy cropping systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bioenergy cropping systems have been proposed as a way to enhance United States energy security. However, research on greenhouse gas emissions from such systems is needed to ensure environmental sustainability in the field. Since soil aeration properties are dynamic, high-resolution data are needed ...

  8. Mechanism of pluronic effect on P-glycoprotein efflux system in blood-brain barrier: contributions of energy depletion and membrane fluidization.

    PubMed

    Batrakova, E V; Li, S; Vinogradov, S V; Alakhov, V Y; Miller, D W; Kabanov, A V

    2001-11-01

    Pluronic block copolymer, P85, inhibits the P-glycoprotein (Pgp) drug efflux system and increases the permeability of a broad spectrum of drugs in the blood-brain barrier (BBB). This study examines the mechanisms by which P85 inhibits Pgp using bovine brain microvessel endothelial cells (BBMEC) as an in vitro model of the BBB. The hypothesis was that simultaneous alterations in intracellular ATP levels and membrane fluidization in BBMEC monolayers by P85 results in inhibition of the drug efflux system. The methods included the use of 1) standard Pgp substrate rhodamine 123 to assay the Pgp efflux system in BBMEC, 2) luciferin/luciferase assay for ATP intracellular levels, and 3) 1,6-diphenyl-1,3,5-hexatriene for membrane microviscosity. Using 3H-labeled P85 and fluorescein-labeled P85 for confocal microscopy, this study suggests that P85 accumulates in the cells and intracellular organelles such as the mitochondria where it can interfere with metabolic processes. Following exposure of BBMEC to P85, the ATP levels were depleted, and microviscosity of the cell membranes was decreased. Furthermore, P85 treatment decreased Pgp ATPase activity in membranes expressing human Pgp. A combination of experiments examining the kinetics, concentration dependence, and directionality of P85 effects on Pgp-mediated efflux in BBMEC monolayers suggests that both energy depletion (decreasing ATP pool available for Pgp) and membrane fluidization (inhibiting Pgp ATPase activity) are critical factors contributing to the activity of the block copolymer in the BBB.

  9. Efflux Pump Blockers in Gram-Negative Bacteria: The New Generation of Hydantoin Based-Modulators to Improve Antibiotic Activity

    PubMed Central

    Otręebska-Machaj, Ewa; Chevalier, Jacqueline; Handzlik, Jadwiga; Szymańska, Ewa; Schabikowski, Jakub; Boyer, Gérard; Bolla, Jean-Michel; Kieć-Kononowicz, Katarzyna; Pagès, Jean-Marie; Alibert, Sandrine

    2016-01-01

    Multidrug resistant (MDR) bacteria are an increasing health problem with the shortage of new active antibiotic agents. Among effective mechanisms that contribute to the spread of MDR Gram-negative bacteria are drug efflux pumps that expel clinically important antibiotic classes out of the cell. Drug pumps are attractive targets to restore the susceptibility toward the expelled antibiotics by impairing their efflux activity. Arylhydantoin derivatives were investigated for their potentiation of activities of selected antibiotics described as efflux substrates in Enterobacter aerogenes expressing or not AcrAB pump. Several compounds increased the bacterial susceptibility toward nalidixic acid, chloramphenicol and sparfloxacin and were further pharmacomodulated to obtain a better activity against the AcrAB producing bacteria. PMID:27199950

  10. Resveratrol counters systemic lupus erythematosus-associated atherogenicity by normalizing cholesterol efflux

    PubMed Central

    Teboul, Isaac; Littlefield, Michael J; Siegart, Nicolle M; Turi, George K; Fazzari, Melissa J; Carsons, Steven E; DeLeon, Joshua; Reiss, Allison B

    2016-01-01

    Resveratrol is a bioactive molecule used in dietary supplements and herbal medicines and consumed worldwide. Numerous investigations by our group and others have indicated cardioprotective and anti-inflammatory properties of resveratrol. The present study explored potential atheroprotective actions of resveratrol on cholesterol efflux in cultured human macrophages exposed to plasma from systemic lupus erythematosus (SLE) patients. These results were confirmed in ApoE−/−Fas−/− double knockout mice, displaying a lupus profile with accelerated atherosclerosis. Resveratrol treatment attenuated atherosclerosis in these mice. THP-1 human macrophages were exposed to 10% pooled or individual plasma from patients who met diagnostic criteria for SLE. Expression of multiple proteins involved in reverse cholesterol transport (ABCA1, ABCG1, SR-B1, and cytochrome P450 27-hydroxylase) was assessed using QRT-PCR and Western blotting techniques. Ten-week-old ApoE−/−Fas−/− double knockout mice (n = 30) were randomly divided into two equal groups of 15, one of which received 0.01% resveratrol for 10 consecutive weeks. Atherosclerosis progression was evaluated in murine aortas. Bone marrow-derived macrophages (BMDM) were cultured and expression of cholesterol efflux proteins was analyzed in each group of mice. Our data indicate that inhibition of cholesterol efflux by lupus plasma in THP-1 human macrophages is rescued by resveratrol. Similarly, administration of resveratrol in a lupus-like murine model reduces plaque formation in vivo and augments cholesterol efflux in BMDM. This study presents evidence for a beneficial role of resveratrol in atherosclerosis in the specific setting of SLE. Therefore, resveratrol may merit investigation as an additional resource available to reduce lipid deposition and atherosclerosis in humans, especially in such vulnerable populations as lupus patients. PMID:27190277

  11. In vitro transport activity of the fully assembled MexAB-OprM efflux pump from Pseudomonas aeruginosa

    NASA Astrophysics Data System (ADS)

    Verchère, Alice; Dezi, Manuela; Adrien, Vladimir; Broutin, Isabelle; Picard, Martin

    2015-04-01

    Antibiotic resistance is a major public health issue and many bacteria responsible for human infections have now developed a variety of antibiotic resistance mechanisms. For instance, Pseudomonas aeruginosa, a disease-causing Gram-negative bacteria, is now resistant to almost every class of antibiotics. Much of this resistance is attributable to multidrug efflux pumps, which are tripartite membrane protein complexes that span both membranes and actively expel antibiotics. Here we report an in vitro procedure to monitor transport by the tripartite MexAB-OprM pump. By combining proteoliposomes containing the MexAB and OprM portions of the complex, we are able to assay energy-dependent substrate translocation in a system that mimics the dual-membrane architecture of Gram-negative bacteria. This assay facilitates the study of pump transport dynamics and could be used to screen pump inhibitors with potential clinical use in restoring therapeutic activity of old antibiotics.

  12. In vitro activity of older and newer fluoroquinolones against efflux-mediated high-level ciprofloxacin-resistant Streptococcus pneumoniae.

    PubMed

    Daporta, Matilde Trigo; Muñoz Bellido, Juan Luis; Guirao, Genoveva Yagüe; Hernández, Manuel Segovia; García-Rodríguez, José Angel

    2004-08-01

    The effect of high-level efflux activity on the MICs of fluoroquinolones against Streptococcus pneumoniae in the absence of topoisomerase mutations leading to fluoroquinolones resistance was investigated. A S. pneumoniae ATCC 46619-derived strain with high-level efflux activity was obtained (SP-25A). Both the parent and obtained strains were tested against efflux substrates acriflavine (Acr) and ethidium bromide (EtBr), and against norfloxacin (NFX), ciprofloxacin (CFX), levofloxacin (LFX), moxifloxacin (MFX), trovafloxacin (TVX) and sitafloxacin (SFX), in presence and absence of the efflux pump inhibitor reserpine. gyrA, gyrB, parC and parE QRDR genes were amplified by PCR and sequenced. MICs of NFX and CFX against SP-25A were 64-fold higher than parent strain MICs (256 mg/L versus 4 mg/L and 64 mg/L versus 1mg/L, respectively). MIC of LFX increased from 1 to 4 mg/L and MICs of MFX, TVX and SFX remained virtually unchanged (0.1-0.2 mg/L). MICs of Acr and EtBr against SP-25A were 8- and 16-fold higher than against parent strains. In both cases, reserpine reverted MICs to the parent strain values (1 and 0.2 mg/L). Only parE showed two mutations leading to a Pro(454) --> Ser and Glu(443) changes, which have previously been shown not to lead to significant fluoroquinolones MIC increases. SP-25A showed a significant increase of MICs of the hydrophilic fluoroquinolones, apparently derived only from efflux activity. Efflux activity, at these high levels, can lead to high-level resistance to older hydrophilic fluoroquinolones, but does affect newer fluoroquinolones such as moxifloxacin, trovafloxacin and sitafloxacin.

  13. Effect of Pluronic P85 on ATPase Activity of Drug Efflux Transporters

    PubMed Central

    Batrakova, Elena V.; Li, Shu; Li, Yili; Alakhov, Valery Yu.; Kabanov, Alexander V.

    2009-01-01

    Purpose Pluronic block copolymers are potent sensitizers of multi-drug resistant (MDR) cancer cells. The sensitization effect by Pluronics is a result of two processes acting in concert: i) intracellular ATP depletion, and ii) inhibition of ATPase activity of drug efflux proteins. This work characterizes effects of Pluronic P85 on ATPase activities of Pgp, MRP1, and MRP2 drug efflux transport proteins and interaction of these proteins with their substrates, vinblastine, and leucotriene C4. Methods Using membranes overexpressing Pgp, MRP1, and MRP2, the current study evaluates effects of Pluronic P85 (P85) on the kinetic parameters (Vmax, Km, Vmax/Km) of ATP hydrolysis by these ATPases. Results The decreases in the maximal reaction rates (Vmax) and increases in apparent Michaelis constants (Km) for these transporters in the presence of various concentrations of P85 were observed. The mechanism of these effects may involve i) conformational changes of the transporter due to membrane fluidization and/or ii) nonspecific steric hindrance of the drug-binding sites by P85 chains embedded into cellular membranes. The extent of these alterations was increased in the row MRP1 < MRP2 << Pgp. Conclusions These data suggest that there are unifying pathways for the inhibition of Pgp and MRPs by the block copolymer. However, the effect of P85 on Pgp ATPase activity is considerably greater compared with the effects on MRP1 and MRP2 ATPases. This may be a reason for greater inhibitory effects of Pluronic in Pgp- compared with MRP-overexpressing cells. PMID:15648254

  14. Altered glycaemia differentially modulates efflux transporter expression and activity in hCMEC/D3 cell line.

    PubMed

    Sajja, Ravi K; Cucullo, Luca

    2015-06-26

    The unique phenotype of blood-brain barrier (BBB) endothelium is partly maintained by abundant expression of ATP-binding cassette superfamily of efflux transporters that strictly restrict the CNS access to toxic substances including xenobiotics in circulation. Previously, we have shown that diabetes-related altered glycemic conditions differentially affect and compromise BBB integrity. However, the impact of diabetes on BBB efflux transporters is less understood. In this study, we examined the effects of single or repeated episodes of hypo-and hyperglycemia on major BBB efflux transporters expression/function in human cerebromicrovascular endothelial cell line (hCMEC/D3). Cells were exposed to normal (5.5 mM), hypo (2.2 mM) or hyper (25 or 35 mM)-glycemic media containing D-glucose for 12h (acute) or two 3h episodes/day of hypo- or hyperglycemia with an intercalated 2h normalglycemic exposure for 3 days ("glycemic variability", see Methods). Acute hypoglycemic exposure (12h) up-regulated BBB endothelial mRNA and protein expression of P-glycoprotein, BCRP and other multidrug resistance associated proteins (MRP1 and 4) paralleled by an increase in transporter-specific efflux activity (∼ 2-fold vs. control). Although, 12h hyperglycemia did not affect the efflux transporter expression (except for MRP4), a significant increase in BCRP activity was observed. By contrast, DNA microarray data revealed that repeated hyperglycemic episodes (but not hypoglycemia) significantly up-regulate P-glycoprotein expression and activity. Thus, this study suggests a differential impact of altered glycemic conditions on major BBB drug efflux transporters expression/function, sensitive to the length of exposure (acute vs. repeated), with an implication for altered CNS drug disposition in diabetic population.

  15. Endomorphins, Met-enkephalin, Tyr-MIF-1, and the P-glycoprotein efflux system.

    PubMed

    Kastin, Abba J; Fasold, Melita B; Zadina, James E

    2002-03-01

    The P-glycoprotein (P-gp) transport system, responsible for the efflux of many therapeutic drugs out of the brain, recently has been shown to transport the endogenous brain opiate endorphin. We used P-gp knockout mice (Mdr1a) and their controls to determine where P-gp is involved in the saturable efflux systems of four other endogenous opiate-modulating peptides across the blood-brain barrier (BBB). After injection of endomorphin-1 (Tyr-Pro-Trp-Phe-NH(2)), endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)), Met-enkephalin (Tyr-Gly-Gly-Phe-Met-OH), and Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH(2)) into the lateral ventricle of the mouse brain, residual radioactivity was measured at 0, 2, 5, 10, and 20 min later. The results showed no difference in the disappearance of any of these peptides from the brains of knockout mice compared with their controls. This demonstrates that unlike endorphin and morphine, P-gp does not seem to be required for the brain-to-blood transport of the endomorphins, Met-enkephalin, or Tyr-MIF-1 across the BBB.

  16. Interactions of Pluronic Block Copolymers on P-gp Efflux Activity: Experience With HIV-1 Protease Inhibitors

    PubMed Central

    SHAIK, NAVEED; PAN, GUOYU; ELMQUIST, WILLIAM F.

    2016-01-01

    The objective was to examine the influence of Pluronic block-copolymers on the interaction between the drug efflux transporter, P-glycoprotein and HIV-1 protease inhibitors (PIs). The ATPase assay determined the effect of various Pluronics on PI-stimulated P-gp ATPase activity. Cellular accumulation studies were conducted using MDCKII and LLC-PK1 cells transfected with human MDR1 to assess Pluronic modulation of PI efflux. Pluronic P85 inhibited both basal and nelfinavir-stimulated P-gp ATPase activity, while Pluronic F127 had no effect. In cell accumulation studies, Pluronic P85 restored the accumulation of nelfinavir in MDCKII-MDR1 cells while Pluronic F127 and F88 had no effect. Pluronic P85 increased saquinavir accumulation in wild-type and MDR1-transfected cells in both the MDCKII and LLC-PK1 cell models, suggesting inhibition of multiple transporters, including MRPs. In conclusion, this study provides evidence that a block-copolymer, Pluronic P85, effectively inhibits the interaction of P-gp with nelfinavir and saquinavir. These data indicate that effective inhibition of HIV-1 PI efflux by Pluronic P85 may influence the distribution of antiretroviral agents to sites protected by efflux mechanisms, such as the blood–brain barrier, and possibly increase the brain exposure of these drugs resulting in suppression of viral replication and reduction in the incidence of drug resistant mutants. PMID:18393290

  17. A Combination Fluorescence Assay Demonstrates Increased Efflux Pump Activity as a Resistance Mechanism in Azole-Resistant Vaginal Candida albicans Isolates

    PubMed Central

    Bhattacharya, Somanon; Sobel, Jack D.

    2016-01-01

    Candida albicans is a pathogenic fungus causing vulvovaginal candidiasis (VVC). Azole drugs, such as fluconazole, are the most common treatment for these infections. Recently, azole-resistant vaginal C. albicans isolates have been detected in patients with recurring and refractory vaginal infections. However, the mechanisms of resistance in vaginal C. albicans isolates have not been studied in detail. In oral and systemic resistant isolates, overexpression of the ABC transporters Cdr1p and Cdr2p and the major facilitator transporter Mdr1p is associated with resistance. Sixteen fluconazole-susceptible and 22 fluconazole-resistant vaginal C. albicans isolates were obtained, including six matched sets containing a susceptible and a resistant isolate, from individual patients. Using quantitative real-time reverse transcriptase PCR (qRT-PCR), 16 of 22 resistant isolates showed overexpression of at least one efflux pump gene, while only 1 of 16 susceptible isolates showed such overexpression. To evaluate the pump activity associated with overexpression, an assay that combined data from two separate fluorescent assays using rhodamine 6G and alanine β-naphthylamide was developed. The qRT-PCR results and activity assay results were in good agreement. This combination of two fluorescent assays can be used to study efflux pumps as resistance mechanisms in clinical isolates. These results demonstrate that efflux pumps are a significant resistance mechanism in vaginal C. albicans isolates. PMID:27431223

  18. Cholesterol and ergosterol affect the activity of Staphylococcus aureus antibiotic efflux pumps.

    PubMed

    Tintino, S R; Oliveira-Tintino, C D M; Campina, F F; Costa, M S; Cruz, R P; Pereira, R L S; Andrade, J C; Sousa, E O; Siqueira-Junior, J P; Coutinho, H D M; Leal-Balbino, T C; Balbino, V Q

    2017-03-01

    The aim of this study is to evaluate the effect of ergosterol on steroids and cholesterol efflux pumps in multidrug resistant strains of S. aureus. Were used RN4220 harboring plasmid pUL5054, which carries the gene encoding the MsrA macrolide efflux protein; and IS-58, which possesses the TetK tetracycline efflux protein; 1199B resists hydrophilic fluoroquinolones via a NorA-mediated mechanism and wild strain 1199B. The Minimal Inhibitory Concentration (MIC) was determined and the evaluation of possible inhibition of efflux pumps by reduction of MIC. Some of the detrimental effects on bacterial cells can be attributed to the detergent properties of cholesterol and ergosterol on account of their amphipathic structure. Besides the cholesterol did not affect directly the pump structure, a synergism was observed, maybe due the interaction with the cell membrane and interference in the lipid bilayer.

  19. Ion Channel Blockers as Antimicrobial Agents, Efflux Inhibitors, and Enhancers of Macrophage Killing Activity against Drug Resistant Mycobacterium tuberculosis

    PubMed Central

    Perdigão, João; Couto, Isabel; Portugal, Isabel; Martins, Marta; Amaral, Leonard; Anes, Elsa; Viveiros, Miguel

    2016-01-01

    Given the ability of M. tuberculosis to survive as an intracellular pathogen and its propensity to develop resistance to the existing antituberculosis drugs, its treatment requires new approaches. Here the antimycobacterial properties of verapamil, thioridazine, chlorpromazine, flupenthixol and haloperidol were investigated against a panel of drug resistant M. tuberculosis strains, both in vitro and on human-infected macrophages. These compounds are efflux inhibitors that share among them the characteristic of being ion channel blockers. In vitro, all compounds exhibited synergistic inhibitory activities when combined with isoniazid and rifampicin, and were able to inhibit active efflux, demonstrating their role as efflux inhibitors. Gene expression analysis showed that M. tuberculosis efflux genes were overexpressed in response to antibiotic exposure, in vitro and within macrophages, irrespective of their resistance pattern. These compounds displayed a rapid and high killing activity against M. tuberculosis, associated with a decrease in intracellular ATP levels demonstrating that the bactericidal action of the ion channel blockers against M. tuberculosis clinical strains is associated with their interference with energy metabolism. The compounds led to a decrease in the intracellular mycobacterial load by increasing phagosome acidification and activating lysosomal hydrolases. The results presented in this study enable us to propose the following mechanism of action for these compounds: a) in the bacteria, the compounds generate a cascade of events involving the inhibition of the respiratory chain complexes and energy production for efflux activity. Indirectly, this reduce the resistance level to antituberculosis drugs potentiating their activity; b) on the host cell, the treatment with the ion channel blockers increases phagosome acidification and induces the expression of phagosomal hydrolases, leading to bacterial growth restriction irrespective of their

  20. Channel-tunnels: outer membrane components of type I secretion systems and multidrug efflux pumps of Gram-negative bacteria.

    PubMed

    Andersen, C

    2003-01-01

    For translocation across the cell envelope of Gram-negative bacteria, substances have to overcome two permeability barriers, the inner and outer membrane. Channel-tunnels are outer membrane proteins, which are central to two distinct export systems: the type I secretion system exporting proteins such as toxins or proteases, and efflux pumps discharging antibiotics, dyes, or heavy metals and thus mediating drug resistance. Protein secretion is driven by an inner membrane ATP-binding cassette (ABC) transporter while drug efflux occurs via an inner membrane proton antiporter. Both inner membrane transporters are associated with a periplasmic accessory protein that recruits an outer membrane channel-tunnel to form a functional export complex. Prototypes of these export systems are the hemolysin secretion system and the AcrAB/TolC drug efflux pump of Escherichia coli, which both employ TolC as an outer membrane component. Its remarkable conduit-like structure, protruding 100 A into the periplasmic space, reveals how both systems are capable of transporting substrates across both membranes directly from the cytosol into the external environment. Proteins of the channel-tunnel family are widespread within Gram-negative bacteria. Their involvement in drug resistance and in secretion of pathogenic factors makes them an interesting system for further studies. Understanding the mechanism of the different export apparatus could help to develop new drugs, which block the efflux pumps or the secretion system.

  1. Nicotinic activation of mesolimbic neurons assessed by rubidium efflux in rat accumbens and ventral tegmentum.

    PubMed

    Rowell, Peter P; Volk, Kelly A

    2004-01-01

    Dopaminergic mesolimbic neurons, with cell bodies in the ventral tegmental area (VTA) projecting to the nucleus accumbens (NAc), have been shown to be involved in the development of drug dependence. The application of nicotine to either the VTA or NAc produces an increase in dopamine release; however, the positive reinforcement produced by the systemic injection of nicotine is primarily due to stimulation of nicotinic acetylcholine receptors (nAChRs) in the VTA. Because the brain levels of nicotine would likely be the same in both brain areas, the nAChRs in the NAc may be less sensitive than those in the VTA. This study was undertaken to make a direct comparison of the native nAChRs in intact slices of NAc and VTA by measuring nicotine-stimulated efflux of (86)Rb(+) in a superfusion assay. The potency of nicotine and several other agonists was similar in both brain areas, but nicotine was somewhat more efficacious in the NAc. The effects of treatment duration, calcium and nicotinic antagonists were also determined. The results suggest that the predominant effect of nicotine in the VTA following systemic administration is due to differences in neuronal circuitry or firing patterns rather than inherent differences in the two nAChR populations.

  2. Escherichia coli populations in unpredictably fluctuating environments evolve to face novel stresses through enhanced efflux activity.

    PubMed

    Karve, S M; Daniel, S; Chavhan, Y D; Anand, A; Kharola, S S; Dey, S

    2015-05-01

    There is considerable understanding about how laboratory populations respond to predictable (constant or deteriorating environment) selection for single environmental variables such as temperature or pH. However, such insights may not apply when selection environments comprise multiple variables that fluctuate unpredictably, as is common in nature. To address this issue, we grew replicate laboratory populations of Escherichia coli in nutrient broth whose pH and concentrations of salt (NaCl) and hydrogen peroxide (H2 O2 ) were randomly changed daily. After ~170 generations, the fitness of the selected populations had not increased in any of the three selection environments. However, these selected populations had significantly greater fitness in four novel environments which have no known fitness-correlation with tolerance to pH, NaCl or H2 O2 . Interestingly, contrary to expectations, hypermutators did not evolve. Instead, the selected populations evolved an increased ability for energy-dependent efflux activity that might enable them to throw out toxins, including antibiotics, from the cell at a faster rate. This provides an alternate mechanism for how evolvability can evolve in bacteria and potentially lead to broad-spectrum antibiotic resistance, even in the absence of prior antibiotic exposure. Given that environmental variability is increasing in nature, this might have serious consequences for public health.

  3. Active efflux as a mechanism of resistance to ciprofloxacin in Streptococcus pneumoniae.

    PubMed Central

    Zeller, V; Janoir, C; Kitzis, M D; Gutmann, L; Moreau, N J

    1997-01-01

    The accumulation of fluoroquinolones (FQs) was studied in a FQ-susceptible laboratory strain of Streptococcus pneumoniae (strain R6). Uptake of FQs was not saturable, was rapidly reversible, and appeared to occur by passive diffusion. In the presence of glucose, which energizes bacteria, the uptake of FQs decreased. Inhibitors of the proton motive force and ATP synthesis increased the uptake of FQs in previously energized bacteria. Similar results were observed with the various FQs tested and may be explained to be a consequence simply of the pH gradient that exists across the cytoplasmic membrane. From a clinical susceptible strain (strain SPn5907) we isolated in vitro on ciprofloxacin an FQ-resistant mutant (strain SPn5929) for which the MICs of hydrophilic molecules were greater than those of hydrophobic molecules, and the mutant was resistant to acriflavine, cetrimide, and ethidium bromide. Strain SPn5929 showed a significantly decreased uptake of ciprofloxacin, and its determinant of resistance to ciprofloxacin was transferred by transformation to susceptible laboratory strain R6 (strain R6tr5929). No mutations in the quinolone resistance-determining regions of the gyrA and parC genes were found. In the presence of arsenate or carbonyl cyanide m-chlorophenylhydrazone, the levels of uptake of ciprofloxacin by the two resistant strains, SPn5929 and R6tr5929, reached the levels of uptake of their susceptible parents. These results suggest an active efflux of ciprofloxacin in strain SPn5929. PMID:9303396

  4. Transcriptional regulation of drug efflux genes by EvgAS, a two-component system in Escherichia coli.

    PubMed

    Eguchi, Yoko; Oshima, Taku; Mori, Hirotada; Aono, Rikizo; Yamamoto, Kaneyoshi; Ishihama, Akira; Utsumi, Ryutaro

    2003-10-01

    A constitutively active mutant of histidine kinase sensor EvgS was found to confer multi-drug resistance (MDR) to an acrA-deficient Escherichia coli, indicating the relationship between the two-component system EvgAS and the expression of the MDR system. The observed MDR also depended on an outer-membrane channel, TolC. Microarray and S1 mapping assays indicated that, in the presence of this constitutive mutant EvgS, the level of transcription increased for some MDR genes, including the drug efflux genes emrKY, yhiUV, acrAB, mdfA and tolC. Transcription in vitro of emrK increased by the addition of phosphorylated EvgA. Transcription activation of tolC by the activated EvgS was, however, dependent on both EvgAS and PhoPQ (Mg(2+)-responsive two-component system), in agreement with the presence of the binding site (PhoP box) for the regulator PhoP in the tolC promoter region. Transcription in vitro of yhiUV also appears to require an as-yet-unidentified additional transcriptional factor besides EvgA. Taken together we propose that the expression of the MDR system is under a complex regulatory network, including the phosphorylated EvgA serving as the master regulator.

  5. Structure-Activity Relationships of a Novel Pyranopyridine Series of Gram-negative Bacterial Efflux Pump Inhibitors

    PubMed Central

    Nguyen, Son T.; Kwasny, Steven M.; Ding, Xiaoyuan; Cardinale, Steven C.; McCarthy, Courtney T.; Kim, Hong-Suk; Nikaido, Hiroshi; Peet, Norton P.; Williams, John D.; Bowlin, Terry L.; Opperman, Timothy J.

    2015-01-01

    Recently we described a novel pyranopyridine inhibitor (MBX2319) of RND-type efflux pumps of the Enterobacteriaceae. MBX2319 (3,3-dimethyl-5-cyano-8-morpholino-6-(phenethylthio)-3,4-dihydro-1H-pyrano[3,4-c]pyridine) is structurally distinct from other known Gram-negative efflux pump inhibitors (EPIs), such as 1-(1-naphthylmethyl)-piperazine (NMP), phenylalanylarginine-β-naphthylamide (PAβN), D13-9001, and the pyridopyrimidine derivatives. Here, we report the synthesis and biological evaluation of 60 new analogs of MBX2319 that were designed to probe the structure activity relationships (SARs) of the pyranopyridine scaffold. The results of these studies produced a molecular activity map of the scaffold, which identifies regions that are critical to efflux inhibitory activities and those that can be modified to improve potency, metabolic stability and solubility. Several compounds, such as 22d–f, 22i and 22k, are significantly more effective than MBX2319 at potentiating the antibacterial activity of levofloxacin and piperacillin against Escherichia coli. PMID:25818767

  6. Host-specific symbiotic requirement of BdeAB, a RegR-controlled RND-type efflux system in Bradyrhizobium japonicum.

    PubMed

    Lindemann, Andrea; Koch, Marion; Pessi, Gabriella; Müller, Andreas J; Balsiger, Sylvia; Hennecke, Hauke; Fischer, Hans-Martin

    2010-11-01

    Multidrug efflux systems not only cause resistance against antibiotics and toxic compounds but also mediate successful host colonization by certain plant-associated bacteria. The genome of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum encodes 24 members of the family of resistance/nodulation/cell division (RND) multidrug efflux systems, of which BdeAB is genetically controlled by the RegSR two-component regulatory system. Phylogenetic analysis of the membrane components of these 24 RND-type transporters revealed that BdeB is more closely related to functionally characterized orthologs in other bacteria, including those associated with plants, than to any of the other 23 paralogs in B. japonicum. A mutant with a deletion of the bdeAB genes was more susceptible to inhibition by the aminoglycosides kanamycin and gentamicin than the wild type, and had a strongly decreased symbiotic nitrogen-fixation activity on soybean, but not on the alternative host plants mungbean and cowpea, and only very marginally on siratro. The host-specific role of a multidrug efflux pump is a novel feature in the rhizobia-legume symbioses. Consistent with the RegSR dependency of bdeAB, a B. japonicum regR mutant was found to have a greater sensitivity against the two tested antibiotics and a symbiotic defect that is most pronounced for soybean.

  7. Effects of atmospheric CO2 enrichment on soil CO2 efflux in a young longleaf pine system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Elevated atmospheric carbon dioxide (CO2) can affect the quantity and quality of plant tissues which will impact carbon (C) cycling and storage in plant/soil systems and the release of CO2 back to the atmosphere. Research is needed to quantify the effects of elevated CO2 on soil CO2 efflux to predi...

  8. Polymyxin Susceptibility in Pseudomonas aeruginosa Linked to the MexXY-OprM Multidrug Efflux System

    PubMed Central

    Lau, Calvin Ho-Fung; Gilmour, Christie; Hao, Youai

    2015-01-01

    The ribosome-targeting antimicrobial, spectinomycin (SPC), strongly induced the mexXY genes of the MexXY-OprM multidrug efflux system in Pseudomonas aeruginosa and increased susceptibility to the polycationic antimicrobials polymyxin B and polymyxin E, concomitant with a decrease in expression of the polymyxin resistance-promoting lipopolysaccharide (LPS) modification loci, arnBCADTEF and PA4773-74. Consistent with the SPC-promoted reduction in arn and PA4773-74 expression being linked to mexXY, expression of these LPS modification loci was moderated in a mutant constitutively expressing mexXY and enhanced in a mutant lacking the efflux genes. Still, the SPC-mediated increase in polymyxin susceptibility was retained in mutants lacking arnB and/or PA4773-74, an indication that their reduced expression in SPC-treated cells does not explain the enhanced polymyxin susceptibility. That the polymyxin susceptibility of a mutant strain lacking mexXY was unaffected by SPC exposure, however, was an indication that the unknown polymyxin resistance ‘mechanism’ is also influenced by the MexXY status of the cell. In agreement with SPC and MexXY influencing polymyxin susceptibility as a result of changes in the LPS target of these agents, SPC treatment yielded a decline in common polysaccharide antigen (CPA) synthesis in wild-type P. aeruginosa but not in the ΔmexXY mutant. A mutant lacking CPA still showed the SPC-mediated decline in polymyxin MICs, however, indicating that the loss of CPA did not explain the SPC-mediated MexXY-dependent increase in polymyxin susceptibility. It is possible, therefore, that some additional change in LPS promoted by SPC-induced mexXY expression impacted CPA synthesis or its incorporation into LPS and that this was responsible for the observed changes in polymyxin susceptibility. PMID:26369970

  9. Benzoic acid and specific 2-oxo acids activate hepatic efflux of glutamate at OAT2.

    PubMed

    Pfennig, Till; Herrmann, Beate; Bauer, Tim; Schömig, Edgar; Gründemann, Dirk

    2013-02-01

    The liver is the principal source of glutamate in blood plasma. Recently we have discovered that efflux of glutamate from hepatocytes is catalyzed by the transporter OAT2 (human gene symbol SLC22A7). Organic anion transporter 2 (OAT2) is an integral membrane protein of the sinusoidal membrane domain; it is primarily expressed in liver and much less in kidney, both in rats and humans. Many years ago, Häussinger and coworkers have demonstrated in isolated perfused rat liver that benzoic acid or specific 2-oxo acid analogs of amino acids like e.g. 2-oxo-4-methyl-pentanoate ('2-oxo-leucine') strongly stimulate release of glutamate (up to 7-fold); '2-oxo-valine' and the corresponding amino acids were without effect. The molecular mechanism of efflux stimulation has remained unclear. In the present study, OAT2 from human and rat were heterologously expressed in 293 cells. Addition of 1 mmol/l benzoic acid to the external medium increased OAT2-specific efflux of glutamate up to 20-fold; '2-oxo-leucine' was also effective, but not '2-oxo-valine'. Similar effects were seen for efflux of radiolabeled orotic acid. Expression of OAT2 did not increase uptake of benzoic acid; thus, benzoic acid is no substrate, and trans-stimulation can be excluded. Instead, further experiments suggest that increased efflux of glutamate is caused by direct interaction of benzoic acid and specific 2-oxo acids with OAT2. We propose that stimulators bind to a distinct extracellular site and thereby accelerate relocation of the empty substrate binding site to the intracellular face. Increased glutamate efflux at OAT2 could be the main benefit of benzoate treatment in patients with urea cycle defects.

  10. Quercetin increases macrophage cholesterol efflux to inhibit foam cell formation through activating PPARγ-ABCA1 pathway.

    PubMed

    Sun, Liqiang; Li, En; Wang, Feng; Wang, Tao; Qin, Zhiping; Niu, Shaohui; Qiu, Chunguang

    2015-01-01

    The accumulation of cholesterol in macrophages could induce the formation of foam cells and increase the risk of developing atherosclerosis. We wonder if quercetin, one of flavonoids with anti-inflammation functions in different cell types, could elevate the development of foam cells formation in atherosclerosis. We treated foam cells derived from oxLDL induced THP-1 cells with quercetin, and evaluated the foam cells formation, cholesterol content and apoptosis of the cells. We found that quercetin induced the expression of ABCA1 in differentiated THP-1 cells, and increased the cholesterol efflux from THP-1 cell derived foam cells. Eventually, cholesterol level and the formation of foam cell derived from THP-1 cells decreased after quercetin treatment. In addition, quercetin activated PPARγ-LXRα pathway to upregulate ABCA1 expression through increasing protein level of PPARγ and its transcriptional activity. Inhibition of PPARγ activity by siRNA knockdown or the addition of chemical inhibitor, GW9662, abolished quercetin induced ABCA1 expression and cholesterol efflux in THP-1 derived macrophages. Our data demonstrated that quercetin increased cholesterol efflux from macrophages through upregulating the expressions of PPARγ and ABCA1. Taken together, increasing uptake of quercetin or quercetin-rich foods would be an effective way to lower the risk of atherosclerosis.

  11. Quercetin increases macrophage cholesterol efflux to inhibit foam cell formation through activating PPARγ-ABCA1 pathway

    PubMed Central

    Sun, Liqiang; Li, En; Wang, Feng; Wang, Tao; Qin, Zhiping; Niu, Shaohui; Qiu, Chunguang

    2015-01-01

    The accumulation of cholesterol in macrophages could induce the formation of foam cells and increase the risk of developing atherosclerosis. We wonder if quercetin, one of flavonoids with anti-inflammation functions in different cell types, could elevate the development of foam cells formation in atherosclerosis. We treated foam cells derived from oxLDL induced THP-1 cells with quercetin, and evaluated the foam cells formation, cholesterol content and apoptosis of the cells. We found that quercetin induced the expression of ABCA1 in differentiated THP-1 cells, and increased the cholesterol efflux from THP-1 cell derived foam cells. Eventually, cholesterol level and the formation of foam cell derived from THP-1 cells decreased after quercetin treatment. In addition, quercetin activated PPARγ-LXRα pathway to upregulate ABCA1 expression through increasing protein level of PPARγ and its transcriptional activity. Inhibition of PPARγ activity by siRNA knockdown or the addition of chemical inhibitor, GW9662, abolished quercetin induced ABCA1 expression and cholesterol efflux in THP-1 derived macrophages. Our data demonstrated that quercetin increased cholesterol efflux from macrophages through upregulating the expressions of PPARγ and ABCA1. Taken together, increasing uptake of quercetin or quercetin-rich foods would be an effective way to lower the risk of atherosclerosis. PMID:26617799

  12. The peptide toxin amylosin of Bacillus amyloliquefaciens from moisture-damaged buildings is immunotoxic, induces potassium efflux from mammalian cells, and has antimicrobial activity.

    PubMed

    Rasimus-Sahari, Stiina; Teplova, Vera V; Andersson, Maria A; Mikkola, Raimo; Kankkunen, Päivi; Matikainen, Sampsa; Gahmberg, Carl G; Andersson, Leif C; Salkinoja-Salonen, Mirja

    2015-04-01

    Amylosin, a heat-stable channel-forming non-ribosomally synthesized peptide toxin produced by strains of Bacillus amyloliquefaciens isolated from moisture-damaged buildings, is shown in this paper to have immunotoxic and cytotoxic effects on human cells as well as antagonistic effects on microbes. Human macrophages exposed to 50 ng of amylosin ml(-1) secreted high levels of cytokines interleukin-1β (IL-1β) and IL-18 within 2 h, indicating activation of the NLRP3 inflammasome, an integral part of the innate immune system. At the same exposure level, expression of IL-1β and IL-18 mRNA increased. Amylosin caused dose-dependent potassium ion efflux from all tested mammalian cells (human monocytes and keratinocytes and porcine sperm cells) at 1 to 2 μM exposure. Amylosin also inhibited the motility of porcine sperm cells and depolarized the mitochondria of human keratinocytes. Amylosin may thus trigger the activation of the NLRP3 inflammasome and subsequently cytokine release by causing potassium efflux from exposed cells. The results of this study indicate that exposure to amylosin activates the innate immune system, which could offer an explanation for the inflammatory symptoms experienced by occupants of moisture-damaged buildings. In addition, the amylosin-producing B. amyloliquefaciens inhibited the growth of both prokaryotic and eukaryotic indoor microbes, and purified amylosin also had an antimicrobial effect. These antimicrobial effects could make amylosin producers dominant and therefore significant causal agents of health problems in some moisture-damaged sites.

  13. Opioid analgesics and P-glycoprotein efflux transporters: a potential systems-level contribution to analgesic tolerance.

    PubMed

    Mercer, Susan L; Coop, Andrew

    2011-01-01

    Chronic clinical pain remains poorly treated. Despite attempts to develop novel analgesic agents, opioids remain the standard analgesics of choice in the clinical management of chronic and severe pain. However, mu opioid analgesics have undesired side effects including, but not limited to, respiratory depression, physical dependence and tolerance. A growing body of evidence suggests that P-glycoprotein (P-gp), an efflux transporter, may contribute a systems-level approach to the development of opioid tolerance. Herein, we describe current in vitro and in vivo methodology available to analyze interactions between opioids and P-gp and critically analyze P-gp data associated with six commonly used mu opioids to include morphine, methadone, loperamide, meperidine, oxycodone, and fentanyl. Recent studies focused on the development of opioids lacking P-gp substrate activity are explored, concentrating on structure-activity relationships to develop an optimal opioid analgesic lacking this systems-level contribution to tolerance development. Continued work in this area will potentially allow for delineation of the mechanism responsible for opioid-related P-gp up-regulation and provide further support for evidence based medicine supporting clinical opioid rotation.

  14. Multidrug efflux systems in Escherichia coli and Enterobacter cloacae obtained from wholesome broiler carcasses

    PubMed Central

    Moreira, Maria Aparecida S.; Rodrigues, Patrícia P.C.F.; Tomaz, Rafael S.; de Moraes, Célia A.

    2009-01-01

    Members of the Enterobacteriaceae family are present in the intestines of man and animals as commensals or are important disease causing agents. Bacteria bearing multidrug efflux systems (MDR) are able to survive adverse ecological niches. Multiresistant Escherichia coli and Enterobacter cloacae isolates from wholesome broiler carcasses were investigated for the presence of MDR. Lowering of Minimal Inhibitory Concentration for antimicrobials in the presence of a proton-motive force (PMF) uncoupler was tested as a potential display of the MDR phenotype. PCR amplification of the genes encoding AcrA and AcrB, components of a MDR system was performed. Diversity of each species was ascertained by Pulsed-Field Gel Electrophoresis (PFGE) of DNA digested with endonuclease XbaI. For all the isolates, except E. coli 1 and E. cloacae 9, lowering of MIC or of the growth rate in the presence of antimicrobials was observed, indicating a PMF dependent resistance mechanism. Expected products of DNA amplification with acrAB derived primers was obtained with all E. coli strains and with two of the five E. cloacae strains. Dendrogram generated shows diverse pulsetypes, confirming the genetic diversity among the strains. An important issue and related public health is the fact that different models and mechanisms of antimicrobial resistance are present in a small number of non-pathogenic strains and isolated from the same origin. These may be sources of resistance genes to others microorganisms, among them, pathogenic strains. PMID:24031352

  15. The BpeAB-OprB Efflux Pump of Burkholderia pseudomallei 1026b Does Not Play a Role in Quorum Sensing, Virulence Factor Production, or Extrusion of Aminoglycosides but Is a Broad-Spectrum Drug Efflux System

    PubMed Central

    Mima, Takehiko; Schweizer, Herbert P.

    2010-01-01

    Most Burkholderia pseudomallei strains are intrinsically aminoglycoside resistant, mainly due to AmrAB-OprA-mediated efflux. Rare naturally occurring or genetically engineered mutants lacking this pump are aminoglycoside susceptible despite the fact that they also encode and express BpeAB-OprB, which was reported to mediate efflux of aminoglycosides in the Singapore strain KHW. To reassess the role of BpeAB-OprB in B. pseudomallei aminoglycoside resistance, we used mutants overexpressing or lacking this pump in either AmrAB-OprA-proficient or -deficient strain 1026b backgrounds. Our data show that BpeAB-OprB does not mediate efflux of aminoglycosides but is a multidrug efflux system which extrudes macrolides, fluoroquinolones, tetracyclines, acriflavine, and, to a lesser extent, chloramphenicol. Phylogenetically, BpeAB-OprB is closely related to Pseudomonas aeruginosa MexAB-OprM, which has a similar substrate spectrum. AmrAB-OprA is most closely related to MexXY, the only P. aeruginosa efflux pump known to extrude aminoglycosides. Since BpeAB-OprB in strain KHW was also implicated in playing a major role in export of acylated homoserine lactone (AHL) quorum-sensing molecules and in expression of diverse virulence factors, we explored whether this was also true in the strain 1026b background. The results showed that BpeAB-OprB was not required for AHL export, and mutants lacking this efflux system exhibited normal swimming motility and siderophore production, which were severely impaired in KHW bpeAB-oprB mutants. Biofilm formation was impaired in 1026b Δ(amrRAB-oprA) and Δ(amrRAB-oprA) Δ(bpeAB-oprB) mutants. At present, we do not know why our BpeAB-OprB susceptibility and virulence factor expression results with 1026b and its derivatives are different from those previously published for Singapore strain KHW. PMID:20498323

  16. The BpeAB-OprB efflux pump of Burkholderia pseudomallei 1026b does not play a role in quorum sensing, virulence factor production, or extrusion of aminoglycosides but is a broad-spectrum drug efflux system.

    PubMed

    Mima, Takehiko; Schweizer, Herbert P

    2010-08-01

    Most Burkholderia pseudomallei strains are intrinsically aminoglycoside resistant, mainly due to AmrAB-OprA-mediated efflux. Rare naturally occurring or genetically engineered mutants lacking this pump are aminoglycoside susceptible despite the fact that they also encode and express BpeAB-OprB, which was reported to mediate efflux of aminoglycosides in the Singapore strain KHW. To reassess the role of BpeAB-OprB in B. pseudomallei aminoglycoside resistance, we used mutants overexpressing or lacking this pump in either AmrAB-OprA-proficient or -deficient strain 1026b backgrounds. Our data show that BpeAB-OprB does not mediate efflux of aminoglycosides but is a multidrug efflux system which extrudes macrolides, fluoroquinolones, tetracyclines, acriflavine, and, to a lesser extent, chloramphenicol. Phylogenetically, BpeAB-OprB is closely related to Pseudomonas aeruginosa MexAB-OprM, which has a similar substrate spectrum. AmrAB-OprA is most closely related to MexXY, the only P. aeruginosa efflux pump known to extrude aminoglycosides. Since BpeAB-OprB in strain KHW was also implicated in playing a major role in export of acylated homoserine lactone (AHL) quorum-sensing molecules and in expression of diverse virulence factors, we explored whether this was also true in the strain 1026b background. The results showed that BpeAB-OprB was not required for AHL export, and mutants lacking this efflux system exhibited normal swimming motility and siderophore production, which were severely impaired in KHW bpeAB-oprB mutants. Biofilm formation was impaired in 1026b Delta(amrRAB-oprA) and Delta(amrRAB-oprA) Delta(bpeAB-oprB) mutants. At present, we do not know why our BpeAB-OprB susceptibility and virulence factor expression results with 1026b and its derivatives are different from those previously published for Singapore strain KHW.

  17. [Bacterial efflux pumps - their role in antibiotic resistance and potential inhibitors].

    PubMed

    Hricová, Kristýna; Kolář, Milan

    2014-12-01

    Efflux pumps capable of actively draining antibiotic agents from bacterial cells may be considered one of potential mechanisms of the development of antimicrobial resistance. The most important group of efflux pumps capable of removing several types of antibiotics include RND (resistance - nodulation - division) pumps. These are three proteins that cross the bacterial cell wall, allowing direct expulsion of the agent out from the bacterial cell. The most investigated efflux pumps are the AcrAB-TolC system in Escherichia coli and the MexAB-OprM system in Pseudomonas aeruginosa. Moreover, efflux pumps are able to export other than antibacterial agents such as disinfectants, thus decreasing their effectiveness. One potential approach to inactivation of an efflux pump is to use the so-called efflux pump inhibitors (EPIs). Potential inhibitors tested in vitro involve, for example, phenylalanyl-arginyl-b-naphthylamide (PAbN), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or agents of the phenothiazine class.

  18. The Asp20-to-Asn Substitution in the Response Regulator AdeR Leads to Enhanced Efflux Activity of AdeB in Acinetobacter baumannii.

    PubMed

    Nowak, Jennifer; Schneiders, Thamarai; Seifert, Harald; Higgins, Paul G

    2016-02-01

    Overexpression of the resistance-nodulation-cell division-type efflux pump AdeABC is often associated with multidrug resistance in Acinetobacter baumannii and has been linked to mutations in the genes encoding the AdeRS two-component system. In a previous study, we reported that the Asp20→Asn amino acid substitution in the response regulator AdeR is associated with adeB overexpression and reduced susceptibility to the antimicrobials levofloxacin, tigecycline, and trimethoprim-sulfamethoxazole. To further characterize the effect of the Asp20→Asn substitution on antimicrobial susceptibility, the expression of the efflux genes adeB, adeJ, and adeG, and substrate accumulation, four plasmid constructs [containing adeR(Asp20)S, adeR(Asn20)S, adeR(Asp20)SABC, and adeR(Asn20)SABC] were introduced into the adeRSABC-deficient A. baumannii isolate NIPH 60. Neither adeRS construct induced changes in antimicrobial susceptibility or substrate accumulation from that for the vector-only control. The adeR(Asp20)SABC transformant showed reduced susceptibility to 6 antimicrobials and accumulated 12% less ethidium than the control, whereas the Asn20 variant showed reduced susceptibility to 6 of 8 antimicrobial classes tested, and its ethidium accumulation was only 72% of that observed for the vector-only construct. adeB expression was 7-fold higher in the adeR(Asn20)SABC transformant than in its Asp20 variant. No changes in adeG or adeJ expression or in acriflavine or rhodamine 6G accumulation were detected. The antimicrobial susceptibility data suggest that AdeRS does not regulate any resistance determinants other than AdeABC. Furthermore, the characterization of the Asp20→Asn20 substitution proves that the reduced antimicrobial susceptibility previously associated with this substitution was indeed caused by enhanced efflux activity of AdeB.

  19. Impact of Lifestyle Intervention on HDL-Induced eNOS Activation and Cholesterol Efflux Capacity in Obese Adolescent

    PubMed Central

    Wesnigk, Jenny; De Guchtenaere, Ann; Fischer, Tina; Schuler, Gerhard; Vrints, Christiaan J.

    2016-01-01

    Background. Endothelial dysfunction occurs in obese children and adolescent and is regarded as a key step in the development of atherosclerosis. Important components for the development of endothelial dysfunction are reduced activity of endothelial nitric oxide synthase (eNOS) and an increase in cholesterol deposition in the vessel wall, due to reduced reverse cholesterol transport (RCT) activity. High density lipoprotein (HDL) exhibits antiatherosclerotic properties including modulation of eNOS activity and cholesterol efflux capacity. Lifestyle intervention programs can modify endothelial dysfunction in obese adolescents, but their impact on HDL-mediated eNOS activation and RCT is unknown so far. Methods. Obese adolescents (15 ± 1 years, BMI > 35 kg/m2) where randomized either to an intervention group (IG, n = 8; restricted diet and exercise) or to a usual care group (UC, n = 8). At the beginning and after 10 months of treatment HDL-mediated eNOS phosphorylation and cholesterol efflux capacity were evaluated. Results. Ten months of treatment resulted in a substantial weight loss (−31%), an improvement of endothelial function, and an increase in HDL-mediated eNOS-Ser1177 phosphorylation and RCT. A correlation between change in eNOS-Ser1177 phosphorylation or RCT and change in endothelial function was noted. Conclusion. A structured lifestyle intervention program improves antiatherosclerotic HDL functions, thereby positively influencing endothelial function. PMID:27965912

  20. The Influence of Efflux Pump Inhibitors on the Activity of Non-Antibiotic NSAIDS against Gram-Negative Rods

    PubMed Central

    Laudy, Agnieszka E.; Mrowka, Agnieszka; Krajewska, Joanna; Tyski, Stefan

    2016-01-01

    Background Most patients with bacterial infections suffer from fever and various pains that require complex treatments with antibiotics, antipyretics, and analgaesics. The most common drugs used to relieve these symptoms are non-steroidal anti-inflammatory drugs (NSAIDs), which are not typically considered antibiotics. Here, we investigate the effects of NSAIDs on bacterial susceptibility to antibiotics and the modulation of bacterial efflux pumps. Methodology The activity of 12 NSAID active substances, paracetamol (acetaminophen), and eight relevant medicinal products was analyzed with or without pump inhibitors against 89 strains of Gram-negative rods by determining the MICs. Furthermore, the effects of NSAIDs on the susceptibility of clinical strains to antimicrobial agents with or without PAβN (Phe-Arg-β-naphtylamide) were measured. Results The MICs of diclofenac, mefenamic acid, ibuprofen, and naproxen, in the presence of PAβN, were significantly (≥4-fold) reduced, decreasing to 25–1600 mg/L, against the majority of the studied strains. In the case of acetylsalicylic acid only for 5 and 7 out of 12 strains of P. mirabilis and E. coli, respectively, a 4-fold increase in susceptibility in the presence of PAβN was observed. The presence of Aspirin resulted in a 4-fold increase in the MIC of ofloxacin against only two strains of E. coli among 48 tested clinical strains, which included species such as E. coli, K. pneumoniae, P. aeruginosa, and S. maltophilia. Besides, the medicinal products containing the following NSAIDs, diclofenac, mefenamic acid, ibuprofen, and naproxen, did not cause the decrease of clinical strains’ susceptibility to antibiotics. Conclusions The effects of PAβN on the susceptibility of bacteria to NSAIDs indicate that some NSAIDs are substrates for efflux pumps in Gram-negative rods. Morever, Aspirin probably induced efflux-mediated resistance to fluoroquinolones in a few E. coli strains. PMID:26771525

  1. SLC30A10 Is a Cell Surface-Localized Manganese Efflux Transporter, and Parkinsonism-Causing Mutations Block Its Intracellular Trafficking and Efflux Activity

    PubMed Central

    Leyva-Illades, Dinorah; Chen, Pan; Zogzas, Charles E.; Hutchens, Steven; Mercado, Jonathan M.; Swaim, Caleb D.; Morrisett, Richard A.; Bowman, Aaron B.

    2014-01-01

    Manganese (Mn) is an essential metal, but elevated cellular levels are toxic and may lead to the development of an irreversible parkinsonian-like syndrome that has no treatment. Mn-induced parkinsonism generally occurs as a result of exposure to elevated Mn levels in occupational or environmental settings. Additionally, patients with compromised liver function attributable to diseases, such as cirrhosis, fail to excrete Mn and may develop Mn-induced parkinsonism in the absence of exposure to elevated Mn. Recently, a new form of familial parkinsonism was reported to occur as a result of mutations in SLC30A10. The cellular function of SLC30A10 and the mechanisms by which mutations in this protein cause parkinsonism are unclear. Here, using a combination of mechanistic and functional studies in cell culture, Caenorhabditis elegans, and primary midbrain neurons, we show that SLC30A10 is a cell surface-localized Mn efflux transporter that reduces cellular Mn levels and protects against Mn-induced toxicity. Importantly, mutations in SLC30A10 that cause familial parkinsonism blocked the ability of the transporter to traffic to the cell surface and to mediate Mn efflux. Although expression of disease-causing SLC30A10 mutations were not deleterious by themselves, neurons and worms expressing these mutants exhibited enhanced sensitivity to Mn toxicity. Our results provide novel insights into the mechanisms involved in the onset of a familial form of parkinsonism and highlight the possibility of using enhanced Mn efflux as a therapeutic strategy for the potential management of Mn-induced parkinsonism, including that occurring as a result of mutations in SLC30A10. PMID:25319704

  2. Saturable Active Efflux by P-Glycoprotein and Breast Cancer Resistance Protein at the Blood-Brain Barrier Leads to Nonlinear Distribution of Elacridar to the Central Nervous System

    PubMed Central

    Sane, Ramola; Agarwal, Sagar; Mittapalli, Rajendar K.

    2013-01-01

    The study objective was to investigate factors that affect the central nervous system (CNS) distribution of elacridar. Elacridar inhibits transport mediated by P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and has been used to study the influence of transporters on brain distribution of chemotherapeutics. Adequate distribution of elacridar across the blood-brain barrier (BBB) and into the brain parenchyma is necessary to target tumor cells in the brain that overexpress transporters and reside behind an intact BBB. We examined the role of P-gp and Bcrp on brain penetration of elacridar using Friend leukemia virus strain B wild-type, Mdr1a/b(−/−), Bcrp1(−/−), and Mdr1a/b(−/−)Bcrp1(−/−) mice. Initially, the mice were administered 2.5 mg/kg of elacridar intravenously, and the plasma and brain concentrations were determined. The brain-to-plasma partition coefficient of elacridar in the wild-type mice was 0.82, as compared with 3.5 in Mdr1a/b(−/−) mice, 6.6 in Bcrp1(−/−) mice, and 15 in Mdr1a/b(−/−)Bcrp1(−/−) mice, indicating that both P-gp and Bcrp limit the brain distribution of elacridar. The four genotypes were then administered increasing doses of elacridar, and the CNS distribution of elacridar was determined. The observed and model predicted maximum brain-to-plasma ratios (Emax) at the highest dose were not significantly different in all genotypes. However, the ED50 was lower for Mdr1a/b(−/−) mice compared with Bcrp1(−/−) mice. These findings correlate with the relative expression of P-gp and Bcrp at the BBB in these mice and demonstrate the quantitative enhancement in elacridar CNS distribution as a function of its dose. Overall, this study provides useful concepts for future applications of elacridar as an adjuvant therapy to improve targeting of chemotherapeutic agents to tumor cells in the brain parenchyma. PMID:23397054

  3. Indole-Induced Activities of β-Lactamase and Efflux Pump Confer Ampicillin Resistance in Pseudomonas putida KT2440

    PubMed Central

    Kim, Jisun; Shin, Bora; Park, Chulwoo; Park, Woojun

    2017-01-01

    Indole, which is widespread in microbial communities, has received attention because of its effects on bacterial physiology. Pseudomonas putida and Pseudomonas aeruginosa can acquire ampicillin (Amp) resistance during growth on indole-Amp agar. Transcriptome, mutant, and inhibitor studies have suggested that Amp resistance induced by indole can be attributed to increased gene expression of ttgAB encoding two genes of RND-type multidrug efflux operons and an ampC encoding β-lactamase. Expression, enzyme activities, and mutational analyses indicated that AmpC β-lactamase is important for acquiring Amp resistance of P. putida in the presence of indole. Here, we show, for the first time, that volatile indole increased Amp-resistant cells. Consistent with results of the volatile indole assay, a low concentration of indole in liquid culture promoted growth initially, but led to mutagenesis after indole was depleted, which could not be observed at high indole concentrations. Interestingly, ttgAB and ampC gene expression levels correlate with the concentration of indole, which might explain the low number of Amp-mutated cells in high indole concentrations. The expression levels of genes involved in mutagenesis, namely rpoS, recA, and mutS, were also modulated by indole. Our data indicates that indole reduces Amp-induced heterogeneity by promoting expression of TtgABC or MexAB-OprM efflux pumps and the indole-induced β-lactamase in P. putida and P. aeruginosa. PMID:28352264

  4. Antibiotic-potentiation activities of four Cameroonian dietary plants against multidrug-resistant Gram-negative bacteria expressing efflux pumps

    PubMed Central

    2014-01-01

    Background The continuous spread of multidrug-resistant (MDR) bacteria, partially due to efflux pumps drastically reduced the efficacy of the antibiotic armory, increasing the frequency of therapeutic failure. The search for new compounds to potentiate the efficacy of commonly used antibiotics is therefore important. The present study was designed to evaluate the ability of the methanol extracts of four Cameroonian dietary plants (Capsicum frutescens L. var. facilulatum, Brassica oleacera L. var. italica, Brassica oleacera L. var. butyris and Basilicum polystachyon (L.) Moench.) to improve the activity of commonly used antibiotics against MDR Gram-negative bacteria expressing active efflux pumps. Methods The qualitative phytochemical screening of the plant extracts was performed using standard methods whilst the antibacterial activity was performed by broth micro-dilution method. Results All the studied plant extracts revealed the presence of alkaloids, phenols, flavonoids, triterpenes and sterols. The minimal inhibitory concentrations (MIC) of the studied extracts ranged from 256-1024 μg/mL. Capsicum frutescens var. facilulatum extract displayed the largest spectrum of activity (73%) against the tested bacterial strains whilst the lower MIC value (256 μg/mL) was recorded with Basilicum polystachyon against E. aerogenes ATCC 13048 and P. stuartii ATCC 29916. In the presence of PAβN, the spectrum of activity of Brassica oleacera var. italica extract against bacteria strains increased (75%). The extracts from Brassica oleacera var. butyris, Brassica oleacera var. italica, Capsicum frutescens var. facilulatum and Basilicum polystachyon showed synergistic effects (FIC ≤ 0.5) against the studied bacteria, with an average of 75.3% of the tested antibiotics. Conclusion These results provide promising information for the potential use of the tested plants alone or in combination with some commonly used antibiotics in the fight against MDR Gram-negative bacteria

  5. Opioids and efflux transporters. Part 1: P-glycoprotein substrate activity of N-substituted analogs of meperidine.

    PubMed

    Mercer, Susan L; Hassan, Hazem E; Cunningham, Christopher W; Eddington, Natalie D; Coop, Andrew

    2007-03-01

    P-Glycoprotein (P-gp) is an efflux transporter which is up-regulated at the blood-brain barrier in both morphine- and oxycodone-tolerant rats. Numerous studies have shown that many clinically employed opioid analgesics are substrates for P-gp, suggesting that up-regulation of P-gp may contribute to the development of central tolerance to opioids. The studies herein focus on the development of SAR for P-gp substrate activity in the meperidine series of compounds, and show that a meperidine analog of greater potency, N-phenylbutyl-N-normeperidine, has low activity as a P-gp substrate and has the potential to be utilized as a tool to study the contribution of P-gp to the development of central tolerance to opioids.

  6. Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells

    SciTech Connect

    Okajima, F.; Sho, K.; Kondo, Y.

    1988-08-01

    Exposure of FRTL-5 thyroid cells to ATP (1 microM to 1 mM) resulted in the stimulation of I- efflux in association with the induction of inositol trisphosphate production and intracellular Ca2+ mobilization. Nonhydrolyzable ATP derivatives, ADP and GTP, were also as effective in magnitude as ATP, whereas neither AMP nor adenosine exerted significant effect on I- efflux, suggesting a P2-purinergic receptor-mediated activation of I- efflux. Treatment of the cells with the islet-activating protein (IAP) pertussis toxin, which ADP-ribosylated a 41,000 mol wt membrane protein, effectively suppressed the phosphoinositide response to ATP in addition to ATP-dependent I- efflux at agonist concentrations below 10 microM. In contrast, the I- efflux stimulated by TSH, A23187, or phorbol myristate acetate was insusceptible to IAP. The IAP substrate, probably GTP-binding protein, is hence proposed to mediate the activation of P2-purinergic receptor-linked phospholipase-C in FRTL-5 cells. However, the responses to ATP, its nonhydrolyzable derivatives, or ADP at the higher agonist concentrations, especially above 100 microM, were only partially inhibited by IAP, even though the IAP substrate was totally ADP ribosylated by the toxin. The responses to GTP in the whole concentration range tested were not influenced by IAP treatment. Thus, signals arising from the P2-receptor might be transduced to phospholipase-C by two different pathways, i.e. IAP-sensitive and insensitive ones, and result in the stimulation of I- efflux.

  7. Regulation of a nickel-cobalt efflux system and nickel homeostasis in a soil actinobacterium Streptomyces coelicolor.

    PubMed

    Kim, Hae Mi; Ahn, Bo-Eun; Lee, Ju-Hyung; Roe, Jung-Hye

    2015-04-01

    In Streptomyces coelicolor, a soil actinobacterium capable of morphological differentiation and complex secondary metabolism, nickel deficiency is sensed by Nur, a Ni-specific Fur family regulator that controls nickel uptake systems (NikABCDE and NikMNOQ) and both Fe-containing and Ni-containing superoxide dismutases (SodF and SodN). On the other hand, the nickel efflux system and its regulator have not been elucidated. In this study, we demonstrate that an ArsR/SmtB family metalloregulator NmtR, a close homologue of NmtR from Mycobacterium tuberculosis, controls a putative efflux pump of P1-type ATPase (NmtA) in S. coelicolor. NmtR binds to the nmtA promoter region to repress its transcription, and is dissociated in the presence of Ni(ii) and Co(ii). Disruption of the nmtA gene makes cells more sensitive to nickel and cobalt, consistent with its predicted role in encoding a Ni-Co-efflux pump. Growth of S. coelicolor in complex YEME medium is only marginally inhibited by up to 0.5 mM Ni(ii), with significant growth retardation at 1 mM. Nur-regulated sodF and nikA genes are repressed at less than 0.1 μM added NiSO4 whereas NmtR-regulated nmtA transcription is induced at 0.5 mM or more Ni(ii). This reveals the extreme sensitivity of S. coelicolor to nickel deficiency as well as tolerance to surplus nickel. How this organism and possibly other actinomycetes have evolved to develop such a highly Ni-tolerant physiology and how the highly sensitive regulator Nur and the obtuse regulator NmtR achieve their characteristic Ni-sensitivity are interesting questions to solve in the future.

  8. Field measurements of soil CO2 efflux in Heteropogon contortus dominated grassland of semi-arid eco-system.

    PubMed

    Saraswathi, S Gnaana; Paliwal, Kailash

    2011-05-01

    Seasonal changes in soil respiration (SR), soil temperature (ST) and soil moisture (SM) were compared between a barren land with no vegetation (control) and grassland dominated by Heteropogon contortus (L.) of a semi-arid eco-system during 2005-2006. A statistically significant (p<0.001) seasonal change in SR was observed between the two sites. The variation characteristics of soil CO2 effiux rates were observed during wet periods along precipitation gradients and it was consistently higher in grasslands than in control.A maximum soil CO2 efflux of 13.35 +/- 0.33 micromol m2 s-1 in grassland and 7.33 +/- 0.8 micromol m2 s- in control was observed during rainy season-ll, i.e., from October to December, a minimum of 1.27 +/- 0.2 micromol m-2 s-1 in grassland and 0.67 +/- 0.5 micromol m-2 s-1 in control during summer season, i.e., from March to June. A positive significant relation observed between soil respiration and soil moisture (r2above 0.8) and no significant relation was observed between soil CO2 efflux and soil temperature (r2 below 0.3). In water-limited semi-arid ecosystem, rewetting of the soil due to precipitation events triggered the increased pulses of soil respiration especially in grassland when compared to the barren land. The observed soil respiration rates during summer and after the subsequent precipitation events strongly indicated that the soil water-deficit conditions reduce the efflux both in barren land (control) and in grassland of semi-arid eco-system.

  9. Identification of Important Compounds Isolated from Natural Sources that Have Activity Against Multidrug-resistant Cancer Cell Lines: Effects on Proliferation, Apoptotic Mechanism and the Efflux Pump Responsible for Multi-resistance Phenotype.

    PubMed

    Amaral, Leonard; Spengler, Gabriella; Molnar, Joseph

    2016-11-01

    The focus of this mini-review is to identify non-toxic compounds isolated from natural sources (plants) that exhibit specific activity against efflux pumps of specific multidrug-resistant (MDR) cancer cell lines, inhibit proliferation of the MDR cancer cell lines and inhibit the activity of overexpressed efflux pumps of the MDR cancer cell line.

  10. Characterisation of SalRAB a salicylic acid inducible positively regulated efflux system of Rhizobium leguminosarum bv viciae 3841.

    PubMed

    Tett, Adrian J; Karunakaran, Ramakrishnan; Poole, Philip S

    2014-01-01

    Salicylic acid is an important signalling molecule in plant-microbe defence and symbiosis. We analysed the transcriptional responses of the nitrogen fixing plant symbiont, Rhizobium leguminosarum bv viciae 3841 to salicylic acid. Two MFS-type multicomponent efflux systems were induced in response to salicylic acid, rmrAB and the hitherto undescribed system salRAB. Based on sequence similarity salA and salB encode a membrane fusion and inner membrane protein respectively. salAB are positively regulated by the LysR regulator SalR. Disruption of salA significantly increased the sensitivity of the mutant to salicylic acid, while disruption of rmrA did not. A salA/rmrA double mutation did not have increased sensitivity relative to the salA mutant. Pea plants nodulated by salA or rmrA strains did not have altered nodule number or nitrogen fixation rates, consistent with weak expression of salA in the rhizosphere and in nodule bacteria. However, BLAST analysis revealed seventeen putative efflux systems in Rlv3841 and several of these were highly differentially expressed during rhizosphere colonisation, host infection and bacteroid differentiation. This suggests they have an integral role in symbiosis with host plants.

  11. The Asp20-to-Asn Substitution in the Response Regulator AdeR Leads to Enhanced Efflux Activity of AdeB in Acinetobacter baumannii

    PubMed Central

    Nowak, Jennifer; Schneiders, Thamarai; Seifert, Harald

    2015-01-01

    Overexpression of the resistance-nodulation-cell division-type efflux pump AdeABC is often associated with multidrug resistance in Acinetobacter baumannii and has been linked to mutations in the genes encoding the AdeRS two-component system. In a previous study, we reported that the Asp20→Asn amino acid substitution in the response regulator AdeR is associated with adeB overexpression and reduced susceptibility to the antimicrobials levofloxacin, tigecycline, and trimethoprim-sulfamethoxazole. To further characterize the effect of the Asp20→Asn substitution on antimicrobial susceptibility, the expression of the efflux genes adeB, adeJ, and adeG, and substrate accumulation, four plasmid constructs [containing adeR(Asp20)S, adeR(Asn20)S, adeR(Asp20)SABC, and adeR(Asn20)SABC] were introduced into the adeRSABC-deficient A. baumannii isolate NIPH 60. Neither adeRS construct induced changes in antimicrobial susceptibility or substrate accumulation from that for the vector-only control. The adeR(Asp20)SABC transformant showed reduced susceptibility to 6 antimicrobials and accumulated 12% less ethidium than the control, whereas the Asn20 variant showed reduced susceptibility to 6 of 8 antimicrobial classes tested, and its ethidium accumulation was only 72% of that observed for the vector-only construct. adeB expression was 7-fold higher in the adeR(Asn20)SABC transformant than in its Asp20 variant. No changes in adeG or adeJ expression or in acriflavine or rhodamine 6G accumulation were detected. The antimicrobial susceptibility data suggest that AdeRS does not regulate any resistance determinants other than AdeABC. Furthermore, the characterization of the Asp20→Asn20 substitution proves that the reduced antimicrobial susceptibility previously associated with this substitution was indeed caused by enhanced efflux activity of AdeB. PMID:26643347

  12. Robust passive and active efflux of cellular cholesterol to a designer functional mimic of high density lipoprotein

    PubMed Central

    Luthi, Andrea J.; Lyssenko, Nicholas N.; Quach, Duyen; McMahon, Kaylin M.; Millar, John S.; Vickers, Kasey C.; Rader, Daniel J.; Phillips, Michael C.; Mirkin, Chad A.; Thaxton, C. Shad

    2015-01-01

    The ability of HDL to support macrophage cholesterol efflux is an integral part of its atheroprotective action. Augmenting this ability, especially when HDL cholesterol efflux capacity from macrophages is poor, represents a promising therapeutic strategy. One approach to enhancing macrophage cholesterol efflux is infusing blood with HDL mimics. Previously, we reported the synthesis of a functional mimic of HDL (fmHDL) that consists of a gold nanoparticle template, a phospholipid bilayer, and apo A-I. In this work, we characterize the ability of fmHDL to support the well-established pathways of cellular cholesterol efflux from model cell lines and primary macrophages. fmHDL received cell cholesterol by unmediated (aqueous) and ABCG1- and scavenger receptor class B type I (SR-BI)-mediated diffusion. Furthermore, the fmHDL holoparticle accepted cholesterol and phospholipid by the ABCA1 pathway. These results demonstrate that fmHDL supports all the cholesterol efflux pathways available to native HDL and thus, represents a promising infusible therapeutic for enhancing macrophage cholesterol efflux. fmHDL accepts cholesterol from cells by all known pathways of cholesterol efflux: unmediated, ABCG1- and SR-BI-mediated diffusion, and through ABCA1. PMID:25652088

  13. Robust passive and active efflux of cellular cholesterol to a designer functional mimic of high density lipoprotein.

    PubMed

    Luthi, Andrea J; Lyssenko, Nicholas N; Quach, Duyen; McMahon, Kaylin M; Millar, John S; Vickers, Kasey C; Rader, Daniel J; Phillips, Michael C; Mirkin, Chad A; Thaxton, C Shad

    2015-05-01

    The ability of HDL to support macrophage cholesterol efflux is an integral part of its atheroprotective action. Augmenting this ability, especially when HDL cholesterol efflux capacity from macrophages is poor, represents a promising therapeutic strategy. One approach to enhancing macrophage cholesterol efflux is infusing blood with HDL mimics. Previously, we reported the synthesis of a functional mimic of HDL (fmHDL) that consists of a gold nanoparticle template, a phospholipid bilayer, and apo A-I. In this work, we characterize the ability of fmHDL to support the well-established pathways of cellular cholesterol efflux from model cell lines and primary macrophages. fmHDL received cell cholesterol by unmediated (aqueous) and ABCG1- and scavenger receptor class B type I (SR-BI)-mediated diffusion. Furthermore, the fmHDL holoparticle accepted cholesterol and phospholipid by the ABCA1 pathway. These results demonstrate that fmHDL supports all the cholesterol efflux pathways available to native HDL and thus, represents a promising infusible therapeutic for enhancing macrophage cholesterol efflux. fmHDL accepts cholesterol from cells by all known pathways of cholesterol efflux: unmediated, ABCG1- and SR-BI-mediated diffusion, and through ABCA1.

  14. Hypotonicity-activated efflux of taurine and myo-inositol in rat inner medullary collecting duct cells: evidence for a major common pathway.

    PubMed

    Ruhfus, B; Kinne, R K

    1996-01-01

    To further characterize the hypotonicity-activated efflux pathways for the organic osmolytes taurine and myo-inositol in inner medullary collecting duct (IMCD) cells tracer fluxes of taurine and myo-inositol were investigated. The time course of activation of both fluxes after exposure of cells isolated at 600 mosm to a hypotonic medium (300 mosm by omission of sucrose) was identical with a major increase of release within the first 10 min. All 'anion channel blockers' employed proved to be strong inhibitors of both fluxes. Inhibition of myo-inositol efflux by 0.5 mM NPPB and 0.1 mM dideoxyforskolin was not significantly different from that of taurine efflux (87.7 +/- 11.4 compared to 94.6 +/- 4.6% and 98.8 +/- 2.0 compared to 95.9 +/- 3.7%). However, SITS (0.5 and 0.01 mM), DIDS (0.5 and 0.01 mM), and niflumic acid (0.5 mM) inhibited myo-inositol efflux more strongly than taurine efflux. The respective values were 65.4 +/- 4 vs. 42.9 +/- 3.6% for 0.01 mM SITS, 65.7 +/- 4.2 vs. 45.8 +/- 2.0% for 0.01 mM DIDS, and 79.5 +/- 3.5 vs. 54.2 +/- 2.5% for 0.5 mM niflumic acid. Taurine as well as myo-inositol efflux were decreased to a similar extent by 10 mM extracellular ATP (26.9 +/- 6.3 vs. 29.8 +/- 17.7% inhibition), by 10 mM extracellular cAMP (52.8 +/- 9.8 vs. 60.1 +/- 17.2% inhibition) and by reduction of the intracellular ATP content employing 2-deoxy-D-glucose (31.9 +/- 5.9 vs. 40.4 +/- 13.6% inhibition). In polarized primary cell cultures taurine and myo-inositol were released during a hypotonic shock primarily across the basal-lateral membrane, the ratio of basolateral versus apical efflux was 4.1 for taurine and 3.9 for myo-inositol. Apical fluxes were more sensitive to 0.01 mM SITS or DIDS; this was particularly evident for apical myo-inositol efflux which was inhibited by 0.01 mM SITS by 84.1 +/- 5.9% compared to 43.5 +/- 13.1% inhibition of the basolateral efflux. Thus, taurine and myo-inositol efflux show to a great extent a similar cellular distribution

  15. Development of Fluorine-18 Labeled Metabolically Activated Tracers for Imaging of Drug Efflux Transporters with Positron Emission Tomography.

    PubMed

    Sander, Kerstin; Galante, Eva; Gendron, Thibault; Yiannaki, Elena; Patel, Niral; Kalber, Tammy L; Badar, Adam; Robson, Mathew; Johnson, Sean P; Bauer, Florian; Mairinger, Severin; Stanek, Johann; Wanek, Thomas; Kuntner, Claudia; Kottke, Tim; Weizel, Lilia; Dickens, David; Erlandsson, Kjell; Hutton, Brian F; Lythgoe, Mark F; Stark, Holger; Langer, Oliver; Koepp, Matthias; Årstad, Erik

    2015-08-13

    Increased activity of efflux transporters, e.g., P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), at the blood-brain barrier is a pathological hallmark of many neurological diseases, and the resulting multiple drug resistance represents a major clinical challenge. Noninvasive imaging of transporter activity can help to clarify the underlying mechanisms of drug resistance and facilitate diagnosis, patient stratification, and treatment monitoring. We have developed a metabolically activated radiotracer for functional imaging of P-gp/BCRP activity with positron emission tomography (PET). In preclinical studies, the tracer showed excellent initial brain uptake and clean conversion to the desired metabolite, although at a sluggish rate. Blocking with P-gp/BCRP modulators led to increased levels of brain radioactivity; however, dynamic PET did not show differential clearance rates between treatment and control groups. Our results provide proof-of-concept for development of prodrug tracers for imaging of P-gp/BCRP function in vivo but also highlight some challenges associated with this strategy.

  16. Activation and inactivation of taurine efflux in hyposmotic and isosmotic swelling in cortical astrocytes: role of ionic strength and cell volume decrease.

    PubMed

    Cardin, V; Peña-Segura, C; Pasantes-Morales, H

    1999-06-15

    A decrease in intracellular ionic strength appears involved in the activation of swelling-elicited 3H-taurine efflux in cortical cultured astrocytes. Hyposmotic (50%) or isosmotic urea-induced swelling leading to a decrease of intracellular ionic strength, activated 3H-taurine efflux from a rate constant of about 0.008 min(-1) to 0.33 min(-1) (hyposmotic) and 0.59 min(-1) (urea). This efflux rate was markedly lower (maximal 0.03 min(-1)) in isosmotic swelling caused by K+ accumulation, where there is no decrease in ionic strength, or in cold (10 degrees C) hyposmotic medium (maximal 0.18 min(-1)), where swelling is reduced and consequently intracellular ionic strength is less affected. Also, astrocytes pretreated with hyperosmotic medium, which recover cell volume by ion accumulation, did not release 3H-taurine when they swelled by switching to isosmotic medium, but when volume was recovered by accumulation of urea, taurine release was restored. These results point to a key role of ionic strength in the activation of osmosensitive 3H-taurine efflux. In contrast, its inactivation was independent of the change in ionic strength but appears related to the reduction in cell volume after swelling, since despite the extent or direction of the change in ionic strength, the 3H-taurine efflux did not inactivate in isosmotic KCl-elicited swelling when cell volume did not recover nor in hyposmotic swelling when RVD was impaired by replacing NaCl in the medium by permeant osmolytes.

  17. The MerR-like regulator BrlR confers biofilm tolerance by activating multidrug efflux pumps in Pseudomonas aeruginosa biofilms.

    PubMed

    Liao, Julie; Schurr, Michael J; Sauer, Karin

    2013-08-01

    A defining characteristic of biofilms is antibiotic tolerance that can be up to 1,000-fold greater than that of planktonic cells. In Pseudomonas aeruginosa, biofilm tolerance to antimicrobial agents requires the biofilm-specific MerR-type transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm tolerance has not been elucidated. Genome-wide transcriptional profiling indicated that brlR was required for maximal expression of genes associated with antibiotic resistance, in particular those encoding the multidrug efflux pumps MexAB-OprM and MexEF-OprN. Chromatin immunoprecipitation (ChIP) analysis revealed a direct regulation of these genes by BrlR, with DNA binding assays confirming BrlR binding to the promoter regions of the mexAB-oprM and mexEF-oprN operons. Quantitative reverse transcriptase PCR (qRT-PCR) analysis further indicated BrlR to be an activator of mexAB-oprM and mexEF-oprN gene expression. Moreover, immunoblot analysis confirmed increased MexA abundance in cells overexpressing brlR. Inactivation of both efflux pumps rendered biofilms significantly more susceptible to five different classes of antibiotics by affecting MIC but not the recalcitrance of biofilms to killing by bactericidal agents. Overexpression of either efflux pump in a ΔbrlR strain partly restored tolerance of ΔbrlR biofilms to antibiotics. Expression of brlR in mutant biofilms lacking both efflux pumps partly restored antimicrobial tolerance of biofilms to wild-type levels. Our results indicate that BrlR acts as an activator of multidrug efflux pumps to confer tolerance to P. aeruginosa biofilms and to resist the action of antimicrobial agents.

  18. The MerR-Like Regulator BrlR Confers Biofilm Tolerance by Activating Multidrug Efflux Pumps in Pseudomonas aeruginosa Biofilms

    PubMed Central

    Liao, Julie; Schurr, Michael J.

    2013-01-01

    A defining characteristic of biofilms is antibiotic tolerance that can be up to 1,000-fold greater than that of planktonic cells. In Pseudomonas aeruginosa, biofilm tolerance to antimicrobial agents requires the biofilm-specific MerR-type transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm tolerance has not been elucidated. Genome-wide transcriptional profiling indicated that brlR was required for maximal expression of genes associated with antibiotic resistance, in particular those encoding the multidrug efflux pumps MexAB-OprM and MexEF-OprN. Chromatin immunoprecipitation (ChIP) analysis revealed a direct regulation of these genes by BrlR, with DNA binding assays confirming BrlR binding to the promoter regions of the mexAB-oprM and mexEF-oprN operons. Quantitative reverse transcriptase PCR (qRT-PCR) analysis further indicated BrlR to be an activator of mexAB-oprM and mexEF-oprN gene expression. Moreover, immunoblot analysis confirmed increased MexA abundance in cells overexpressing brlR. Inactivation of both efflux pumps rendered biofilms significantly more susceptible to five different classes of antibiotics by affecting MIC but not the recalcitrance of biofilms to killing by bactericidal agents. Overexpression of either efflux pump in a ΔbrlR strain partly restored tolerance of ΔbrlR biofilms to antibiotics. Expression of brlR in mutant biofilms lacking both efflux pumps partly restored antimicrobial tolerance of biofilms to wild-type levels. Our results indicate that BrlR acts as an activator of multidrug efflux pumps to confer tolerance to P. aeruginosa biofilms and to resist the action of antimicrobial agents. PMID:23687276

  19. Exogenously Applied 24-Epibrassinolide (EBL) Ameliorates Detrimental Effects of Salinity by Reducing K+ Efflux via Depolarization-Activated K+ Channels.

    PubMed

    Azhar, Nazila; Su, Nana; Shabala, Lana; Shabala, Sergey

    2017-03-02

    This study has investigated mechanisms conferring beneficial effects of exogenous application of 24-epibrassinolides (EBL) on plant growth and performance under saline conditions. Barley seedlings treated with 0.25 mg l-1 EBL showed significant improvements in root hair length, shoot length, shoot fresh weight and relative water content when grown in the presence of 150 mM NaCl in the growth medium. In addition, EBL treatment significantly decreased the Na+ content in both shoots (by approximately 50%) and roots. Electrophysiological experiments revealed that pre-treatment with EBL for 1 and 24 h suppressed or completely prevented the NaCl-induced K+ leak in the elongation zone of barley roots, but did not affect root sensitivity to oxidative stress. Further experiments using Arabidopsis loss-of-function gork1-1 (lacking functional depolarization-activated outward-rectifying K+ channels in the root epidermal cells) and akt1 (lacking inward-rectifying K+ uptake channel) mutants showed that NaCl-induced K+ loss in the elongation zone of roots was reduced by EBL pre-treatment 50- to 100-fold in wild-type Col-0 and akt1, but only 10-fold in the gork1-1 mutant. At the same time, EBL treatment shifted vanadate-sensitive H+ flux towards net efflux. Taken together, these data indicate that exogenous application of EBL effectively improves plant salinity tolerance by prevention of K+ loss via regulating depolarization-activated K+ channels.

  20. Contribution of Resistance-Nodulation-Cell Division Efflux Systems to Antibiotic Resistance and Biofilm Formation in Acinetobacter baumannii

    PubMed Central

    Yoon, Eun-Jeong; Nait Chabane, Yassine; Goussard, Sylvie; Snesrud, Erik; Courvalin, Patrice; Dé, Emmanuelle

    2015-01-01

    ABSTRACT Acinetobacter baumannii is a nosocomial pathogen of increasing importance due to its multiple resistance to antibiotics and ability to survive in the hospital environment linked to its capacity to form biofilms. To fully characterize the contribution of AdeABC, AdeFGH, and AdeIJK resistance-nodulation-cell division (RND)-type efflux systems to acquired and intrinsic resistance, we constructed, from an entirely sequenced susceptible A. baumannii strain, a set of isogenic mutants overexpressing each system following introduction of a point mutation in their cognate regulator or a deletion for the pump by allelic replacement. Pairwise comparison of every derivative with the parental strain indicated that AdeABC and AdeFGH are tightly regulated and contribute to acquisition of antibiotic resistance when overproduced. AdeABC had a broad substrate range, including β-lactams, fluoroquinolones, tetracyclines-tigecycline, macrolides-lincosamides, and chloramphenicol, and conferred clinical resistance to aminoglycosides. Importantly, when combined with enzymatic resistance to carbapenems and aminoglycosides, this pump contributed in a synergistic fashion to the level of resistance of the host. In contrast, AdeIJK was expressed constitutively and was responsible for intrinsic resistance to the same major drug classes as AdeABC as well as antifolates and fusidic acid. Surprisingly, overproduction of AdeABC and AdeIJK altered bacterial membrane composition, resulting in decreased biofilm formation but not motility. Natural transformation and plasmid transfer were diminished in recipients overproducing AdeABC. It thus appears that alteration in the expression of efflux systems leads to multiple changes in the relationship between the host and its environment, in addition to antibiotic resistance. PMID:25805730

  1. Calcium Efflux from Barnacle Muscle Fibers

    PubMed Central

    Russell, J. M.; Blaustein, M. P.

    1974-01-01

    Calcium-45 was injected into single giant barnacle muscle fibers, and the rate of efflux was measured under a variety of conditions. The rate constant (k) for 45Ca efflux into standard seawater averaged 17 x 10–4 min–1 which corresponds to an efflux of about 1–2 pmol/cm2·s. Removal of external Ca (Cao) reduced the efflux by 50%. In most fibers about 40% of the 45Ca efflux into Ca-free seawater was dependent on external Na (Nao); treatment with 3.5 mM caffeine increased the magnitude of the Nao-dependent efflux. In a few fibers removal of Nao, in the absence of Cao, either had no effect or increased k; caffeine (2–3.5 mM) unmasked an Nao-dependent efflux in these fibers. The Nao-dependent Ca efflux had a Q10 of about 3.7. The data are consistent with the idea that a large fraction of the Ca efflux may be carrier-mediated, and may involve both Ca-Ca and Na-Ca counterflow. The relation between the Nao-dependent Ca efflux and the external Na concentration is sigmoid, and suggests that two, or more likely three, external Na+ ions may activate the efflux of one Ca+2. With a three-for-one Na-Ca exchange, the Na electrochemical gradient may be able to supply sufficient energy to maintain the Ca gradient in these fibers. Other, more complex models are not excluded, however, and may be required to explain some puzzling features of the Ca efflux such as the variable Nao-dependence. PMID:4812633

  2. Carbon dioxide efflux from soil with poultry litter applications in conventional and conservation tillage systems in northern Alabama.

    PubMed

    Roberson, T; Reddy, K C; Reddy, S S; Nyakatawa, E Z; Raper, R L; Reeves, D W; Lemunyon, J

    2008-01-01

    Increased CO2 release from soils resulting from agricultural practices such as tillage has generated concerns about contributions to global warming. Maintaining current levels of soil C and/or sequestering additional C in soils are important mechanisms to reduce CO2 in the atmosphere through production agriculture. We conducted a study in northern Alabama from 2003 to 2006 to measure CO2 efflux and C storage in long-term tilled and non-tilled cotton (Gossypium hirsutum L.) plots receiving poultry litter or ammonium nitrate (AN). Treatments were established in 1996 on a Decatur silt loam (clayey, kaolinitic thermic, Typic Paleudults) and consisted of conventional-tillage (CT), mulch-tillage (MT), and no-tillage (NT) systems with winter rye [Secale cereale (L.)] cover cropping and AN and poultry litter (PL) as nitrogen sources. Cotton was planted in 2003, 2004, and 2006. Corn was planted in 2005 as a rotation crop using a no-till planter in all plots, and no fertilizer was applied. Poultry litter application resulted in higher CO2 emission from soil compared with AN application regardless of tillage system. In 2003 and 2006, CT (4.39 and 3.40 micromol m(-2) s(-1), respectively) and MT (4.17 and 3.39 micromol m(-2) s(-1), respectively) with PL at 100 kg N ha(-1) (100 PLN) recorded significantly higher CO2 efflux compared with NT with 100 PLN (2.84 and 2.47 micromol m(-2) s(-1), respectively). Total soil C at 0- to 15-cm depth was not affected by tillage but significantly increased with PL application and winter rye cover cropping. In general, cotton produced with NT conservation tillage in conjunction with PL and winter rye cover cropping reduced CO2 emissions and sequestered more soil C compared with control treatments.

  3. Consecutive salinomycin treatment reduces doxorubicin resistance of breast tumor cells by diminishing drug efflux pump expression and activity.

    PubMed

    Hermawan, Adam; Wagner, Ernst; Roidl, Andreas

    2016-03-01

    Chemoresistance is a major challenge for the successful therapy of breast cancer. The discovery of salinomycin as an anticancer stem cell drug provides progress in overcoming chemoresistance. However, it remains to be elucidated whether salinomycin treatment is able to sensitize cancer cells to chemotherapeutic drugs. In the present study, we consecutively treated epithelial MCF-7 and BT-474 breast cancer cells as well as mesenchymal MDA-MB 231 and MDA-MB 436 cells with salinomycin, and analyzed the gene expression of the two prominent multiple drug resistance (MDR) genes, MDR1 and BCRP1. We found that repeated treatment with salinomycin generated resistance against this drug in all cell lines and increased the chemosensitivity towards doxorubicin. Drug efflux pump gene expression and pump activity of MDR1 and BCRP1 were downregulated in almost all cell lines, except for MDR1 in the MDA-MB 231 cells. Consequently, the intracellular doxorubicin accumulation was increased compared to the respective parental cells. Our findings suggest a novel treatment option for MDR tumors by sensitizing these tumors via salinomycin pretreatment.

  4. Rheumatoid Arthritis Disease Activity Is Determinant for ABCB1 and ABCG2 Drug-Efflux Transporters Function

    PubMed Central

    Atisha-Fregoso, Yemil; Lima, Guadalupe; Pascual-Ramos, Virginia; Baños-Peláez, Miguel; Fragoso-Loyo, Hilda; Jakez-Ocampo, Juan; Contreras-Yáñez, Irazú; Llorente, Luis

    2016-01-01

    Objective To compare drug efflux function of ABCB1 and ABCG2 transporters in rheumatoid arthritis (RA) patients with active disease and in remission. Methods Twenty two active RA patients (DAS28 ≥3.2) and 22 patients in remission (DAS28<2.6) were selected from an early RA clinic. All patients were evaluated at study inclusion and six months later. ABCB1 and ABCG2 functional activity was measured in peripheral lymphocytes by flow cytometry. The percentage of cells able to extrude substrates for ABCB1 and ABCG2 was recorded. Results Active patients had higher ABCB1 and ABCG2 activity compared with patients in remission (median [interquartile range]): 3.9% (1.4–22.2) vs (1.3% (0.6–3.2), p = 0.003 and 3.9% (1.1–13.3) vs 0.9% (0.5–1.9) p = 0.006 respectively. Both transporters correlated with disease activity assessed by DAS28, rho = 0.45, p = 0.002 and rho = 0.47, p = 0.001 respectively. Correlation was observed between the time from the beginning of treatment and transporter activity: rho = 0.34, p = 0.025 for ABCB1 and rho = 0.35, p = 0.018 for ABCG2. The linear regression model showed that DAS28 and the time from the onset of treatment are predictors of ABCB1 and ABCG2 functional activity, even after adjustment for treatment. After six months we calculated the correlation between change in DAS28 and change in the functional activity in both transporters and found a moderate and significant correlation for ABCG2 (rho = 0.28, p = 0.04) and a non-significant correlation for ABCB1 (rho = 0.22, p = 0.11). Conclusions Patients with active RA have an increased function of ABCB1 and ABCG2, and disease activity is the main determinant of this phenomena. PMID:27442114

  5. Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes.

    PubMed

    Ghisalberti, Didier; Masi, Muriel; Pagès, Jean-Marie; Chevalier, Jacqueline

    2005-03-25

    Chloramphenicol has been reported to act as an inducer of the multidrug resistance in Escherichia coli. A resistant variant able to grow on plates containing 64 microg/ml chloramphenicol was obtained from the Enterobacter aerogenes ATCC 13048-type strain. Chloramphenicol resistance was due to an active efflux of this antibiotic and it was associated with resistance to fluoroquinolones and tetracycline, but not to aminoglycoside or beta-lactam antibiotics. MDR in the chloramphenicol-resistant variant is linked to the overexpression of the major AcrAB-TolC efflux system. This overexpression seems unrelated to the global Mar and the local AcrR regulatory pathways.

  6. Intracellular sodium, potassium and magnesium concentration, ouabain-sensitive 86rubidium-uptake and sodium-efflux and Na+, K+-cotransport activity in erythrocytes of normal male subjects studied on two occasions.

    PubMed

    Lijnen, P; Hespel, P; Lommelen, G; Laermans, M; M'Buyamba-Kabangu, J R; Amery, A

    1986-09-01

    The red cell Na+,K+-ATPase pump activity estimated by the ouabain-sensitive 86Rb-uptake or Na+-efflux, the Na+,K+-cotransport activity measured either by the furosemide-sensitive K+- or Na+- efflux or by the ethacrynic acid-sensitive 86Rb-uptake as well as the intraerythrocyte concentration of sodium, potassium and magnesium were studied in 29 normal male subjects with one to three weeks interval between the first and second blood sampling. Both the red cell sodium and potassium concentration, the erythrocyte ouabain-sensitive 86Rb-uptake and Na+-efflux, the furosemide-sensitive Na+- and K+-efflux and the ethacrynic acid-sensitive 86Rb-uptake are stable over time in the same individual. The furosemide-sensitive Na+-and K+-efflux is significantly related to the ethacrynic acid-sensitive 86Rb-uptake. The intraerythrocyte Na+ concentration was negatively related to the ouabain-sensitive 86Rb-uptake, but not to the ouabain-sensitive Na+-efflux; it was, however, negatively related to the rate constant for the ouabain-sensitive Na+-efflux.

  7. Hydrophilicity of quinolones is not an exclusive factor for decreased activity in efflux-mediated resistant mutants of Staphylococcus aureus.

    PubMed

    Takenouchi, T; Tabata, F; Iwata, Y; Hanzawa, H; Sugawara, M; Ohya, S

    1996-08-01

    The elevated expression of the norA gene is responsible for efflux-mediated resistance to quinolones in Staphylococcus aureus (E.Y.W. Ng, M. Trucksis, and D.C. Hooper, Antimicrob. Agents Chemother. 38:1345-1355, 1994). For S. aureus transformed with a plasmid containing the cloned norA gene, SA113(pTUS20) (H. Yoshida, M. Bogaki, S. Nakamura, K. Ubukata, and M. Konno, J. Bacteriol. 172:6942-6949, 1990), and an overexpressed mutant, SA-1199B (G.W. Kaatz, S.M. Seo, and C.A. Ruble, J. Infect. Dis. 163:1080-1086, 1991), the MICs of norfloxacin increased 16 and 64 times compared with its MICs for the recipient and wild-type strains, SA113 and SA-1199, respectively. MICs of CS-940, however, increased only two and eight times, even though these two fluoroquinolones are similarly hydrophilic (apparent logPs of approximately -1). No good correlation was found, among 15 developed and developing quinolones, between the increment ratio in MICs and hydrophobicity (r = 0.61). Analysis of the quantitative structure-activity relationship among 40 fluoroquinolones revealed that the MIC increment ratio was significantly correlated with the bulkiness of the C-7 substituent and bulkiness and hydrophobicity of the C-8 substituent of fluoroquinolones (r = 0.87) and not with its molecular hydrophobicity (r = 0.47). Cellular accumulation of norfloxacin in SA-1199B was significantly lower than that in SA-1199, and it was increased by addition of carbonyl cyanide m-chlorophenyl hydrazone. On the other hand, accumulations of CS-940 in these strains were nearly identical, and they were not affected by addition of the protonophore.

  8. Activation of GPR55 Receptors Exacerbates oxLDL-Induced Lipid Accumulation and Inflammatory Responses, while Reducing Cholesterol Efflux from Human Macrophages

    PubMed Central

    Lanuti, Mirko; Talamonti, Emanuela; Maccarrone, Mauro; Chiurchiù, Valerio

    2015-01-01

    The G protein-coupled receptor GPR55 has been proposed as a new cannabinoid receptor associated with bone remodelling, nervous system excitability, vascular homeostasis as well as in several pathophysiological conditions including obesity and cancer. However, its physiological role and underlying mechanism remain unclear. In the present work, we demonstrate for the first time its presence in human macrophages and its increased expression in ox-LDL-induced foam cells. In addition, pharmacological activation of GPR55 by its selective agonist O-1602 increased CD36- and SRB-I-mediated lipid accumulation and blocked cholesterol efflux by downregulating ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, as well as enhanced cytokine- and pro-metalloprotease-9 (pro-MMP-9)-induced proinflammatory responses in foam cells. Treatment with cannabidiol, a selective antagonist of GPR55, counteracted these pro-atherogenic and proinflammatory O-1602-mediated effects. Our data suggest that GPR55 could play deleterious role in ox-LDL-induced foam cells and could be a novel pharmacological target to manage atherosclerosis and other related cardiovascular diseases. PMID:25970609

  9. Two-component regulatory systems in Pseudomonas aeruginosa: an intricate network mediating fimbrial and efflux pump gene expression

    PubMed Central

    Sivaneson, Melissa; Mikkelsen, Helga; Ventre, Isabelle; Bordi, Christophe; Filloux, Alain

    2011-01-01

    Pseudomonas aeruginosa is responsible for chronic and acute infections in humans. Chronic infections are associated with production of fimbriae and the formation of a biofilm. The two-component system Roc1 is named after its role in the regulation of cup genes, which encode components of a machinery allowing assembly of fimbriae. A non-characterized gene cluster, roc2, encodes components homologous to the Roc1 system. We show that cross-regulation occurs between the Roc1 and Roc2 signalling pathways. We demonstrate that the sensors RocS2 and RocS1 converge on the response regulator RocA1 to control cupC gene expression. This control is independent of the response regulator RocA2. Instead, we show that these sensors act via the RocA2 response regulator to repress the mexAB-oprM genes. These genes encode a multidrug efflux pump and are upregulated in the rocA2 mutant, which is less susceptible to antibiotics. It has been reported that in cystic fibrosis lungs, in which P. aeruginosa adopts the biofilm lifestyle, most isolates have an inactive MexAB-OprM pump. The concomitant RocS2-dependent upregulation of cupC genes (biofilm formation) and downregulation of mexAB-oprM genes (antibiotic resistance) is in agreement with this observation. It suggests that the Roc systems may sense the environment in the cystic fibrosis lung. PMID:21205015

  10. Genotypic and phenotypic detection of efflux pump in Rhodococcus equi

    PubMed Central

    Gressler, Letícia Trevisan; de Vargas, Agueda Castagna; da Costa, Mateus Matiuzzi; Pötter, Luciana; da Silveira, Bibiana Petri; Sangioni, Luis Antônio; de Avila Botton, Sônia

    2014-01-01

    The req_39680 gene, associated to a putative efflux system, was detected in 60% (54/90) of R. equi isolates by PCR. The phenotypic expression of efflux mechanism was verified in 20% of the isolates using ethidium bromide. For the first time, the expression of efflux mechanism was demonstrated in R. equi. PMID:25242956

  11. How to Measure Export via Bacterial Multidrug Resistance Efflux Pumps

    PubMed Central

    Blair, Jessica M. A.

    2016-01-01

    ABSTRACT Bacterial multidrug resistance (MDR) efflux pumps are an important mechanism of antibiotic resistance and are required for many pathogens to cause infection. They are also being harnessed to improve microbial biotechnological processes, including biofuel production. Therefore, scientists of many specialties must be able to accurately measure efflux activity. However, myriad methodologies have been described and the most appropriate method is not always clear. Within the scientific literature, many methods are misused or data arising are misinterpreted. The methods for measuring efflux activity can be split into two groups, (i) those that directly measure efflux and (ii) those that measure the intracellular accumulation of a substrate, which is then used to infer efflux activity. Here, we review the methods for measuring efflux and explore the most recent advances in this field, including single-cell or cell-free technologies and mass spectrometry, that are being used to provide more detailed information about efflux pump activity. PMID:27381291

  12. Functional components of the bacterial CzcCBA efflux system reduce cadmium uptake and accumulation in transgenic tobacco plants.

    PubMed

    Nesler, Andrea; DalCorso, Giovanni; Fasani, Elisa; Manara, Anna; Di Sansebastiano, Gian Pietro; Argese, Emanuele; Furini, Antonella

    2017-03-25

    Cadmium (Cd) is a toxic trace element released into the environment by industrial and agricultural practices, threatening the health of plants and contaminating the food/feed chain. Biotechnology can be used to develop plant varieties with a higher capacity for Cd accumulation (for use in phytoremediation programs) or a lower capacity for Cd accumulation (to reduce Cd levels in food and feed). Here we generated transgenic tobacco plants expressing components of the Pseudomonas putida CzcCBA efflux system. Plants were transformed with combinations of the CzcC, CzcB and CzcA genes, and the impact on Cd mobilization was analysed. Plants expressing PpCzcC showed no differences in Cd accumulation, whereas those expressing PpCzcB or PpCzcA accumulated less Cd in the shoots, but more Cd in the roots. Plants expressing both PpCzcB and PpCzcA accumulated less Cd in the shoots and roots compared to controls, whereas plants expressing all three genes showed a significant reduction in Cd levels only in shoots. These results show that components of the CzcCBA system can be expressed in plants and may be useful for developing plants with a reduced capacity to accumulate Cd in the shoots, potentially reducing the toxicity of food/feed crops cultivated in Cd-contaminated soils.

  13. Population pharmacokinetic modelling of non-linear brain distribution of morphine: influence of active saturable influx and P-glycoprotein mediated efflux

    PubMed Central

    Groenendaal, D; Freijer, J; de Mik, D; Bouw, M R; Danhof, M; de Lange, E C M

    2007-01-01

    Background and purpose: Biophase equilibration must be considered to gain insight into the mechanisms underlying the pharmacokinetic-pharmacodynamic (PK-PD) correlations of opioids. The objective was to characterise in a quantitative manner the non-linear distribution kinetics of morphine in brain. Experimental approach: Male rats received a 10-min infusion of 4 mg kg−1 of morphine, combined with a continuous infusion of the P-glycoprotein (Pgp) inhibitor GF120918 or vehicle, or 40 mg kg−1 morphine alone. Unbound extracellular fluid (ECF) concentrations obtained by intracerebral microdialysis and total blood concentrations were analysed using a population modelling approach. Key results: Blood pharmacokinetics of morphine was best described with a three-compartment model and was not influenced by GF120918. Non-linear distribution kinetics in brain ECF was observed with increasing dose. A one compartment distribution model was developed, with separate expressions for passive diffusion, active saturable influx and active efflux by Pgp. The passive diffusion rate constant was 0.0014 min−1. The active efflux rate constant decreased from 0.0195 min−1 to 0.0113 min−1 in the presence of GF120918. The active influx was insensitive to GF120918 and had a maximum transport (Nmax/Vecf) of 0.66 ng min−1 ml−1 and was saturated at low concentrations of morphine (C50=9.9 ng ml−1). Conclusions and implications: Brain distribution of morphine is determined by three factors: limited passive diffusion; active efflux, reduced by 42% by Pgp inhibition; low capacity active uptake. This implies blood concentration-dependency and sensitivity to drug-drug interactions. These factors should be taken into account in further investigations on PK-PD correlations of morphine. PMID:17471182

  14. Endomorphins exit the brain by a saturable efflux system at the basolateral surface of cerebral endothelial cells.

    PubMed

    Somogyvari-Vigh, Aniko; Kastin, Abba J; Liao, Jie; Zadina, James E; Pan, Weihong

    2004-05-01

    Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are two highly selective mu-opiate receptor agonists. We recently demonstrated that EM-1 and EM-2 have a saturable transport system from brain-to-blood in vivo. Since the endothelial cells are the main component of the non-fenestrated microvessels of the blood-brain barrier (BBB), we examined whether these endogenous tetrapeptides have a saturable transport system in cultured cerebral endothelial cells. EM-1 and EM-2 binding and transport were studied in a transwell system in which primary mouse endothelial cells were co-cultured with rat glioma cells. We found that binding of both endomorphins was greater on the basolateral than the apical cell surface. Flux of EM-1 and EM-2 occurred predominantly in the basolateral to apical direction, each showing self-inhibition with an excess of the respective endomorphin. Transport was not influenced by the addition of the P-glycoprotein inhibitor, cyclosporin A. Neither the mu-opiate receptor agonist DAMGO nor the delta-opiate receptor agonist DPDPE had any effect on the transport. Thus, the results show that a saturable transport system for EM-1 and EM-2 occurs at the level of endothelial cells of the BBB, and unlike beta-endorphin and morphine, P-glycoprotein is not needed for the brain-to-blood transport. Cross-inhibition of the transport of each endomorphin by the other suggests a shared transport system that is different from mu- or delta-opiate receptors. As endormorphins are mainly produced in the CNS, the presence of the efflux system at the BBB could play an important role in pain modulation and neuroendocrine control.

  15. RND type efflux pump system MexAB-OprM of pseudomonas aeruginosa selects bacterial languages, 3-oxo-acyl-homoserine lactones, for cell-to-cell communication

    PubMed Central

    2012-01-01

    Background Bacteria release a wide variety of small molecules including cell-to-cell signaling compounds. Gram-negative bacteria use a variety of self-produced autoinducers such as acylated homoserine lactones (acyl-HSLs) as signal compounds for quorum sensing (QS) within and between bacterial species. QS plays a significant role in the pathogenesis of infectious diseases and in beneficial symbiosis by responding to acyl-HSLs in Pseudomonas aeruginosa. It is considered that the selection of bacterial languages is necessary to regulate gene expression and thus it leads to the regulation of virulence and provides a growth advantage in several environments. In this study, we hypothesized that RND-type efflux pump system MexAB-OprM of P. aeruginosa might function in the selection of acyl-HSLs, and we provide evidence to support this hypothesis. Results Loss of MexAB-OprM due to deletion of mexB caused increases in QS responses, as shown by the expression of gfp located downstream of the lasB promoter and LasB elastase activity, which is regulated by a LasR-3-oxo-C12-HSL complex. Either complementation with a plasmid containing wild-type mexB or the addition of a LasR-specific inhibitor, patulin, repressed these high responses to 3-oxo-acyl-HSLs. Furthermore, it was shown that the acyl-HSLs-dependent response of P. aeruginosa was affected by the inhibition of MexB transport activity and the mexB mutant. The P. aeruginosa MexAB-OprM deletion mutant showed a strong QS response to 3-oxo-C10-HSL produced by Vibrio anguillarum in a bacterial cross-talk experiment. Conclusion This work demonstrated that MexAB-OprM does not control the binding of LasR to 3-oxo-Cn-HSLs but rather accessibility of non-cognate acyl-HSLs to LasR in P. aeruginosa. MexAB-OprM not only influences multidrug resistance, but also selects acyl-HSLs and regulates QS in P. aeruginosa. The results demonstrate a new QS regulation mechanism via the efflux system MexAB-OprM in P. aeruginosa. PMID:22574700

  16. [Significance of efflux pumps in multidrug resistance of Gram-negative bacteria].

    PubMed

    Wiercińska, Olga; Chojecka, Agnieszka; Kanclerski, Krzysztof; Rőhm-Rodowald, Ewa; Jakimiak, Bożenna

    2015-01-01

    The phenomenon of multidrug. resistance of bacteria is a serious problem of modern medicine. This resistance largely is a consequence of abuse and improper use of antibacterial substances, especially antibiotics and chemotherapeutics in hospital settings. Multidrug resistance is caused by a number of interacting mechanisms of resistance. Recent studies have indicated that efflux pumps and systems of efflux pumps are an important determinant of this phenomenon. Contribute to this particular RND efflux systems of Gram-negative bacteria, which possess a wide range of substrates such as antibiotics, dyes, detergents, toxins and active substances of disinfectants and antiseptics. These transporters are usually encoded on bacterial chromosomes. Genes encoding efflux pumps' proteins may also be carried on plasmids and other mobile genetic elements. Such pumps are usually specific to a small group of substrates, but as an additional mechanism of resistance may contribute to the multidrug resistance.

  17. Role of different Escherichia coli hydrogenases in H+ efflux and F₁F(o)-ATPase activity during glycerol fermentation at different pH values.

    PubMed

    Blbulyan, Syuzanna; Avagyan, Arev; Poladyan, Anna; Trchounian, Armen

    2011-06-01

    Escherichia coli is able to ferment glycerol and produce H2 by different Hyds (hydrogenases). Wild-type whole cells were shown to extrude H+ through the F1Fo-ATPase and by other means with a lower rate compared with that under glucose fermentation. At pH 7.5, H+ efflux was stimulated in fhlA mutant (with defective transcriptional activator of Hyd-3 or Hyd-4) and was lowered in hyaB or hybC mutants (with defective Hyd-1 or Hyd-2) and hyaB hybC double mutant; DCCD (dicyclohexylcarbodi-imide)-sensitive H+ efflux was observed. At pH 5.5, H+ efflux in wild-type was lower compared with that at pH 7.5; it was increased in fhlA mutant and absent in hyaB hybC mutant. Membrane vesicle ATPase activity was lower in wild-type glycerol-fermented cells at pH 7.5 compared with that in glucose-fermented cells; 100 mM K+ did not stimulate ATPase activity. The latter at pH 7.5, compared with that in wild-type, was lower in hyaB and less in hybC mutants, stimulated in the hyaB hybC mutant and suppressed in the fhlA mutant; DCCD inhibited ATPase activity. At pH 5.5, the ATPase activities of hyaB and hybC mutants had similar values and were higher compared with that in wild-type; ATPase activity was suppressed in hyaB hybC and fhlA mutants. The results indicate that during glycerol fermentation, H+ was expelled also via F1Fo. At pH 7.5 Hyd-1 and Hyd-2 but not FhlA or Hyd-4 might be related to F1Fo or have their own H+-translocating ability. At pH 5.5, both Hyd-1 and Hyd-2 more than F1Fo might be involved in H+ efflux.

  18. Distribution of Gefitinib to the Brain Is Limited by P-glycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2)-Mediated Active Efflux

    PubMed Central

    Agarwal, Sagar; Sane, Ramola; Gallardo, Jose L.; Ohlfest, John R.

    2010-01-01

    Gefitinib is an orally active inhibitor of the epidermal growth factor receptor approved for use in patients with locally advanced or metastatic non–small cell lung cancer. It has also been evaluated in several clinical trials for treatment of brain tumors such as high-grade glioma. In this study, we investigated the influence of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) on distribution of gefitinib to the central nervous system. In vitro studies conducted in Madin-Darby canine kidney II cells indicate that both P-gp and BCRP effectively transport gefitinib, limiting its intracellular accumulation. In vivo studies demonstrated that transport of gefitinib across the blood-brain barrier (BBB) is significantly limited. Steady-state brain-to-plasma (B/P) concentration ratios were 70-fold higher in the Mdr1a/b(−/−) Bcrp1(−/−) mice (ratio of approximately 7) compared with wild-type mice (ratio of approximately 0.1). The B/P ratio after oral administration increased significantly when gefitinib was coadministered with the dual P-gp and BCRP inhibitor elacridar. We investigated the integrity of tight junctions in the Mdr1a/b(−/−) Bcrp1(−/−) mice and found no difference in the brain inulin and sucrose space between the wild-type and Mdr1a/b(−/−) Bcrp1(−/−) mice. This suggested that the dramatic enhancement in the brain distribution of gefitinib is not due to a leakier BBB in these mice. These results show that brain distribution of gefitinib is restricted due to active efflux by P-gp and BCRP. This finding is of clinical significance for therapy in brain tumors such as glioma, where concurrent administration of a dual inhibitor such as elacridar can increase delivery and thus enhance efficacy of gefitinib. PMID:20421331

  19. Transcriptional Analysis of MexAB-OprM Efflux Pumps System of Pseudomonas aeruginosa and Its Role in Carbapenem Resistance in a Tertiary Referral Hospital in India.

    PubMed

    Choudhury, Debarati; Das Talukdar, Anupam; Dutta Choudhury, Manabendra; Maurya, Anand Prakash; Paul, Deepjyoti; Dhar Chanda, Debadatta; Chakravorty, Atanu; Bhattacharjee, Amitabha

    2015-01-01

    Carbapenem resistance presents severe threat to the treatment of multidrug resistant Pseudomonas aeruginosa infections. The study was undertaken to investigate the role of efflux pumps in conferring meropenem resistance and effect of single dose exposure of meropenem on transcription level of mexA gene in clinical isolates of P. aeruginosa from a tertiary referral hospital of India. Further, in this investigation an effort was made to assess whether different components of MexAB-OprM operon expresses in the same manner and the extent of contributions of those components in meropenem resistance in its natural host (P. aeruginosa) and in a heterologous host (E. coli). Out of 83 meropenem nonsusceptible isolates, 22 isolates were found to possess efflux pump activity phenotypically. Modified hodge test and multiplex PCR confirmed the absence of carbapenemase genes in those isolates. All of them were of multidrug resistant phenotype and were resistant to all the carbepenem drug tested. MexAB-OprM efflux pump was found to be overexpressed in all the study isolates. It could be observed that single dose exposure meropenem could give rise to trivial increase in transcription of mexA gene. Different constructs of MexAB-OprM (mexR-mexA-mexB-OprM; mexA-mexB-OprM; mexA-mexB) could be expressed in both its natural (P. aeruginosa PAO1) and heterologous host (E. coli JM107) but transcription level of mexA gene varied in both the hosts before and after single dose exposure of meropenem. Different components of the operon failed to enhance meropenem resistance in E. coli JM107 and P. aeruginosa PAO1. This study could prove that MexAB-OprM efflux pump can significantly contribute to meropenem resistance in hospital isolates of P. aeruginosa where an acquired resistant mechanism is absent. Thus, equal importance should be given for diagnosis of intrinsic resistance mechanism so as to minimize treatment failure. As meropenem could not enhance mexA transcriptions significantly, there

  20. Interplay between Three RND Efflux Pumps in Doxycycline-Selected Strains of Burkholderia thailandensis

    PubMed Central

    Biot, Fabrice Vincent; Lopez, Mélanie Monique; Poyot, Thomas; Neulat-Ripoll, Fabienne; Lignon, Sabrina; Caclard, Arnaud; Thibault, François Michel; Peinnequin, Andre; Pagès, Jean-Marie; Valade, Eric

    2013-01-01

    Background Efflux systems are involved in multidrug resistance in most Gram-negative non-fermentative bacteria. We have chosen Burkholderia thailandensis to dissect the development of multidrug resistance phenotypes under antibiotic pressure. Methodology/Principal Findings We used doxycycline selection to obtain several resistant B. thailandensis variants. The minimal inhibitory concentrations of a large panel of structurally unrelated antibiotics were determined ± the efflux pump inhibitor phenylalanine-arginine ß-naphthylamide (PAßN). Membrane proteins were identified by proteomic method and the expressions of major efflux pumps in the doxycycline selected variants were compared to those of the parental strains by a quantitative RT-PCR analysis. Doxycycline selected variants showed a multidrug resistance in two major levels corresponding to the overproduction of two efflux pumps depending on its concentration: AmrAB-OprA and BpeEF-OprC. The study of two mutants, each lacking one of these pumps, indicated that a third pump, BpeAB-OprB, could substitute for the defective pump. Surprisingly, we observed antagonistic effects between PAßN and aminoglycosides or some ß-lactams. PAßN induced the overexpression of AmrAB-OprA and BpeAB-OprB pump genes, generating this unexpected effect. Conclusions/Significance These results may account for the weak activity of PAßN in some Gram-negative species. We clearly demonstrated two antagonistic effects of this molecule on bacterial cells: the blocking of antibiotic efflux and an increase in efflux pump gene expression. Thus, doxycycline is a very efficient RND efflux pump inducer and PAßN may promote the production of some efflux pumps. These results should be taken into account when considering antibiotic treatments and in future studies on efflux pump inhibitors. PMID:24386333

  1. Binary and ternary combinations of anti-HIV protease inhibitors: effect on gene expression and functional activity of CYP3A4 and efflux transporters

    PubMed Central

    Kwatra, Deep; Vadlapudi, Aswani Dutt; Vadlapatla, Ramya Krishna; Khurana, Varun; Pal, Dhananjay; Mitra, Ashim K.

    2015-01-01

    Background The purpose of this study is to identify the effect of binary and ternary combinations of anti-HIV protease inhibitors (PIs) on the expression of metabolizing enzyme (CYP3A4) and efflux transporters [multidrug resistance-associated protein 2 (MRP2), P-glycoprotein (P-gp) and breast cancer resistant protein (BCRP)] in a model intestinal cell line (LS-180). Methods LS-180 cells were treated with various combinations of PIs (amprenavir, indinavir, saquinavir and lopinavir), and the mRNA expression levels of metabolizing enzyme and efflux transporters were measured using quantitative reverse transcription polymerase chain reaction. The alteration of gene expression was further correlated to the expression of nuclear hormone receptor PXR. Uptake of fluorescent and radioactive substrates was carried out to study the functional activity of these proteins. Cytotoxicity and adenosine triphosphate (ATP) assays were carried out to measure stress responses. Results Binary and ternary combinations of PIs appeared to modulate the expression of CYP3A4, MRP2, P-gp and BCRP in a considerable manner. Unlike the individual PIs, their binary combinations showed much greater induction of metabolizing enzyme and efflux proteins. However, such pronounced induction was not observed in the presence of ternary combinations. The observed trend of altered mRNA expression was found to correlate well with the change in expression levels of PXR. The gene expression was found to correlate with activity assays. Lack of cytotoxicity and ATP activity was observed in the treatment samples, suggesting that these alterations in expression levels were probably not stress responses. Conclusions In the present study, we demonstrated that combinations of drugs can have serious consequences toward the treatment of HIV infection by altering their bioavailability and disposition. PMID:24399676

  2. The TetR-Type MfsR Protein of the Integrative and Conjugative Element (ICE) ICEclc Controls both a Putative Efflux System and Initiation of ICE Transfer

    PubMed Central

    Pradervand, Nicolas; Delavat, François; Sulser, Sandra; Miyazaki, Ryo

    2014-01-01

    Integrative and conjugating elements (ICE) are self-transferable DNAs widely present in bacterial genomes, which often carry a variety of auxiliary genes of potential adaptive benefit. One of the model ICE is ICEclc, an element originally found in Pseudomonas knackmussii B13 and known for its propensity to provide its host with the capacity to metabolize chlorocatechols and 2-aminophenol. In this work, we studied the mechanism and target of regulation of MfsR, a TetR-type repressor previously found to exert global control on ICEclc horizontal transfer. By using a combination of ICEclc mutant and transcriptome analysis, gene reporter fusions, and DNA binding assays, we found that MfsR is a repressor of both its own expression and that of a gene cluster putatively coding for a major facilitator superfamily efflux system on ICEclc (named mfsABC). Phylogenetic analysis suggests that mfsR was originally located immediately adjacent to the efflux pump genes but became displaced from its original cis target DNA by a gene insertion. This resulted in divergence of the original bidirectional promoters into two separated individual regulatory units. Deletion of mfsABC did not result in a strong phenotype, and despite screening a large number of compounds and conditions, we were unable to define the precise current function or target of the putative efflux pump. Our data reconstruct how the separation of an ancestor mfsR-mfsABC system led to global control of ICEclc transfer by MfsR. PMID:25182498

  3. Overexpression of the Multidrug Efflux Operon acrEF by Insertional Activation with IS1 or IS10 Elements in Salmonella enterica Serovar Typhimurium DT204 acrB Mutants Selected with Fluoroquinolones

    PubMed Central

    Olliver, Anne; Vallé, Michel; Chaslus-Dancla, Elisabeth; Cloeckaert, Axel

    2005-01-01

    High-level fluoroquinolone (FQ) resistance in Salmonella enterica serovar Typhimurium phage type DT204 has been previously shown to be essentially due to both multiple target gene mutations and active efflux by the AcrAB-TolC efflux system. In this study we show that in intermediatly resistant acrB-inactivated serovar Typhimurium DT204 mutants, high-level resistance to FQs can be restored on in vitro selection with FQs. In each FQ- resistant mutant selected from serovar Typhimurium DT204 acrB mutant strains, an insertion sequence (IS1 or IS10) was found integrated upstream of the acrEF operon, coding for AcrEF, an efflux pump highly homologous to AcrAB. In one of the strains, transposition of IS1 caused partial deletion of acrS, the putative local repressor gene of the acrEF operon. Sequence analysis showed that both IS1 and IS10 elements contain putative promoter sequences that might alter the expression of adjacent acrEF genes. Indeed, reverse transcription-PCR experiments showed an 8- to 10-fold increase in expression of acrF in these insertional mutants, relative to their respective parental strain, which correlated well with the resistance levels observed to FQs and other unrelated drugs. It is noteworthy that AcrEF did not contribute to the intrinsic drug resistance of serovar Typhimurium, since acrF deletion in wild-type strains did not result in any increase in drug susceptibility. Moreover, deletion of acrS did not cause any acrF overexpression or any decrease in drug susceptibility, suggesting that acrEF overexpression is mediated solely by the IS1 and IS10 promoter sequences and not by inactivity of AcrS. Southern blot experiments showed that the number of chromosomal IS1 and IS10 elements in the serovar Typhimurium DT204 genome was about 5 and 15 respectively. None were detected in epidemic serovar Typhimurium DT104 strains or in the serovar Typhimurium reference strain LT2. Carrying IS1 and/or IS10 elements in their chromosome may thus be a selective

  4. Tunicamycin Depresses P-Glycoprotein Glycosylation Without an Effect on Its Membrane Localization and Drug Efflux Activity in L1210 Cells

    PubMed Central

    Šereš, Mário; Cholujová, Dana; Bubenčíkova, Tatiana; Breier, Albert; Sulová, Zdenka

    2011-01-01

    P-glycoprotein (P-gp), also known as ABCB1, is a member of the ABC transporter family of proteins. P-gp is an ATP-dependent drug efflux pump that is localized to the plasma membrane of mammalian cells and confers multidrug resistance in neoplastic cells. P-gp is a 140-kDa polypeptide that is glycosylated to a final molecular weight of 170 kDa. Our experimental model used two variants of L1210 cells in which overexpression of P-gp was achieved: either by adaptation of parental cells (S) to vincristine (R) or by transfection with the human gene encoding P-gp (T). R and T cells were found to differ from S cells in transglycosylation reactions in our recent studies. The effects of tunicamycin on glycosylation, drug efflux activity and cellular localization of P-gp in R and T cells were examined in the present study. Treatment with tunicamycin caused less concentration-dependent cellular damage to R and T cells compared with S cells. Tunicamycin inhibited P-gp N-glycosylation in both of the P-gp-positive cells. However, tunicamycin treatment did not alter either the P-gp cellular localization to the plasma membrane or the P-gp transport activity. The present paper brings evidence that independently on the mode of P-gp expression (selection with drugs or transfection with a gene encoding P-gp) in L1210 cells, tunicamycin induces inhibition of N-glycosylation of this protein, without altering its function as plasma membrane drug efflux pump. PMID:22174631

  5. Importance of influx and efflux systems and xenobiotic metabolizing enzymes in intratumoral disposition of anticancer agents.

    PubMed

    Rochat, B

    2009-08-01

    In this review, intratumoral drug disposition will be integrated into the wide range of resistance mechanisms to anticancer agents with particular emphasis on targeted protein kinase inhibitors. Six rules will be established: 1. There is a high variability of extracellular/intracellular drug level ratios; 2. There are three main systems involved in intratumoral drug disposition that are composed of SLC, ABC and XME enzymes; 3. There is a synergistic interplay between these three systems; 4. In cancer subclones, there is a strong genomic instability that leads to a highly variable expression of SLC, ABC or XME enzymes; 5. Tumor-expressed metabolizing enzymes play a role in tumor-specific ADME and cell survival and 6. These three systems are involved in the appearance of resistance (transient event) or in the resistance itself. In addition, this article will investigate whether the overexpression of some ABC and XME systems in cancer cells is just a random consequence of DNA/chromosomal instability, hypo- or hypermethylation and microRNA deregulation, or a more organized modification induced by transposable elements. Experiments will also have to establish if these tumor-expressed enzymes participate in cell metabolism or in tumor-specific ADME or if they are only markers of clonal evolution and genomic deregulation. Eventually, the review will underline that the fate of anticancer agents in cancer cells should be more thoroughly investigated from drug discovery to clinical studies. Indeed, inhibition of tumor expressed metabolizing enzymes could strongly increase drug disposition, specifically in the target cells resulting in more efficient therapies.

  6. Peptide mediators of cholesterol efflux

    DOEpatents

    Bielicki, John K.; Johansson, Jan

    2013-04-09

    The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABAC1 that parallels that of full-length apolipoproteins. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

  7. Molecular Components of Nitrate and Nitrite Efflux in Yeast

    PubMed Central

    Cabrera, Elisa; González-Montelongo, Rafaela; Giraldez, Teresa; de la Rosa, Diego Alvarez

    2014-01-01

    Some eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeast Hansenula polymorpha was used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking either SSU2 or NAR1 along with the nitrate reductase gene YNR1 showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-known Saccharomyces cerevisiae sulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation. PMID:24363367

  8. Spatial and temporal effects of drought on soil CO2 efflux in a cacao agroforestry system in Sulawesi, Indonesia

    NASA Astrophysics Data System (ADS)

    van Straaten, O.; Veldkamp, E.; Köhler, M.; Anas, I.

    2010-04-01

    Climate change induced droughts pose a serious threat to ecosystems across the tropics and sub-tropics, particularly to those areas not adapted to natural dry periods. In order to study the vulnerability of cacao (Theobroma cacao) - Gliricidia sepium agroforestry plantations to droughts a large scale throughfall displacement roof was built in Central Sulawesi, Indonesia. In this 19-month experiment, we compared soil surface CO2 efflux (soil respiration) from three roof plots with three adjacent control plots. Soil respiration rates peaked at intermediate soil moisture conditions and decreased under increasingly dry conditions (drought induced), or increasingly wet conditions (as evidenced in control plots). The roof plots exhibited a slight decrease in soil respiration compared to the control plots (average 13% decrease). The strength of the drought effect was spatially variable - while some measurement chamber sites reacted strongly (responsive) to the decrease in soil water content (up to R2=0.70) (n=11), others did not react at all (non-responsive) (n=7). A significant correlation was measured between responsive soil respiration chamber sites and sap flux density ratios of cacao (R=0.61) and Gliricidia (R=0.65). Leaf litter CO2 respiration decreased as conditions became drier. The litter layer contributed approximately 3-4% of the total CO2 efflux during dry periods and up to 40% during wet periods. Within days of roof opening soil CO2 efflux rose to control plot levels. Thereafter, CO2 efflux remained comparable between roof and control plots. The cumulative effect on soil CO2 emissions over the duration of the experiment was not significantly different: the control plots respired 11.1±0.5 Mg C ha-1 yr-1, while roof plots respired 10.5±0.5 Mg C ha-1 yr-1. The relatively mild decrease measured in soil CO2 efflux indicates that this agroforestry ecosystem is capable of mitigating droughts with only minor stress symptoms.

  9. Streptococcus pneumoniae and Streptococcus pyogenes resistant to macrolides but sensitive to clindamycin: a common resistance pattern mediated by an efflux system.

    PubMed Central

    Sutcliffe, J; Tait-Kamradt, A; Wondrack, L

    1996-01-01

    Macrolide-resistant Streptococcus pyogenes isolates from Finland, Australia, and the United Kingdom and, more recently, Streptococcus pneumoniae and S. pyogenes strains from the United States were shown to have an unusual resistance pattern to macrolides, lincosamides, and streptogramin B antibiotics. This pattern, referred to as M resistance, consists of susceptibility to clindamycin and streptogramin B antibiotics but resistance to 14- and 15-membered macrolides. An evaluation of the macrolide-lincosamide-streptogramin B resistance phenotypes among our streptococcal strains collected from 1993 to 1995 suggested that this unusual resistance pattern is not rare. Eighty-five percent (n = 66) of the S. pneumoniae and 75% (n = 28) of the S. pyogenes strains in our collection had an M phenotype. The mechanism of M resistance was not mediated by target modification, as isolated ribosomes from a pneumococcal strain bearing the M phenotype were fully sensitive to erythromycin. Further, the presence of an erm methylase was excluded with primers specific for an erm consensus sequence. However, results of studies that determined the uptake and incorporation of radiolabeled erythromycin into cells were consistent with the presence of a macrolide efflux determinant. The putative efflux determinant in streptococci seems to be distinct from the multicomponent macrolide efflux system in coagulase-negative staphylococci. The recognition of the prevalence of the M phenotype in streptococci has implications for sensitivity testing and may have an impact on the choice of antibiotic therapy in clinical practice. PMID:8843287

  10. First evidence for the presence of efflux pump in the earthworm Eisenia andrei.

    PubMed

    Hackenberger, Branimir K; Velki, Mirna; Stepić, Sandra; Hackenberger, Davorka K

    2012-01-01

    Efflux pumps are transport proteins involved in the extrusion of toxic substrates from cells to the external environment. Activities of efflux pumps have been found in many organisms, however such activity has not been evidenced in earthworms. Adult Eisenia andrei earthworms were exposed to efflux modulators - verapamil (a known inhibitor of efflux pump protein) and dexamethasone (a known inducer of efflux activity) - and the amount of absorbed fluorescent dye rhodamine B was measured. The results showed that verapamil inhibited efflux activity and decreased removal of rhodamine B, whereas dexamethasone induced efflux activity and increased removal of rhodamine B. This is the first evidence of the presence of efflux pump in earthworm Eisenia andrei. Since earthworms are often used as test organisms due to their sensitive reactions towards environmental influences, the discovery of efflux pump activity can contribute to the better understanding of toxicity of certain pollutants.

  11. Microdamage induced calcium efflux from bone matrix activates intracellular calcium signaling in osteoblasts via L-type and T-type voltage-gated calcium channels.

    PubMed

    Jung, Hyungjin; Best, Makenzie; Akkus, Ozan

    2015-07-01

    Mechanisms by which bone microdamage triggers repair response are not completely understood. It has been shown that calcium efflux ([Ca(2+)]E) occurs from regions of bone undergoing microdamage. Such efflux has also been shown to trigger intracellular calcium signaling ([Ca(2+)]I) in MC3T3-E1 cells local to damaged regions. Voltage-gated calcium channels (VGCCs) are implicated in the entry of [Ca(2+)]E to the cytoplasm. We investigated the involvement of VGCC in the extracellular calcium induced intracellular calcium response (ECIICR). MC3T3-E1 cells were subjected to one dimensional calcium efflux from their basal aspect which results in an increase in [Ca(2+)]I. This increase was concomitant with membrane depolarization and it was significantly reduced in the presence of Bepridil, a non-selective VGCC inhibitor. To identify specific type(s) of VGCC in ECIICR, the cells were treated with selective inhibitors for different types of VGCC. Significant changes in the peak intensity and the number of [Ca(2+)]I oscillations were observed when L-type and T-type specific VGCC inhibitors (Verapamil and NNC55-0396, respectively) were used. So as to confirm the involvement of L- and T-type VGCC in the context of microdamage, cells were seeded on devitalized notched bone specimen, which were loaded to induce microdamage in the presence and absence of Verapamil and NNC55-0396. The results showed significant decrease in [Ca(2+)]I activity of cells in the microdamaged regions of bone when L- and T-type blockers were applied. This study demonstrated that extracellular calcium increase in association with damage depolarizes the cell membrane and the calcium ions enter the cell cytoplasm by L- and T-type VGCCs.

  12. Identification of a multidrug efflux pump in Mycobacterium smegmatis.

    PubMed

    Bansal, Ankita; Mallik, Dhriti; Kar, Debasish; Ghosh, Anindya S

    2016-07-01

    Cell wall impermeability and active efflux of drugs are among the primary reasons for drug resistance in mycobacteria. Efflux pumps are tripartite membrane localized transport proteins that expel drug molecules outside the cells. Several of such efflux pumps are annotated in mycobacteria, but few have been characterized, like MSMEG_2991, a putative efflux pump permease of Mycobacterium smegmatis To substantiate this, we overexpressed MSMEG_2991 protein in Escherichia coli 2443. Expression of MSMEG_2991 elevated the resistance towards structurally unrelated groups of antibiotics. An active antibiotic efflux pump nature of MSMEG_2991 was revealed by assessing the acquisition of ciprofloxacin in the absence and presence of the efflux pump inhibitor, carbonyl cyanide m-chlorophenyl hydrazone, indicating the involvement of proton-motive force (pmf) during the efflux activity. MSMEG_2991 expression elevated biofilm formation in E. coli by 4-fold, keeping parity to some of the earlier reported efflux pumps. In silico analysis suggested the presence of 12 transmembrane helices in MSMEG_2991 resembling EmrD efflux pump of E. coli Based on in vivo and in silico analyses, MSMEG_2991 may be designated as a pmf-mediated multidrug efflux pump protein that expels diverse groups of antibiotics and might as well be involved in the biofilm enhancement.

  13. A new automatic chamber system for measuring soil surface CO2 and its isotopic (13CO2) effluxes in New Jersey Pinelands

    NASA Astrophysics Data System (ADS)

    Xu, M.

    2006-05-01

    A new automatic chamber system was developed to measure soil surface CO2 efflux with 16 pairs of chambers at hourly interval in an oak forest in New Jersey Pinelands. The trench method was applied to separate root respiration from the total soil respiration. I found that soil temperature and moisture alternatively control root and soil heterotrophic respiration in different seasons based on the data collected in about 3 years. The isotopic flux of soil 13CO2 was also measured at hourly interval using the chamber system with a new laser-based stable carbon isotope analyzer.

  14. ArsP: a methylarsenite efflux permease

    PubMed Central

    Chen, Jian; Madegowda, Mahendra; Bhattacharjee, Hiranmoy; Rosen, Barry P.

    2015-01-01

    Trivalent organoarsenic compounds are far more toxic than either pentavalent organoarsenicals or inorganic arsenite. Many microbes methylate inorganic arsenite (As(III)) to more toxic and carcinogenic methylarsenite (MAs(III)). Additionally, monosodium methylarsenate (MSMA or MAs(V)) has been used widely as an herbicide and is reduced by microbial communities to MAs(III). Roxarsone (3-nitro-4-hydroxybenzenearsonic acid) is a pentavalent aromatic arsenical that is used as antimicrobial growth promoter for poultry and swine, and its active form is the trivalent species Rox(III). A bacterial permease, ArsP, from Campylobacter jejuni, was recently shown to confer resistance to roxarsone. In this study C. jejuni arsP was expressed in Escherichia coli and shown to confer resistance to MAs(III) and Rox(III) but not to inorganic As(III) or pentavalent organoarsenicals. Cells of E. coli expressing arsP did not accumulate trivalent organoarsenicals. Everted membrane vesicles from those cells accumulated MAs(III)>Rox(III) with energy supplied by NADH oxidation, reflecting efflux from cells. The vesicles did not transport As(III), MAs(V) or pentavalent roxarsone. Mutation or modification of the two conserved cysteine residues resulted in loss of transport activity, suggesting that they play a role in ArsP function. Thus ArsP is the first identified efflux system specific for trivalent organoarsenicals. PMID:26234817

  15. A Simple Method for Assessment of MDR Bacteria for Over-Expressed Efflux Pumps

    PubMed Central

    Martins, Marta; McCusker, Matthew P; Viveiros, Miguel; Couto, Isabel; Fanning, Séamus; Pagès, Jean-Marie; Amaral, Leonard

    2013-01-01

    It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer cell membrane (in the case of Gram-negative bacteria). In addition, adaptation to the environment, such as quorum sensing and biofilm formation can also contribute to bacterial persistence. Due to the rapid emergence and spread of bacterial isolates showing resistance to several classes of antibiotics, methods that can rapidly and efficiently identify isolates whose resistance is due to active efflux have been developed. However, there is still a need for faster and more accurate methodologies. Conventional methods that evaluate bacterial efflux pump activity in liquid systems are available. However, these methods usually use common efflux pump substrates, such as ethidium bromide or radioactive antibiotics and therefore, require specialized instrumentation, which is not available in all laboratories. In this review, we will report the results obtained with the Ethidium Bromide-agar Cartwheel method. This is an easy, instrument-free, agar based method that has been modified to afford the simultaneous evaluation of as many as twelve bacterial strains. Due to its simplicity it can be applied to large collections of bacteria to rapidly screen for multi-drug resistant isolates that show an over-expression of their efflux systems. The principle of the method is simple and relies on the ability of the bacteria to expel a fluorescent molecule that is substrate for most efflux pumps, ethidium bromide. In this approach, the higher the concentration of ethidium bromide required to

  16. A dynamic soil chamber system coupled with a tunable diode laser for online measurements of delta13C, delta18O, and efflux rate of soil-respired CO2.

    PubMed

    Powers, Heath H; Hunt, John E; Hanson, David T; McDowell, Nate G

    2010-02-01

    High frequency observations of the stable isotopic composition of CO(2) effluxes from soil have been sparse due in part to measurement challenges. We have developed an open-system method that utilizes a flow-through chamber coupled to a tunable diode laser (TDL) to quantify the rate of soil CO(2) efflux and its delta(13)C and delta(18)O values (delta(13)C(R) and delta(18)O(R), respectively). We tested the method first in the laboratory using an artificial soil test column and then in a semi-arid woodland. We found that the CO(2) efflux rates of 1.2 to 7.3 micromol m(-2) s(-1) measured by the chamber-TDL system were similar to measurements made using the chamber and an infrared gas analyzer (IRGA) (R(2) = 0.99) and compared well with efflux rates generated from the soil test column (R(2) = 0.94). Measured delta(13)C and delta(18)O values of CO(2) efflux using the chamber-TDL system at 2 min intervals were not significantly different from source air values across all efflux rates after accounting for diffusive enrichment. Field measurements during drought demonstrated a strong dependency of CO(2) efflux and isotopic composition on soil water content. Addition of water to the soil beneath the chamber resulted in average changes of +6.9 micromol m(-2) s(-1), -5.0 per thousand, and -55.0 per thousand for soil CO(2) efflux, delta(13)C(R) and delta(18)O(R), respectively. All three variables initiated responses within 2 min of water addition, with peak responses observed within 10 min for isotopes and 20 min for efflux. The observed delta(18)O(R) was more enriched than predicted from temperature-dependent H(2)O-CO(2) equilibration theory, similar to other recent observations of delta(18)O(R) from dry soils (Wingate L, Seibt U, Maseyk K, Ogee J, Almeida P, Yakir D, Pereira JS, Mencuccini M. Global Change Biol. 2008; 14: 2178). The soil chamber coupled with the TDL was found to be an effective method for capturing soil CO(2) efflux and its stable isotope composition at high

  17. A dynamic soil chamber system coupled with a tunable diode laser for online measurements of delta-13C, delta-18O, and efflux rate of soil respired CO2

    SciTech Connect

    Powers, Heath H; Mcdowell, Nate; Hanson, David; Hunt, John

    2009-01-01

    High frequency observations of the stable isotopic composition of CO(2) effluxes from soil have been sparse due in part to measurement challenges. We have developed an open-system method that utilizes a flow-through chamber coupled to a tunable diode laser (TDL) to quantify the rate of soil CO(2) efflux and its delta(13)C and delta(18)O values (delta(13)C(R) and delta(18)O(R), respectively). We tested the method first in the laboratory using an artificial soil test column and then in a semi-arid woodland. We found that the CO(2) efflux rates of 1.2 to 7.3 micromol m(-2) s(-1) measured by the chamber-TDL system were similar to measurements made using the chamber and an infrared gas analyzer (IRGA) (R(2) = 0.99) and compared well with efflux rates generated from the soil test column (R(2) = 0.94). Measured delta(13)C and delta(18)O values of CO(2) efflux using the chamber-TDL system at 2 min intervals were not significantly different from source air values across all efflux rates after accounting for diffusive enrichment. Field measurements during drought demonstrated a strong dependency of CO(2) efflux and isotopic composition on soil water content. Addition of water to the soil beneath the chamber resulted in average changes of +6.9 micromol m(-2) s(-1), -5.0 per thousand, and -55.0 per thousand for soil CO(2) efflux, delta(13)C(R) and delta(18)O(R), respectively. All three variables initiated responses within 2 min of water addition, with peak responses observed within 10 min for isotopes and 20 min for efflux. The observed delta(18)O(R) was more enriched than predicted from temperature-dependent H(2)O-CO(2) equilibration theory, similar to other recent observations of delta(18)O(R) from dry soils (Wingate L, Seibt U, Maseyk K, Ogee J, Almeida P, Yakir D, Pereira JS, Mencuccini M. Global Change Biol. 2008; 14: 2178). The soil chamber coupled with the TDL was found to be an effective method for capturing soil CO(2) efflux and its stable isotope composition at high

  18. Alternative to antibiotics against Pseudomonas aeruginosa: Effects of Glycyrrhiza glabra on membrane permeability and inhibition of efflux activity and biofilm formation in Pseudomonas aeruginosa and its in vitro time-kill activity.

    PubMed

    Chakotiya, Ankita Singh; Tanwar, Ankit; Narula, Alka; Sharma, Rakesh Kumar

    2016-09-01

    The multi-drug resistance offered by Pseudomonas aeruginosa to antibiotics can be attributed towards its propensity to develop biofilm, modification in cell membrane and to efflux antibacterial drugs. The present study explored the activity of Glycyrrhiza glabra and one of its pure compounds, glycyrrhizic acid against P. aeruginosa and their mechanism of action in terms of the effect on membrane permeability, efflux activity, and biofilm formation were determined. Minimum inhibitory concentrations were determined by using broth dilution technique. The minimum bactericidal concentrations were assessed on agar plate. The MIC of the extract and glycyrrhizic acid was found to be 200 and 100 μg ml(-1), respectively. The MBC was found to be 800 and 400 μg ml(-1) in the case of extract and glycyrrhizic acid, respectively. Time -dependent killing efficacy was also estimated. Flowcytometric analysis with staining methods was used to determine the effect of extract and glycyrrhizic acid at 2 × MIC on different physiological parameters and compared it with the standard (antibiotic). The growth of P. aeruginosa was significantly inhibited by extract and the pure compound. The herbal extract and the glycyrrhic acid were also found to effective in targeting the physiological parameters of the bacteria that involve cell membrane permeabilization, efflux activity, and biofilm formation. This study reports the antipseudomonal action of Glycyrrhiza glabra and one of its compound and provides insight into their mode of action.

  19. Differential requirement of the transcription factor Mcm1 for activation of the Candida albicans multidrug efflux pump MDR1 by its regulators Mrr1 and Cap1.

    PubMed

    Mogavero, Selene; Tavanti, Arianna; Senesi, Sonia; Rogers, P David; Morschhäuser, Joachim

    2011-05-01

    Overexpression of the multidrug efflux pump Mdr1 causes increased fluconazole resistance in the pathogenic yeast Candida albicans. The transcription factors Mrr1 and Cap1 mediate MDR1 upregulation in response to inducing stimuli, and gain-of-function mutations in Mrr1 or Cap1, which render the transcription factors hyperactive, result in constitutive MDR1 overexpression. The essential MADS box transcription factor Mcm1 also binds to the MDR1 promoter, but its role in inducible or constitutive MDR1 upregulation is unknown. Using a conditional mutant in which Mcm1 can be depleted from the cells, we investigated the importance of Mcm1 for MDR1 expression. We found that Mcm1 was dispensable for MDR1 upregulation by H2O2 but was required for full MDR1 induction by benomyl. A C-terminally truncated, hyperactive Cap1 could upregulate MDR1 expression both in the presence and in the absence of Mcm1. In contrast, a hyperactive Mrr1 containing a gain-of-function mutation depended on Mcm1 to cause MDR1 overexpression. These results demonstrate a differential requirement for the coregulator Mcm1 for Cap1- and Mrr1-mediated MDR1 upregulation. When activated by oxidative stress or a gain-of-function mutation, Cap1 can induce MDR1 expression independently of Mcm1, whereas Mrr1 requires either Mcm1 or an active Cap1 to cause overexpression of the MDR1 efflux pump. Our findings provide more detailed insight into the molecular mechanisms of drug resistance in this important human fungal pathogen.

  20. Apolipoprotein A-I configuration and cell cholesterol efflux activity of discoidal lipoproteins depend on the reconstitution process.

    PubMed

    Cuellar, Luz Ángela; Prieto, Eduardo Daniel; Cabaleiro, Laura Virginia; Garda, Horacio Alberto

    2014-01-01

    Discoidal high-density lipoproteins (D-HDL) are critical intermediates in reverse cholesterol transport. Most of the present knowledge of D-HDL is based on studies with reconstituted lipoprotein complexes of apolipoprotein A-I (apoA-I) obtained by cholate dialysis (CD). D-HDL can also be generated by the direct microsolubilization (DM) of phospholipid vesicles at the gel/fluid phase transition temperature, a process mechanistically similar to the "in vivo" apoAI lipidation via ABCA1. We compared the apoA-I configuration in D-HDL reconstituted with dimyristoylphosphatidylcholine by both procedures using fluorescence resonance energy transfer measurements with apoA-I tryptophan mutants and fluorescently labeled cysteine mutants. Results indicate that apoA-I configuration in D-HDL depends on the reconstitution process and are consistent with a "double belt" molecular arrangement with different helix registry. As reported by others, a configuration with juxtaposition of helices 5 of each apoAI monomer (5/5 registry) predominates in D-HDL obtained by CD. However, a configuration with helix 5 of one monomer juxtaposed with helix 2 of the other (5/2 registry) would predominate in D-HDL generated by DM. Moreover, we also show that the kinetics of cholesterol efflux from macrophage cultures depends on the reconstitution process, suggesting that apoAI configuration is important for this HDL function.

  1. Natural and Synthetic Polymers as Inhibitors of Drug Efflux Pumps

    PubMed Central

    2007-01-01

    Inhibition of efflux pumps is an emerging approach in cancer therapy and drug delivery. Since it has been discovered that polymeric pharmaceutical excipients such as Tweens® or Pluronics® can inhibit efflux pumps, various other polymers have been investigated regarding their potential efflux pump inhibitory activity. Among them are polysaccharides, polyethylene glycols and derivatives, amphiphilic block copolymers, dendrimers and thiolated polymers. In the current review article, natural and synthetic polymers that are capable of inhibiting efflux pumps as well as their application in cancer therapy and drug delivery are discussed. PMID:17896100

  2. The (95)(Δ)G mutation in the 5'untranslated region of the norA gene increases efflux activity in Staphylococcus epidermidis isolates.

    PubMed

    García-Gómez, Elizabeth; Jaso-Vera, Marcos E; Juárez-Verdayes, Marco A; Alcántar-Curiel, María D; Zenteno, Juan C; Betanzos-Cabrera, Gabriel; Peralta, Humberto; Rodríguez-Martínez, Sandra; Cancino-Díaz, Mario E; Jan-Roblero, Janet; Cancino-Diaz, Juan C

    2017-02-01

    In the Staphylococcus aureus ATCC25923 strain, the flqB mutation in the 5'untranslated region (5'UTR) of the norA gene causes increased norA mRNA expression and high efflux activity (HEA). The involvement of the norA gene 5'UTR in HEA has not been explored in S. epidermidis; therefore, we examined the function of this region in S. epidermidis clinical isolates. The selection of isolates with HEA was performed based on ethidium bromide (EtBr) MIC values and efflux efficiency (EF) using the semi-automated fluorometric method. The function of the 5'UTR was studied by quantifying the levels of norA expression (RT-qPCR) and by identifying 5'UTR mutations by sequence analysis. Only 10 isolates from a total of 165 (6.1%) had HEA (EtBr MIC = 300 μg/ml and EF ranged from 48.4 to 97.2%). Eight of 10 isolates with HEA had the 5'UTR (95)(Δ)G mutation. Isolates carrying the (95)(Δ)G mutation had higher levels of norA expression compared with those that did not. To corroborate that the (95)(Δ)G mutation is involved in HEA, a strain adapted to EtBr was obtained in vitro. This strain also presented the (95)(Δ)G mutation and had a high level of norA expression and EF, indicating that the (95)(Δ)G mutation is important for the HEA phenotype. The (95)(Δ)G mutation produces a different structure in the Shine-Dalgarno region, which may promote better translation of norA mRNA. To our knowledge, this is the first report to demonstrate the participation of the 5'UTR (95)(Δ)G mutation of the norA gene in the HEA phenotype of S. epidermidis isolates. Here, we propose that the efflux of EtBr is caused by an increment in the transcription and/or translation of the norA gene.

  3. Targeting efflux pumps to overcome antifungal drug resistance.

    PubMed

    Holmes, Ann R; Cardno, Tony S; Strouse, J Jacob; Ivnitski-Steele, Irena; Keniya, Mikhail V; Lackovic, Kurt; Monk, Brian C; Sklar, Larry A; Cannon, Richard D

    2016-08-01

    Resistance to antifungal drugs is an increasingly significant clinical problem. The most common antifungal resistance encountered is efflux pump-mediated resistance of Candida species to azole drugs. One approach to overcome this resistance is to inhibit the pumps and chemosensitize resistant strains to azole drugs. Drug discovery targeting fungal efflux pumps could thus result in the development of azole-enhancing combination therapy. Heterologous expression of fungal efflux pumps in Saccharomyces cerevisiae provides a versatile system for screening for pump inhibitors. Fungal efflux pumps transport a range of xenobiotics including fluorescent compounds. This enables the use of fluorescence-based detection, as well as growth inhibition assays, in screens to discover compounds targeting efflux-mediated antifungal drug resistance. A variety of medium- and high-throughput screens have been used to identify a number of chemical entities that inhibit fungal efflux pumps.

  4. Effect of Transcriptional Activators SoxS, RobA, and RamA on Expression of Multidrug Efflux Pump AcrAB-TolC in Enterobacter cloacae

    PubMed Central

    Pérez, Astrid; Poza, Margarita; Aranda, Jesús; Latasa, Cristina; Medrano, Francisco Javier; Tomás, María; Romero, Antonio; Lasa, Iñigo

    2012-01-01

    Control of membrane permeability is a key step in regulating the intracellular concentration of antibiotics. Efflux pumps confer innate resistance to a wide range of toxic compounds such as antibiotics, dyes, detergents, and disinfectants in members of the Enterobacteriaceae. The AcrAB-TolC efflux pump is involved in multidrug resistance in Enterobacter cloacae. However, the underlying mechanism that regulates the system in this microorganism remains unknown. In Escherichia coli, the transcription of acrAB is upregulated under global stress conditions by proteins such as MarA, SoxS, and Rob. In the present study, two clinical isolates of E. cloacae, EcDC64 (a multidrug-resistant strain overexpressing the AcrAB-TolC efflux pump) and Jc194 (a strain with a basal AcrAB-TolC expression level), were used to determine whether similar global stress responses operate in E. cloacae and also to establish the molecular mechanisms underlying this response. A decrease in susceptibility to erythromycin, tetracycline, telithromycin, ciprofloxacin, and chloramphenicol was observed in clinical isolate Jc194 and, to a lesser extent in EcDC64, in the presence of salicylate, decanoate, tetracycline, and paraquat. Increased expression of the acrAB promoter in the presence of the above-described conditions was observed by flow cytometry and reverse transcription-PCR, by using a reporter fusion protein (green fluorescent protein). The expression level of the AcrAB promoter decreased in E. cloacae EcDC64 derivates deficient in SoxS, RobA, and RamA. Accordingly, the expression level of the AcrAB promoter was higher in E. cloacae Jc194 strains overproducing SoxS, RobA, and RamA. Overall, the data showed that SoxS, RobA, and RamA regulators were associated with the upregulation of acrAB, thus conferring antimicrobial resistance as well as a stress response in E. cloacae. In summary, the regulatory proteins SoxS, RobA, and RamA were cloned and sequenced for the first time in this species. The

  5. Pathways of Arsenic Uptake and Efflux

    PubMed Central

    Yang, Hung-Chi; Fu, Hsueh-Liang; Lin, Yung-Feng; Rosen, Barry P.

    2015-01-01

    Arsenic is the most prevalent environmental toxic substance and ranks first on the U.S. Environmental Protection Agency’s Superfund List. Arsenic is a carcinogen and a causative agent of numerous human diseases. Paradoxically arsenic is used as a chemotherapeutic agent for treatment of acute promyelocytic leukemia. Inorganic arsenic has two biological important oxidation states: As(V) (arsenate) and As(III) (arsenite). Arsenic uptake is adventitious because the arsenate and arsenite are chemically similar to required nutrients. Arsenate resembles phosphate and is a competitive inhibitor of many phosphate-utilizing enzymes. Arsenate is taken up by phosphate transport systems. In contrast, at physiological pH, the form of arsenite is As(OH)3, which resembles organic molecules such as glycerol. Consequently, arsenite is taken into cells by aquaglyceroporin channels. Arsenic efflux systems are found in nearly every organism and evolved to rid cells of this toxic metalloid. These efflux systems include members of the multidrug resistance protein family and the bacterial exchangers Acr3 and ArsB. ArsB can also be a subunit of the ArsAB As(III)-translocating ATPase, an ATP-driven efflux pump. The ArsD metallochaperone binds cytosolic As(III) and transfers it to the ArsA subunit of the efflux pump. Knowledge of the pathways and transporters for arsenic uptake and efflux is essential for understanding its toxicity and carcinogenicity and for rational design of cancer chemotherapeutic drugs. PMID:23046656

  6. Inactivation of SmeSyRy Two-Component Regulatory System Inversely Regulates the Expression of SmeYZ and SmeDEF Efflux Pumps in Stenotrophomonas maltophilia

    PubMed Central

    Lin, Yi-Tsung; Ning, Hsiao-Chen; Yang, Tsuey-Ching

    2016-01-01

    SmeYZ efflux pump is a critical pump responsible for aminoglycosides resistance, virulence-related characteristics (oxidative stress susceptibility, motility, and secreted protease activity), and virulence in Stenotrophomonas maltophilia. However, the regulatory circuit involved in SmeYZ expression is little known. A two-component regulatory system (TCS), smeRySy, transcribed divergently from the smeYZ operon is the first candidate to be considered. To assess the role of SmeRySy in smeYZ expression, the smeRySy isogenic deletion mutant, KJΔRSy, was constructed by gene replacement strategy. Inactivation of smeSyRy correlated with a higher susceptibility to aminoglycosides concomitant with an increased resistance to chloramphenicol, ciprofloxacin, tetracycline, and macrolides. To elucidate the underlying mechanism responsible for the antimicrobials susceptibility profiles, the SmeRySy regulon was firstly revealed by transcriptome analysis and further confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and promoter transcription fusion constructs assay. The results demonstrate that inactivation of smeRySy decreased the expression of SmeYZ pump and increased the expression of SmeDEF pump, which underlies the ΔsmeSyRy-mediated antimicrobials susceptibility profile. To elucidate the cognate relationship between SmeSy and SmeRy, a single mutant, KJΔRy, was constructed and the complementation assay of KJΔRSy with smeRy were performed. The results support that SmeSy-SmeRy TCS is responsible for the regulation of smeYZ operon; whereas SmeSy may be cognate with another unidentified response regulator for the regulation of smeDEF operon. The impact of inverse expression of SmeYZ and SmeDEF pumps on physiological functions was evaluated by mutants construction, H2O2 susceptibility test, swimming, and secreted protease activity assay. The increased expression of SmeDEF pump in KJΔRSy may compensate, to some extents, the SmeYZ downexpression

  7. Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification

    PubMed Central

    Keating, David H.; Zhang, Yaoping; Ong, Irene M.; McIlwain, Sean; Morales, Eduardo H.; Grass, Jeffrey A.; Tremaine, Mary; Bothfeld, William; Higbee, Alan; Ulbrich, Arne; Balloon, Allison J.; Westphall, Michael S.; Aldrich, Josh; Lipton, Mary S.; Kim, Joonhoon; Moskvin, Oleg V.; Bukhman, Yury V.; Coon, Joshua J.; Kiley, Patricia J.; Bates, Donna M.; Landick, Robert

    2014-01-01

    Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5-hydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. PMID:25177315

  8. Mycorrhiza-induced lower oxidative burst is related with higher antioxidant enzyme activities, net H2O2 effluxes, and Ca2+ influxes in trifoliate orange roots under drought stress.

    PubMed

    Zou, Ying-Ning; Huang, Yong-Ming; Wu, Qiang-Sheng; He, Xin-Hua

    2015-02-01

    Mechanisms of arbuscular mycorrhiza (AM)-induced lower oxidative burst of host plants under drought stress (DS) are not elucidated. A noninvasive microtest technology (NMT) was used to investigate the effects of Funneliformis mosseae on net fluxes of root hydrogen peroxide (H2O2) and calcium ions (Ca2+) in 5-month-old Poncirus trifoliata, in combination with catalase (CAT) and superoxide dismutase (SOD) activities as well as tissue superoxide radical (O2•-) and H2O2 concentrations under DS and well-watered (WW) conditions. A 2-month DS (55% maximum water holding capacity of growth substrates) significantly inhibited AM fungal root colonization, while AM symbiosis significantly increased plant biomass production, irrespective of water status. F. mosseae inoculation generally increased SOD and CAT activity but decreased O2•- and H2O2 concentrations in leaves and roots under WW and DS. Compared with non-AM seedlings, roots of AM seedlings had significantly higher net H2O2 effluxes and net Ca2+ influxes, especially in the meristem zone, but lower net H2O2 efflux in the elongation zone. Net Ca2+ influxes into roots were significantly positively correlated with root net H2O2 effluxes but negatively with root H2O2 concentrations. Results from this study suggest that AM-induced lower oxidative burst is related with higher antioxidant enzyme activities, root net H2O2 effluxes, and Ca2+ influxes under WW and DS.

  9. Snow-melting season CO2 efflux along the trans-Alaska pipeline

    NASA Astrophysics Data System (ADS)

    Kim, Y.; Nakai, T.

    2011-12-01

    This research was conducted to estimate CO2 effluxes in exposed and snow-covered soils along the trans-Alaska pipeline (ca. 660 km) during snow-melting seasons of April 2010 and April-May 2011. In-situ CO2 efflux was measured with a dynamic chamber system that consisted of a chamber (22 cm in diameter and 6 cm high), pump, NDIR (CO2 analyzer), and a laptop computer. Soil temperature and snow depth were measured with a portable thermocouple and from snow pit-wall. The difference in snow-melting season CO2 efflux was remarkably showed in exposed and snow-covered soils of boreal forest and tundra, suggesting the distinctly latitudinal CO2 efflux gradient. Mean CO2 efflux was 0.88±0.51 and 2.4±3.4 gCO2-C/m2/day in soil temperature of -1.8±4.0 and -1.1±3.4 °C during the snow-melting period of 2010 and 2011, respectively. When the snow was disappeared, mean CO2 efflux was 1.3±0.3 and 5.4±3.7 gCO2-C/m2/day for 2010 and 2011; on the other hand, when the seasonal covered snow was melting, mean CO2 efflux was 0.2±0.2 and 0.3±0.3 gCO2-C/m2/day for both years. However, the coastal site near Arctic sea was not still melted, showing much lower CO2 efflux was 0.02±0.02 and 0.08±0.12 gCO2-C/m2/day in soil temperature of -12.4±2.2 and -12.9±3.4 °C for 2010 and 2011, respectively. A relationship between mean CO2 efflux at each site and mean soil temperature at 5 cm below the surface along the trans-Alaska pipeline is a good exponential, which the equation is as follows: CO2 efflux = 885×exp(0.335×Ts) (R2=0.86; p<0.001) and CO2 efflux = 888×exp(0.337×Ts) (R2=0.92; p<0.001) for 2010 and 2011, respectively. CO2 efflux in a white spruce forest during the snow-thawing season was measured in four directions from the bottom stem, suggesting that distinct differences of CO2 efflux between the exposed soil and the snow-covered soil in the four directions. This may be due to the fast decomposition of soil organic carbon and/or active root respiration in the exposed soil

  10. Membrane fluidization by ether, other anesthetics, and certain agents abolishes P-glycoprotein ATPase activity and modulates efflux from multidrug-resistant cells.

    PubMed

    Regev, R; Assaraf, Y G; Eytan, G D

    1999-01-01

    The anesthetics benzyl alcohol and the nonaromatic chloroform and diethyl ether, abolish P-glycoprotein (Pgp) ATPase activity in a mode that does not fit classical competitive, noncompetitive, or uncompetitive inhibition. At concentrations similar to those required for inhibition of ATPase activity, these anesthetics fluidize membranes leading to twofold acceleration of doxorubicin flip-flop across lipid membranes and prevent photoaffinity labeling of Pgp with [125I]-iodoarylazidoprazosin. Similar concentrations of ether proved nontoxic and modulated efflux from Pgp-overexpressing cells. A similar twofold acceleration of doxorubicin flip-flop rate across membranes was observed with neutral mild detergents, including Tween 20, Nonidet P-40 and Triton X-100, and certain Pgp modulators, such as verapamil and progesterone. Concentrations of these agents, similar to those required for membrane fluidization, inhibited Pgp ATPase activity in a mode similar to that observed with the anesthetics. The mode of inhibition, i.e. lack of evidence for classical enzyme inhibition and the correlation of Pgp ATPase inhibition with membrane fluidization over a wide range of concentrations and structures of drugs favors the direct inhibition of Pgp ATPase activity by membrane fluidization. The unusual sensitivity of Pgp to membrane fluidization, as opposed to acceleration of ATPase activity of ion transporters, could fit the proposed function of Pgp as a 'flippase', which is in close contact with the membrane core.

  11. An overview of bacterial efflux pumps and computational approaches to study efflux pump inhibitors.

    PubMed

    Jamshidi, Shirin; Sutton, J Mark; Rahman, Khondaker M

    2016-01-01

    Micro-organisms express a wide range of transmembrane pumps known as multidrug efflux pumps that improve the micro-organism's ability to survive in severe environments and contribute to resistance against antibiotic and antimicrobial agents. There is significant interest in developing efflux inhibitors as an adjunct to treatment with current and next generation of antibiotics. A greater understanding of drug recognition and transport by multidrug efflux pumps is needed to develop clinically useful inhibitors, given the breadth of molecules that can be effluxed by these systems. We summarize some structural and functional data that could provide insights into the inhibition of transport mechanisms of these intricate molecular nanomachines with a focus on the advances in computational approaches.

  12. Overexpression of MexAB-OprM efflux pump in carbapenem-resistant Pseudomonas aeruginosa.

    PubMed

    Pan, Ya-Ping; Xu, Yuan-Hong; Wang, Zhong-Xin; Fang, Ya-Ping; Shen, Ji-Lu

    2016-08-01

    Efflux pump systems are one of the most important mechanisms conferring multidrug resistance in Pseudomonas aeruginosa. MexAB-OprM efflux pump is one of the largest multi-drug resistant efflux pumps with high-level expression, which is controlled by regulatory genes mexR, nalC, and nalD. This study investigated the role of efflux pump MexAB-OprM in 75 strains of carbapenem-resistant P. aeruginosa and evaluated the influence of point mutation of the regulatory genes. The minimum inhibitory concentrations of imipenem and meropenem, with or without MC207110, an efflux pump inhibitor, were determined by agar dilution method to select the positive strains for an overexpressed active efflux pump. Carba NP test and EDTA-disk synergy test were used for the detection of carbapenemase and metallo-β-lactamases, respectively. The gene mexA, responsible for the fusion protein structure, and the reference gene rpoD of the MexAB-OprM pump were amplified by real-time PCR. The quantity of relative mRNA expression was determined simultaneously. By PCR method, the efflux regulatory genes mexR, nalC, and nalD and outer membrane protein OprD2 were amplified for the strains showing overexpression of MexAB-OprM and subsequently analyzed by BLAST. Among the 75 P. aeruginosa strains, the prevalence of efflux pump-positive phenotype was 17.3 % (13/75). Carba NP test and EDTA-disk synergy test were all negative in the 13 strains. PCR assay results showed that ten strains overexpressed the MexAB-OprM efflux pump and were all positive for the regulatory genes mexR, nalC, and nalD. Sequence analysis indicated that of the ten isolates, nine had a mutation (Gly → Glu) at 71st amino acid position in NalC, and eight also had a mutation (Ser → Arg) at 209th position in NalC. Only one strain had a mutation (Thr → Ile) at the 158th amino acid position in NalD, whereas eight isolates had mutations in MexR. In conclusion, overexpression of efflux pump MexAB-OprM plays an important role in

  13. CO2 efflux along the trans-Alaska pipeline in snow-thawing season

    NASA Astrophysics Data System (ADS)

    Kim, Y.

    2010-12-01

    This research was conducted to estimate CO2 efflux along the trans-Alaska pipeline (ca. 660 km) before and during the snow-thawing season (April 7 to 23) of 2010. The sampling sites are in white and black spruce forest soils, which cover more than 60% of Alaska’s terrestrial ecosystem. CO2 efflux was measured with a dynamic chamber system that consisted of a chamber (22 cm in diameter and 6 cm high), pump, NDIR (CO2 analyzer), and a laptop computer. Soil temperature and soil moisture were measured with a portable thermocouple and a soil moisture sensor. Remarkably, before and during the snow-thawing season, mean CO2 efflux between both seasons appeared to show the magnitude of an order. The efflux ranged from 21±4 mgCO2/m2/day near coastal tundra to 1670±240 mgCO2/m2/day in white spruce forest during the snow-thawing season. A relationship between mean CO2 efflux at each site and mean soil temperature at 5 cm below the surface along the trans-Alaska pipeline is a good exponential, which the equation is as follows: CO2 efflux = 885×exp(0.335×Ts) (R2=0.86; p<0.001). CO2 efflux in a white spruce forest during the snow-thawing season is measured in four directions from the bottom stem. The measurements show apparent differences of CO2 efflux between the exposed soil and the snow-covered soil in the four directions. This may be due to the fast decomposition of soil organic carbon and/or active root respiration in the exposed soil caused by strong radiation in the spring. The efflux increases in the order of east, south, west, and north at 60 cm from the stem. Although the snow-thawing period is relatively short, CO2 efflux during that season in white and black spruce forest soils of Alaska should not be overlooked.

  14. The Deviant ATP-binding Site of the Multidrug Efflux Pump Pdr5 Plays an Active Role in the Transport Cycle*

    PubMed Central

    Furman, Christopher; Mehla, Jitender; Ananthaswamy, Neeti; Arya, Nidhi; Kulesh, Bridget; Kovach, Ildiko; Ambudkar, Suresh V.; Golin, John

    2013-01-01

    Pdr5 is the founding member of a large subfamily of evolutionarily distinct, clinically important fungal ABC transporters containing a characteristic, deviant ATP-binding site with altered Walker A, Walker B, Signature (C-loop), and Q-loop residues. In contrast to these motifs, the D-loops of the two ATP-binding sites have similar sequences, including a completely conserved aspartate residue. Alanine substitution mutants in the deviant Walker A and Signature motifs retain significant, albeit reduced, ATPase activity and drug resistance. The D-loop residue mutants D340A and D1042A showed a striking reduction in plasma membrane transporter levels. The D1042N mutation localized properly had nearly WT ATPase activity but was defective in transport and was profoundly hypersensitive to Pdr5 substrates. Therefore, there was a strong uncoupling of ATPase activity and drug efflux. Taken together, the properties of the mutants suggest an additional, critical intradomain signaling role for deviant ATP-binding sites. PMID:24019526

  15. The Role of Efflux and Physiological Adaptation in Biofilm Tolerance and Resistance.

    PubMed

    Van Acker, Heleen; Coenye, Tom

    2016-06-10

    Microbial biofilms demonstrate a decreased susceptibility to antimicrobial agents. Various mechanisms have been proposed to be involved in this recalcitrance. We focus on two of these factors. Firstly, the ability of sessile cells to actively mediate efflux of antimicrobial compounds has a profound impact on resistance and tolerance, and several studies point to the existence of biofilm-specific efflux systems. Secondly, biofilm-specific stress responses have a marked influence on cellular physiology, and contribute to the occurrence of persister cells. We provide an overview of the data that demonstrate that both processes are important for survival following exposure to antimicrobial agents.

  16. Drought effects on soil COefflux in a cacao agroforestry system in Sulawesi, Indonesia

    NASA Astrophysics Data System (ADS)

    van Straaten, O.; Veldkamp, E.; Köhler, M.; Anas, I.

    2009-12-01

    Climate change induced droughts pose a serious threat to ecosystems across the tropics and sub-tropics, particularly to those areas not adapted to natural dry periods. In order to study the vulnerability of cacao (Theobroma cacao) - Gliricidia sepium agroforestry plantations to droughts a large scale throughfall displacement roof was built in Central Sulawesi, Indonesia. In this 19-month replicated experiment, we measured soil surface CO2 efflux (soil respiration) in three simulated drought plots compared with three adjacent control plots. Soil respiration rates peaked at intermediate soil moisture and decreased under increasingly dry conditions (drought induced), but also decreased when soils became water saturated, as evidenced in control plots. The simulated drought plots exhibited a slight decrease in soil respiration compared to the control plots (average 13% decrease). The strength of the drought effect was spatially variable - while some measurement chamber sites reacted strongly ("responsive") to the decrease in soil water content (up to R2=0.70) (n=11), others did not react at all ("non-responsive") (n=7). The degree of soil CO2 respiration drought response was highest around cacao tree stems and decreased with distance from the stem (R2=0.22). A significant correlation was measured between "responsive" soil respiration chamber sites and sap flux density ratios of cacao (R=0.61) and Gliricidia (R=0.65). Leaf litter CO2 respiration decreased as conditions became drier. During dry periods the litter layer contributed approximately 3-4% of the total CO2 efflux and up to 40% during wet periods. A CO2 flush was recorded during the rewetting phase that lasted for approximately two weeks, during which time accumulated labile carbon stocks mineralized. The net effect on soil CO2 emissions over the duration of the experiment was neutral, control plots respired 11.1±0.5 Mg C ha-1 yr-1, while roof plots respired 10.5±0.5 Mg C ha-1 yr-1.

  17. Functional characterization of MexXY and OpmG in aminoglycoside efflux in Pseudomonas aeruginosa.

    PubMed

    Chuanchuen, Rungtip; Wannaprasat, Wechsiri; Schweizer, Herbert P

    2008-01-01

    MexXY is an active efflux system that contributes to intrinsic resistance to aminoglycosides in Pseudomonas aeruginosa. MexXY can function in combination with OprM in aminoglycoside efflux but may also functionally associate with another as yet unidentified outer membrane channel. The possible role of OpmG as a third component of MexXY in aminoglycoside efflux was investigated by construction of unmarked opmG mutants. Loss of OpmG did not have any impact on minimum inhibitory concentrations for aminoglycosides regardless of the presence of oprM, indicating that MexXY does not interact with OpmG in aminoglycoside efflux. In a clinical isolate PAJ010, (mexXY) enhanced streptomycin susceptibility but neither oprM nor opmG could, suggesting that MexXY functionally associates with an unidentified outer membrane protein for aminoglycoside efflux. Expression of an opmG-lacZ transcriptional fusion revealed that OpmG expression was neither constitutive nor inducible by gentamicin. Growth rates of wildtype P. aeruginosa and opmG mutant derivatives were not different, indicating that expression of opmG is not essential for P. aeruginosa growth.

  18. The Arabidopsis AtPP2CA Protein Phosphatase Inhibits the GORK K+ Efflux Channel and Exerts a Dominant Suppressive Effect on Phosphomimetic-activating Mutations*

    PubMed Central

    Lefoulon, Cécile; Boeglin, Martin; Moreau, Bertrand; Véry, Anne-Aliénor; Szponarski, Wojciech; Dauzat, Myriam; Michard, Erwan; Gaillard, Isabelle; Chérel, Isabelle

    2016-01-01

    The regulation of the GORK (Guard Cell Outward Rectifying) Shaker channel mediating a massive K+ efflux in Arabidopsis guard cells by the phosphatase AtPP2CA was investigated. Unlike the gork mutant, the atpp2ca mutants displayed a phenotype of reduced transpiration. We found that AtPP2CA interacts physically with GORK and inhibits GORK activity in Xenopus oocytes. Several amino acid substitutions in the AtPP2CA active site, including the dominant interfering G145D mutation, disrupted the GORK-AtPP2CA interaction, meaning that the native conformation of the AtPP2CA active site is required for the GORK-AtPP2CA interaction. Furthermore, two serines in the GORK ankyrin domain that mimic phosphorylation (Ser to Glu) or dephosphorylation (Ser to Ala) were mutated. Mutations mimicking phosphorylation led to a significant increase in GORK activity, whereas mutations mimicking dephosphorylation had no effect on GORK. In Xenopus oocytes, the interaction of AtPP2CA with “phosphorylated” or “dephosphorylated” GORK systematically led to inhibition of the channel to the same baseline level. Single-channel recordings indicated that the GORK S722E mutation increases the open probability of the channel in the absence, but not in the presence, of AtPP2CA. The dephosphorylation-independent inactivation mechanism of GORK by AtPP2CA is discussed in relation with well known conformational changes in animal Shaker-like channels that lead to channel opening and closing. In plants, PP2C activity would control the stomatal aperture by regulating both GORK and SLAC1, the two main channels required for stomatal closure. PMID:26801610

  19. Possible involvement of cationic-drug sensitive transport systems in the blood-to-brain influx and brain-to-blood efflux of amantadine across the blood-brain barrier.

    PubMed

    Suzuki, Toyofumi; Fukami, Toshiro; Tomono, Kazuo

    2015-03-01

    The purpose of this study was to characterize the brain-to-blood efflux transport of amantadine across the blood-brain barrier (BBB). The apparent in vivo efflux rate constant for [(3) H]amantadine from the rat brain (keff ) was found to be 1.53 × 10(-2) min(-1) after intracerebral microinjection using the brain efflux index method. The efflux of [(3) H]amantadine was inhibited by 1-methyl-4-phenylpyridinium (MPP(+) ), a cationic neurotoxin, suggesting that amantadine transport from the brain to the blood across the BBB potentially involves the rat plasma membrane monoamine transporter (rPMAT). On the other hand, other selected substrates for organic cation transporters (OCTs) and organic anion transporters (OATs), as well as inhibitors of P-glycoprotein (P-gp), did not affect the efflux transport of [(3) H]amantadine. In addition, in vitro studies using an immortalized rat brain endothelial cell line (GPNT) showed that the uptake and retention of [(3) H]amantadine by the cells was not changed by the addition of cyclosporin, which is an inhibitor of P-gp. However, cyclosporin affected the uptake and retention of rhodamine123. Finally, the initial brain uptake of [(3) H]amantadine was determined using an in situ mouse brain perfusion technique. Notably, the brain uptake clearance for [(3) H]amantadine was significantly decreased with the co-perfusion of quinidine or verapamil, which are cationic P-gp inhibitors, while MPP(+) did not have a significant effect. It is thus concluded that while P-gp is not involved, it is possible that rPMAT and the cationic drug-sensitive transport system participate in the brain-to-blood efflux and the blood-to-brain influx of amantadine across the BBB, respectively.

  20. Differential roles of RND efflux pumps in antimicrobial drug resistance of sessile and planktonic Burkholderia cenocepacia cells.

    PubMed

    Buroni, Silvia; Matthijs, Nele; Spadaro, Francesca; Van Acker, Heleen; Scoffone, Viola C; Pasca, Maria Rosalia; Riccardi, Giovanna; Coenye, Tom

    2014-12-01

    Burkholderia cenocepacia is notorious for causing respiratory tract infections in people with cystic fibrosis. Infections with this organism are particularly difficult to treat due to its high level of intrinsic resistance to most antibiotics. Multidrug resistance in B. cenocepacia can be ascribed to different mechanisms, including the activity of efflux pumps and biofilm formation. In the present study, the effects of deletion of the 16 operons encoding resistance-nodulation-cell division (RND)-type efflux pumps in B. cenocepacia strain J2315 were investigated by determining the MICs of various antibiotics and by investigating the antibiofilm effect of these antibiotics. Finally, the expression levels of selected RND genes in treated and untreated cultures were investigated using reverse transcriptase quantitative PCR (RT-qPCR). Our data indicate that the RND-3 and RND-4 efflux pumps are important for resistance to various antimicrobial drugs (including tobramycin and ciprofloxacin) in planktonic B. cenocepacia J2315 populations, while the RND-3, RND-8, and RND-9 efflux systems protect biofilm-grown cells against tobramycin. The RND-8 and RND-9 efflux pumps are not involved in ciprofloxacin resistance. Results from the RT-qPCR experiments on the wild-type strain B. cenocepacia J2315 suggest that there is little regulation at the level of mRNA expression for these efflux pumps under the conditions tested.

  1. Osmotolerance in Escherichia coli is improved by activation of copper efflux genes or supplementation with sulfur containing amino acids.

    PubMed

    Xiao, Mengyong; Zhu, Xinna; Fan, Feiyu; Xu, Hongtao; Tang, Jinlei; Qin, Ying; Ma, Yanhe; Zhang, Xueli

    2017-01-23

    Improvement in the osmotolerance of Escherichia coli is essential for the production of high titers of various bioproducts. In this work, a cusS mutation identified in the previously constructed high succinate-producing E. coli strain HX024 was investigated for its effect on osmotolerance. CusS is part of the two-component system CusSR that protects cell from Ag(I) and Cu(I) toxicity. Changing cusS from strain HX024 back to its original sequence led to a 24% decrease in cell mass and succinate titer under osmotic stress (12% glucose). When cultivated with a high initial glucose concentration (12%), introduction of the cusS mutation into parental strain Suc-T110 led to a 21% increase in cell mass and 40% increase in succinate titer. When the medium was supplemented with 30 g/L disodium succinate, the cusS mutation led to a 120% increase in cell mass and 492% increase in succinate titer. Introducing the cusS mutation into the wild-type strain ATCC 8739 led to increases in cell mass of 87% with 20% glucose and 36% using 30 g/L disodium succinate. The cusS mutation increased expression of cusCFBA, and gene expression levels were found to be positively related to osmotolerance abilities. Because high osmotic stress has been associated with deleterious accumulation of Cu(I) in the periplasm, activation of CusCFBA might alleviate this effect by transporting Cu(I) out of the cells. This hypothesis was confirmed by supplementing sulfur-containing amino acids that could chelate Cu(I). Adding methionine or cysteine to the medium increased the osmotolerance of E. coli under anaerobic conditions.

  2. Methoxypolyethylene glycol-block-polycaprolactone diblock copolymers reduce P-glycoprotein efflux in the absence of a membrane fluidization effect while stimulating P-glycoprotein ATPase activity.

    PubMed

    Zastre, Jason; Jackson, John K; Wong, Wesley; Burt, Helen M

    2007-04-01

    We have previously shown that amphiphilic diblock copolymers composed of methoxypolyethylene glycol-b-polycaprolactone (MePEG-b-PCL) increased the cellular accumulation and reduced the basolateral to apical flux of the P-glycoprotein substrate, rhodamine 123 (R-123) in caco-2 cells. The purpose of this study was to investigate membrane perturbation effects of MePEG-b-PCL diblock copolymers with erythrocyte membranes and caco-2 cells and the effect on P-gp ATPase activity. The diblock copolymer MePEG(17)-b-PCL(5) induced increasing erythrocyte hemolysis at concentrations which correlated with increasing accumulation of R-123 into caco-2 cells. However, no increase in cellular accumulation of R-123 by non-P-gp expressing cells was observed, suggesting that diblock did not enhance the transmembrane passive diffusion of R-123, but that the accumulation enhancement effect of the diblock in caco-2 cells was likely mediated primarily via P-gp inhibition. Fluorescence anisotropy measurements of membrane fluidity and P-gp ATPase activity demonstrated that MePEG(17)-b-PCL(5) decreased caco-2 membrane fluidity while stimulating ATPase activity approximately threefold at concentrations that maximally enhanced R-123 caco-2 accumulation. These results suggest that inhibition of P-gp efflux by MePEG(17)-b-PCL(5) does not appear to be related to increases in membrane fluidity or through inhibition in P-gp ATPase activities, which are two commonly reported cellular effects for P-gp inhibition mediated by surfactants.

  3. Adaptation to benzalkonium chloride and ciprofloxacin affects biofilm formation potential, efflux pump and haemolysin activity of Escherichia coli of dairy origin.

    PubMed

    Pagedar, Ankita; Singh, Jitender; Batish, Virender K

    2012-11-01

    The present study investigates the effect of adaptive resistance to ciprofloxacin (Cip) and benzalkonium chloride (BC) on biofilm formation potential (BFP), efflux pump activity (EPA) and haemolysin activity of Escherichia coli isolates of dairy origin. All the isolates, irrespective of antimicrobial susceptibility, developed significant adaptive resistance (P < 0·05). All the resistant phenotypes (antibiotic resistant: AR; & biocide resistant: BR) were stronger biofilm former and post-adaptation, an insignificant change was observed in their BFP. Whereas, post-adaptation, non-resistant isolates (antibiotic non-resistant: ANR; biocide non-resistant: BNR) transformed from poor or moderate to strong biofilm formers. Post-adaptive percentage increase in EPA was highly significant in non-resistant categories (P < 0·01) and significant at P < 0·05 in BR category. Interestingly, post-adaptive increase in EPA in BR isolates was more than that in AR yet, the latter exhibited greater adaptive resistance than the former. These findings indicated prevalence of some other specific resistance mechanism/s responsible for adaptive resistance against Cip. Strain specific variations were observed for stability of adaptive resistance and haemolysin activity for all the categories. Our findings especially in reference to post-adaptation upgradation of BFP status of non-resistant isolates seems to be providing an insight into the process of conversion of non-resistant isolate into resistant ones with enhanced BFP. These observations emphasize the serious implications of sub-lethal residual levels of antimicrobials in food environments and suggest a role of food chain in emergence of antimicrobial resistances.

  4. Charged Amino Acids (R83, E567, D617, E625, R669, and K678) of CusA Are Required for Metal Ion Transport in the Cus Efflux System

    SciTech Connect

    Su, Chih-Chia; Long, Feng; Lei, Hsiang-Ting; Reddy Bolla, Jani; Do, Sylvia V.; Rajashankar, Kanagalaghatta R.; Yu, Edward W.

    2012-10-23

    Gram-negative bacteria expel various toxic chemicals via tripartite efflux pumps belonging to the resistance-nodulation-cell division superfamily. These pumps span both the inner and outer membranes of the cell. The three components of these tripartite systems are an inner-membrane, substrate-binding transporter (or pump); a periplasmic membrane fusion protein (or adaptor); and an outer-membrane-anchored channel. These three efflux proteins interact in the periplasmic space to form the three-part complexes. We previously presented the crystal structures of both the inner-membrane transporter CusA and membrane fusion protein CusB of the CusCBA tripartite efflux system from Escherichia coli. We also described the co-crystal structure of the CusBA adaptor-transporter, revealing that the trimeric CusA efflux pump assembles with six CusB protein molecules to form the complex CusB{sub 6}-CusA{sub 3}. We here report three different conformers of the crystal structures of CusBA-Cu(I), suggesting a mechanism on how Cu(I) binding initiates a sequence of conformational transitions in the transport cycle. Genetic analysis and transport assays indicate that charged residues, in addition to the methionine pairs and clusters, are essential for extruding metal ions out of the cell.

  5. Effluxing ABC Transporters in Human Corneal Epithelium

    PubMed Central

    Vellonen, Kati-Sisko; Mannermaa, Eliisa; Turner, Helen; Häkli, Marika; Wolosin, J. Mario; Tervo, Timo; Honkakoski, Paavo; Urtti, Arto

    2010-01-01

    ATP-binding cassette (ABC) transporters are able to efflux their substrate drugs from the cells. We compared expression of efflux proteins in normal human corneal epithelial tissue, primary human corneal epithelial cells (HCEpiC), and corneal epithelial cell culture model (HCE model) based on human immortal cell line. Expression of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1–6 (MRP1–6) and breast cancer resistance protein (BCRP) was studied using quantitative RT-PCR, Western blot, and immunohistochemistry. Only MRP1, MRP5, and BCRP were expressed in the freshly excised human corneal epithelial tissue. Expression of MRP1 and MRP5 was localized predominantly in the basal cells of the central cornea and limbus. Functional efflux activity was shown in the cell models, but they showed over-expression of most efflux transporters compared to that of normal corneal epithelium. In conclusion, MRP1, MRP5, and BCRP are expressed in the corneal epithelium, but MDR1, MRP2, MRP3, MRP4, and MRP6 are not significantly expressed. HCE cell model and commercially available primary cells deviate from this expression profile. PMID:19623615

  6. CO2 efflux from subterranean nests of ant communities in a seasonal tropical forest, Thailand.

    PubMed

    Hasin, Sasitorn; Ohashi, Mizue; Yamada, Akinori; Hashimoto, Yoshiaki; Tasen, Wattanachai; Kume, Tomonori; Yamane, Seiki

    2014-10-01

    Many ant species construct subterranean nests. The presence of their nests may explain soil respiration "hot spots", an important factor in the high CO2 efflux from tropical forests. However, no studies have directly measured CO2 efflux from ant nests. We established 61 experimental plots containing 13 subterranean ant species to evaluate the CO2 efflux from subterranean ant nests in a tropical seasonal forest, Thailand. We examined differences in nest CO2 efflux among ant species. We determined the effects of environmental factors on nest CO2 efflux and calculated an index of nest structure. The mean CO2 efflux from nests was significantly higher than those from the surrounding soil in the wet and dry seasons. The CO2 efflux was species-specific, showing significant differences among the 13 ant species. The soil moisture content significantly affected nest CO2 efflux, but there was no clear relationship between nest CO2 efflux and nest soil temperature. The diameter of the nest entrance hole affected CO2 efflux. However, there was no significant difference in CO2 efflux rates between single-hole and multiple-hole nests. Our results suggest that in a tropical forest ecosystem the increase in CO2 efflux from subterranean ant nests is caused by species-specific activity of ants, the nest soil environment, and nest structure.

  7. Direct measurement of efflux in Pseudomonas aeruginosa using an environment-sensitive fluorescent dye.

    PubMed

    Iyer, Ramkumar; Erwin, Alice L

    2015-01-01

    Resistance-Nodulation-Division (RND) family pumps AcrB and MexB are the major efflux routes in Escherichia coli and Pseudomonas aeruginosa respectively. Fluorescent environment-sensitive dyes provide a means to study efflux pump function in live bacterial cells in real-time. Recently, we demonstrated the utility of this approach using the dye Nile Red to quantify AcrB-mediated efflux and measured the ability of antibiotics and other efflux pump substrates to compete with efflux of Nile Red, independent of antibacterial activity. Here, we extend this method to P. aeruginosa and describe a novel application that permits the comparison and rank-ordering of bacterial strains by their inherent efflux potential. We show that glucose and l-malate re-energize Nile Red efflux in P. aeruginosa, and we highlight differences in the glucose dependence and kinetics of efflux between P. aeruginosa and E. coli. We quantify the differences in efflux among a set of P. aeruginosa laboratory strains, which include PAO1, the hyper-sensitive strain ATCC 35151 and its parent, ATCC 12055. Efflux of Nile Red in P. aeruginosa is mediated by MexAB-OprM and is slower than in E. coli. In conclusion, we describe an efflux measurement tool for use in antibacterial drug discovery and basic research on P. aeruginosa efflux pumps.

  8. CO2 efflux from subterranean nests of ant communities in a seasonal tropical forest, Thailand

    PubMed Central

    Hasin, Sasitorn; Ohashi, Mizue; Yamada, Akinori; Hashimoto, Yoshiaki; Tasen, Wattanachai; Kume, Tomonori; Yamane, Seiki

    2014-01-01

    Many ant species construct subterranean nests. The presence of their nests may explain soil respiration “hot spots”, an important factor in the high CO2 efflux from tropical forests. However, no studies have directly measured CO2 efflux from ant nests. We established 61 experimental plots containing 13 subterranean ant species to evaluate the CO2 efflux from subterranean ant nests in a tropical seasonal forest, Thailand. We examined differences in nest CO2 efflux among ant species. We determined the effects of environmental factors on nest CO2 efflux and calculated an index of nest structure. The mean CO2 efflux from nests was significantly higher than those from the surrounding soil in the wet and dry seasons. The CO2 efflux was species-specific, showing significant differences among the 13 ant species. The soil moisture content significantly affected nest CO2 efflux, but there was no clear relationship between nest CO2 efflux and nest soil temperature. The diameter of the nest entrance hole affected CO2 efflux. However, there was no significant difference in CO2 efflux rates between single-hole and multiple-hole nests. Our results suggest that in a tropical forest ecosystem the increase in CO2 efflux from subterranean ant nests is caused by species-specific activity of ants, the nest soil environment, and nest structure. PMID:25505521

  9. CpxR Activates MexAB-OprM Efflux Pump Expression and Enhances Antibiotic Resistance in Both Laboratory and Clinical nalB-Type Isolates of Pseudomonas aeruginosa

    PubMed Central

    Yi, Xue-Xian; O’Gara, Fergal; Wang, Yi-Ping

    2016-01-01

    Resistance-Nodulation-Division (RND) efflux pumps are responsible for multidrug resistance in Pseudomonas aeruginosa. In this study, we demonstrate that CpxR, previously identified as a regulator of the cell envelope stress response in Escherichia coli, is directly involved in activation of expression of RND efflux pump MexAB-OprM in P. aeruginosa. A conserved CpxR binding site was identified upstream of the mexA promoter in all genome-sequenced P. aeruginosa strains. CpxR is required to enhance mexAB-oprM expression and drug resistance, in the absence of repressor MexR, in P. aeruginosa strains PA14. As defective mexR is a genetic trait associated with the clinical emergence of nalB-type multidrug resistance in P. aeruginosa during antibiotic treatment, we investigated the involvement of CpxR in regulating multidrug resistance among resistant isolates generated in the laboratory via antibiotic treatment and collected in clinical settings. CpxR is required to activate expression of mexAB-oprM and enhances drug resistance, in the absence or presence of MexR, in ofloxacin-cefsulodin-resistant isolates generated in the laboratory. Furthermore, CpxR was also important in the mexR-defective clinical isolates. The newly identified regulatory linkage between CpxR and the MexAB-OprM efflux pump highlights the presence of a complex regulatory network modulating multidrug resistance in P. aeruginosa. PMID:27736975

  10. Role of the Mmr Efflux Pump in Drug Resistance in Mycobacterium tuberculosis

    PubMed Central

    Rodrigues, Liliana; Villellas, Cristina; Bailo, Rebeca; Viveiros, Miguel

    2013-01-01

    Efflux pumps are membrane proteins capable of actively transporting a broad range of substrates from the cytoplasm to the exterior of the cell. Increased efflux activity in response to drug treatment may be the first step in the development of bacterial drug resistance. Previous studies showed that the efflux pump Mmr was significantly overexpressed in strains exposed to isoniazid. In the work to be described, we constructed mutants lacking or overexpressing Mmr in order to clarify the role of this efflux pump in the development of resistance to isoniazid and other drugs in M. tuberculosis. The mmr knockout mutant showed an increased susceptibility to ethidium bromide, tetraphenylphosphonium, and cetyltrimethylammonium bromide (CTAB). Overexpression of mmr caused a decreased susceptibility to ethidium bromide, acriflavine, and safranin O that was obliterated in the presence of the efflux inhibitors verapamil and carbonyl cyanide m-chlorophenylhydrazone. Isoniazid susceptibility was not affected by the absence or overexpression of mmr. The fluorometric method allowed the detection of a decreased efflux of ethidium bromide in the knockout mutant, whereas the overexpressed strain showed increased efflux of this dye. This increased efflux activity was inhibited in the presence of efflux inhibitors. Under our experimental conditions, we have found that efflux pump Mmr is mainly involved in the susceptibility to quaternary compounds such as ethidium bromide and disinfectants such as CTAB. The contribution of this efflux pump to isoniazid resistance in Mycobacterium tuberculosis still needs to be further elucidated. PMID:23165464

  11. Opioids and efflux transporters. Part 3: P-glycoprotein substrate activity of 3-hydroxyl addition to meperidine analogs.

    PubMed

    Mercer, Susan L; Cunningham, Christopher W; Eddington, Natalie D; Coop, Andrew

    2008-06-15

    Numerous studies have shown that many clinically employed opioid analgesics are substrates for P-glycoprotein (P-gp), suggesting that up-regulation of P-gp may contribute to the development of central tolerance to opioids. The studies herein focus on the development of SAR for P-gp substrate activity in the meperidine series of opioids. Addition of a 3-OH to meperidine and the ketone analog of meperidine yielding bemidone and ketobemidone, respectively, significantly increased P-gp substrate affinity. The results of this study have implications in the development of novel analgesics to be utilized as tools to study the contribution of P-gp on the development of central tolerance to opioids.

  12. Oral and inhaled corticosteroids: Differences in P-glycoprotein (ABCB1) mediated efflux

    SciTech Connect

    Crowe, Andrew Tan, Ai May

    2012-05-01

    There is concern that P-glycoprotein mediated efflux contributes to steroid resistance. Therefore, this study examined bidirectional corticosteroid transport and induction capabilities for P-glycoprotein (P-gp) to understand which of the systemic and inhaled corticosteroids interacted with P-gp to the greatest extent. Hydrocortisone, prednisolone, prednisone, methylprednisolone, and dexamethasone represented systemically active drugs, while fluticasone propionate, beclomethasone dipropionate, ciclesonide and budesonide represented inhaled corticosteroids. Aldosterone and fludrocortisone represented mineralocorticoids. All drugs were detected using individually optimised HPLC protocols. Transport studies were conducted through Caco-2 monolayers. Hydrocortisone and aldosterone had efflux ratios below 1.5, while prednisone showed a P-gp mediated efflux ratio of only 1.8 compared to its active drug, prednisolone, with an efflux ratio of 4.5. Dexamethasone and beclomethasone had efflux ratios of 2.1 and 3.3 respectively, while this increased to 5.1 for methylprednisolone. Fluticasone showed an efflux ratio of 2.3. Protein expression studies suggested that all of the inhaled corticosteroids were able to induce P-gp expression, from 1.6 to 2 times control levels. Most of the systemic corticosteroids had higher passive permeability (> 20 × 10{sup −6} cm/s) compared to the inhaled corticosteroids (> 5 × 10{sup −6} cm/s), except for budesonide, with permeability similar to the systemic corticosteroids. Inhaled corticosteroids are not transported by P-gp to the same extent as systemic corticosteroids. However, they are able to induce P-gp production. Thus, inhaled corticosteroids may have greater interactions with other P-gp substrates, but P-gp itself is less likely to influence resistance to the drugs. -- Highlights: ► Inhaled corticosteroids are only weak substrates for P-gp, including budesonide. ► Inhaled corticosteroid potent P-gp inducers especially

  13. The multidrug efflux pump MdtEF protects against nitrosative damage during the anaerobic respiration in Escherichia coli.

    PubMed

    Zhang, Yiliang; Xiao, Minfeng; Horiyama, Tsukasa; Zhang, Yinfeng; Li, Xuechen; Nishino, Kunihiko; Yan, Aixin

    2011-07-29

    Drug efflux represents an important protection mechanism in bacteria to withstand antibiotics and environmental toxic substances. Efflux genes constitute 6-18% of all transporters in bacterial genomes, yet the expression and functions of only a handful of them have been studied. Among the 20 efflux genes encoded in the Escherichia coli K-12 genome, only the AcrAB-TolC system is constitutively expressed. The expression, activities, and physiological functions of the remaining efflux genes are poorly understood. In this study we identified a dramatic up-regulation of an additional efflux pump, MdtEF, under the anaerobic growth condition of E. coli, which is independent of antibiotic exposure. We found that expression of MdtEF is up-regulated more than 20-fold under anaerobic conditions by the global transcription factor ArcA, resulting in increased efflux activity and enhanced drug tolerance in anaerobically grown E. coli. Cells lacking mdtEF display a significantly decreased survival rate under the condition of anaerobic respiration of nitrate. Deletion of the genes responsible for the biosynthesis of indole, tnaAB, or replacing nitrate with fumarate as the terminal electron acceptor during the anaerobic respiration restores the decreased survival of ΔmdtEF cells. Moreover, ΔmdtEF cells are susceptible to indole nitrosative derivatives, a class of toxic byproducts formed and accumulated within E. coli when the bacterium respires nitrate under anaerobic conditions. Taken together, we conclude that the multidrug efflux pump MdtEF is up-regulated during the anaerobic physiology of E. coli to protect the bacterium from nitrosative damage through expelling the nitrosyl indole derivatives out of the cells.

  14. The Acinetobacter baumannii Two-Component System AdeRS Regulates Genes Required for Multidrug Efflux, Biofilm Formation, and Virulence in a Strain-Specific Manner

    PubMed Central

    Richmond, Grace E.; Evans, Laura P.; Anderson, Michele J.; Wand, Matthew E.; Bonney, Laura C.; Ivens, Alasdair; Chua, Kim Lee; Webber, Mark A.; Sutton, J. Mark; Peterson, Marnie L.

    2016-01-01

    ABSTRACT The opportunistic pathogen Acinetobacter baumannii is able to persist in the environment and is often multidrug resistant (MDR), causing difficulties in the treatment of infections. Here, we show that the two-component system AdeRS, which regulates the production of the AdeABC multidrug resistance efflux pump, is required for the formation of a protective biofilm in an ex vivo porcine mucosal model, which mimics a natural infection of the human epithelium. Interestingly, deletion of adeB impacted only on the ability of strain AYE to form a biofilm on plastic and only on the virulence of strain Singapore 1 for Galleria mellonella. RNA-Seq revealed that loss of AdeRS or AdeB significantly altered the transcriptional landscape, resulting in the changed expression of many genes, notably those associated with antimicrobial resistance and virulence interactions. For example, A. baumannii lacking AdeRS displayed decreased expression of adeABC, pil genes, com genes, and a pgaC-like gene, whereas loss of AdeB resulted in increased expression of pil and com genes and decreased expression of ferric acinetobactin transport system genes. These data define the scope of AdeRS-mediated regulation, show that changes in the production of AdeABC mediate important phenotypes controlled by AdeRS, and suggest that AdeABC is a viable target for antimicrobial drug and antibiofilm discovery. PMID:27094331

  15. Synthetic interaction between the TipN polarity factor and an AcrAB-family efflux pump implicates cell polarity in bacterial drug resistance.

    PubMed

    Kirkpatrick, Clare L; Viollier, Patrick H

    2014-05-22

    Quinolone antibiotics are clinically important drugs that target bacterial DNA replication and chromosome segregation. Although the AcrAB-family efflux pumps generally protect bacteria from such drugs, the physiological role of these efflux systems and their interplay with other cellular events are poorly explored. Here, we report an intricate relationship between antibiotic resistance and cell polarity in the model bacterium Caulobacter crescentus. We show that a polarity landmark protein, TipN, identified by virtue of its ability to direct flagellum placement to the new cell pole, protects cells from toxic misregulation of an AcrAB efflux pump through a cis-encoded nalidixic acid-responsive transcriptional repressor. Alongside the importance of polarity in promoting the inheritance and activity of virulence functions including motility, we can now ascribe to it an additional role in drug resistance that is distinct from classical efflux mechanisms.

  16. Quercetin up-regulates expressions of peroxisome proliferator-activated receptor γ, liver X receptor α, and ATP binding cassette transporter A1 genes and increases cholesterol efflux in human macrophage cell line.

    PubMed

    Lee, Seung-Min; Moon, Jiyoung; Cho, Yoonsu; Chung, Ji Hyung; Shin, Min-Jeong

    2013-02-01

    Cholesterol-laden macrophages trigger accumulation of foam cells and increase the risk of developing atherosclerosis. We hypothesized that quercetin could lower the content of cholesterol in macrophages by regulating the expression of the ATP binding cassette transporter A1 (ABCA1) gene in differentiated human acute monocyte leukemia cell line (THP-1) cells and thereby reducing the chance of forming foam cells. Quercetin, in concentrations up to 30 μM, was not cytotoxic to differentiated THP-1 cells. Quercetin up-regulated both ABCA1 messenger RNA and protein expression in differentiated THP-1 cells, and its maximum effects were demonstrated at 0.3 μM for 4 to 8 hours in incubation. In addition, quercetin increased protein levels of peroxisome proliferator-activated receptor γ (PPARγ) and liver X receptor α (LXRα) within 2 hours of treatment. Because PPARγ and LXRα are important transcriptional factors for ABCA1, quercetin-induced up-regulation of ABCA1 may be mediated by increased expression levels of the PPARγ and LXRα genes. Furthermore, quercetin-enhanced cholesterol efflux from differentiated THP-1 cells to both high-density lipoprotein (HDL) and apolipoprotein A1. Quercetin at the dose of 0.15 μM elevated the cholesterol efflux only for HDL. At the dose of 0.3 μM, quercetin demonstrated effects both on HDL and apolipoprotein A1. Our data demonstrated that quercetin increased the expressions of PPARγ, LXRα, and ABCA1 genes and cholesterol efflux from THP-1 macrophages. Quercetin-induced expression of PPARγ and LXRα might subsequently affect up-regulation of their target gene ABCA1. Taken together, ingestion of quercetin or quercetin-rich foods could be an effective way to improve cholesterol efflux from macrophages, which would contribute to lowering the risk of atherosclerosis.

  17. Potent and selective mediators of cholesterol efflux

    DOEpatents

    Bielicki, John K; Johansson, Jan

    2015-03-24

    The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABAC1 that parallels that of full-length apolipoproteins. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

  18. Two coregulated efflux transporters modulate intracellular heme and protoporphyrin IX availability in Streptococcus agalactiae.

    PubMed

    Fernandez, Annabelle; Lechardeur, Delphine; Derré-Bobillot, Aurélie; Couvé, Elisabeth; Gaudu, Philippe; Gruss, Alexandra

    2010-04-22

    Streptococcus agalactiae is a major neonatal pathogen whose infectious route involves septicemia. This pathogen does not synthesize heme, but scavenges it from blood to activate a respiration metabolism, which increases bacterial cell density and is required for full virulence. Factors that regulate heme pools in S. agalactiae are unknown. Here we report that one main strategy of heme and protoporphyrin IX (PPIX) homeostasis in S. agalactiae is based on a regulated system of efflux using two newly characterized operons, gbs1753 gbs1752 (called pefA pefB), and gbs1402 gbs1401 gbs1400 (called pefR pefC pefD), where pef stands for 'porphyrin-regulated efflux'. In vitro and in vivo data show that PefR, a MarR-superfamily protein, is a repressor of both operons. Heme or PPIX both alleviate PefR-mediated repression. We show that bacteria inactivated for both Pef efflux systems display accrued sensitivity to these porphyrins, and give evidence that they accumulate intracellularly. The DeltapefR mutant, in which both pef operons are up-regulated, is defective for heme-dependent respiration, and attenuated for virulence. We conclude that this new efflux regulon controls intracellular heme and PPIX availability in S. agalactiae, and is needed for its capacity to undergo respiration metabolism, and to infect the host.

  19. AcrB, AcrD, and MdtABC multidrug efflux systems are involved in enterobactin export in Escherichia coli.

    PubMed

    Horiyama, Tsukasa; Nishino, Kunihiko

    2014-01-01

    Escherichia coli produces the iron-chelating compound enterobactin to enable growth under iron-limiting conditions. After biosynthesis, enterobactin is released from the cell. However, the enterobactin export system is not fully understood. Previous studies have suggested that the outer membrane channel TolC is involved in enterobactin export. There are several multidrug efflux transporters belonging to resistance-nodulation-cell division (RND) family that require interaction with TolC to function. Therefore, several RND transporters may be responsible for enterobactin export. In this study, we investigated whether RND transporters are involved in enterobactin export using deletion mutants of multidrug transporters in E. coli. Single deletions of acrB, acrD, mdtABC, acrEF, or mdtEF did not affect the ability of E. coli to excrete enterobactin, whereas deletion of tolC did affect enterobactin export. We found that multiple deletion of acrB, acrD, and mdtABC resulted in a significant decrease in enterobactin export and that plasmids carrying the acrAB, acrD, or mdtABC genes restored the decrease in enterobactin export exhibited by the ΔacrB acrD mdtABC mutant. These results indicate that AcrB, AcrD, and MdtABC are required for the secretion of enterobactin.

  20. Cystathionine γ-lyase(CSE)/hydrogen sulfide system is regulated by miR-216a and influences cholesterol efflux in macrophages via the PI3K/AKT/ABCA1 pathway.

    PubMed

    Gong, Duo; Cheng, Hai-peng; Xie, Wei; Zhang, Min; Liu, Dan; Lan, Gang; Huang, Chong; Zhao, Zhen-wang; Chen, Ling-yan; Yao, Feng; Tan, Yu-lin; Li, Liang; Xia, Xiao-dan; Zheng, Xi-long; Wang, Zong-bao; Tang, Chao-ke

    2016-01-29

    This study was designed to evaluate whether CSE/H2S system, which is regulated by miR-216a, regulated ABCA1-mediated cholesterol efflux and cholesterol contents in THP-1 macrophages-derived foam cells. Our qPCR and western blotting results showed that CSE/H2S significantly up-regulated the expression of ATP-binding cassette transporter A1 (ABCA1) mRNA and protein via PI3K/AKT pathway in foam cells derived from human THP-1 macrophages. The miR-216a directly targeted 3' untranslated region of CSE. It significantly reduced CSE and ABCA1 expression, and also decreased the phosphorylation of PI3K and AKT. Additionally, cholesterol efflux decreased, and cholesterol levels increased in THP-1 macrophage-derived foam cells in response to treatment with miR-216a. Our study demonstrates that CSE/H2S system is regulated by miR-216a, and regulates ABCA1-mediated cholesterol efflux and cholesterol levels through the PI3K/AKT pathway.

  1. Seasonal dynamics of CO2 efflux in soils amended with composted and thermally-dried sludge as affected by soil tillage systems in a semi-arid agroecosystem

    NASA Astrophysics Data System (ADS)

    García-Gil, Juan Carlos; Soler-Rovira, Pedro; López-de-Sa, Esther G.; Polo, Alfredo

    2014-05-01

    In semi-arid agricultural soils, seasonal dynamic of soil CO2 efflux (SCE) is highly variable. Based on soil respiration measurements the effects of different management systems (moldboard plowing, chisel and no-tillage) and the application of composted sludge (CS) and thermally-dried sewage sludge (TSS) was investigated in a long-term field experiment (28 years) conducted on a sandy-loam soil at the experimental station 'La Higueruela' (40o 03'N, 4o 24'W). Both organic amendments were applied at a rate of 30 Mg ha-1 prior to tillage practices. Unamended soils were used as control for each tillage system. SCE was moderate in late spring (2.2-11.8 μmol CO2 m-2 s-1) when amendments were applied and tillage was performed, markedly decreased in summer (0.4-3.2 μmol CO2 m-2 s-1), following a moderate increase in autumn (3.4-14.1 μmol CO2 m-2 s-1), rising sharply in October (5.6-39.8 μmol CO2 m-2 s-1 ). In winter, SCE was low (0.6-6.5 μmol CO2 m-2 s-1). In general, SCE was greater in chisel and moldboard tilled soils, and in CS and particularly TSS-amended soils, due to the addition of labile C with these amendments, meanwhile no-tillage soils exhibited smaller increases in C efflux throughout the seasons. Soil temperature controlled the seasonal variations of SCE. In summer, when drought occurs, a general decrease of SCE was observed due to a deficit in soil water content. After drought period SCE jumped to high values in response to rain events ('Birch effect') that changed soil moisture conditions. Soil drying in summer and rewetting in autumn may promotes some changes on the structure of soil microbial community, affecting associated metabolic processes, and enhancing a rapid mineralization of water-soluble organic C compounds and/or dead microbial biomass that acts as an energy source for soil microorganisms. To assess the effects of tillage and amendments on SCE, Q10 values were calculated. Data were grouped into three groups according to soil moisture (0

  2. Physico-chemical factors affect chloramphenicol efflux and EmhABC efflux pump expression in Pseudomonas fluorescens cLP6a.

    PubMed

    Adebusuyi, Abigail; Foght, Julia

    2013-01-01

    Protein synthesis inhibitors such as chloramphenicol and tetracycline may be inducers of efflux pumps such as MexY in Pseudomonas aeruginosa, complicating their use for the treatment of bacterial infections. We previously determined that chloramphenicol, a substrate of the EmhABC efflux pump in Pseudomonas fluorescens cLP6a, did not induce emhABC expression. In this study, we determined the effect of physico-chemical factors on chloramphenicol efflux by EmhABC, and the expression of emhABC. Efflux assays measuring accumulation of (14)C-chloramphenicol in cell pellets showed that chloramphenicol efflux is dependent on growth temperature, pH and concentration of Mg(2+). These physico-chemical factors modulated the efflux of chloramphenicol by 26 to >50%. All conditions tested that decreased the efflux of chloramphenicol unexpectedly induced transcription of emhABC efflux genes. EmhABC activity also effectively suppressed the deleterious effect of chloramphenicol on the cell membrane of strain cLP6a, which may explain why chloramphenicol is not an inducer of emhABC. Our results suggest that the detrimental effect of an antibiotic on cell membrane integrity and fatty acid composition may be the signal that induces emhABC expression, and that inducers of other bacterial efflux pumps may include environmental factors rather than their substrates per se.

  3. Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport

    PubMed Central

    Long, Feng; Su, Chih-Chia; Zimmermann, Michael T.; Boyken, Scott E.; Rajashankar, Kanagalaghatta R.; Jernigan, Robert L.; Yu, Edward W.

    2010-01-01

    Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel diverse toxic compounds from the cell.1,2 The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions.3,4 No prior structural information was available for the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here we describe the crystal structures of the inner membrane transporter CusA in the absence and presence of bound Cu(I) or Ag(I). These CusA structures provide important new structural information about the HME sub-family of RND efflux pumps. The structures suggest that the metal binding sites, formed by a three-methionine cluster, are located within the cleft region of the periplasmic domain. Intriguingly, this cleft is closed in the apo-CusA form but open in the CusA-Cu(I) and CusA-Ag(I) structures, which directly suggests a plausible pathway for ion export. Binding of Cu(I) and Ag(I) triggers significant conformational changes in both the periplasmic and transmembrane domains. The crystal structure indicates that CusA has, in addition to the three-methionine metal binding site, four methionine pairs - three located in the transmembrane region and one in the periplasmic domain. Genetic analysis and transport assays suggest that CusA is capable of actively picking up metal ions from the cytosol, utilizing these methionine pairs/clusters to bind and export metal ions. These structures suggest a stepwise shuttle mechanism for transport between these sites. PMID:20865003

  4. Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport

    SciTech Connect

    Long, Feng; Su, Chih-Chia; Zimmermann, Michael T.; Boyken, Scott E.; Rajashankar, Kanagalaghatta R.; Jernigan, Robert L.; Yu, Edward W.

    2010-09-23

    Gram-negative bacteria, such as Escherichia coli, frequently use tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel various toxic compounds from the cell. The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. No previous structural information was available for the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here we describe the crystal structures of the inner-membrane transporter CusA in the absence and presence of bound Cu(I) or Ag(I). These CusA structures provide new structural information about the HME subfamily of RND efflux pumps. The structures suggest that the metal-binding sites, formed by a three-methionine cluster, are located within the cleft region of the periplasmic domain. This cleft is closed in the apo-CusA form but open in the CusA-Cu(I) and CusA-Ag(I) structures, which directly suggests a plausible pathway for ion export. Binding of Cu(I) and Ag(I) triggers significant conformational changes in both the periplasmic and transmembrane domains. The crystal structure indicates that CusA has, in addition to the three-methionine metal-binding site, four methionine pairs - three located in the transmembrane region and one in the periplasmic domain. Genetic analysis and transport assays suggest that CusA is capable of actively picking up metal ions from the cytosol, using these methionine pairs or clusters to bind and export metal ions. These structures suggest a stepwise shuttle mechanism for transport between these sites.

  5. Continuous monitoring of soil gas efflux with Forced Diffusion (FD) chamber technique in a tundra ecosystem, Alaska

    NASA Astrophysics Data System (ADS)

    Kim, Y.; Park, S. J.; Lee, B. Y.

    2015-12-01

    Continuous measurements of soil carbon dioxide (CO2) efflux provide essential information about the soil carbon budget in response to an abruptly changing climate at Arctic and Subarctic scales. The Forced Diffusion (FD) chamber technique has a gas permeable membrane, which passively regulates the mixing of atmosphere and soil air in the chamber, in place of the active pumping system inside a regular dynamics efflux chamber system (Risk et al., 2011). Here the system has been modified the sampling routine to eliminate the problem of sensor drift. After that, we deployed the FD chamber system in a tundra ecosystem over the discontinuous permafrost regime of Council, Alaska. The representative understory plants are tussock (17 %), lichen (32 %), and moss (51 %), within a 40 נ40 m plot at an interval of five meters (81 points total) for efflux-measurement by dynamic chamber. The FD chamber monitored soil CO2 efflux from moss, lichen, and tussock regimes at an interval of 30 min during the growing season of 2015. As the results, mean soil CO2 effluxes in sphagnum moss, lichen, and tussock were 1.98 ± 1.10 (coefficient of variance: 55.8 %), 3.34 ± 0.84 (CV: 25.0 %), and 5.32 ± 1.48 (CV: 27.8 %) gCO2/m2/d, respectively. The difference between the 30-min efflux interval and the average efflux of three 10-min intervals is not significant for sphagnum (n = 196), lichen (n = 918), and tussock (n = 918) under a 95 % confidence level. The deploying interval was then set to 30 min and synchronized with eddy covariance tower data. During the deployment period of 2015, soil CO2 efflux over moss, lichen, and tussock using the FD chamber system were 44 ± 24, 73 ± 18, and 117 ± 33 gCO2/m2/period, respectively. Using the dynamic chamber, mean ecosystem respiration (Re) ranges for moss, lichen, and tussock were 2.2-2.6, 1.8-2.0, and 3.3-3.6 gCO2/m2/d, respectively, during June and July of 2015. These techniques provide the representativeness of spatiotemporal variation of soil

  6. Engineering bacterial efflux pumps for solar-powered bioremediation of surface waters.

    PubMed

    Kapoor, Vikram; Wendell, David

    2013-05-08

    Antibiotics are difficult to selectively remove from surface waters by present treatment methods. Bacterial efflux pumps have evolved the ability to discriminately expel antibiotics and other noxious agents via proton and ATP driven pathways. Here, we describe light-dependent removal of antibiotics by engineering the bacterial efflux pump AcrB into a proteovesicle system. We have created a chimeric protein with the requisite proton motive force by coupling AcrB to the light-driven proton pump Delta-rhodopsin (dR) via a glycophorin A transmembrane domain. This creates a solar powered protein material capable of selectively capturing antibiotics from bulk solutions. Using environmental water and direct sunlight, our AcrB-dR vesicles removed almost twice as much antibiotic as the treatment standard, activated carbon. Altogether, the AcrB-dR system provides an effective means of extracting antibiotics from surface waters as well as potential antibiotic recovery through vesicle solubilization.

  7. Root growth inhibition by NH(4)(+) in Arabidopsis is mediated by the root tip and is linked to NH(4)(+) efflux and GMPase activity.

    PubMed

    Li, Qing; Li, Bao-Hai; Kronzucker, Herbert J; Shi, Wei-Ming

    2010-09-01

    Root growth in higher plants is sensitive to excess ammonium (NH(4)(+)). Our study shows that contact of NH(4)(+) with the primary root tip is both necessary and sufficient to the development of arrested root growth under NH(4)(+) nutrition in Arabidopsis. We show that cell elongation and not cell division is the principal target in the NH(4)(+) inhibition of primary root growth. Mutant and expression analyses using DR5:GUS revealed that the growth inhibition is furthermore independent of auxin and ethylene signalling. NH(4)(+) fluxes along the primary root, measured using the Scanning Ion-selective Electrode Technique, revealed a significant stimulation of NH(4)(+) efflux at the elongation zone following treatment with elevated NH(4)(+), coincident with the inhibition of root elongation. Stimulation of NH(4)(+) efflux and inhibition of cell expansion were significantly more pronounced in the NH(4)(+)-hypersensitive mutant vtc1-1, deficient in the enzyme GDP-mannose pyrophosphorylase (GMPase). We conclude that both restricted transmembrane NH(4)(+) fluxes and proper functioning of GMPase in roots are critical to minimizing the severity of the NH(4)(+) toxicity response in Arabidopsis.

  8. Thiazole-valine peptidomimetic (TTT-28) antagonizes multidrug resistance in vitro and in vivo by selectively inhibiting the efflux activity of ABCB1

    PubMed Central

    Wang, Yi-Jun; Patel, Bhargav A.; Anreddy, Nagaraju; Zhang, Yun-Kai; Zhang, Guan-Nan; Alqahtani, Saeed; Singh, Satyakam; Shukla, Suneet; Kaddoumi, Amal; Ambudkar, Suresh V.; Talele, Tanaji T.; Chen, Zhe-Sheng

    2017-01-01

    Multidrug resistance (MDR) attenuates the chemotherapy efficacy and increases the probability of cancer recurrence. The accelerated drug efflux mediated by ATP-binding cassette (ABC) transporters is one of the major MDR mechanisms. This study investigated if TTT-28, a newly synthesized thiazole-valine peptidomimetic, could reverse ABCB1-mediated MDR in vitro and in vivo. TTT-28 reversed the ABCB1-mediated MDR and increased the accumulation of [3H]-paclitaxel in ABCB1 overexpressing cells by selectively blocking the efflux function of ABCB1, but not interfering with the expression level and localization of ABCB1. Animal study revealed that TTT-28 enhanced the intratumoral concentration of paclitaxel and promoted apoptosis, thereby potently inhibiting the growth of ABCB1 overexpressing tumors. But TTT-28 did not induce the toxicity (cardiotoxicity/myelosuppression) of paclitaxel in mice. In this study, we synthesized and evaluated a novel selective inhibitor of ABCB1 (TTT-28) with high efficacy and low toxicity. The identification and characterization of this new thiazole-valine peptidomimetic will facilitate design and synthesis of a new generation of ABCB1 inhibitors, leading to further research on multidrug resistance and combination chemotherapy. Furthermore, the strategy that co-administer MDR-ABCB1 inhibitor to overcome the resistance of one FDA approved, widely used chemotherapeutic paclitaxel, may be promising direction for the field of adjuvant chemotherapy. PMID:28181548

  9. Chlorinated phenols control the expression of the multi-drug resistance efflux pump MexAB-OprM in Pseudomonas aeruginosa by activating NalC

    PubMed Central

    Ghosh, Sudeshna; Cremers, Claudia M.; Jakob, Ursula; Love, Nancy G.

    2011-01-01

    Summary NalC is a TetR type regulator that represses the multidrug efflux pump MexAB-OprM in Pseudomonas aeruginosa. Here we explain the mechanism of NalC mediated regulation of MexAB-OprM. We show that NalC non-covalently binds chlorinated phenols and chemicals containing chlorophenol sidechains such as triclosan. NalC-chlorinated phenol binding results in its dissociation from promoter DNA and up-regulation of NalC’s downstream targets, including the MexR antirepressor ArmR. ArmR up-regulation and MexR-ArmR complex formation have previously been shown to upregulate MexAB-OprM. In vivo mexB and armR expression analyses were used to corroborate in vitro NalC chlorinated phenol binding. We also show that the interaction between chlorinated phenols and NalC is reversible, such that removal of these chemicals restored NalC promoter DNA binding. Thus, the NalC-chlorinated phenol interaction is likely a pertinent physiological mechanism that P. aeruginosa uses to control expression of the MexAB-OprM efflux pump. PMID:21231970

  10. Regulation of ABC Efflux Transporters at Blood-Brain Barrier in Health and Neurological Disorders

    PubMed Central

    Qosa, Hisham; Miller, David S.; Pasinelli, Piera; Trotti, Davide

    2015-01-01

    The strength of the blood-brain barrier (BBB) in providing protection to the central nervous system from exposure to circulating chemicals is maintained by tight junctions between endothelial cells and by a broad range of transporter proteins that regulate exchange between CNS and blood. The most important transporters that restrict the permeability of large number of toxins as well as therapeutic agents are the ABC transporters. Among them, P-gp, BCRP, MRP1 and MRP2 are the utmost studied. These efflux transporters are neuroprotective, limiting the brain entry of neurotoxins; however, they could also restrict the entry of many therapeutics and contribute to CNS pharmacoresistance. Characterization of several regulatory pathways that govern expression and activity of ABC efflux transporters in the endothelium of brain capillaries have led to an emerging consensus that these processes are complex and contain several cellular and molecular elements. Alterations in ABC efflux transporters expression and/or activity occur in several neurological diseases. Here, we review the signaling pathways that regulate expression and transport activity of P-gp, BCRP, MRP1 and MRP2 as well as how their expression/activity changes in neurological diseases. PMID:26187753

  11. Effect of ABCG2/BCRP Expression on Efflux and Uptake of Gefitinib in NSCLC Cell Lines

    PubMed Central

    Galetti, Maricla; Petronini, Pier Giorgio; Fumarola, Claudia; Cretella, Daniele; La Monica, Silvia; Bonelli, Mara; Cavazzoni, Andrea; Saccani, Francesca; Caffarra, Cristina; Andreoli, Roberta; Mutti, Antonio; Tiseo, Marcello; Ardizzoni, Andrea; Alfieri, Roberta R.

    2015-01-01

    Background BCRP/ABCG2 emerged as an important multidrug resistance protein, because it confers resistance to several classes of cancer chemotherapeutic agents and to a number of novel molecularly-targeted therapeutics such as tyrosine kinase inhibitors. Gefitinib is an orally active, selective EGFR tyrosine kinase inhibitor used in the treatment of patients with advanced non small cell lung cancer (NSCLC) carrying activating EGFR mutations. Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism. Aim The present study, performed in a panel of NSCLC cell lines expressing different ABCG2 plasma membrane levels, was designed to investigate the effect of the efflux transporter ABCG2 on intracellular gefitinib accumulation, by dissecting the contribution of uptake and efflux processes. Methods and Results Our findings indicate that gefitinib, in lung cancer cells, inhibits ABCG2 activity, as previously reported. In addition, we suggest that ABCG2 silencing or overexpression affects intracellular gefitinib content by modulating the uptake rather than the efflux. Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake. Conclusions Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells. PMID:26536031

  12. CC-Chemokine Ligand 2 (CCL2) Suppresses High Density Lipoprotein (HDL) Internalization and Cholesterol Efflux via CC-Chemokine Receptor 2 (CCR2) Induction and p42/44 Mitogen-activated Protein Kinase (MAPK) Activation in Human Endothelial Cells *

    PubMed Central

    Sun, Run-Lu; Huang, Can-Xia; Bao, Jin-Lan; Jiang, Jie-Yu; Zhang, Bo; Zhou, Shu-Xian; Cai, Wei-Bin; Wang, Hong; Wang, Jing-Feng; Zhang, Yu-Ling

    2016-01-01

    High density lipoprotein (HDL) has been proposed to be internalized and to promote reverse cholesterol transport in endothelial cells (ECs). However, the mechanism underlying these processes has not been studied. In this study, we aim to characterize HDL internalization and cholesterol efflux in ECs and regulatory mechanisms. We found mature HDL particles were reduced in patients with coronary artery disease (CAD), which was associated with an increase in CC-chemokine ligand 2 (CCL2). In cultured primary human coronary artery endothelial cells and human umbilical vein endothelial cells, we determined that CCL2 suppressed the binding (4 °C) and association (37 °C) of HDL to/with ECs and HDL cellular internalization. Furthermore, CCL2 inhibited [3H]cholesterol efflux to HDL/apoA1 in ECs. We further found that CCL2 induced CC-chemokine receptor 2 (CCR2) expression and siRNA-CCR2 reversed CCL2 suppression on HDL binding, association, internalization, and on cholesterol efflux in ECs. Moreover, CCL2 induced p42/44 mitogen-activated protein kinase (MAPK) phosphorylation via CCR2, and p42/44 MAPK inhibition reversed the suppression of CCL2 on HDL metabolism in ECs. Our study suggests that CCL2 was elevated in CAD patients. CCL2 suppressed HDL internalization and cholesterol efflux via CCR2 induction and p42/44 MAPK activation in ECs. CCL2 induction may contribute to impair HDL function and form atherosclerosis in CAD. PMID:27458015

  13. CC-Chemokine Ligand 2 (CCL2) Suppresses High Density Lipoprotein (HDL) Internalization and Cholesterol Efflux via CC-Chemokine Receptor 2 (CCR2) Induction and p42/44 Mitogen-activated Protein Kinase (MAPK) Activation in Human Endothelial Cells.

    PubMed

    Sun, Run-Lu; Huang, Can-Xia; Bao, Jin-Lan; Jiang, Jie-Yu; Zhang, Bo; Zhou, Shu-Xian; Cai, Wei-Bin; Wang, Hong; Wang, Jing-Feng; Zhang, Yu-Ling

    2016-09-09

    High density lipoprotein (HDL) has been proposed to be internalized and to promote reverse cholesterol transport in endothelial cells (ECs). However, the mechanism underlying these processes has not been studied. In this study, we aim to characterize HDL internalization and cholesterol efflux in ECs and regulatory mechanisms. We found mature HDL particles were reduced in patients with coronary artery disease (CAD), which was associated with an increase in CC-chemokine ligand 2 (CCL2). In cultured primary human coronary artery endothelial cells and human umbilical vein endothelial cells, we determined that CCL2 suppressed the binding (4 °C) and association (37 °C) of HDL to/with ECs and HDL cellular internalization. Furthermore, CCL2 inhibited [(3)H]cholesterol efflux to HDL/apoA1 in ECs. We further found that CCL2 induced CC-chemokine receptor 2 (CCR2) expression and siRNA-CCR2 reversed CCL2 suppression on HDL binding, association, internalization, and on cholesterol efflux in ECs. Moreover, CCL2 induced p42/44 mitogen-activated protein kinase (MAPK) phosphorylation via CCR2, and p42/44 MAPK inhibition reversed the suppression of CCL2 on HDL metabolism in ECs. Our study suggests that CCL2 was elevated in CAD patients. CCL2 suppressed HDL internalization and cholesterol efflux via CCR2 induction and p42/44 MAPK activation in ECs. CCL2 induction may contribute to impair HDL function and form atherosclerosis in CAD.

  14. Mechanisms and physiological roles of K+ efflux from root cells.

    PubMed

    Demidchik, Vadim

    2014-05-15

    Potassium is the most abundant macronutrient, which is involved in a multitude of physiological processes. Potassium uptake in roots is crucial for plants; however, K(+) efflux can also occur and has important functions. Potassium efflux from roots is mainly induced by stresses, such as pathogens, salinity, freezing, oxidants and heavy metals. Reactive oxygen species (ROS) and exogenous purines also cause this reaction. The depolarisation and activation of cation channels are required for K(+) efflux from plant roots. Potassium channels and nonselective cation channels (NSCCs) are involved in this process. Some of them are 'constitutive', while the others require a chemical agent for activation. In Arabidopsis, there are 77 genes that can potentially encode K(+)-permeable channels. Potassium-selective channel genes include 9 Shaker and 6 Tandem-Pore K(+) channels. Genes of NSCCs are more abundant and present by 20 cyclic nucleotide gated channels, 20 ionotropic glutamate receptors, 1 two-pore channel, 10 mechanosensitive-like channels, 2 mechanosensitive 'Mid1-Complementing Activity' channels, 1 mechanosensitive Piezo channel, and 8 annexins. Two Shakers (SKOR and GORK) and several NSCCs are expressed in root cell plasma membranes. SKOR mediates K(+) efflux from xylem parenchyma cells to xylem vessels while GORK is expressed in the epidermis and functions in K(+) release. Both these channels are activated by ROS. The GORK channel activity is stimulated by hydroxyl radicals that are generated in a Ca(2+)-dependent manner in stress conditions, such as salinity or pathogen attack, resulting in dramatic K(+) efflux from root cells. Potassium loss simulates cytosolic proteases and endonucleases, leading to programmed cell death. Other physiological functions of K(+) efflux channels include repolarisation of the plasma membrane during action potentials and the 'hypothetical' function of a metabolic switch, which provides inhibition of energy-consuming biosyntheses and

  15. Chromate Efflux by Means of the ChrA Chromate Resistance Protein from Pseudomonas aeruginosa

    PubMed Central

    Alvarez, Angel H.; Moreno-Sánchez, Rafael; Cervantes, Carlos

    1999-01-01

    Everted membrane vesicles of Pseudomonas aeruginosa PAO1 harboring plasmid pCRO616, expressing the ChrA chromate resistance protein, accumulated four times more 51CrO42− than vesicles from plasmidless cells, indicating that a chromate efflux system functions in the resistant strain. Chromate uptake showed saturation kinetics with an apparent Km of 0.12 mM chromate and a Vmax of 0.5 nmol of chromate/min per mg of protein. Uptake of chromate by vesicles was dependent on NADH oxidation and was abolished by energy inhibitors and by the chromate analog sulfate. The mechanism of resistance to chromate determined by ChrA appears to be based on the active efflux of chromate driven by the membrane potential. PMID:10572148

  16. Tripartite assembly of RND multidrug efflux pumps

    NASA Astrophysics Data System (ADS)

    Daury, Laetitia; Orange, François; Taveau, Jean-Christophe; Verchère, Alice; Monlezun, Laura; Gounou, Céline; Marreddy, Ravi K. R.; Picard, Martin; Broutin, Isabelle; Pos, Klaas M.; Lambert, Olivier

    2016-02-01

    Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB-OprM and Escherichia coli AcrAB-TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA-MexB-TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components.

  17. ABCB1 and ABCC4 efflux transporters are involved in methyl parathion detoxification in ZFL cells.

    PubMed

    Nornberg, Bruna Félix; Batista, Carolina Reyes; Almeida, Daniela Volcan; Trindade, Gilma Santos; Marins, Luis Fernando

    2015-02-01

    The multi-xenobiotics resistance (MXR) mechanisms are the first line of defense against toxic substances in aquatic organisms and present great importance in the adaptation related to contaminated environments. Methyl parathion (MP) is a widely used organophosphate pesticide, which has been associated to various toxic effects in organisms. In the present work, we studied the main genes related to efflux transporters in zebrafish liver (ZFL) cells exposed to MP with and without an inhibitor of ABC transporters (verapamil). The results concerning transporters activity showed that the MXR mechanism is activated to detoxify from methyl parathion. The toxic effects of MP on ZFL cells were increased in the presence of the efflux transporter inhibitor, once cell viability was significantly decreased in co-exposure experiments. The combined exposure to MP and the inhibitor caused an increase in gene expression of P-gp1 (Abcb1) and MRP4 (Abcc4), suggesting that these transporters isoforms are associated with MP efflux. In general, the expression of genes related to the antioxidant defense system (ADS) was significantly increased in ZFL cells co-exposed to MP and verapamil. These data provide useful insights for better understanding of MP detoxification mechanism in fish hepatocytes.

  18. Efflux Pump Control Alters Synthetic Gene Circuit Function.

    PubMed

    Diao, Junchen; Charlebois, Daniel A; Nevozhay, Dmitry; Bódi, Zoltán; Pál, Csaba; Balázsi, Gábor

    2016-07-15

    Synthetic biology aims to design new biological systems for predefined purposes, such as the controlled secretion of biofuels, pharmaceuticals, or other chemicals. Synthetic gene circuits regulating an efflux pump from the ATP-binding cassette (ABC) protein family could achieve this. However, ABC efflux pumps can also drive out intracellular inducer molecules that control the gene circuits. This will introduce an implicit feedback that could alter gene circuit function in ways that are poorly understood. Here, we used two synthetic gene circuits inducible by tetracycline family molecules to regulate the expression of a yeast ABC pump (Pdr5p) that pumps out the inducer. Pdr5p altered the dose-responses of the original gene circuits substantially in Saccharomyces cerevisiae. While one aspect of the change could be attributed to the efflux pumping function of Pdr5p, another aspect remained unexplained. Quantitative modeling indicated that reduced regulator gene expression in addition to efflux pump function could fully explain the altered dose-responses. These predictions were validated experimentally. Overall, we highlight how efflux pumps can alter gene circuit dynamics and demonstrate the utility of mathematical modeling in understanding synthetic gene circuit function in new circumstances.

  19. Modulation of the Cellular Accumulation and Intracellular Activity of Daptomycin towards Phagocytized Staphylococcus aureus by the P-Glycoprotein (MDR1) Efflux Transporter in Human THP-1 Macrophages and Madin-Darby Canine Kidney Cells▿

    PubMed Central

    Lemaire, Sandrine; Van Bambeke, Françoise; Mingeot-Leclercq, Marie-Paule; Tulkens, Paul M.

    2007-01-01

    P-glycoprotein (P-gp; MDR1), a major efflux transporter, recognizes various antibiotics and is present in macrophages. We have examined its effect on the modulation of the intracellular accumulation and activity of daptomycin towards phagocytized Staphylococcus aureus (ATCC 25923) in human THP-1 macrophages, in comparison with MDCK epithelial cells (wild type and MDCK-MDR1 overexpressing P-gp; the bulk of the protein was immunodetected at the surface of all three cell types). Daptomycin displayed concentration-dependent intracellular activity (Hill equation pattern) in THP-1 and MDCK cells with (i) 50% effective drug extracellular concentration (EC50; relative potency) and static concentrations at 9 to 10 times the MIC and (ii) maximal efficacy (Emax; CFU decrease at infinite extracellular drug concentration) at 1.6 to 2 log compared to that of the postphagocytosis inoculum. Verapamil (100 μM) and elacridar (GF 120918; 0.5 μM), two known inhibitors of P-gp, decreased daptomycin EC50 (about threefold) in THP-1 and MDCK cells without affecting Emax. Daptomycin EC50 was about three- to fourfold higher and accumulation in MDCK-MDR1 commensurately lower than in wild-type cells. In THP-1 macrophages, (i) verapamil and ATP depletion increased, and ouabain (an inducer of mdr1 [the gene encoding P-gp] expression) decreased the accumulation of daptomycin in parallel with that of DiOC2 (a known substrate of P-gp); (ii) silencing mdr1 with duplex human mdr1 siRNAs reduced the cell content in immunoreactive P-gp to 15 to 30% of controls and caused an eight- to 13-fold increase in daptomycin accumulation. We conclude that daptomycin is subject to efflux from THP-1 macrophages and MDCK cells by P-gp, which reduces its intracellular activity against phagocytized S. aureus. PMID:17548493

  20. Multidrug Efflux Pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus Bacterial Food Pathogens

    PubMed Central

    Andersen, Jody L.; He, Gui-Xin; Kakarla, Prathusha; KC, Ranjana; Kumar, Sanath; Lakra, Wazir Singh; Mukherjee, Mun Mun; Ranaweera, Indrika; Shrestha, Ugina; Tran, Thuy; Varela, Manuel F.

    2015-01-01

    Foodborne illnesses caused by bacterial microorganisms are common worldwide and constitute a serious public health concern. In particular, microorganisms belonging to the Enterobacteriaceae and Vibrionaceae families of Gram-negative bacteria, and to the Staphylococcus genus of Gram-positive bacteria are important causative agents of food poisoning and infection in the gastrointestinal tract of humans. Recently, variants of these bacteria have developed resistance to medically important chemotherapeutic agents. Multidrug resistant Escherichia coli, Salmonella enterica, Vibrio cholerae, Enterobacter spp., and Staphylococcus aureus are becoming increasingly recalcitrant to clinical treatment in human patients. Of the various bacterial resistance mechanisms against antimicrobial agents, multidrug efflux pumps comprise a major cause of multiple drug resistance. These multidrug efflux pump systems reside in the biological membrane of the bacteria and actively extrude antimicrobial agents from bacterial cells. This review article summarizes the evolution of these bacterial drug efflux pump systems from a molecular biological standpoint and provides a framework for future work aimed at reducing the conditions that foster dissemination of these multidrug resistant causative agents through human populations. PMID:25635914

  1. Multidrug efflux pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus bacterial food pathogens.

    PubMed

    Andersen, Jody L; He, Gui-Xin; Kakarla, Prathusha; K C, Ranjana; Kumar, Sanath; Lakra, Wazir Singh; Mukherjee, Mun Mun; Ranaweera, Indrika; Shrestha, Ugina; Tran, Thuy; Varela, Manuel F

    2015-01-28

    Foodborne illnesses caused by bacterial microorganisms are common worldwide and constitute a serious public health concern. In particular, microorganisms belonging to the Enterobacteriaceae and Vibrionaceae families of Gram-negative bacteria, and to the Staphylococcus genus of Gram-positive bacteria are important causative agents of food poisoning and infection in the gastrointestinal tract of humans. Recently, variants of these bacteria have developed resistance to medically important chemotherapeutic agents. Multidrug resistant Escherichia coli, Salmonella enterica, Vibrio cholerae, Enterobacter spp., and Staphylococcus aureus are becoming increasingly recalcitrant to clinical treatment in human patients. Of the various bacterial resistance mechanisms against antimicrobial agents, multidrug efflux pumps comprise a major cause of multiple drug resistance. These multidrug efflux pump systems reside in the biological membrane of the bacteria and actively extrude antimicrobial agents from bacterial cells. This review article summarizes the evolution of these bacterial drug efflux pump systems from a molecular biological standpoint and provides a framework for future work aimed at reducing the conditions that foster dissemination of these multidrug resistant causative agents through human populations.

  2. Two Coregulated Efflux Transporters Modulate Intracellular Heme and Protoporphyrin IX Availability in Streptococcus agalactiae

    PubMed Central

    Fernandez, Annabelle; Lechardeur, Delphine; Derré-Bobillot, Aurélie; Couvé, Elisabeth; Gaudu, Philippe; Gruss, Alexandra

    2010-01-01

    Streptococcus agalactiae is a major neonatal pathogen whose infectious route involves septicemia. This pathogen does not synthesize heme, but scavenges it from blood to activate a respiration metabolism, which increases bacterial cell density and is required for full virulence. Factors that regulate heme pools in S. agalactiae are unknown. Here we report that one main strategy of heme and protoporphyrin IX (PPIX) homeostasis in S. agalactiae is based on a regulated system of efflux using two newly characterized operons, gbs1753 gbs1752 (called pefA pefB), and gbs1402 gbs1401 gbs1400 (called pefR pefC pefD), where pef stands for ‘porphyrin-regulated efflux’. In vitro and in vivo data show that PefR, a MarR-superfamily protein, is a repressor of both operons. Heme or PPIX both alleviate PefR-mediated repression. We show that bacteria inactivated for both Pef efflux systems display accrued sensitivity to these porphyrins, and give evidence that they accumulate intracellularly. The ΔpefR mutant, in which both pef operons are up-regulated, is defective for heme-dependent respiration, and attenuated for virulence. We conclude that this new efflux regulon controls intracellular heme and PPIX availability in S. agalactiae, and is needed for its capacity to undergo respiration metabolism, and to infect the host. PMID:20421944

  3. Active optical zoom system

    DOEpatents

    Wick, David V.

    2005-12-20

    An active optical zoom system changes the magnification (or effective focal length) of an optical imaging system by utilizing two or more active optics in a conventional optical system. The system can create relatively large changes in system magnification with very small changes in the focal lengths of individual active elements by leveraging the optical power of the conventional optical elements (e.g., passive lenses and mirrors) surrounding the active optics. The active optics serve primarily as variable focal-length lenses or mirrors, although adding other aberrations enables increased utility. The active optics can either be LC SLMs, used in a transmissive optical zoom system, or DMs, used in a reflective optical zoom system. By appropriately designing the optical system, the variable focal-length lenses or mirrors can provide the flexibility necessary to change the overall system focal length (i.e., effective focal length), and therefore magnification, that is normally accomplished with mechanical motion in conventional zoom lenses. The active optics can provide additional flexibility by allowing magnification to occur anywhere within the FOV of the system, not just on-axis as in a conventional system.

  4. Sesamin enhances cholesterol efflux in RAW264.7 macrophages.

    PubMed

    Liu, Nan; Wu, Chongming; Sun, Lizhong; Zheng, Jun; Guo, Peng

    2014-06-06

    Foam cells formation as a result of the uncontrolled cytophagy of modified cholesterol by macrophages plays a key role in the occurrence and development of atherosclerosis. Sesamin is an active constituent of Sesamum indicum which has been shown to possess multiple pharmacological activities. In this work, we investigated the effects of sesamin on foam cell formation and cholesterol efflux in RAW264.7 macrophages. Sesamin dose-dependently inhibited the enhanced cholesterol accumulation elicited by oxidized low-density lipoprotein cholesterol (oxLDL) in RAW264.7 cells. Treatment with sesamin (10 μM) significantly enhanced cholesterol efflux mediated by high-density lipoprotein (HDL). Realtime quantitative PCR and luciferase assays showed that sesamin significantly increased the mRNA levels of PPARγ, LXRα, and ABCG1, and increased the transcriptional activity of PPARγ. The stimulating effect of sesamin on cholesterol efflux was substantially inhibited by the co-treatment with GW9662, a potent inhibitor of PPARγ. These results suggest that sesamin is a new inhibitor of foam cell formation that may stimulate cholesterol efflux through upregulation of the PPARγ-LXRα-ABCG1 pathway.

  5. Rifaximin resistance in Escherichia coli associated with inflammatory bowel disease correlates with prior rifaximin use, mutations in rpoB, and activity of Phe-Arg-β-naphthylamide-inhibitable efflux pumps.

    PubMed

    Kothary, Vishesh; Scherl, Ellen J; Bosworth, Brian; Jiang, Zhi-Dong; Dupont, Herbert L; Harel, Josee; Simpson, Kenneth W; Dogan, Belgin

    2013-02-01

    Escherichia coli is implicated in the pathogenesis of inflammatory bowel disease (IBD). Rifaximin, a nonabsorbable derivative of rifampin effective against E. coli, improves symptoms in mild-to-moderate IBD. However, rifaximin resistance can develop in a single step in vitro. We examined the prevalence and mechanisms of rifaximin resistance in 62 strains of E. coli isolated from the ileal mucosa of 50 patients (19 with ileal Crohn's disease [L1+L3], 6 with colonic Crohn's disease [L2], 13 with ulcerative colitis [UC], 4 with symptomatic non-IBD diagnoses [NI], and 8 healthy [H]). Resistance (MIC > 1,024 mg/liter) was present in 12/48 IBD-associated ileal E. coli strains. Resistance correlated with prior rifaximin treatment (P < 0.00000001) but not with the presence of ileal inflammation (P = 0.73) or E. coli phylogroup. Mutations in a 1,057-bp region of rpoB, which encodes the bacterial target of rifaximin, were identified in 10/12 resistant strains versus 0/50 sensitive strains (P < 0.000000001) and consisted of seven amino acid substitutions. The efflux pump inhibitor Phe-Arg-β-naphthylamide (PAβN) lowered the MIC of 9/12 resistant strains 8- to 128-fold. Resistance was stable in the absence of rifaximin in 10/12 resistant strains after 30 passages. We conclude that IBD-associated ileal E. coli frequently manifest resistance to rifaximin that correlates with prior rifaximin use, amino acid substitutions in rpoB, and activity of PAβN-inhibitable efflux pumps, but not with the presence of ileal inflammation or E. coli phylogroup. These findings have significant implications for treatment trials targeting IBD-associated E. coli.

  6. Bacterial Multidrug Efflux Pumps of the Major Facilitator Superfamily as Targets for Modulation.

    PubMed

    Kumar, Sanath; He, Guixin; Kakarla, Prathusha; Shrestha, Ugina; Ranjana, K C; Ranaweera, Indrika; Willmon, T Mark; Barr, Sharla R; Hernandez, Alberto J; Varela, Manuel F

    2016-01-01

    Causative agents of infectious disease that are multidrug resistant bacterial pathogens represent a serious public health concern due to the increasingly difficult nature of achieving efficacious clinical treatments. Of the various acquired and intrinsic antimicrobial agent resistance determinants, integral-membrane multidrug efflux pumps of the major facilitator superfamily constitute a major mechanism of bacterial resistance. The major facilitator superfamily (MFS) encompasses thousands of known related secondary active and passive solute transporters, including multidrug efflux pumps, from bacteria to humans. This review article addresses recent developments involving the targeting by various modulators of bacterial multidrug efflux pumps from the major facilitator superfamily. It is currently of tremendous interest to modulate bacterial multidrug efflux pumps in order to eventually restore the clinical efficacy of therapeutic agents against recalcitrant bacterial infections. Such MFS multidrug efflux pumps are good targets for modulation.

  7. Effects of Efflux Transporter Genes on Susceptibility of Escherichia coli to Tigecycline (GAR-936)

    PubMed Central

    Hirata, Takahiro; Saito, Asami; Nishino, Kunihiko; Tamura, Norihisa; Yamaguchi, Akihito

    2004-01-01

    The activity of tigecycline, 9-(t-butylglycylamido)-minocycline, against Escherichia coli KAM3 (acrB) strains harboring plasmids encoding various tetracycline-specific efflux transporter genes, tet(B), tet(C), and tet(K), and multidrug transporter genes, acrAB, acrEF, and bcr, was examined. Tigecycline showed potent activity against all three Tet-expressing, tetracycline-resistant strains, with the MICs for the strains being equal to that for the host strain. In the Tet(B)-containing vesicle study, tigecycline did not significantly inhibit tetracycline efflux-coupled proton translocation and at 10 μM did not cause proton translocation. This suggests that tigecycline is not recognized by the Tet efflux transporter at a low concentration; therefore, it exhibits significant antibacterial activity. These properties can explain its potent activity against bacteria with a Tet efflux resistance determinant. Tigecycline induced the Tet(B) protein approximately four times more efficiently than tetracycline, as determined by Western blotting, indicating that it is at least recognized by a TetR repressor. The MICs for multidrug efflux proteins AcrAB and AcrEF were increased fourfold. Tigecycline inhibited active ethidium bromide efflux from intact E. coli cells overproducing AcrAB. Therefore, tigecycline is a possible substrate of AcrAB and its close homolog, AcrEF, which are resistance-modulation-division-type multicomponent efflux transporters. PMID:15155219

  8. Calcium Efflux from Internally Dialyzed Squid Giant Axons

    PubMed Central

    Dipolo, Reinaldo

    1973-01-01

    Calcium efflux has been studied in squid giant axons under conditions in which the internal composition was controlled by means of a dialysis perfusion technique. The mean calcium efflux from axons dialyzed with 0.3 µM calcium and 5 mM ATP was 0.26 pmol/cm2·s at 22°C. The curve relating the Ca efflux with the internal Ca concentration had a slope of about one for [Ca]i lower than 0.3µM and a slope smaller than one for higher concentrations. Under the above conditions replacement of [Na]o and [Ca]o by Tris and Mg causes an 80% fall in the calcium efflux. When the axons were dialyzed with a medium free of ATP and containing 2 mM cyanide plus 5µg/ml oligomycin, analysis of the perfusion effluent gave values of 1–4 µM ATP. Under this low ATP condition, replacement of external sodium and calcium causes the same drop in the calcium efflux. The same effect was observed at higher [Ca]i, (80 µM). These results suggest that the Na-Ca exchange component of the calcium efflux is apparently not dependent on the amounts of ATP in the axoplasm. Axons previously depleted of ATP show a significant transient drop in the calcium efflux when ATP is added to the dialysis medium. This effect probably represents the sequestering of calcium by the mitochondrial system. The consumption of calcium by the mitochondria of the axoplasm in dialyzed axons was determined to be of the order of 6.0 x 10-7 mol Ca++/mg of protein with an initial rate of 2.6 x 10-8 mol Ca++/min·mg of protein. Axons dialyzed with 2 mM cyanide after 8–10-min delays show a rise in the calcium efflux in the presence of "normal" amounts of exogenous ATP. This effect seems to indicate that cyanide, per se, can release calcium ions from internal sources. PMID:4751386

  9. Contribution of Efflux to the Emergence of Isoniazid and Multidrug Resistance in Mycobacterium tuberculosis

    PubMed Central

    Machado, Diana; Couto, Isabel; Perdigão, João; Rodrigues, Liliana; Portugal, Isabel; Baptista, Pedro; Veigas, Bruno; Amaral, Leonard; Viveiros, Miguel

    2012-01-01

    Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment. PMID:22493700

  10. Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis.

    PubMed

    Machado, Diana; Couto, Isabel; Perdigão, João; Rodrigues, Liliana; Portugal, Isabel; Baptista, Pedro; Veigas, Bruno; Amaral, Leonard; Viveiros, Miguel

    2012-01-01

    Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.

  11. Efflux Of Nitrate From Hydroponically Grown Wheat

    NASA Technical Reports Server (NTRS)

    Huffaker, R. C.; Aslam, M.; Ward, M. R.

    1992-01-01

    Report describes experiments to measure influx, and efflux of nitrate from hydroponically grown wheat seedlings. Ratio between efflux and influx greater in darkness than in light; increased with concentration of nitrate in nutrient solution. On basis of experiments, authors suggest nutrient solution optimized at lowest possible concentration of nitrate.

  12. A tetracycline efflux gene on Bacteroides transposon Tn4400 does not contribute to tetracycline resistance.

    PubMed Central

    Speer, B S; Salyers, A A

    1990-01-01

    Previously, we demonstrated that the Bacteroides transposon Tn4351, which confers tetracycline resistance only on aerobically grown Escherichia coli, carries a gene that codes for a tetracycline-inactivating enzyme (B. S. Speer and A. A. Salyers, J. Bacteriol. 170:1423-1429, 1988). However, Park et al. (B. H. Park, M. Hendricks, M. H. Malamy, F. P. Tally, and S. B. Levy, Antimicrob. Agents Chemother. 31:1739-1743, 1987) showed that E. coli carrying a closely related transposon, Tn4400, exhibits energy-dependent efflux of tetracycline as well as tetracycline-inactivating activity (B. H. Park and S. B. Levy, Antimicrob. Agents Chemother. 32:1797-1800, 1988). This result raised the question of whether efflux or inactivation or a combination of the two was necessary for resistance conferred by both transposons. We showed that cells carrying Tn4351 did not exhibit the clear-cut efflux activity seen with cells carrying Tn4400 but rather exhibited a tetracycline accumulation profile which could be explained solely on the basis of inactivation of tetracycline in the cytoplasm and rapid diffusion of altered tetracycline out of the cell. Additionally, we were able to clone the efflux and tetracycline-modifying genes of Tn4400 separately. The region carrying the efflux gene spanned one of the two regions in which Tn4400 differs from Tn4351. A clone containing the corresponding region of Tn4351 did not exhibit efflux. Thus, it appears that Tn4351 does not have the efflux gene and that efflux makes no contribution to the resistance conferred by Tn4351. The MIC for cells carrying the subclone from Tn4400 that contained only the gene for tetracycline inactivation was the same that for cells carrying both the inactivation and efflux genes. Cells carrying only the gene for tetracycline efflux were tetracycline sensitive. This was true even when the efflux gene was on a high-copy-number plasmid which increased the level of efflux to that associated with the Tcr gene on pBR328. These

  13. Fluid transport by the cornea endothelium is dependent on buffering lactic acid efflux.

    PubMed

    Li, Shimin; Kim, Edward; Bonanno, Joseph A

    2016-07-01

    Maintenance of corneal hydration is dependent on the active transport properties of the corneal endothelium. We tested the hypothesis that lactic acid efflux, facilitated by buffering, is a component of the endothelial fluid pump. Rabbit corneas were perfused with bicarbonate-rich (BR) or bicarbonate-free (BF) Ringer of varying buffering power, while corneal thickness was measured. Perfusate was collected and analyzed for lactate efflux. In BF with no added HEPES, the maximal corneal swelling rate was 30.0 ± 4.1 μm/h compared with 5.2 ± 0.9 μm/h in BR. Corneal swelling decreased directly with [HEPES], such that with 60 mM HEPES corneas swelled at 7.5 ± 1.6 μm/h. Perfusate [lactate] increased directly with [HEPES]. Similarly, reducing the [HCO3 (-)] increased corneal swelling and decreased lactate efflux. Corneal swelling was inversely related to Ringer buffering power (β), whereas lactate efflux was directly related to β. Ouabain (100 μM) produced maximal swelling and reduction in lactate efflux, whereas carbonic anhydrase inhibition and an monocarboxylic acid transporter 1 inhibitor produced intermediate swelling and decreases in lactate efflux. Conversely, 10 μM adenosine reduced the swelling rate to 4.2 ± 0.8 μm/h and increased lactate efflux by 25%. We found a strong inverse relation between corneal swelling and lactate efflux (r = 0.98, P < 0.0001). Introducing lactate in the Ringer transiently increased corneal thickness, reaching a steady state (0 ± 0.6 μm/h) within 90 min. We conclude that corneal endothelial function does not have an absolute requirement for bicarbonate; rather it requires a perfusing solution with high buffering power. This facilitates lactic acid efflux, which is directly linked to water efflux, indicating that lactate flux is a component of the corneal endothelial pump.

  14. Recent advances toward a molecular mechanism of efflux pump inhibition

    PubMed Central

    Opperman, Timothy J.; Nguyen, Son T.

    2015-01-01

    Multidrug resistance (MDR) in Gram-negative pathogens, such as the Enterobacteriaceae and Pseudomonas aeruginosa, poses a significant threat to our ability to effectively treat infections caused by these organisms. A major component in the development of the MDR phenotype in Gram-negative bacteria is overexpression of Resistance-Nodulation-Division (RND)-type efflux pumps, which actively pump antibacterial agents and biocides from the periplasm to the outside of the cell. Consequently, bacterial efflux pumps are an important target for developing novel antibacterial treatments. Potent efflux pump inhibitors (EPIs) could be used as adjunctive therapies that would increase the potency of existing antibiotics and decrease the emergence of MDR bacteria. Several potent inhibitors of RND-type efflux pump have been reported in the literature, and at least three of these EPI series were optimized in a pre-clinical development program. However, none of these compounds have been tested in the clinic. One of the major hurdles to the development of EPIs has been the lack of biochemical, computational, and structural methods that could be used to guide rational drug design. Here, we review recent reports that have advanced our understanding of the mechanism of action of several potent EPIs against RND-type pumps. PMID:25999939

  15. ABCG1 is involved in vitamin E efflux.

    PubMed

    Olivier, Maryline; BottG, Remain; Frisdal, Eric; Nowick, Marion; Plengpanich, Wanee; Desmarchelier, Charles; Roi, Stéphanie; Quinn, Carmel M; Gelissen, Ingrid; Jessup, Wendy; Van Eck, Miranda; Guérin, Maryse; Le Goff, Wilfried; Reboul, Emmanuelle

    2014-12-01

    Vitamin E membrane transport has been shown to involve the cholesterol transporters SR-BI, ABCA1 and NPC1L1. Our aim was to investigate the possible participation of another cholesterol transporter in cellular vitamin E efflux: ABCG1. In Abcgl-deficient mice, vitamin E concentration was reduced in plasma lipoproteins whereas most tissues displayed a higher vitamin E content compared to wild-type mice. α- and γ-tocopherol efflux was increased in CHO cells overexpressing human ABCG1 compared to control cells. Conversely, α- and γ- tocopherol efflux was decreased in ABCG1-knockdown human cells (Hep3B hepatocytes and THP-1 macro- phages). Interestingly, α- and γ-tocopherol significantly downregulated ABCG1 and ABCA1 expression levels in Hep3B and THP-1, an effect confirmed in vivo in rats given vitamin E for 5 days. This was likely due to reduced LXR activation by oxysterols, as Hep3B cells and rat liver treated with vitamin E displayed a significantly reduced content in oxysterols compared to their respective controls. Overall, the present study reveals for the first time that ABCG1 is involved in cellular vitamin E efflux.

  16. 45Ca efflux from anterior byssus retractor muscle in phasic and catch contraction.

    PubMed

    Bloomquist, E; Curtis, B A

    1975-11-01

    Phasic or catch contractions in Mytilus anterior byssus retractor muscle (ABRM) were activated by acetylcholine (ACh) and catch relaxation was initiated by 5-hydroxytryptamine (5HT). During phasic contraction and early in catch there is a brief increase in 45Ca efflux. When catch occurs, there is a subsequent drop in 45Ca efflux which then slowly recovers as catch tension declines. With catch relaxation by 5HT there is a biphasic increase in 45Ca efflux, identical to that seen when 5HT is applied to resting muscle. Compartment analyses based on the magnitude of pairs of these responses at varying times of the washout indicated that the increase in 45Ca efflux with activation originates from a compartment with the same time constant as the intermediate (80--100 min) compartment already described by previous resting efflux experiments. The decrease in 45Ca efflux during catch also involves this compartment. The increase in 45Ca efflux with 5HT originates from a more slowly exchanging Ca store with a time constant of approximately200 min.

  17. Identification of Acinetobacter baumannii serum-associated antibiotic efflux pump inhibitors.

    PubMed

    Blanchard, Catlyn; Barnett, Pamela; Perlmutter, Jessamyn; Dunman, Paul M

    2014-11-01

    Adaptive antibiotic resistance is a newly described phenomenon by which Acinetobacter baumannii induces efflux pump activity in response to host-associated environmental cues that may, in part, account for antibiotic treatment failures against clinically defined susceptible strains. To that end, during adaptation to growth in human serum, the organism induces approximately 22 putative efflux-associated genes and displays efflux-mediated minocycline tolerance at antibiotic concentrations corresponding to patient serum levels. Here, we show that in addition to minocycline, growth in human serum elicits A. baumannii efflux-mediated tolerance to the antibiotics ciprofloxacin, meropenem, tetracycline, and tigecycline. Moreover, using a whole-cell high-throughput screen and secondary assays, we identified novel serum-associated antibiotic efflux inhibitors that potentiated the activities of antibiotics toward serum-grown A. baumannii. Two compounds, Acinetobacter baumannii efflux pump inhibitor 1 (ABEPI1) [(E)-4-((4-chlorobenzylidene)amino)benezenesulfonamide] and ABEPI2 [N-tert-butyl-2-(1-tert-butyltetrazol-5-yl)sulfanylacetamide], were shown to lead to minocycline accumulation within A. baumannii during serum growth and inhibit the efflux potential of the organism. While both compounds also inhibited the antibiotic efflux properties of the bacterial pathogen Pseudomonas aeruginosa, they did not display significant cytotoxicity toward human cells or mammalian Ca(2+) channel inhibitory effects, suggesting that ABEPI1 and ABEPI2 represent promising structural scaffolds for the development of new classes of bacterial antibiotic efflux pump inhibitors that can be used to potentiate the activities of current and future antibiotics for the therapeutic intervention of Gram-negative bacterial infections.

  18. Identification of Acinetobacter baumannii Serum-Associated Antibiotic Efflux Pump Inhibitors

    PubMed Central

    Blanchard, Catlyn; Barnett, Pamela; Perlmutter, Jessamyn

    2014-01-01

    Adaptive antibiotic resistance is a newly described phenomenon by which Acinetobacter baumannii induces efflux pump activity in response to host-associated environmental cues that may, in part, account for antibiotic treatment failures against clinically defined susceptible strains. To that end, during adaptation to growth in human serum, the organism induces approximately 22 putative efflux-associated genes and displays efflux-mediated minocycline tolerance at antibiotic concentrations corresponding to patient serum levels. Here, we show that in addition to minocycline, growth in human serum elicits A. baumannii efflux-mediated tolerance to the antibiotics ciprofloxacin, meropenem, tetracycline, and tigecycline. Moreover, using a whole-cell high-throughput screen and secondary assays, we identified novel serum-associated antibiotic efflux inhibitors that potentiated the activities of antibiotics toward serum-grown A. baumannii. Two compounds, Acinetobacter baumannii efflux pump inhibitor 1 (ABEPI1) [(E)-4-((4-chlorobenzylidene)amino)benezenesulfonamide] and ABEPI2 [N-tert-butyl-2-(1-tert-butyltetrazol-5-yl)sulfanylacetamide], were shown to lead to minocycline accumulation within A. baumannii during serum growth and inhibit the efflux potential of the organism. While both compounds also inhibited the antibiotic efflux properties of the bacterial pathogen Pseudomonas aeruginosa, they did not display significant cytotoxicity toward human cells or mammalian Ca2+ channel inhibitory effects, suggesting that ABEPI1 and ABEPI2 represent promising structural scaffolds for the development of new classes of bacterial antibiotic efflux pump inhibitors that can be used to potentiate the activities of current and future antibiotics for the therapeutic intervention of Gram-negative bacterial infections. PMID:25114126

  19. Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling

    PubMed Central

    Shekhar-Guturja, Tanvi; Gunaherath, G. M. Kamal B.; Kithsiri Wijeratne, E. M.; Lambert, Jean-Philippe; Averette, Anna F.; Lee, Soo Chan; Kim, Taeyup; Bahn, Yong-Sun; Tripodi, Farida; Ammar, Ron; Döhl, Katja; Niewola-Staszkowska, Karolina; Schmitt, Lutz; Loewith, Robbie J.; Roth, Frederick P.; Sanglard, Dominique; Andes, David; Nislow, Corey; Coccetti, Paola; Gingras, Anne-Claude; Heitman, Joseph; Leslie Gunatilaka, A. A.; Cowen, Leah E.

    2016-01-01

    There is an urgent need for new strategies to treat invasive fungal infections, which are a leading cause of human mortality. We establish two activities of the natural product beauvericin, which potentiates the activity of the most widely deployed class of antifungal against the leading human fungal pathogens, blocks the emergence of drug resistance, and renders resistant pathogens responsive to treatment in mammalian infection models. Harnessing genome sequencing of beauvericin-resistant mutants, affinity purification of a biotinylated beauvericin analog, and biochemical and genetic assays reveals that beauvericin blocks multidrug efflux and inhibits the global regulator TORC1 kinase, thereby activating protein kinase CK2 and inhibiting the molecular chaperone Hsp90. Substitutions in the multidrug transporter Pdr5 that enable beauvericin efflux impair antifungal efflux, thereby impeding resistance to the drug combination. Thus, dual targeting of multidrug efflux and TOR signaling provides a powerful, broadly effective therapeutic strategy for fungal infectious disease that evades resistance. PMID:27571477

  20. Chloroquinolines block antibiotic efflux pumps in antibiotic-resistant Enterobacter aerogenes isolates.

    PubMed

    Ghisalberti, Didier; Mahamoud, Abdallah; Chevalier, Jacqueline; Baitiche, Milad; Martino, Michèle; Pagès, Jean-Marie; Barbe, Jacques

    2006-06-01

    Efflux mechanisms protect bacterial cells by pumping out toxic compounds and actively contribute to bacterial multidrug resistance. Agents inhibiting efflux pumps are of interest for the control of multidrug-resistant bacterial infections. Herein we report the effects of new chloroquinoline derivatives that render resistant Enterobacter aerogenes isolates noticeably more susceptible to structurally unrelated antibiotics. In addition, some of these chloroquinolines increase the intracellular concentration of chloramphenicol. Some of the molecules tested in this work are able to inhibit the main efflux pump (AcrAB-TolC), which is involved in E. aerogenes antibiotic resistance.

  1. Anion-coupled Na efflux mediated by the human red blood cell Na/K pump

    PubMed Central

    1990-01-01

    The red cell Na/K pump is known to continue to extrude Na when both Na and K are removed from the external medium. Because this ouabain- sensitive flux occurs in the absence of an exchangeable cation, it is referred to as uncoupled Na efflux. This flux is also known to be inhibited by 5 mM Nao but to a lesser extent than that inhibitable by ouabain. Uncoupled Na efflux via the Na/K pump therefore can be divided into a Nao-sensitive and Nao-insensitive component. We used DIDS- treated, SO4-equilibrated human red blood cells suspended in HEPES- buffered (pHo 7.4) MgSO4 or (Tris)2SO4, in which we measured 22Na efflux, 35SO4 efflux, and changes in the membrane potential with the fluorescent dye, diS-C3 (5). A principal finding is that uncoupled Na efflux occurs electroneurally, in contrast to the pump's normal electrogenic operation when exchanging Nai for Ko. This electroneutral uncoupled efflux of Na was found to be balanced by an efflux of cellular anions. (We were unable to detect any ouabain-sensitive uptake of protons, measured in an unbuffered medium at pH 7.4 with a Radiometer pH-STAT.) The Nao-sensitive efflux of Nai was found to be 1.95 +/- 0.10 times the Nao-sensitive efflux of (SO4)i, indicating that the stoichiometry of this cotransport is two Na+ per SO4=, accounting for 60-80% of the electroneutral Na efflux. The remainder portion, that is, the ouabain-sensitive Nao-insensitive component, has been identified as PO4-coupled Na transport and is the subject of a separate paper. That uncoupled Na efflux occurs as a cotransport with anions is supported by the result, obtained with resealed ghosts, that when internal and external SO4 was substituted by the impermeant anion, tartrate i,o, the efflux of Na was inhibited 60-80%. This inhibition could be relieved by the inclusion, before DIDS treatment, of 5 mM Cli,o. Addition of 10 mM Ko to tartrate i,o ghosts, with or without Cli,o, resulted in full activation of Na/K exchange and the pump's electrogenicity

  2. A multi-layer, closed-loop system for continuous measurement of CO2 concentrations and its isotopic signature in forest soils as a basis for CO2 efflux calculation and for revealing its controls

    NASA Astrophysics Data System (ADS)

    Jochheim, Hubert; Wirth, Stephan

    2015-04-01

    We present a setup of measurement devices that allows the application of the soil CO2 gradient approach for CO2 efflux calculation in combination with the analysis of isotopic signature (δ13C). Vertical profiles of CO2 concentrations in air-filled pores of soil were measured using miniature NDIR sensors within a 16-channel closed-loop system where equilibrium with soil air can be achieved using hydrophobic, gas-permeable porous polypropylene tubes circulating gas using peristaltic pumps. A 16-position multiplexer allows the connection to an isotopic CO2 analyser. This setup was applied at two ICP Forest intensive monitoring sites, a beech and a pine forest on sandy soils located in Brandenburg, Germany. CO2 concentrations in air-filled pores of soils were measured on top of soil surface, below the humus layer, and in 10cm, 20cm, 30cm and 100 cm depths every 30 min. At both sites, soil moisture and temperature were measured continuously in the respective soil depths in identical time intervals. Isotopic signatures of soil CO2 was detected by measurement campaigns. After two years of measurements, our results provided evidence for distinct seasonal dynamics and vertical gradients of soil CO2 concentration and δ13C values. Varying impacts of soil temperature and moisture on CO2 concentration were revealed, highlighting its impact on soil physical and soil biological controls. Higher levels of CO2 concentration and a more distinct seasonal dynamics were detected at the beech site compared to the pine site. The collected data provide a suitable database for calculation of CO2 efflux and modelling of soil respiration.

  3. Time lag between photosynthesis and CO2 efflux from soil

    NASA Astrophysics Data System (ADS)

    Kuzyakov, Y.; Gavrichkova, O.

    2009-04-01

    Important part of CO2 efflux from planted soils is root-derived CO2, meaning that it originates directly and indirectly from roots: directly from root respiration, and indirectly from respiration of rhizosphere microorganisms decomposing organic substances released by roots into the soil (rhizodeposits). Recent studies have shown that apart of well studied effect of soil temperature and soil water content, the C supply of assimilates from photosynthetically active plant organs have a significant effect on the root-derived CO2. In fact, the effect of photosynthesis on root-derived CO2 is often masked by temperature because root biomass typically peaks in summer. However, roots can only respire the C that was allocated belowground, and so the effect of temperature on root respiration is likely to be constrained by photosynthesis. If models of soil respiration are to incorporate photosynthetic C inputs it is necessary to understand how these two fluxes are coupled and what are the factors affecting the time lag between C uptake and its following respiration by roots and associated microorganisms. We reviewed literature and own studies relevant for estimation of the delay of C assimilation by photosynthesis and CO2 efflux from soil. The most of the studies were based on pulse labeling of annual plants in the atmosphere with 14CO2 or 13CO2 and subsequent chase of 14C or 13C in the CO2 efflux from soil. We analyzed the dynamics of the CO2 efflux curves and evaluated 3 parameters: 1) the first appearance of labeled CO2 from soil, 2) maximum of labeled CO2, and 3) disappearance of the labeled CO2 from the total CO2 efflux from soil. Numerous studies showed that newly assimilated C cycles quickly within the ecosystem, being found in root respiration already some minutes after its assimilation. Reported time lags in situ and laboratory experiments varied from minutes to days. For annual and perennial grasses the first appearance of labeled CO2 from soil was measured within

  4. Inducer expulsion in Streptococcus pyogenes: properties and mechanism of the efflux reaction

    SciTech Connect

    Sutrina, S.L.; Reizer, J.; Saier, M.H Jr.

    1988-04-01

    Expulsion of preaccumulated methyl-..beta..-D-thiogalactoside-phosphate (TMG-P) from Streptococcus pyogenes is a two-step process comprising intracellular dephosphorylation of TMG-P followed by rapid efflux of the intracellularly formed free galactoside. The present study identifies the mechanism and the order and characterizes the temperature dependency of the efflux step. Unidirectional efflux of the intracellularly formed (/sup 14/C)TMG was only slightly affected when measured in the presence of unlabeled TMG (25 to 400 mM) in the extracellular medium. In contrast, pronounced inhibition of net efflux was observed in the presence of relatively low concentrations (1 to 16 mM) of extracellular (/sup 14/C)TMG. Since net efflux was nearly arrested when the external concentration of (/sup 14/C)TMG approached the intracellular concentration of this sugar, we propose that a facilitated diffusion mechanism is responsible for efflux and equilibration of TMG between the intracellular and extracellular milieus. The exit reaction was markedly dependent upon temperature, exhibited a high energy of activation (23 kcal (ca. 96 kJ) per mol), and followed first-order kinetics, indicating that the permease mediating this efflux was not saturated under the conditions of expulsion employed.

  5. In vitro activity of levofloxacin against planktonic and biofilm Stenotrophomonas maltophilia lifestyles under conditions relevant to pulmonary infection in cystic fibrosis, and relationship with SmeDEF multidrug efflux pump expression.

    PubMed

    Pompilio, Arianna; Crocetta, Valentina; Verginelli, Fabio; Bonaventura, Giovanni Di

    2016-07-01

    The activity of levofloxacin against planktonic and biofilm Stenotrophomonas maltophilia cells and the role played by the multidrug efflux pump SmeDEF were evaluated under conditions relevant to the cystic fibrosis (CF) lung. MIC, MBC and MBEC of levofloxacin were assessed, against five CF strains, under 'standard' (CLSI-recommended) and 'CF-like' (pH 6.8, 5% CO2, in a synthetic CF sputum) conditions. Levofloxacin was tested against biofilms at concentrations (10, 50 and 100 μg mL(-1)) corresponding to achievable serum levels and sputum levels by aerosolisation. smeD expression was evaluated, under both conditions, in planktonic and biofilm cells by RT-PCR. The bactericidal effect of levofloxacin was decreased, in three out of five strains tested, under 'CF-like' conditions (MBC: 2-4 vs 8-16 μg mL(-1), under 'standard' and 'CF-like' conditions, respectively). Biofilm was intrinsically resistant to levofloxacin, regardless of conditions tested (MBECs ≥ 100 μg mL(-1) for all strains). Only under 'CF-like' conditions, smeD expression increased during planktonic-to-biofilm transition, and in biofilm cells compared to stationary planktonic cells. Our findings confirmed that S. maltophilia biofilm is intrinsically resistant to therapeutic concentrations of levofloxacin. Under conditions relevant to CF, smeD overexpression could contribute to levofloxacin resistance. Further studies are warranted to define the clinical relevance of our findings.

  6. Active optical zoom system.

    PubMed

    Wang, Di; Wang, Qiong-Hua; Shen, Chuan; Zhou, Xin; Liu, Chun-Mei

    2014-11-01

    In this work, we propose an active optical zoom system. The zoom module of the system is formed by a liquid lens and a spatial light modulator (SLM). By controlling the focal lengths of the liquid lens and the encoded digital lens on the SLM panel, we can change the magnification of an image without mechanical moving parts and keep the output plane stationary. The magnification can change from 1/3 to 3/2 as the focal length of the encoded lens on the SLM changes from infinity to 24 cm. The proposed active zoom system is simple and flexible, and has widespread application in optical communications, imaging systems, and displays.

  7. Imaging nervous system activity.

    PubMed

    Fields, Douglas R; Shneider, Neil; Mentis, George Z; O'Donovan, Michael J

    2009-10-01

    This unit describes methods for loading ion- and voltage-sensitive dyes into neurons, with a particular focus on the spinal cord as a model system. In addition, we describe the use of these dyes to visualize neural activity. Although the protocols described here concern spinal networks in culture or an intact in vitro preparation, they can be, and have been, widely used in other parts of the nervous system.

  8. Kinetics and stoichiometry of coupled Na efflux and Ca influx (Na/Ca exchange) in barnacle muscle cells

    PubMed Central

    1989-01-01

    Coupled Na+ exit/Ca2+ entry (Na/Ca exchange operating in the Ca2+ influx mode) was studied in giant barnacle muscle cells by measuring 22Na+ efflux and 45Ca2+ influx in internally perfused, ATP-fueled cells in which the Na+ pump was poisoned by 0.1 mM ouabain. Internal free Ca2+, [Ca2+]i, was controlled with a Ca-EGTA buffering system containing 8 mM EGTA and varying amounts of Ca2+. Ca2+ sequestration in internal stores was inhibited with caffeine and a mitochondrial uncoupler (FCCP). To maximize conditions for Ca2+ influx mode Na/Ca exchange, and to eliminate tracer Na/Na exchange, all of the external Na+ in the standard Na+ sea water (NaSW) was replaced by Tris or Li+ (Tris-SW or LiSW, respectively). In both Na-free solutions an external Ca2+ (Cao)-dependent Na+ efflux was observed when [Ca2+]i was increased above 10(-8) M; this efflux was half-maximally activated by [Ca2+]i = 0.3 microM (LiSW) to 0.7 microM (Tris-SW). The Cao-dependent Na+ efflux was half-maximally activated by [Ca2+]o = 2.0 mM in LiSW and 7.2 mM in Tris-SW; at saturating [Ca2+]o, [Ca2+]i, and [Na+]i the maximal (calculated) Cao-dependent Na+ efflux was approximately 75 pmol#cm2.s. This efflux was inhibited by external Na+ and La3+ with IC50's of approximately 125 and 0.4 mM, respectively. A Nai-dependent Ca2+ influx was also observed in Tris-SW. This Ca2+ influx also required [Ca2+]i greater than 10(-8) M. Internal Ca2+ activated a Nai-independent Ca2+ influx from LiSW (tracer Ca/Ca exchange), but in Tris-SW virtually all of the Cai-activated Ca2+ influx was Nai-dependent (Na/Ca exchange). Half-maximal activation was observed with [Na+]i = 30 mM. The fact that internal Ca2+ activates both a Cao-dependent Na+ efflux and a Nai- dependent Ca2+ influx in Tris-SW implies that these two fluxes are coupled; the activating (intracellular) Ca2+ does not appear to be transported by the exchanger. The maximal (calculated) Nai-dependent Ca2+ influx was -25 pmol/cm2.s. At various [Na+]i between 6 and 106 m

  9. Efflux pumps expression and its association with porin down-regulation and β-lactamase production among Pseudomonas aeruginosa causing bloodstream infections in Brazil

    PubMed Central

    2010-01-01

    Background Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile. Results Aztreonam exhibited the highest in vitro activity against the P. aeruginosa isolates studied (64.4% susceptibility), whereas susceptibility rates of imipenem and meropenem were both 47.5%. The MexXY-OprM and MexAB-OprM efflux systems were overexpressed in 50.8% and 27.1% of isolates studied, respectively. Overexpression of the MexEF-OprN and MexCD-OprJ systems was not observed. AmpC β-lactamase was overexpressed in 11.9% of P. aeruginosa isolates. In addition, decreased oprD expression was also observed in 69.5% of the whole collection, and in 87.1% of the imipenem non-susceptible P. aeruginosa clinical isolates. The MBL-encoding genes blaSPM-1 and blaIMP-1 were detected in 23.7% and 1.7% P. aeruginosa isolates, respectively. The blaGES-1 was detected in 5.1% of the isolates, while blaGES-5 and blaCTX-M-2 were observed in 1.7% of the isolates evaluated. In the present study, we have observed that efflux systems represent an adjuvant mechanism for antimicrobial resistance. Conclusions Efflux systems in association of distinct mechanisms such as the porin down-regulation, AmpC overproduction and secondary β-lactamases play also an important role in the multi-drug resistance phenotype among P. aeruginosa clinical isolates. PMID:20704733

  10. The effect of a hyposmotic shock and purinergic agonists on K+(Rb+) efflux from cultured human breast cancer cells.

    PubMed

    Gow, I F; Thomson, J; Davidson, J; Shennan, D B

    2005-06-15

    The effect of a hyposmotic shock and extracellular ATP on the efflux of K(+)(Rb(+)) from human breast cancer cell lines (MDA-MB-231 and MCF-7) has been examined. A hyposmotic shock increased the fractional efflux of K(+)(Rb(+)) from MDA-MB-231 cells via a pathway which was unaffected by Cl(-) replacement. Apamin, charybdotoxin or removing extracellular Ca(2+) had no effect on volume-activated K(+)(Rb(+)) efflux MDA-MB-231 cells. An osmotic shock also stimulated K(+)(Rb(+)) efflux from MCF-7 cells but to a much lesser extent than found with MDA-MB-231 cells. ATP-stimulated K(+)(Rb(+)) efflux from MDA-MB-231 cells in a dose-dependent fashion but had little effect on K(+)(Rb(+)) release from MCF-7 cells. ATP-stimulated K(+)(Rb(+)) efflux was only inhibited slightly by replacing Cl(-) with NO(3)(-). Removal of external Ca(2+) during treatment with ATP reduced the fractional efflux of K(+)(Rb(+)) in a manner suggesting a role for cellular Ca(2+) stores. Charybdotoxin, but neither apamin nor iberiotoxin, inhibited ATP-stimulated K(+)(Rb(+)) release from MDA-MB-231 cells. Suramin inhibited the ATP-activated efflux of K(+)(Rb(+)). UTP also stimulated K(+)(Rb(+)) efflux from MDA-MB-231 cells whereas ADP, AMP and adenosine were without effect. A combination of an osmotic shock and ATP increased the fractional efflux of K(+)(Rb(+)) to a level greater than the sum of the individual treatments. It appears that the hyposmotically-activated and ATP-stimulated K(+) efflux pathways are separate entities. However, there may be a degree of 'crosstalk' between the two pathways.

  11. The effect of NO-donors on chloride efflux, intracellular Ca(2+) concentration and mRNA expression of CFTR and ENaC in cystic fibrosis airway epithelial cells.

    PubMed

    Oliynyk, Igor; Hussain, Rashida; Amin, Ahmad; Johannesson, Marie; Roomans, Godfried M

    2013-06-01

    Since previous studies showed that the endogenous bronchodilator, S-nitrosglutathione (GSNO), caused a marked increase in CFTR-mediated chloride (Cl(-)) efflux and improved the trafficking of CFTR to the plasma membrane, and that also the nitric oxide (NO)-donor GEA3162 had a similar, but smaller, effect on Cl(-) efflux, it was investigated whether the NO-donor properties of GSNO were relevant for its effect on Cl(-) efflux from airway epithelial cells. Hence, the effect of a number of other NO-donors, sodium nitroprusside (SNP), S-nitroso-N-acetyl-DL-penicillamine (SNAP), diethylenetriamine/nitric oxide adduct (DETA-NO), and diethylenetriamine/nitric oxide adduct (DEA-NONOate) on Cl(-) efflux from CFBE (∆F508/∆F508-CFTR) airway epithelial cells was tested. Cl(-) efflux was determined using the fluorescent N-(ethoxycarbonylmethyl)-6-methoxyquinoliniu bromide (MQAE)-technique. Possible changes in the intracellular Ca(2+) concentration were tested by the fluorescent fluo-4 method in a confocal microscope system. Like previously with GSNO, after 4 h incubation with the NO-donor, an increased Cl(-) efflux was found (in the order SNAP>DETA-NO>SNP). The effect of DEA-NONOate on Cl(-) efflux was not significant, and the compound may have (unspecific) deleterious effects on the cells. Again, as with GSNO, after a short (5 min) incubation, SNP had no significant effect on Cl(-) efflux. None of the NO-donors that had a significant effect on Cl(-) efflux caused significant changes in the intracellular Ca(2+) concentration. After 4 h preincubation, SNP caused a significant increase in the mRNA expression of CFTR. SNAP and DEA-NONOate decreased the mRNA expression of all ENaC subunits significantly. DETA-NO caused a significant decrease only in α-ENaC expression. After a short preincubation, none of the NO-donors had a significant effect, neither on the expression of CFTR, nor on that of the ENaC subunits in the presence and absence of L-cysteine. It can be concluded that

  12. Active control system trends

    NASA Technical Reports Server (NTRS)

    Yore, E. E.; Gunderson, D. C.

    1976-01-01

    The active control concepts which achieve the benefit of improved mission performance and lower cost and generate system trends towards improved dynamic performance, more integration, and digital fly by wire mechanization are described. Analytical issues and implementation requirements and tools and approaches developed to address the analytical and implementation issues are briefly discussed.

  13. Production Systems. Laboratory Activities.

    ERIC Educational Resources Information Center

    Gallaway, Ann, Ed.

    This production systems guide provides teachers with learning activities for secondary students. Introductory materials include an instructional planning outline and worksheet, an outline of essential elements, domains and objectives, a course description, and a content outline. The guide contains 30 modules on the following topics: production…

  14. Communication Systems. Laboratory Activities.

    ERIC Educational Resources Information Center

    Sutherland, Barbara, Ed.

    This communication systems guide provides teachers with learning activities for secondary students. Introductory materials include an instructional planning outline and worksheet, an outline of essential elements, a list of objectives, a course description, and a content outline. The guide contains 32 modules on the following topics: story…

  15. Pyrazinoic acid efflux rate in Mycobacterium tuberculosis is a better proxy of pyrazinamide resistance.

    PubMed

    Zimic, Mirko; Fuentes, Patricia; Gilman, Robert H; Gutiérrez, Andrés H; Kirwan, Daniela; Sheen, Patricia

    2012-01-01

    Pyrazinamide is one of the most important drugs in the treatment of latent Mycobacterium tuberculosis infection. The emergence of strains resistant to pyrazinamide represents an important public health problem, as both first- and second-line treatment regimens include pyrazinamide. The accepted mechanism of action states that after the conversion of pyrazinamide into pyrazinoic acid by the bacterial pyrazinamidase enzyme, the drug is expelled from the bacteria by an efflux pump. The pyrazinoic acid is protonated in the extracellular environment and then re-enters the mycobacterium, releasing the proton and causing a lethal disruption of the membrane. Although it has been shown that mutations causing significant loss of pyrazinamidase activity significantly contribute to pyrazinamide resistance, the mechanism of resistance is not completely understood. The pyrazinoic acid efflux rate may depend on multiple factors, including pyrazinamidase activity, intracellular pyrazinamidase concentration, and the efficiency of the efflux pump. Whilst the importance of the pyrazinoic acid efflux rate to the susceptibility to pyrazinamide is recognized, its quantitative effect remains unknown. Thirty-four M. tuberculosis clinical isolates and a Mycobacterium smegmatis strain (naturally resistant to PZA) were selected based on their susceptibility to pyrazinamide, as measured by Bactec 460TB and the Wayne method. For each isolate, the initial velocity at which pyrazinoic acid is released from the bacteria and the initial velocity at which pyrazinamide enters the bacteria were estimated. The data indicated that pyrazinoic acid efflux rates for pyrazinamide-susceptible M. tuberculosis strains fell within a specific range, and M. tuberculosis strains with a pyrazinoic acid efflux rate below this range appeared to be resistant. This finding contrasts with the high pyrazinoic acid efflux rate for M. smegmatis, which is innately resistant to pyrazinamide: its pyrazinoic acid efflux

  16. Landscape of Resistance-Nodulation-Cell Division (RND)-Type Efflux Pumps in Enterobacter cloacae Complex.

    PubMed

    Guérin, François; Lallement, Claire; Isnard, Christophe; Dhalluin, Anne; Cattoir, Vincent; Giard, Jean-Christophe

    2016-04-01

    In Gram-negative bacteria, the active efflux is an important mechanism of antimicrobial resistance, but little is known about theEnterobacter cloacaecomplex (ECC). It is mediated primarily by pumps belonging to the RND (resistance-nodulation-cell division) family, and only AcrB, part of the AcrAB-TolC tripartite system, was characterized in ECC. However, detailed genome sequence analysis of the strainE. cloacaesubsp.cloacaeATCC 13047 revealed to us that 10 other genes putatively coded for RND-type transporters. We then characterized the role of all of these candidates by construction of corresponding deletion mutants, which were tested for their antimicrobial susceptibility to 36 compounds, their virulence in the invertebrateGalleria mellonellamodel of infection, and their ability to form biofilm. Only the ΔacrBmutant displayed significantly different phenotypes compared to that of the wild-type strain: 4- to 32-fold decrease of MICs of several antibiotics, antiseptics, and dyes, increased production of biofilm, and attenuated virulence inG. mellonella In order to identify specific substrates of each pump, we individually expressed intransall operons containing an RND pump-encoding gene into the ΔacrBhypersusceptible strain. We showed that three other RND-type efflux systems (ECL_00053-00055, ECL_01758-01759, and ECL_02124-02125) were able to partially restore the wild-type phenotype and to superadd to and even enlarge the broad range of antimicrobial resistance. This is the first global study assessing the role of all RND efflux pumps chromosomally encoded by the ECC, which confirms the major role of AcrB in both pathogenicity and resistance and the potential involvement of other RND-type members in acquired resistance.

  17. Landscape of Resistance-Nodulation-Cell Division (RND)-Type Efflux Pumps in Enterobacter cloacae Complex

    PubMed Central

    Guérin, François; Lallement, Claire; Isnard, Christophe; Dhalluin, Anne; Giard, Jean-Christophe

    2016-01-01

    In Gram-negative bacteria, the active efflux is an important mechanism of antimicrobial resistance, but little is known about the Enterobacter cloacae complex (ECC). It is mediated primarily by pumps belonging to the RND (resistance-nodulation-cell division) family, and only AcrB, part of the AcrAB-TolC tripartite system, was characterized in ECC. However, detailed genome sequence analysis of the strain E. cloacae subsp. cloacae ATCC 13047 revealed to us that 10 other genes putatively coded for RND-type transporters. We then characterized the role of all of these candidates by construction of corresponding deletion mutants, which were tested for their antimicrobial susceptibility to 36 compounds, their virulence in the invertebrate Galleria mellonella model of infection, and their ability to form biofilm. Only the ΔacrB mutant displayed significantly different phenotypes compared to that of the wild-type strain: 4- to 32-fold decrease of MICs of several antibiotics, antiseptics, and dyes, increased production of biofilm, and attenuated virulence in G. mellonella. In order to identify specific substrates of each pump, we individually expressed in trans all operons containing an RND pump-encoding gene into the ΔacrB hypersusceptible strain. We showed that three other RND-type efflux systems (ECL_00053-00055, ECL_01758-01759, and ECL_02124-02125) were able to partially restore the wild-type phenotype and to superadd to and even enlarge the broad range of antimicrobial resistance. This is the first global study assessing the role of all RND efflux pumps chromosomally encoded by the ECC, which confirms the major role of AcrB in both pathogenicity and resistance and the potential involvement of other RND-type members in acquired resistance. PMID:26856831

  18. A medium-throughput functional assay of KCNQ2 potassium channels using rubidium efflux and atomic absorption spectrometry.

    PubMed

    Scott, Clay W; Wilkins, Deidre E; Trivedi, Shephali; Crankshaw, Denis J

    2003-08-15

    Heterologous expression of KCNQ2 (Kv7.2) results in the formation of a slowly activating, noninactivating, voltage-gated potassium channel. Using a cell line that stably expresses KCNQ2, we developed a rubidium flux assay to measure the functional activity and pharmacological modulation of this ion channel. Rubidium flux was performed in a 96-well microtiter plate format; rubidium was quantified using an automated atomic absorption spectrometer to enable screening of 1000 data points/day. Cells accumulated rubidium at 37 degrees C in a monoexponential manner with t(1/2)=40min. Treating cells with elevated extracellular potassium caused membrane depolarization and stimulation of rubidium efflux through KCNQ2. The rate of rubidium efflux increased with increasing extracellular potassium: the t(1/2) at 50mM potassium was 5.1 min. Potassium-stimulated efflux was potentiated by the anticonvulsant drug retigabine (EC(50)=0.5 microM). Both potassium-induced and retigabine-facilitated efflux were blocked by TEA (IC(50)s=0.4 and 0.3mM, respectively) and the neurotransmitter release enhancers and putative cognition enhancers linopirdine (IC(50)s=2.3 and 7.1 microM, respectively) and XE991 (IC(50)s=0.3 and 0.9 microM, respectively). Screening a collection of ion channel modulators revealed additional inhibitors including clofilium (IC(50) = 27 microM). These studies extend the pharmacological profile of KCNQ2 and demonstrate the feasibility of using this assay system to rapidly screen for compounds that modulate the function of KCNQ2.

  19. Efflux of hydraulically lifted water from mycorrhizal fungal hyphae during imposed drought.

    PubMed

    Egerton-Warburton, Louise M; Querejeta, José Ignacio; Allen, Michael F

    2008-01-01

    Apart from improving plant and soil water status during drought, it has been suggested that hydraulic lift (HL) could enhance plant nutrient capture through the flow of mineral nutrients directly from the soil to plant roots, or by maintaining the functioning of mycorrhizal fungi. We evaluated the extent to which the diel cycle of water availability created by HL covaries with the efflux of HL water from the tips of extramatrical (external) mycorrhizal hyphae, and the possible effects on biogeochemical processes. Phenotypic mycorrhizal fungal variables, such as total and live hyphal lengths, were positively correlated with HL efflux from hyphae, soil water potential (dawn), and plant response variables (foliar (15)N). The efflux of HL water from hyphae was also correlated with bacterial abundance and soil enzyme activity (P), and the moistening of soil organic matter. Such findings indicate that the efflux of HL water from the external mycorrhizal mycelia may be a complementary explanation for plant nutrient acquisition and survival during drought.

  20. RAGE Suppresses ABCG1-Mediated Macrophage Cholesterol Efflux in Diabetes

    PubMed Central

    Daffu, Gurdip; Shen, Xiaoping; Senatus, Laura; Thiagarajan, Devi; Abedini, Andisheh; Hurtado del Pozo, Carmen; Rosario, Rosa; Song, Fei; Friedman, Richard A.; Ramasamy, Ravichandran

    2015-01-01

    Diabetes exacerbates cardiovascular disease, at least in part through suppression of macrophage cholesterol efflux and levels of the cholesterol transporters ATP binding cassette transporter A1 (ABCA1) and ABCG1. The receptor for advanced glycation end products (RAGE) is highly expressed in human and murine diabetic atherosclerotic plaques, particularly in macrophages. We tested the hypothesis that RAGE suppresses macrophage cholesterol efflux and probed the mechanisms by which RAGE downregulates ABCA1 and ABCG1. Macrophage cholesterol efflux to apolipoprotein A1 and HDL and reverse cholesterol transport to plasma, liver, and feces were reduced in diabetic macrophages through RAGE. In vitro, RAGE ligands suppressed ABCG1 and ABCA1 promoter luciferase activity and transcription of ABCG1 and ABCA1 through peroxisome proliferator–activated receptor-γ (PPARG)–responsive promoter elements but not through liver X receptor elements. Plasma levels of HDL were reduced in diabetic mice in a RAGE-dependent manner. Laser capture microdissected CD68+ macrophages from atherosclerotic plaques of Ldlr−/− mice devoid of Ager (RAGE) displayed higher levels of Abca1, Abcg1, and Pparg mRNA transcripts versus Ager-expressing Ldlr−/− mice independently of glycemia or plasma levels of total cholesterol and triglycerides. Antagonism of RAGE may fill an important therapeutic gap in the treatment of diabetic macrovascular complications. PMID:26253613

  1. ADASY (Active Daylighting System)

    NASA Astrophysics Data System (ADS)

    Vázquez-Moliní, Daniel; González-Montes, Mario; Fernández-Balbuena, Antonio Á.; Bernabéu, Eusebio; García-Botella, Ángel; García-Rodríguez, Lucas; Pohl, Wilfried

    2009-08-01

    The main objective of ADASY (Active Daylighting System) work is to design a façade static daylighting system oriented to office applications, mainly. The goal of the project is to save energy by guiding daylight into a building for lighting purpose. With this approach we can reduce the electrical load for artificial lighting, completing it with sustainable energy. The collector of the system is integrated on a vertical façade and its distribution guide is always horizontal inside of the false ceiling. ADASY is designed with a specific patent pending caption system, a modular light-guide and light extractor luminaire system. Special care has been put on the final cost of the system and its building integration purpose. The current ADASY configuration is able to illuminate 40 m2 area with a 300lx-400lx level in the mid time work hours; furthermore it has a good enough spatial uniformity distribution and a controlled glare. The data presented in this study are the result of simulation models and have been confirmed by a physical scaled prototype. ADASY's main advantages over regular illumination systems are: -Low maintenance; it has not mobile pieces and therefore it lasts for a long time and require little attention once installed. - No energy consumption; solar light continue working even if there has been a power outage. - High quality of light: the colour rendering of light is very high - Psychological benefits: People working with daylight get less stress and more comfort, increasing productivity. - Health benefits

  2. Influence of overexpression of efflux proteins on the function and gene expression of endogenous peptide transporters in MDR-transfected MDCKII cell lines

    PubMed Central

    Wang, Zhiying; Pal, Dhananjay; Patel, Ashaben; Kwatra, Deep; Mitra, Ashim K.

    2013-01-01

    The objective of this study is to delineate whether overexpression of human efflux transporters (P-gp, MRP2, and BCRP) in transfected MDCK cells affect the functional activities, and gene and protein expression of endogenous influx peptide transporter system (PepT). Real-time PCR, immunoblotting, uptake and permeability studies of [3H]Gly-Sar were conducted on transfected MDCKII and wild-type cells to investigate functional differences. Cellular [3H]Gly-Sar accumulation was significantly lower in transfected MDCKII cell lines compared to wild-type cells. Transport efficiency of apical peptide transporters was markedly reduced to around 25%, 30%, and 40% in P-gp-, MRP2-, and BCRP-overexpressed MDCK cell lines, respectively. With ascending cell-passage, transport efficiency was enhanced. A significantly higher Gly-Sar permeability was observed across parental cell-monolayers over transfected cells at all pHs. Levels of mRNA for both canine PepT1 and PepT2 were substantially reduced when efflux transporters overexpressed but enhanced when mRNA-levels of efflux genes diminished with ascending cell-passage of transfected cells. An inverse correlation was evident between endogenous PepT and exogenous efflux transporters in transfected MDCKII cells. Results of protein expression also supported these findings. Overexpression of MDR genes can affect endogenous PepT function which might be due to the phenomenon of transporter-compensation resulting in down-regulation of endogenous genes. PMID:23262422

  3. The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis

    PubMed Central

    Hürlimann, Lea M.; Corradi, Valentina; Hohl, Michael; Bloemberg, Guido V.; Tieleman, D. Peter

    2016-01-01

    Nosocomial infections with Enterococcus faecalis are an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters of E. faecalis that are annotated as drug efflux pumps. Deletion of ef0789-ef0790 on the chromosome of E. faecalis resulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context of E. faecalis. In contrast, heterologous expression in Lactococcus lactis revealed that EfrAB, EfrCD, and the product of ef2226-ef2227 (EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression in L. lactis. Since all seven transporters were purified as heterodimers after overexpression in L. lactis, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell. PMID:27381387

  4. Modifications on the hydrogen bond network by mutations of Escherichia coli copper efflux oxidase affect the process of proton transfer to dioxygen leading to alterations of enzymatic activities

    SciTech Connect

    Kajikawa, Takao; Kataoka, Kunishige; Sakurai, Takeshi

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer Proton transfer pathway to dioxygen in CueO was identified. Black-Right-Pointing-Pointer Glu506 is the key amino acid to transport proton. Black-Right-Pointing-Pointer The Ala mutation at Glu506 formed a compensatory proton transfer pathway. Black-Right-Pointing-Pointer The Ile mutation at Glu506 shut down the hydrogen bond network. -- Abstract: CueO has a branched hydrogen bond network leading from the exterior of the protein molecule to the trinuclear copper center. This network transports protons in the four-electron reduction of dioxygen. We replaced the acidic Glu506 and Asp507 residues with the charged and uncharged amino acid residues. Peculiar changes in the enzyme activity of the mutants relative to the native enzyme indicate that an acidic amino acid residue at position 506 is essential for effective proton transport. The Ala mutation resulted in the formation of a compensatory hydrogen bond network with one or two extra water molecules. On the other hand, the Ile mutation resulted in the complete shutdown of the hydrogen bond network leading to loss of enzymatic activities of CueO. In contrast, the hydrogen bond network without the proton transport function was constructed by the Gln mutation. These results exerted on the hydrogen bond network in CueO are discussed in comparison with proton transfers in cytochrome oxidase.

  5. HDL Cholesterol Efflux Capacity: Cardiovascular Risk Factor and Potential Therapeutic Target.

    PubMed

    Bhatt, Anish; Rohatgi, Anand

    2016-01-01

    Low high-density lipoprotein cholesterol (HDL-C) levels are associated with incident cardiovascular events; however, many therapies targeting increases in HDL-C have failed to show consistent clinical benefit. Thus, focus has recently shifted toward measuring high-density lipoprotein (HDL) function. HDL is the key mediator of reverse cholesterol transport, the process of cholesterol extraction from foam cells, and eventual excretion into the biliary system. Cholesterol efflux from peripheral macrophages to HDL particles has been associated with atherosclerosis in both animals and humans. We review the mechanism of cholesterol efflux and the emerging evidence on the association between cholesterol efflux capacity and cardiovascular disease in human studies. We also focus on the completed and ongoing trials of novel therapies targeting different aspects of HDL cholesterol efflux.

  6. Efficiency of N use by wheat as a function of influx and efflux of NO3

    NASA Technical Reports Server (NTRS)

    Huffaker, R. C.; Aslam, M.; Ward, M. R.

    1990-01-01

    Since N assimilation is one of the most costly functions of a plant, its efflux before assimilation results in a serious energy cost and loss in efficiency which could decrease yields. Efficient crop production is critical to the Closed Ecology Life Support System (CELSS). The objective is to determine the extent of efflux of the N species NO3(-), NH4(+), NO2(-), and urea after uptake, and possible means of regulation. Researchers found that NO3 efflux became serious as its substrate level increased. Efflux/Influx (E/I) of NO3(-) was greater in darkness (35 percent) than in light (14 percent), and the ratio greatly increased with substrate NO3 (-), (up to 45 percent at 10 mM). It seems advantageous to use the lowest possible nutrient concentration of NO3(-). The feasibility of using ClO3(-) was assessed and its toxicity determined.

  7. Old carbon efflux from tropical peat swamp drainage waters

    NASA Astrophysics Data System (ADS)

    Vihermaa, Leena; Waldron, Susan; Evers, Stephanie; Garnett, Mark; Newton, Jason

    2014-05-01

    Tropical peatlands constitute ~12% of the global peatland carbon pool, and of this 10% is in Malaysia1. Due to rising demand for food and biofuels, large areas of peat swamp forest ecosystems have been converted to plantation in Southeast Asia and are being subjected to degradation, drainage and fire, changing their carbon fluxes eg.2,3. Dissolved organic carbon (DOC) lost from disturbed tropical peat can be derived from deep within the peat column and be aged from centuries to millennia4 contributing to aquatic release and cycling of old carbon. Here we present the results of a field campaign to the Raja Musa Peat Swamp Forest Reserve in N. Selangor Malaysia, which has been selectively logged for 80 years before being granted timber reserve status. We measured CO2 and CH4efflux rates from drainage systems with different treatment history, and radiocarbon dated the evasion CO2 and associated [DOC]. We also collected water chemistry and stable isotope data from the sites. During our sampling in the dry season CO2 efflux rates ranged from 0.8 - 13.6 μmol m-2 s-1. Sediments in the channel bottom contained CH4 that appeared to be primarily lost by ebullition, leading to sporadic CH4 efflux. However, dissolved CH4 was also observed in water samples collected from these systems. The CO2 efflux was aged up to 582±37 years BP (0 BP = AD 1950) with the associated DOC aged 495±35 years BP. Both DOC and evasion CO2 were most 14C-enriched (i.e. younger) at the least disturbed site, and implied a substantial component of recently fixed carbon. In contrast, CO2 and DOC from the other sites had older 14C ages, indicating disturbance as the trigger for the loss of old carbon. 1Page et al., 2010 2Hooijer et al., 2010 3Kimberly et al., 2012 4Moore et al., 2013

  8. Rasagiline and selegiline suppress calcium efflux from mitochondria by PK11195-induced opening of mitochondrial permeability transition pore: a novel anti-apoptotic function for neuroprotection.

    PubMed

    Wu, Yuqiu; Kazumura, Kimiko; Maruyama, Wakako; Osawa, Toshihiko; Naoi, Makoto

    2015-10-01

    Rasagiline and selegiline, inhibitors of type B monoamine oxidase (MAO-B), protect neurons from cell death in cellular and animal models. Suppression of mitochondrial membrane permeabilization and subsequent activation of apoptosis cascade, and induction of anti-apoptotic, pro-survival genes are proposed to contribute the anti-apoptotic function. Rasagiline suppresses neurotoxin- and oxidative stress-induced membrane permeabilization in isolated mitochondria, but the mechanism has been not fully clarified. In this paper, regulation of the mitochondrial permeability transition pore by rasagiline and selegiline was examined in apoptosis induced by PK11195, a ligand of the outer membrane translocator protein 18 kDa (TSPO) in SH-SY5Y cells. The pore opening was quantitatively measured using a simultaneous monitoring system for calcium (Ca(2+)) and superoxide (O2(-)) (Ishibashi et al. in Biochem Biophys Res Commun 344:571-580, 2006). The association of the pore opening with Ca(2+) efflux and ROS increase was proved by the inhibition of Bcl-2 overexpression and cyclosporine A treatment. Potency to release Ca(2+) was correlated with the cytotoxicity of TSPO antagonists, PK11195, FGIN-1-27 and protoporphyrin IX, whereas a TSPO agonist, 4-chloro-diazepamine, did not significantly increase Ca(2+) or cause cell death. Rasagiline and selegiline inhibited mitochondrial Ca(2+) efflux through the mitochondrial permeability transition pore dose dependently. Ca(2+) efflux was confirmed as the initial signal in mitochondrial apoptotic cascade, and the suppression of Ca(2+) efflux may account for the neuroprotective function of rasagiline and selegiline. The quantitative measurement of Ca(2+) efflux can be applied to determine anti-apoptotic activity of neuroprotective compounds. The role of mitochondrial Ca(2+) release in neuronal death and also in neuroprotection by MAO-B inhibitors is discussed.

  9. Induction of Efflux-Mediated Macrolide Resistance in Streptococcus pneumoniae ▿

    PubMed Central

    Chancey, Scott T.; Zhou, Xiaoliu; Zähner, Dorothea; Stephens, David S.

    2011-01-01

    The antimicrobial efflux system encoded by the operon mef(E)-mel on the mobile genetic element MEGA in Streptococcus pneumoniae and other Gram-positive bacteria is inducible by macrolide antibiotics and antimicrobial peptides. Induction may affect the clinical response to the use of macrolides. We developed mef(E) reporter constructs and a disk diffusion induction and resistance assay to determine the kinetics and basis of mef(E)-mel induction. Induction occurred rapidly, with a >15-fold increase in transcription within 1 h of exposure to subinhibitory concentrations of erythromycin. A spectrum of environmental conditions, including competence and nonmacrolide antibiotics with distinct cellular targets, did not induce mef(E). Using 16 different structurally defined macrolides, induction was correlated with the amino sugar attached to C-5 of the macrolide lactone ring, not with the size (e.g., 14-, 15- or 16-member) of the ring or with the presence of the neutral sugar cladinose at C-3. Macrolides with a monosaccharide attached to C-5, known to block exit of the nascent peptide from the ribosome after the incorporation of up to eight amino acids, induced mef(E) expression. Macrolides with a C-5 disaccharide, which extends the macrolide into the ribosomal exit tunnel, disrupting peptidyl transferase activity, did not induce it. The induction of mef(E) did not require macrolide efflux, but the affinity of macrolides for the ribosome determined the availability for efflux and pneumococcal susceptibility. The induction of mef(E)-mel expression by inducing macrolides appears to be based on specific interactions of the macrolide C-5 saccharide with the ribosome that alleviate transcriptional attenuation of mef(E)-mel. PMID:21537010

  10. Evaluation of efflux pumps gene expression in resistant Pseudomonas aeruginosa isolates in an Iranian referral hospital

    PubMed Central

    Pourakbari, Babak; Yaslianifard, Sahar; Yaslianifard, Somaye; Mahmoudi, Shima; Keshavarz-Valian, Sepideh; Mamishi, Setareh

    2016-01-01

    Background and Objectives: Pseudomonas aeruginosa (PA) is one of the most important causes of nosocomial infections and has an intrinsic resistance to many antibiotics. Among all the resistance-nodulation-division (RND) pumps of P. aeruginosa, MexAB-OprM is the first efflux pump found to target multiple classes of antibiotics. This study was aimed to evaluate the expression level of genes expressing MexAB-OprM in clinical isolates of P. aeruginosa. Materials and Methods: In this study, 45 P. aeruginosa strains were isolated from patients admitted to Children’s Medical Center Hospital, an Iranian referral hospital. Disk diffusion and Minimum Inhibitory Concentration (MIC) methods were used for determination of the patterns of resistance to antibiotics. Real-time PCR was used to investigate the expression level of genes of MexAB-OprM efflux pump. Results: Among 45 resistant PA isolates, the frequency of genes overexpression was as follows: MexA (n=25, 55.5%), MexB (n=24, 53.3%) and OprM (n=16, 35.5%). In addition, in 28 strains (62%) overexpression was observed in one of the studied three genes of MexAB-OprM efflux pump. Conclusion: In our study 28 isolates (62%) had increased expression level of efflux pumps genes, MexAB-OprM. Although the efflux pumps play important roles in increasing the resistance towards different antibiotics but the role of other agents and mechanisms in evolution of resistance should not be ignored. Since the concomitant overproduction of other Mex efflux systems might have additive effects on antibiotic resistance, the co-expressing of a multicomponent efflux pump is recommended. On the other hand, the concomitant overproduction of two Mex pumps might have additive effects on resistance to antibiotic. Therefore co-expressing of Mex efflux systems is recommended. PMID:28210464

  11. Impact of repeated dry-wet cycles on soil CO2 efflux in a beech forest

    NASA Astrophysics Data System (ADS)

    Leitner, Sonja; Saronjic, Nermina; Kobler, Johannes; Holtermann, Christian; Zechmeister-Boltenstern, Sophie; Zimmermann, Michael

    2015-04-01

    Climate change research predicts that both frequency and intensity of weather extremes such as severe droughts and heavy rainfall events will increase in mid Europe over the next decades. Because soil moisture is one of the major factors controlling microbially-driven soil processes, a changed moisture regime will impact soil organic matter (SOM) decomposition and nutrient cycling. This in turn can lead to feedback effects between altered precipitation and changed soil CO2 fluxes which can intensify climate change. Soil microorganisms can go into a state of dormancy or form inactive cysts to protect themselves from osmotic stress during soil drying. However, severe droughts increase microbial mortality which slows down SOM decomposition and decreases soil CO2 efflux. The rewetting of dry soil, on the other hand, causes large CO2 emissions, which is also known as the "Birch effect". Until today it is not clear whether these CO2 peaks outweigh the drought-induced decrease of total CO2 efflux. To investigate the impact of repeated dry-wet cycles on soil CO2 efflux we are conducting a precipitation manipulation experiment in a temperate Austrian beech forest. Roofs exclude rainfall and simulate drought periods, and heavy rainfall events are simulated with a sprinkler system. We apply repeated dry-wet cycles in two intensities: one treatment receives 6 cycles of 1 month drought followed by 75mm irrigation, and a parallel treatment receives 3 cycles of 2 months drought followed by 150mm irrigation. Soil CO2 efflux is constantly monitored with an automated flux chamber system, and environmental parameters are recorded via dataloggers. Our results show that droughts significantly reduce soil CO2 effluxes, and that the reductions depend on the length of the drought periods, with longer droughts leading to stronger reductions of CO2 effluxes. In the first 24 to 48h after rewetting, CO2 emissions strongly increased, and then slowly decreased again. Soil CO2 efflux was

  12. Imaging nervous system activity.

    PubMed

    Fields, R D; O'Donovan, M J

    2001-05-01

    Optical imaging methods rely upon visualization of three types of signals: (1) intrinsic optical signals, including light scattering and reflectance, birefringence, and spectroscopic changes of intrinsic molecules, such as NADH or oxyhemoglobin; (2) changes in fluorescence or absorbance of voltage-sensitive membrane dyes; and (3) changes in fluorescence or absorbance of calcium-sensitive indicator dyes. Of these, the most widely used approach is fluorescent microscopy of calcium-sensitive dyes. This unit describes protocols for the use of calcium-sensitive dyes and voltage-dependent dyes for studies of neuronal activity in culture, tissue slices, and en-bloc preparations of the central nervous system.

  13. Neutron activation analysis system

    DOEpatents

    Taylor, M.C.; Rhodes, J.R.

    1973-12-25

    A neutron activation analysis system for monitoring a generally fluid media, such as slurries, solutions, and fluidized powders, including two separate conduit loops for circulating fluid samples within the range of radiation sources and detectors is described. Associated with the first loop is a neutron source that emits s high flux of slow and thermal neutrons. The second loop employs a fast neutron source, the flux from which is substantially free of thermal neutrons. Adjacent to both loops are gamma counters for spectrographic determination of the fluid constituents. Other gsmma sources and detectors are arranged across a portion of each loop for deterMining the fluid density. (Official Gazette)

  14. Mind the gap: non-biological processes contributing to soil CO2 efflux.

    PubMed

    Rey, Ana

    2015-05-01

    Widespread recognition of the importance of soil CO2 efflux as a major source of CO2 to the atmosphere has led to active research. A large soil respiration database and recent reviews have compiled data, methods, and current challenges. This study highlights some deficiencies for a proper understanding of soil CO2 efflux focusing on processes of soil CO2 production and transport that have not received enough attention in the current soil respiration literature. It has mostly been assumed that soil CO2 efflux is the result of biological processes (i.e. soil respiration), but recent studies demonstrate that pedochemical and geological processes, such as geothermal and volcanic CO2 degassing, are potentially important in some areas. Besides the microbial decomposition of litter, solar radiation is responsible for photodegradation or photochemical degradation of litter. Diffusion is considered to be the main mechanism of CO2 transport in the soil, but changes in atmospheric pressure and thermal convection may also be important mechanisms driving soil CO2 efflux greater than diffusion under certain conditions. Lateral fluxes of carbon as dissolved organic and inorganic carbon occur and may cause an underestimation of soil CO2 efflux. Traditionally soil CO2 efflux has been measured with accumulation chambers assuming that the main transport mechanism is diffusion. New techniques are available such as improved automated chambers, CO2 concentration profiles and isotopic techniques that may help to elucidate the sources of carbon from soils. We need to develop specific and standardized methods for different CO2 sources to quantify this flux on a global scale. Biogeochemical models should include biological and non-biological CO2 production processes before we can predict the response of soil CO2 efflux to climate change. Improving our understanding of the processes involved in soil CO2 efflux should be a research priority given the importance of this flux in the global

  15. Applicability of soil column incubation experiments to measure CO2 efflux

    NASA Astrophysics Data System (ADS)

    Guo, Linlin; Nishimura, Taku; Imoto, Hiromi; Sun, Zhigang

    2015-10-01

    Accurate measurements of CO2 efflux from soils are essential to understand dynamic changes in soil carbon storage. Column incubation experiments are commonly used to study soil water and solute transport; however, the use of column incubation experiments to study soil CO2 efflux has seldom been reported. In this study, a 150-day greenhouse experiment with two treatments (no-tillage and tillage soils) was conducted to evaluate the applicability of soil column incubation experiments to study CO2 efflux. Both the chamber measurement and the gradient method were used, and results from the two methods were consistent: tillage increased soil cumulative CO2 efflux during the incubation period. Compared with fieldwork, incubation experiments can create or precisely control experimental conditions and thus have advantages for investigating the influence of climate factors or human activities on CO2 efflux. They are superior to bottle incubation because soil column experiments maintain a soil structure that is almost the same as that in the field, and thus can facilitate analyses on CO2 behaviour in the soil profile and more accurate evaluations of CO2 efflux. Although some improvements are still required for column incubation experiments, wider application of this method to study soil CO2 behaviour is expected.

  16. Burkholderia pseudomallei resistance to antibiotics in biofilm-induced conditions is related to efflux pumps.

    PubMed

    Sirijant, Nopphasul; Sermswan, Rasana W; Wongratanacheewin, Surasakdi

    2016-11-01

    Burkholderia pseudomallei, the causative agent of melioidosis, has been found to increase its resistance to antibiotics when growing as a biofilm. The resistance is related to several mechanisms. One of the possible mechanisms is the efflux pump. Using bioinformatics analysis, it was found that BPSL1661, BPSL1664 and BPSL1665 were orthologous genes of the efflux transporter encoding genes for biofilm-related antibiotic resistance, PA1874-PA1877 genes in Pseudomonas aeruginosa strain PAO1. Expression of selected encoding genes for the efflux transporter system during biofilm formation were investigated. Real-time reverse transcriptase PCR expression of amrB, cytoplasmic membrane protein of AmrAB-OprA efflux transporter encoding gene, was slightly increased, while BPSL1665 was significantly increased during growth of bacteria in biofilm formation. Minimum biofilm inhibition concentration and minimum biofilm eradication concentration (MBEC) of ceftazidime (CTZ), doxycycline (DOX) and imipenem were found to be 2- to 1024-times increased when compared to their MICs for of planktonic cells. Inhibition of the efflux transporter by adding phenylalanine arginine β-napthylamide (PAβN), a universal efflux inhibitor, decreased 2 to 16 times as much as MBEC in B. pseudomallei biofilms with CTZ and DOX. When the intracellular accumulation of antibiotics was tested to reveal the pump inhibition, only the concentrations of CTZ and DOX increased in PAβN treated biofilm. Taken together, these results indicated that BPSL1665, a putative precursor of the efflux pump gene, might be related to the adaptation of B. pseudomallei in biofilm conditions. Inhibition of efflux pumps may lead to a decrease of resistance to CTZ and DOX in biofilm cells.

  17. /sup 3/H-imipramine uptake into rat striatal slices and imipramine-induced /sup 3/H-dopamine efflux

    SciTech Connect

    Saito, R.; Kawasaki, K.; Ono, N.; Kamiya, H.

    1983-04-01

    The effect of imipramine on spontaneous efflux of radiolabelled dopamine (DA) from slices of rat striatum was examined by a superfusion method. Imipramine at concentrations of 10 - 100 microM enhanced the efflux of DA accumulated in a high-affinity uptake system in a concentration-dependent manner. This efflux of /sup 3/H-DA was not affected by conditions (Ca/sup 2 +/-free medium, 100 microM bretylium and 30 microM tetrodotoxin) which inhibited the release of /sup 3/H-DA by electrical stimulation. Furthermore, this imipramine-induced /sup 3/H-DA efflux was temperature-dependent. The uptake of /sup 3/H-imipramine into striatal slices was determined. This uptake was concentration- and temperature-dependent and increased linearly. These results are discussed in relation to the hypothesis that /sup 3/H-DA efflux by imipramine is connected with uptake of imipramine.

  18. Top consumer abundance influences lake methane efflux.

    PubMed

    Devlin, Shawn P; Saarenheimo, Jatta; Syväranta, Jari; Jones, Roger I

    2015-11-04

    Lakes are important habitats for biogeochemical cycling of carbon. The organization and structure of aquatic communities influences the biogeochemical interactions between lakes and the atmosphere. Understanding how trophic structure regulates ecosystem functions and influences greenhouse gas efflux from lakes is critical to understanding global carbon cycling and climate change. With a whole-lake experiment in which a previously fishless lake was divided into two treatment basins where fish abundance was manipulated, we show how a trophic cascade from fish to microbes affects methane efflux to the atmosphere. Here, fish exert high grazing pressure and remove nearly all zooplankton. This reduction in zooplankton density increases the abundance of methanotrophic bacteria, which in turn reduce CH4 efflux rates by roughly 10 times. Given that globally there are millions of lakes emitting methane, an important greenhouse gas, our findings that aquatic trophic interactions significantly influence the biogeochemical cycle of methane has important implications.

  19. Effect of multidrug-efflux transporter genes on dipeptide resistance and overproduction in Escherichia coli.

    PubMed

    Hayashi, Mikiro; Tabata, Kazuhiko; Yagasaki, Makoto; Yonetani, Yoshiyuki

    2010-03-01

    L-Alanyl-L-glutamine (Ala-Gln) is a clinically and nutritionally important dipeptide. We have already shown a novel method for the fermentative production of Ala-Gln using an Escherichia coli strain expressing L-amino acid alpha-ligase (Lal), which catalyzes the formation of dipeptides by combining two amino acids. In the course of Ala-Gln-producing strain development, it was revealed that Lal expression caused growth inhibition. We also found that the addition of some dipeptides, including Ala-Gln, inhibited the growth of a multiple peptidase-deficient strain. To further increase the productivity by overcoming the inhibitory effect of dipeptides, we focused on dipeptide transport systems. The four genes (bcr, norE, ydeE and yeeO) were selected from 34 genes encoding a multidrug-efflux transporter of E. coli as those conferring resistance to growth inhibitory dipeptides. Intracellular concentration of Ala-Gln was reduced by overexpressing these genes in a multiple peptidase-deficient strain. Furthermore, overexpression of each gene in the dipeptide-producing strains resulted in the increase of Ala-Gln and L-alanyl-L-branched chain amino acids titers. These results indicate that some multidrug-efflux transporters of E. coli can transport dipeptides and that enhancement of their activities is effective for fermentative production of dipeptides.

  20. Influence of efflux pump inhibitors on the multidrug resistance of Helicobacter pylori

    PubMed Central

    Zhang, Zhan; Liu, Zhi-Qiang; Zheng, Peng-Yuan; Tang, Fu-Ai; Yang, Ping-Chang

    2010-01-01

    AIM: To evaluate the effect of efflux pump inhibitors (EPIs) on multidrug resistance of Helicobacter pylori (H. pylori). METHODS: H. pylori strains were isolated and cultured on Brucella agar plates with 10% sheep’s blood. The multidrug resistant (MDR) H. pylori were obtained with the inducer chloramphenicol by repeated doubling of the concentration until no colony was seen, then the susceptibilities of the MDR strains and their parents to 9 antibiotics were assessed with agar dilution tests. The present study included periods before and after the advent of the EPIs, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), reserpine and pantoprazole), and the minimum inhibitory concentrations (MICs) were determined accordingly. In the same way, the effects of 5 proton pump inhibitors (PPIs), used in treatment of H. pylori infection, on MICs of antibiotics were evaluated. RESULTS: Four strains of MDR H. pylori were induced successfully, and the antibiotic susceptibilities of MDR strains were partly restored by CCCP and pantoprazole, but there was little effect of reserpine. Rabeprazole was the most effective of the 5 PPIs which could decrease the MICs of antibiotics for MDR H. pylori significantly. CONCLUSION: In vitro, some EPIs can strengthen the activities of different antibiotics which are the putative substrates of the efflux pump system in H. pylori. PMID:20222174

  1. Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump

    SciTech Connect

    Abdali, Narges; Parks, Jerry M.; Haynes, Keith M.; Chaney, Julie L.; Green, Adam T.; Wolloscheck, David; Walker, John K.; Rybenkov, Valentin V.; Baudry, Jerome; Smith, Jeremy C.; Zgurskaya, Helen I.

    2016-10-21

    Antibiotic resistance is a major threat to human welfare. Inhibitors of multidrug efflux pumps (EPIs) are promising alternative therapeutics that could revive activities of antibiotics and reduce bacterial virulence. Identification of new druggable sites for inhibition is critical for developing effective EPIs, especially in light of constantly emerging resistance. We describe new EPIs that interact with and possibly inhibit the function of periplasmic membrane fusion proteins, critical components of efflux pumps that are responsible for the activation of the transporter and the recruitment of the outer-membrane channel. The discovered EPIs bind to AcrA, a component of the prototypical AcrAB-TolC pump, change its structure in vivo, inhibit efflux of fluorescent probes and potentiate the activities of antibiotics in Escherichia coli cells. These findings expand the chemical and mechanistic diversity of EPIs, suggest the mechanism for regulation of the efflux pump assembly and activity, and provide a promising path for reviving the activities of antibiotics in resistant bacteria.

  2. Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump

    DOE PAGES

    Abdali, Narges; Parks, Jerry M.; Haynes, Keith M.; ...

    2016-10-21

    Antibiotic resistance is a major threat to human welfare. Inhibitors of multidrug efflux pumps (EPIs) are promising alternative therapeutics that could revive activities of antibiotics and reduce bacterial virulence. Identification of new druggable sites for inhibition is critical for developing effective EPIs, especially in light of constantly emerging resistance. We describe new EPIs that interact with and possibly inhibit the function of periplasmic membrane fusion proteins, critical components of efflux pumps that are responsible for the activation of the transporter and the recruitment of the outer-membrane channel. The discovered EPIs bind to AcrA, a component of the prototypical AcrAB-TolC pump,more » change its structure in vivo, inhibit efflux of fluorescent probes and potentiate the activities of antibiotics in Escherichia coli cells. These findings expand the chemical and mechanistic diversity of EPIs, suggest the mechanism for regulation of the efflux pump assembly and activity, and provide a promising path for reviving the activities of antibiotics in resistant bacteria.« less

  3. On the physics of multidrug efflux through a biomolecular complex

    NASA Astrophysics Data System (ADS)

    Mishima, Hirokazu; Oshima, Hiraku; Yasuda, Satoshi; Amano, Ken-ichi; Kinoshita, Masahiro

    2013-11-01

    Insertion and release of a solute into and from a vessel comprising biopolymers is a fundamental function in a biological system. A typical example is found in a multidrug efflux transporter. "Multidrug efflux" signifies that solutes such as drug molecules with diverse properties can be handled. In our view, the mechanism of the multidrug efflux is not chemically specific but rather has to be based on a physical factor. In earlier works, we showed that the spatial distribution of the solute-vessel potential of mean force (PMF) induced by the solvent plays imperative roles in the insertion/release process. The PMF can be decomposed into the energetic and entropic components. The entropic component, which originates from the translational displacement of solvent molecules, is rather insensitive to the solute-solvent and vessel inner surface-solvent affinities. This feature is not shared with the energetic component. When the vessel inner surface is neither solvophobic nor solvophilic, the solvents within the vessel cavity and in the bulk offer almost the same environment to any solute with solvophobicity or solvophilicity, and the energetic component becomes much smaller than the entropic component (i.e., the latter predominates over the former). Our idea is that the multidrug efflux can be realized if the insertion/release process is accomplished by the entropic component exhibiting the insensitivity to the solute properties. However, we have recently argued that the entropic release of the solute is not feasible as long as the vessel geometry is fixed. Here we consider a model of TolC, a cylindrical vessel possessing an entrance at one end and an exit at the other end for the solute. The spatial distribution of the PMF is calculated by employing the three-dimensional integral equation theory with rigid-body models in which the constituents interact only through hard-body potentials. Since the behavior of these models is purely entropic in origin, our analysis is

  4. Arctigenin promotes cholesterol efflux from THP-1 macrophages through PPAR-γ/LXR-α signaling pathway

    SciTech Connect

    Xu, Xiaolin; Li, Qian; Pang, Liewen; Huang, Guoqian; Huang, Jiechun; Shi, Meng; Sun, Xiaotian; Wang, Yiqing

    2013-11-15

    Highlights: •Arctigenin enhanced cholesterol efflux in oxLDL-loaded THP-1 macrophages. •The expression of ABCA1, ABCG1 and apoE was upregulated in arctigenin-treated cells. •Arctigenin promoted the expression of PPAR-γ and LXR-α. •Inhibition of PPAR-γ or LXR-α reversed arctigenin-mediated biological effects. •Arctigenin promotes cholesterol efflux via activation of PPAR-γ/LXR-α/ABCA1 pathway. -- Abstract: Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α.

  5. Efflux inhibition with verapamil potentiates bedaquiline in Mycobacterium tuberculosis.

    PubMed

    Gupta, Shashank; Cohen, Keira A; Winglee, Kathryn; Maiga, Mamoudou; Diarra, Bassirou; Bishai, William R

    2014-01-01

    Drug efflux is an important resistance mechanism in Mycobacterium tuberculosis. We found that verapamil, an efflux inhibitor, profoundly decreases the MIC of bedaquiline and clofazimine to M. tuberculosis by 8- to 16-fold. This exquisite susceptibility was noted among drug-susceptible and drug-resistant clinical isolates. Thus, efflux inhibition is an important sensitizer of bedaquiline and clofazimine, and efflux may emerge as a resistance mechanism to these drugs.

  6. Sequential estrogen-progestin replacement therapy in healthy postmenopausal women: effects on cholesterol efflux capacity and key proteins regulating high-density lipoprotein levels.

    PubMed

    Ulloa, Natalia; Arteaga, Eugenio; Bustos, Paulina; Durán-Sandoval, Daniel; Schulze, Kim; Castro, Graciela; Jauhiainen, Matti; Fruchart, Jean Charles; Calvo, Carlos

    2002-11-01

    Thirty healthy postmenopausal women were randomized into 2 groups that received a sequential combined hormone-replacement therapy (HRT) (n = 18; conjugated equine estrogen 0.625 mg/d for 28 days and 5 mg of medroxyprogesterone acetate during the last 14 days) or placebo (n = 12). Plasma samples were collected before and during treatment (days 0, 15, 43, 71). High-density lipoprotein (HDL) lipid content, lipoprotein (Lp)A-I and LpA-I:LpA-II concentration, lecithin:cholesterol acyl transferase activity (LCAT), phospholipid transfer protein (PLTP) activity, and the plasma capacity to carry out cholesterol efflux from Fu5AH cells were measured. Most significant changes were found within the first 15 days after HRT. After 71 days of HRT, we found an increase in LpA-I lipoparticles (27%) and the following HDL lipids: phospholipids (21%), triglycerides (45%), and free cholesterol (43%), as well as an increase in cholesterol efflux (12.5%). PLTP activity, on the other hand, decreased 21% after 71 days of treatment. No significant changes in LCAT activity, HDL-cholesterol ester or LpA-I:LpA-II particles were found. Positive correlation between cholesterol efflux and the variables LpA-I and HDL-phospholipids were observed. PLTP was negatively correlated with apolipoprotein (apo) A-I, LpA-I, and LpA-I:LpA-II. In summary, our study, performed during 3 hormonal cycles, shows that HRT not only modifies HDL-cholesterol level, but also its lipid composition and HDL lipoparticle distribution. HRT enhances the plasma capacity to carry out cholesterol efflux from the Fu5AH system and decreases the activity of PLTP, a key protein regulating HDL levels. Considering the protocol sampling, these results represent mainly the estrogenic effect of HRT.

  7. CO2 Efflux from Cleared Mangrove Peat

    PubMed Central

    Lovelock, Catherine E.; Ruess, Roger W.; Feller, Ilka C.

    2011-01-01

    Background CO2 emissions from cleared mangrove areas may be substantial, increasing the costs of continued losses of these ecosystems, particularly in mangroves that have highly organic soils. Methodology/Principal Findings We measured CO2 efflux from mangrove soils that had been cleared for up to 20 years on the islands of Twin Cays, Belize. We also disturbed these cleared peat soils to assess what disturbance of soils after clearing may have on CO2 efflux. CO2 efflux from soils declines from time of clearing from ∼10 600 tonnes km−2 year−1 in the first year to 3000 tonnes km2 year−1 after 20 years since clearing. Disturbing peat leads to short term increases in CO2 efflux (27 umol m−2 s−1), but this had returned to baseline levels within 2 days. Conclusions/Significance Deforesting mangroves that grow on peat soils results in CO2 emissions that are comparable to rates estimated for peat collapse in other tropical ecosystems. Preventing deforestation presents an opportunity for countries to benefit from carbon payments for preservation of threatened carbon stocks. PMID:21738628

  8. Modified host cells with efflux pumps

    DOEpatents

    Dunlop, Mary J.; Keasling, Jay D.; Mukhopadhyay, Aindrila

    2016-08-30

    The present invention provides for a modified host cell comprising a heterologous expression of an efflux pump capable of transporting an organic molecule out of the host cell wherein the organic molecule at a sufficiently high concentration reduces the growth rate of or is lethal to the host cell.

  9. The EmhABC efflux pump decreases the efficiency of phenanthrene biodegradation by Pseudomonas fluorescens strain LP6a.

    PubMed

    Adebusuyi, Abigail A; Smith, Angela Y; Gray, Murray R; Foght, Julia M

    2012-08-01

    Pseudomonas fluorescens strain LP6a, designated here as strain WEN (wild-type PAH catabolism, efflux positive), utilizes the polycyclic aromatic hydrocarbon phenanthrene as a carbon source but also extrudes it into the extracellular medium using the efflux pump EmhABC. Because phenanthrene is considered a nontoxic carbon source for P. fluorescens WEP, its energy-dependent efflux seems counter-productive. We hypothesized that the efflux of phenanthrene would decrease the efficiency of its biodegradation. Indeed, an emhB disruptant strain, wild-type PAH catabolism, efflux negative (WEN), biodegraded 44% more phenanthrene than its parent strain WEP during a 6-day incubation. To determine whether efflux affected the degree of oxidation of phenanthrene, we quantified the conversion of ¹⁴C-phenanthrene to radiolabeled polar metabolites and ¹⁴CO₂. The emhB⁻ WEN strain produced approximately twice as much ¹⁴CO₂ and radiolabeled water-soluble metabolites as the WEP strain. In contrast, the mineralization of ¹⁴C-glucose, which is not a known EmhB efflux substrate, was equivalent in both strains. An early open-ring metabolite of phenanthrene, trans-4-(1-hydroxynaphth-2-yl)-2-oxo-3-butenoic acid, also was found to be a substrate of the EmhABC pump and accumulated in the supernatant of WEP but not WEN cultures. The analogous open-ring metabolite of dibenzothiophene, a heterocyclic analog of phenanthrene, was extruded by EmhABC plus a putative alternative efflux pump, whereas the end product 3-hydroxy-2-formylbenzothiophene was not actively extruded from either WEP or WEN cells. These results indicate that the active efflux of phenanthrene and its early metabolite(s) decreases the efficiency of phenanthrene degradation by the WEP strain. This activity has implications for the bioremediation and biocatalytic transformation of polycyclic aromatic hydrocarbons and heterocycles.

  10. Peptides having reduced toxicity that stimulate cholesterol efflux

    DOEpatents

    Bielicki, John K.; Johansson, Jan; Danho, Waleed

    2016-08-16

    The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABCA1 that parallels that of full-length apolipoproteins. Further, the peptides of the invention have little or no toxicity when administered at therapeutic and higher doses. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

  11. Technology Systems. Laboratory Activities.

    ERIC Educational Resources Information Center

    Brame, Ray; And Others

    This guide contains 43 modules of laboratory activities for technology education courses. Each module includes an instructor's resource sheet and the student laboratory activity. Instructor's resource sheets include some or all of the following elements: module number, course title, activity topic, estimated time, essential elements, objectives,…

  12. Functional and Genetic Characterization of the Tap Efflux Pump in Mycobacterium bovis BCG

    PubMed Central

    Mick, Virginie; Dainese, Elisa; Martín, Carlos; Thompson, Charles J.; De Rossi, Edda; Manganelli, Riccardo; Aínsa, José A.

    2012-01-01

    Efflux pumps extrude a wide variety of chemically unrelated compounds conferring multidrug resistance and participating in numerous physiological processes. Mycobacterium tuberculosis possesses many efflux pumps, and their roles in drug resistance and physiology are actively investigated. In this work we found that tap mutant cells showed changes in morphology and a progressive loss of viability upon subcultivation in liquid medium. Transcriptome analysis in Mycobacterium bovis BCG revealed that disruption of the Rv1258c gene, encoding the Tap efflux pump, led to an extensive change in gene expression patterns during stationary phase, with no changes during exponential growth. In stationary phase, Tap inactivation triggered a general stress response and led to a general repression of genes involved in cell wall biosynthesis, in particular the formation of the peptidoglycan; this suggested the accumulation of an unknown Tap substrate that reaches toxic concentrations during stationary phase. We also found that both disruption and overexpression of tap altered susceptibility to many clinically approved antibiotics in M. bovis BCG. Acriflavine and tetracycline accumulation assays and carbonyl cyanide m-chlorophenylhydrazone (CCCP) potentiation experiments demonstrated that this phenotype was due to an active efflux mechanism. These findings emphasize the important role of the Tap efflux pump in bacterial physiology and intrinsic drug resistance. PMID:22232275

  13. Contribution of the Ade Resistance-Nodulation-Cell Division-Type Efflux Pumps to Fitness and Pathogenesis of Acinetobacter baumannii

    PubMed Central

    Yoon, Eun-Jeong; Balloy, Viviane; Fiette, Laurence; Chignard, Michel; Courvalin, Patrice

    2016-01-01

    ABSTRACT Overexpression of chromosomal resistance-nodulation-cell division (RND)-type efflux systems with broad substrate specificity contributes to multidrug resistance (MDR) in Acinetobacter baumannii. We have shown that modulation of expression of the structural genes for the efflux systems AdeABC and AdeIJK confers MDR and results in numerous alterations of membrane-associated cellular functions, in particular biofilm formation. However, the contribution of these RND pumps to cell fitness and virulence has not yet been studied. The biological cost of an antibiotic resistance mechanism is a key parameter in determining its stability and dissemination. From an entirely sequenced susceptible clinical isolate, we have generated a set of isogenic derivatives having single point mutations resulting in overexpression of each efflux system or with every pump deleted by allelic replacement. We found that overproduction of the pumps results in a significant decrease in fitness of the bacterial host when measured by competition experiments in vitro. Fitness and virulence were also evaluated in vivo both in systemic and pulmonary infection models in immunocompetent mice. A diminished competitiveness of the AdeABC-overexpressing mutant was observed only after intraperitoneal inoculation, but not after intranasal inoculation, the latter mimicking the most frequent type of human A. baumannii infection. However, in mice infected intranasally, this mutant was more virulent and stimulated an enhanced neutrophil activation in the lungs. Altogether, these data account for the observation that adeABC overexpression is common in MDR A. baumannii frequently found in ventilator-associated pneumonia. PMID:27247231

  14. Activation of ion transport systems during cell volume regulation

    SciTech Connect

    Eveloff, J.L.; Warnock, D.G.

    1987-01-01

    This review discusses the activation of transport pathways during volume regulation, including their characteristics, the possible biochemical pathways that may mediate the activation of transport pathways, and the relations between volume regulation and transepithelial transport in renal cells. Many cells regulate their volume when exposed to an anisotonic medium. The changes in cell volume are caused by activation of ion transport pathways, plus the accompanying osmotically driven water movement such that cell volume returns toward normal levels. The swelling of hypertonically shrunken cells is termed regulatory volume increase (RVI) and involves an influx of NaCl into the cell via either activation of Na-Cl, Na-K-2Cl cotransport systems, or Na/sup +/-H/sup +/ and Cl/sup -/-HCO/sub 3//sup -/ exchangers. The reshrinking of hypotonically swollen cells is termed regulatory volume decrease (RVD) and involves an efflux of KCl and water from the cell by activation of either separate K/sup +/ and Cl/sup -/ conductances, a K-Cl cotransport system, or parallel K/sup +/-H/sup +/ and Cl/sup -/-HCO/sub 3//sup -/ exchangers. The biochemical mechanisms involved in the activation of transport systems are largely unknown, however, the phosphoinositide pathway may be implicated in RVI; phorbol esters, cGMP, and Ca/sup 2 +/ affect the process of volume regulation. Renal tubular cells, as well as the blood cells that transverse the medulla, are subjected to increasing osmotic gradients from the corticomedullary junction to the papillary tip, as well as changing interstitial and tubule fluid osmolarity, depending on the diuretic state of the animal. Medullary cells from the loop of Henle and the papilla can volume regulate by activating Na-K-2Cl cotransport or Na/sup +/-H/sup +/ and Cl/sup -/-HCO/sub 3//sup -/ exchange systems.

  15. Interaction of gatifloxacin with efflux transporters: a possible mechanism for drug resistance

    PubMed Central

    Kwatra, Deep; Vadlapatla, Ramya Krishna; Vadlapudi, Aswani Dutt; Pal, Dhananjay; Mitra, Ashim K.

    2010-01-01

    The purpose of the study is to screen the interactions of fourth generation fluoroquinolone-gatifloxacin with efflux pumps i.e. P-gp, MRP2 and BCRP. Mechanism of gatifloxacin interaction with efflux transporters may explain its acquired resistance. Such clarification may lead to the development of strategies to overcome efflux and enhance its bioavailability at target site. This process will aid in the reduction of dose volume, further eliminating the chances of systemic toxicity from topical gatifloxacin eye drops. MDCK cell lines transfected with the targeted efflux transporters were used for this study. [14C] Erythromycin was selected as a model substrate for P-gp and MRP2 whereas Hoechst 33342 was employed as a substrate for BCRP. Uptake and transport studies of these substrates were performed in the presence of gatifloxacin to delineate its interaction with efflux transporters. Further the efflux ratio in the presence of gatifloxacin was calculated from bidirectional transport studies. The concentration of [14C] erythromycin and Hoechst 33342 were measured using scintillation counter and fluorescence plate reader respectively. A concentration dependent inhibition effect in the presence of gatifloxacin was revealed on [14C] erythromycin uptake. The efflux ratio (BL-AP/AP-BL) of substrates was found to approach unity at higher gatifloxacin concentrations. Increased concentration of gatifloxacin did not elevate uptake of Hoechst 33342. All these studies were validated with known inhibitors as positive control. Uptake and transport studies support the hypothesis that gatifloxacin is a substrate for P-gp, MRP2 but not for BCRP. Possible interactions of gatifloxacin with P-gp and MRP2 may be a possible mechanism for acquired resistance of gatifloxacin. This information can be further extended to design prodrugs or formulations in order to prevent development of acquired resistance and improve therapeutic efficacy with its reduction in side effects. PMID:20573570

  16. Contribution of the biological crust to the soil CO2 efflux in a Mediterranean ecosystem

    NASA Astrophysics Data System (ADS)

    Morillas, Lourdes; Bellucco, Veronica; Lo Cascio, Mauro; Marras, Serena; Spano, Donatella; Mereu, Simone

    2016-04-01

    Lately, the important role of the soil biological crust (hereafter biocrust) in Mediterranean ecosystems is emerging from a multitude of articles. It is becoming apparent that the biocrust has an important role in regulating ecosystem functions and that it interacts with the woody and herbaceous vegetation to a degree depending on the availability of water among other factors. Here we present the first results of a wider project and focus on the contribution of the biocrust to soil CO2 efflux, and on how the respiration of the biocrust responds to soil water content and temperature. A manipulative experiment was performed in a Mediterranean shrubland ecosystem in Sardinia (Italy) to assess the contribution of the bicocrust to soil CO2 efflux and to identify the main environmental drivers of the CO2 efflux in this ecosystem. For 19 months,in situ soil CO2 efflux was measured over three different surfaces: soil deprived of biocrust (hereafter Soil), biocrust (hereafter BC) and intact soil (hereafter Soil+BC). For these surfaces, three different approaches were used to investigate the dependency of CO2 efflux on soil temperature and soil water content, e.g. a simple linear regression, a multi-linear equation, and a modified version of the most common used Lloyd and Taylor model (Lloyd and Taylor, 1994). Results showed that CO2 effluxes emitted by Soil, BC and Soil+BC were differently driven by soil moisture and temperature: BC respiration was mainly controlled by soil moisture at 5 cm depth, whereas both soil temperature and water content at 20 cm depth determined Soil CO2 efflux. Soil temperature and water content at 5 cm depth drove Soil+BC respiration. We also found that biocrust can contribute substantially (up to 60%) to the total soil respiration depending on its moisture content. This contribution persists even in periods in which deeper soil layers are inactive, as small water pulses can activate lichens, mosses and cyanobacteria associated to the biocrust as

  17. Facies and depositional architecture according to a jet efflux model of a late Paleozoic tidewater grounding-line system from the Itararé Group (Paraná Basin), southern Brazil

    NASA Astrophysics Data System (ADS)

    Aquino, Carolina Danielski; Buso, Victoria Valdez; Faccini, Ubiratan Ferrucio; Milana, Juan Pablo; Paim, Paulo Sergio Gomes

    2016-04-01

    -laden nature of the flows. At the transitional zone, a hydraulic jump produces a rapid shift of conglomeratic to sandy facies with associated scour features. Towards the distal zones, the jet detaches to originate a vertical turbulent jet characterized by more diluted flows. Discussion of fan facies and architecture within a framework of jet-efflux dynamics provides an improved understanding of grounding-line fans systems that produce coarse-grained strata commonly enclosed by fine-grained rocks. The results have clear implication in terms of prediction of facies tract and geometry of oil and gas reservoirs deposited under similar conditions. And also can be useful to identifying the position of a glacial terminus through time.

  18. Baicalin promotes cholesterol efflux by regulating the expression of SR-BI in macrophages

    PubMed Central

    Yu, Renchao; Lv, Yuexia; Wang, Juanling; Pan, Nana; Zhang, Rui; Wang, Xiaxia; Yu, Haichu; Tan, Lijuan; Zhao, Yunhe; Li, Bo

    2016-01-01

    Intake of a high dosage of baicalin has previously been shown to attenuate hyperlipidemia induced by a high-fat diet. Baicalin functions as an activator of peroxisome proliferator-activated receptor-γ (PPAR-γ), which is the key regulator of reverse cholesterol transport (RCT). The present study aimed to test the hypothesis that baicalin could promote cholesterol efflux in macrophages through activating PPAR-γ. Phorbol 12-myristate 13-acetate-stimulated THP-1 cells were treated with oxidized low-density lipoprotein and (3H)-cholesterol for 24 h, and the effects of baicalin on cholesterol efflux were evaluated in the presence of apolipoprotein A-1 (ApoA-1), or high-density lipoprotein subfraction 2 (HDL2) or subfraction 3 (HDL3). The expression levels of scavenger receptor class B type I (SR-BI), PPAR-γ and liver X receptor-α (LXRα) were detected and specific inhibitors or activators of SR-BI, PPAR-γ and LXRα were applied to investigate the mechanism. Treatment of THP-1 macrophages with baicalin significantly accelerated HDL-mediated, but not ApoA-1-mediated cholesterol efflux. However, baicalin treatment increased the expression of SR-BI at the mRNA and protein levels in a dose- and time-dependent manner, and pre-treatment with the SR-BI inhibitor BLT-1 and SR-BI small interfering RNA significantly inhibited baicalin-induced cholesterol efflux. Furthermore, baicalin increased the expression of PPAR-γ and LXRα, and the application of specific agonists and inhibitors of PPAR-γ and LXRα changed the expression of SR-BI, as well as cholesterol efflux. It may be concluded that baicalin induced cholesterol efflux from THP-1 macrophages via the PPAR-γ/LXRα/SR-BI pathway. PMID:28105139

  19. Baicalin promotes cholesterol efflux by regulating the expression of SR-BI in macrophages.

    PubMed

    Yu, Renchao; Lv, Yuexia; Wang, Juanling; Pan, Nana; Zhang, Rui; Wang, Xiaxia; Yu, Haichu; Tan, Lijuan; Zhao, Yunhe; Li, Bo

    2016-12-01

    Intake of a high dosage of baicalin has previously been shown to attenuate hyperlipidemia induced by a high-fat diet. Baicalin functions as an activator of peroxisome proliferator-activated receptor-γ (PPAR-γ), which is the key regulator of reverse cholesterol transport (RCT). The present study aimed to test the hypothesis that baicalin could promote cholesterol efflux in macrophages through activating PPAR-γ. Phorbol 12-myristate 13-acetate-stimulated THP-1 cells were treated with oxidized low-density lipoprotein and ((3)H)-cholesterol for 24 h, and the effects of baicalin on cholesterol efflux were evaluated in the presence of apolipoprotein A-1 (ApoA-1), or high-density lipoprotein subfraction 2 (HDL2) or subfraction 3 (HDL3). The expression levels of scavenger receptor class B type I (SR-BI), PPAR-γ and liver X receptor-α (LXRα) were detected and specific inhibitors or activators of SR-BI, PPAR-γ and LXRα were applied to investigate the mechanism. Treatment of THP-1 macrophages with baicalin significantly accelerated HDL-mediated, but not ApoA-1-mediated cholesterol efflux. However, baicalin treatment increased the expression of SR-BI at the mRNA and protein levels in a dose- and time-dependent manner, and pre-treatment with the SR-BI inhibitor BLT-1 and SR-BI small interfering RNA significantly inhibited baicalin-induced cholesterol efflux. Furthermore, baicalin increased the expression of PPAR-γ and LXRα, and the application of specific agonists and inhibitors of PPAR-γ and LXRα changed the expression of SR-BI, as well as cholesterol efflux. It may be concluded that baicalin induced cholesterol efflux from THP-1 macrophages via the PPAR-γ/LXRα/SR-BI pathway.

  20. Inhibition of the multidrug efflux pump LmrS from Staphylococcus aureus by cumin spice Cuminum cyminum.

    PubMed

    Kakarla, Prathusha; Floyd, Jared; Mukherjee, MunMun; Devireddy, Amith R; Inupakutika, Madhuri A; Ranweera, Indrika; Kc, Ranjana; 'Shrestha, Ugina; Cheeti, Upender Rao; Willmon, Thomas Mark; Adams, Jaclyn; Bruns, Merissa; Gunda, Shravan Kumar; Varela, Manuel F

    2017-04-01

    Staphylococcus aureus is a serious causative agent of infectious disease. Multidrug-resistant strains like methicillin-resistant S. aureus compromise treatment efficacy, causing significant morbidity and mortality. Active efflux represents a major antimicrobial resistance mechanism. The proton-driven multidrug efflux pump, LmrS, actively exports structurally distinct antimicrobials. To circumvent resistance and restore clinical efficacy of antibiotics, efflux pump inhibitors are necessary, and natural edible spices like cumin are potential candidates. The mode of cumin antibacterial action and underlying mechanisms behind drug resistance inhibition, however, are unclear. We tested the hypothesis that cumin inhibits LmrS drug transport. We found that cumin inhibited bacterial growth and LmrS ethidium transport in a dosage-dependent manner. We demonstrate that cumin is antibacterial toward a multidrug-resistant host and that resistance modulation involves multidrug efflux inhibition.

  1. Multidrug Efflux Pumps Attenuate the Effect of MGMT Inhibitors.

    PubMed

    Tomaszowski, Karl-Heinz; Schirrmacher, Ralf; Kaina, Bernd

    2015-11-02

    Various mechanisms of drug resistance attenuate the effectiveness of cancer therapeutics, including drug transport and DNA repair. The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is a key factor determining the resistance against alkylating anticancer drugs inducing the genotoxic DNA lesions O(6)-methylguanine and O(6)-chloroethylguanine, and MGMT inactivation or depletion renders cells more susceptible to treatment with methylating and chloroethylating agents. Highly specific and efficient inhibitors of the repair protein MGMT were designed, including O(6)-benzylguanine (O(6)BG) and O(6)-(4-bromothenyl)guanine (O(6)BTG) that are nontoxic on their own. Unfortunately, these inhibitors do not select between MGMT in normal and cancer cells, causing nontarget effects in the healthy tissue. Therefore, a targeting strategy for MGMT inhibitors is required. Here, we used O(6)BG and O(6)BTG conjugated to β-d-glucose (O(6)BG-Glu and O(6)BTG-Glu, respectively) in order to selectively inhibit MGMT in tumors, harnessing their high demand for glucose. Both glucose conjugates efficiently inhibited MGMT in several cancer cell lines, but with different extents of sensitization to DNA alkylating agents, with lomustine being more effective than temozolomide. We further show that the glucose conjugates are subject to ATP-binding cassette (ABC) transporter mediated efflux, involving P-glycoprotein, MRP1, and BCRP, which impacts the efficiency of MGMT inhibition. Surprisingly, also O(6)BG and O(6)BTG were subject to an active transport out of the cell. We also show that pharmacological inhibition of efflux transporters increases the induction of cell death following treatment with these MGMT inhibitors and temozolomide. We conclude that strategies of attenuating the efflux by ABC transporters are required for achieving successful MGMT targeting.

  2. Genomic potential for arsenic efflux and methylation varies among global Prochlorococcus populations.

    PubMed

    Saunders, Jaclyn K; Rocap, Gabrielle

    2016-01-01

    The globally significant picocyanobacterium Prochlorococcus is the main primary producer in oligotrophic subtropical gyres. When phosphate concentrations are very low in the marine environment, the mol:mol availability of phosphate relative to the chemically similar arsenate molecule is reduced, potentially resulting in increased cellular arsenic exposure. To mediate accidental arsenate uptake, some Prochlorococcus isolates contain genes encoding a full or partial efflux detoxification pathway, consisting of an arsenate reductase (arsC), an arsenite-specific efflux pump (acr3) and an arsenic-related repressive regulator (arsR). This efflux pathway was the only previously known arsenic detox pathway in Prochlorococcus. We have identified an additional putative arsenic mediation strategy in Prochlorococcus driven by the enzyme arsenite S-adenosylmethionine methyltransferase (ArsM) which can convert inorganic arsenic into more innocuous organic forms and appears to be a more widespread mode of detoxification. We used a phylogenetically informed approach to identify Prochlorococcus linked arsenic genes from both pathways in the Global Ocean Sampling survey. The putative arsenic methylation pathway is nearly ubiquitously present in global Prochlorococcus populations. In contrast, the complete efflux pathway is only maintained in populations which experience extremely low PO4:AsO4, such as regions in the tropical and subtropical Atlantic. Thus, environmental exposure to arsenic appears to select for maintenance of the efflux detoxification pathway in Prochlorococcus. The differential distribution of these two pathways has implications for global arsenic cycling, as their associated end products, arsenite or organoarsenicals, have differing biochemical activities and residence times.

  3. Arctigenin promotes cholesterol efflux from THP-1 macrophages through PPAR-γ/LXR-α signaling pathway.

    PubMed

    Xu, Xiaolin; Li, Qian; Pang, Liewen; Huang, Guoqian; Huang, Jiechun; Shi, Meng; Sun, Xiaotian; Wang, Yiqing

    2013-11-15

    Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α.

  4. Differential regulation of ABCA1 and macrophage cholesterol efflux by elaidic and oleic acids.

    PubMed

    Shao, Fei; Ford, David A

    2013-08-01

    Trans fatty acid consumption is associated with an increased risk of coronary heart disease. This increased risk has been attributed to decreased levels of HDL cholesterol and increased levels of LDL cholesterol. However, the mechanism by which trans fatty acid modulates cholesterol transit remains poorly defined. ATP-binding cassette transporter A1 (ABCA1)-mediated macrophage cholesterol efflux is the rate-limiting step initiating apolipoprotein A-I lipidation. In this study, elaidic acid, the most abundant trans fatty acid in partially hydrogenated vegetable oil, was shown to stabilize macrophage ABCA1 protein levels in comparison to that of its cis fatty acid isomer, oleic acid. The mechanism responsible for the disparate effects of oleic and elaidic acid on ABCA1 levels was through accelerated ABCA1 protein degradation in cells treated with oleic acid. In contrast, no apparent differences were observed in ABCA1 mRNA levels, and only minor changes were observed in Liver X receptor/Retinoic X receptor promoter activity in cells treated with elaidic and oleic acid. Efflux of both tracers and cholesterol mass revealed that elaidic acid slightly increased ABCA1-mediated cholesterol efflux, while oleic acid led to decreased ABCA1-mediated efflux. In conclusion, these studies show that cis and trans structural differences in 18 carbon n-9 monoenoic fatty acids variably impact cholesterol efflux through disparate effects on ABCA1 protein degradation.

  5. Beyond Antimicrobial Resistance: Evidence for a Distinct Role of the AcrD Efflux Pump in Salmonella Biology

    PubMed Central

    Buckner, Michelle M. C.; La Ragione, Roberto M.; Newcombe, Jane; Dwyer, Daniel J.; Ivens, Alasdair

    2016-01-01

    ABSTRACT For over 20 years, bacterial multidrug resistance (MDR) efflux pumps have been studied because of their impact on resistance to antimicrobials. However, critical questions remain, including why produce efflux pumps under non-antimicrobial treatment conditions, and why have multiple pumps if their only purpose is antimicrobial efflux? Salmonella spp. possess five efflux pump families, including the resistance-nodulation-division (RND) efflux pumps. Notably, the RND efflux pump AcrD has a unique substrate profile, distinct from other Salmonella efflux pumps. Here we show that inactivation of acrD results in a profoundly altered transcriptome and modulation of pathways integral to Salmonella biology. The most significant transcriptome changes were central metabolism related, with additional changes observed in pathogenicity, environmental sensing, and stress response pathway expression. The extent of tricarboxylic acid cycle and fumarate metabolism expression changes led us to hypothesize that acrD inactivation may result in motility defects due to perturbation of metabolite concentrations, such as fumarate, for which a role in motility has been established. Despite minimal detectable changes in flagellar gene expression, we found that an acrD mutant Salmonella enterica serovar Typhimurium isolate was significantly impaired for swarming motility, which was restored by addition of fumarate. The acrD mutant outcompeted the wild type in fitness experiments. The results of these diverse experiments provide strong evidence that the AcrD efflux pump is not simply a redundant system providing response resilience, but also has distinct physiological functions. Together, these data indicate that the AcrD efflux pump has a significant and previously underappreciated impact on bacterial biology, despite only minor perturbations of antibiotic resistance profiles. PMID:27879336

  6. The effects of extracellular pH and buffer concentration on the efflux of lactate from frog sartorius muscle

    PubMed Central

    Mainwood, G. W.; Worsley-Brown, Pauline

    1975-01-01

    1. The rate of efflux of lactate from isolated frog sartorius muscles is measured with a superfusion technique. Efflux curves are followed after raising the internal lactate level of the muscles by repetitive electrical stimulation over a 200 sec period. 2. With an external pH of 7·0 or below the measured efflux rates following stimulation reach 100-150 n-mole/g.min. Increasing the pH of the superfusion fluid to 8·0 results in a two or threefold increase in the peak efflux rate. The effect is independent of the buffer system used and occurs fairly rapidly when the pH of the superfusion fluid is changed. This suggests that the effect of pH on lactate efflux is extracellular. 3. The increase in efflux rate due to an increase in pH is dependent on buffer concentration. This fact together with measurements of surface pH changes in muscles following arrest of superfusion indicates that a pH gradient exists through the muscle thickness during lactate efflux. 4. The low lactate efflux rate seen at a low buffer concentration (1 mM) is reduced to an even lower level by depolarization with potassium sulphate suggesting a membrane potential dependent component. At pH 8·0 with a high buffer concentration (25 mM) potassium sulphate only reduces efflux rate slightly. The observations are interpreted as indicating that a fraction of lactate lost is in the form of undissociated acid and that this fraction increases with increasing external pH. 5. Conditions which favour loss of hydrogen ions and lactate from muscle are also associated with improved recovery of twitch tension. ImagesFig. 1 PMID:16992502

  7. Beyond Antimicrobial Resistance: Evidence for a Distinct Role of the AcrD Efflux Pump in Salmonella Biology.

    PubMed

    Buckner, Michelle M C; Blair, Jessica M A; La Ragione, Roberto M; Newcombe, Jane; Dwyer, Daniel J; Ivens, Alasdair; Piddock, Laura J V

    2016-11-22

    For over 20 years, bacterial multidrug resistance (MDR) efflux pumps have been studied because of their impact on resistance to antimicrobials. However, critical questions remain, including why produce efflux pumps under non-antimicrobial treatment conditions, and why have multiple pumps if their only purpose is antimicrobial efflux? Salmonella spp. possess five efflux pump families, including the resistance-nodulation-division (RND) efflux pumps. Notably, the RND efflux pump AcrD has a unique substrate profile, distinct from other Salmonella efflux pumps. Here we show that inactivation of acrD results in a profoundly altered transcriptome and modulation of pathways integral to Salmonella biology. The most significant transcriptome changes were central metabolism related, with additional changes observed in pathogenicity, environmental sensing, and stress response pathway expression. The extent of tricarboxylic acid cycle and fumarate metabolism expression changes led us to hypothesize that acrD inactivation may result in motility defects due to perturbation of metabolite concentrations, such as fumarate, for which a role in motility has been established. Despite minimal detectable changes in flagellar gene expression, we found that an acrD mutant Salmonella enterica serovar Typhimurium isolate was significantly impaired for swarming motility, which was restored by addition of fumarate. The acrD mutant outcompeted the wild type in fitness experiments. The results of these diverse experiments provide strong evidence that the AcrD efflux pump is not simply a redundant system providing response resilience, but also has distinct physiological functions. Together, these data indicate that the AcrD efflux pump has a significant and previously underappreciated impact on bacterial biology, despite only minor perturbations of antibiotic resistance profiles.

  8. Ascorbate Efflux as a New Strategy for Iron Reduction and Transport in Plants*

    PubMed Central

    Grillet, Louis; Ouerdane, Laurent; Flis, Paulina; Hoang, Minh Thi Thanh; Isaure, Marie-Pierre; Lobinski, Ryszard; Curie, Catherine; Mari, Stéphane

    2014-01-01

    Iron (Fe) is essential for virtually all living organisms. The identification of the chemical forms of iron (the speciation) circulating in and between cells is crucial to further understand the mechanisms of iron delivery to its final targets. Here we analyzed how iron is transported to the seeds by the chemical identification of iron complexes that are delivered to embryos, followed by the biochemical characterization of the transport of these complexes by the embryo, using the pea (Pisum sativum) as a model species. We have found that iron circulates as ferric complexes with citrate and malate (Fe(III)3Cit2Mal2, Fe(III)3Cit3Mal1, Fe(III)Cit2). Because dicotyledonous plants only transport ferrous iron, we checked whether embryos were capable of reducing iron of these complexes. Indeed, embryos did express a constitutively high ferric reduction activity. Surprisingly, iron(III) reduction is not catalyzed by the expected membrane-bound ferric reductase. Instead, embryos efflux high amounts of ascorbate that chemically reduce iron(III) from citrate-malate complexes. In vitro transport experiments on isolated embryos using radiolabeled 55Fe demonstrated that this ascorbate-mediated reduction is an obligatory step for the uptake of iron(II). Moreover, the ascorbate efflux activity was also measured in Arabidopsis embryos, suggesting that this new iron transport system may be generic to dicotyledonous plants. Finally, in embryos of the ascorbate-deficient mutants vtc2-4, vtc5-1, and vtc5-2, the reducing activity and the iron concentration were reduced significantly. Taken together, our results identified a new iron transport mechanism in plants that could play a major role to control iron loading in seeds. PMID:24347170

  9. Molecular Expression and Functional Evidence of a Drug Efflux Pump (BCRP) in Human Corneal Epithelial Cells

    PubMed Central

    Karla, Pradeep K.; Earla, Ravinder; Boddu, Sagar H.; Johnston, Thomas P.; Pal, Dhananjay; Mitra, Ashim

    2015-01-01

    Purpose Breast Cancer Resistance Protein (BCRP) belongs to the family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify the presence and possible role of BCRP as a bar-rier for ocular drug resistance. Methods Transfected human corneal epithelial cells (SV40-HCEC) were selected as an in vitro model for corneal epithelium with MDCKII-BCRP as positive control. [3H]-Mitoxantrone ([3H]-MTX), which is a proven substrate for organic anion transporter like BCRP, was selected as a model drug for functional expression studies. Fumetremorgin C (FTC), a known specific inhibitor for BCRP and GF120918, an inhibitor for BCRP and P-gp, were added to inhibit BCRP-mediated efflux. PGP-4008, a specific inhibitor of P-gp was used to delineate the contribution of P-gp. The mRNA extracted from cells was used for RT-PCR analysis and gene expression. Membrane fractions of SV40-HCEC and MDCKII-BCRP were used for immunoprecipitation followed by Western blot analysis. Results Efflux was inhibited significantly in the presence of FTC and GF120918. Dose-dependent inhibition of efflux by BCRP was noticed in SV40-HCEC and MDCKII-BCRP in the presence of FTC and GF120918, and the efflux was ATP-dependent. The metabolic inhibitor, 2,4-DNP, significantly inhibited efflux. No pH-dependent efflux was noticed except at pH 5.5. RT-PCR analysis indicated a unique and distinct band at ~429 bp, corresponding to BCRP in SV40-HCEC and MDCKII-BCRP cells. Western Blot analysis indicated a specific band at ~70 kDa in the membrane fraction of SV40-HCEC and MDCKII-BCRP cells. Conclusions We have demonstrated the expression of BCRP in human corneal epithelial cells and, for the first time, demonstrated its functional activity leading to drug efflux. RT-PCR and Western blot analysis further confirmed this finding. PMID:19172464

  10. Temperature versus plant effects on diel dynamics of soil CO2 production and efflux: a controlled environment study

    NASA Astrophysics Data System (ADS)

    Reinthaler, David; Roy, Jacques; Landais, Damien; Piel, Clement; Resco de Dios, Victor; Bahn, Michael

    2015-04-01

    Soil respiration (Rs) is the biggest source of CO2 emitted from terrestrial ecosystems to the atmosphere. Therefore the understanding of its drivers is of major importance for models of carbon cycling. Next to temperature as a major abiotic factor, photosynthesis has been suggested as an important driver influencing diel patterns in Rs. Under natural conditions it is difficult to disentangle abiotic and biotic effects on soil CO2 production, as fluctuating light intensity affects both photosynthetic activity and soil temperature. To analyse individual and combined effects of soil temperature and light on the dynamics of soil CO2 production and efflux, we performed a controlled environment study at the ECOTRON facility in Montpellier. The study manipulated temperature and photosynthetically active radiation independently and was carried out in large macrocosms, hosting canopies of either a woody (cotton) or a herbaceous (bean) crop. In each macrocosm membrane tubes had been installed across the soil profile for continuous measurement of soil CO2 concentrations. In addition, an automated soil respiration system was installed in each macrocosm, whose data were also used for validating a model of soil CO2 production and transport based on the concentration profiles. Both for cotton and for bean canopies, under conditions of naturally fluctuating temperature and light conditions, soil CO2 production and efflux followed a clear diel pattern. Under constantly dark conditions (excluding immediate effects of photosynthesis) and constant temperature, no significant diel changes in Rs could be observed. Furthermore, soil CO2 production and efflux did not increase significantly upon exposure of previously darkened macrocosms to light. Under constant temperature and fluctuating light conditions, we observed a dampened diel pattern of Rs, which did not match diurnal solar cycles. A detailed residual analysis accounting for temporal trends in soil moisture suggested a significant

  11. CO₂ efflux from shrimp ponds in Indonesia.

    PubMed

    Sidik, Frida; Lovelock, Catherine E

    2013-01-01

    The conversion of mangrove forest to aquaculture ponds has been increasing in recent decades. One of major concerns of this habitat loss is the release of stored 'blue' carbon from mangrove soils to the atmosphere. In this study, we assessed carbon dioxide (CO₂) efflux from soil in intensive shrimp ponds in Bali, Indonesia. We measured CO₂ efflux from the floors and walls of shrimp ponds. Rates of CO₂ efflux within shrimp ponds were 4.37 kg CO₂ m⁻² y⁻¹ from the walls and 1.60 kg CO₂ m⁻² y⁻¹ from the floors. Combining our findings with published data of aquaculture land use in Indonesia, we estimated that shrimp ponds in this region result in CO₂ emissions to the atmosphere between 5.76 and 13.95 Tg y⁻¹. The results indicate that conversion of mangrove forests to aquaculture ponds contributes to greenhouse gas emissions that are comparable to peat forest conversion to other land uses in Indonesia. Higher magnitudes of CO₂ emission may be released to atmosphere where ponds are constructed in newly cleared mangrove forests. This study indicates the need for incentives that can meet the target of aquaculture industry without expanding the converted mangrove areas, which will lead to increased CO₂ released to atmosphere.

  12. Macrolide Efflux in Streptococcus pneumoniae Is Mediated by a Dual Efflux Pump (mel and mef) and Is Erythromycin Inducible

    PubMed Central

    Ambrose, Karita D.; Nisbet, Rebecca; Stephens, David S.

    2005-01-01

    Macrolide resistance in Streptococcus pneumoniae due to efflux has emerged as an important worldwide clinical problem over the past decade. Efflux is mediated by the genes of the genetic element mega (macrolide efflux genetic assembly) and related elements, such as Tn1207.1. These elements contain two adjacent genes, mef (mefE or mefA) and the closely related mel gene (msrA homolog), encoding a proton motive force pump and a putative ATP-binding cassette transporter homolog, and are transcribed as an operon (M. Del Grosso et al., J. Clin. Microbiol. 40:774-778, 2004; K. Gay and D. S. Stephens, J. Infect. Dis. 184:56-65, 2001; and M. Santagati et al., Antimicrob. Agents Chemother. 44:2585-2587, 2000). Previous studies have shown that Mef is required for macrolide resistance in S. pneumoniae; however, the contribution of Mel has not been fully determined. Independent deletions were constructed in mefE and mel in the serotype 14 macrolide-resistant strains GA16638 (erythromycin [Em] MIC, 8 to 16 μg/ml) and GA17719 (Em MIC, 2 to 4 μg/ml), which contain allelic variations in the mega element. The MICs to erythromycin were significantly reduced for the independent deletion mutants of both mefE and mel compared to those of the parent strains and further reduced threefold to fourfold to Em MICs of <0.15 μg/ml with mefE mel double mutants. Using quantitative reverse transcription-PCR, the expression of mefE in the mel deletion mutants was increased more than 10-fold. However, in the mefE deletion mutants, the expression of mel did not differ significantly from the parent strains. The expression of both mefE and mel was inducible by erythromycin. These data indicate a requirement for both Mef and Mel in the novel efflux-mediated macrolide resistance system in S. pneumoniae and other gram-positive bacteria and that the system is inducible by macrolides. PMID:16189099

  13. Portable active interrogation system.

    SciTech Connect

    Moss, C. E.; Brener, M. W.; Hollas, C. L.; Myers, W. L.

    2004-01-01

    The system consists of a pulsed DT neutron generator (5 x 10{sup 7} n/s) and a portable but high intrinsic efficiency, custom-designed, polyethylene-moderated {sup 3}He neutron detector. A multichannel scaler card in a ruggedized laptop computer acquires the data. A user-friendly LabVIEW program analyzes and displays the data. The program displays a warning message when highly enriched uranium or any other fissionable materials is detected at a specified number of sigmas above background in the delayed region between pulses. This report describes the system and gives examples of the response of the system to highly enriched uranium and some other fissionable materials, at several distances and with various shielding materials.

  14. Action of cholecalciferol and alpha-tocopherol on Staphylococcus aureus efflux pumps

    PubMed Central

    Tintino, Saulo R.; Morais-Tintino, Cícera D.; Campina, Fábia F.; Pereira, Raimundo L.; Costa, Maria do S.; Braga, Maria Flaviana B.M.; Limaverde, Paulo W.; Andrade, Jacqueline C.; Siqueira-Junior, José P.; Coutinho, Henrique Douglas Melo; Balbino, Valdir Q.; Leal-Balbino, Tereza C.; Ribeiro-Filho, Jaime; Quintans-Júnior, Lucindo J.

    2016-01-01

    Alpha-tocopherol is one the most abundant and biologically active isoforms of vitamin E. This compound is a potent antioxidant and one of most studied isoforms of vitamin E. Vitamin D3 (cholecalciferol) is an important nutrient for calcium homeostasis and bone health, that has also been recognized as a potent modulator of the immune response. Methicillin-resistant Staphylococcus aureus (MRSA) is the most important causative agent of both nosocomial and community-acquired infections. The aim of this study was to evaluate the inhibitory effect of alpha-tocopherol and cholecalciferol on both S. aureus and multidrug resistant S. aureus efflux pumps. The RN4220 strain has the plasmid pUL5054 that is the carrier of gene that encodes the macrolide resistance protein (an efflux pump) MsrA; the IS-58 strain possesses the TetK tetracycline efflux protein in its genome and the 1199B strain resists to hydrophilic fluoroquinolones via a NorA-mediated mechanism. The antibacterial activity was evaluated by determining the Minimal Inhibitory Concentration (MIC) and a possible inhibition of efflux pumps was associated to a reduction of the MIC. In this work we observed that in the presence of the treatments there was a decrease in the MIC for the RN4220 and IS-58 strains, suggesting that the substances presented an inhibitory effect on the efflux pumps of these strains. Significant efforts have been done to identify efflux pump inhibitors (EPIs) from natural sources and, therefore, the antibacterial properties of cholecalciferol and alpha-tocopherol might be attributed to a direct effect on the bacterial cell depending on their amphipathic structure. PMID:27298617

  15. Action of cholecalciferol and alpha-tocopherol on Staphylococcus aureus efflux pumps.

    PubMed

    Tintino, Saulo R; Morais-Tintino, Cícera D; Campina, Fábia F; Pereira, Raimundo L; Costa, Maria do S; Braga, Maria Flaviana B M; Limaverde, Paulo W; Andrade, Jacqueline C; Siqueira-Junior, José P; Coutinho, Henrique Douglas Melo; Balbino, Valdir Q; Leal-Balbino, Tereza C; Ribeiro-Filho, Jaime; Quintans-Júnior, Lucindo J

    2016-01-01

    Alpha-tocopherol is one the most abundant and biologically active isoforms of vitamin E. This compound is a potent antioxidant and one of most studied isoforms of vitamin E. Vitamin D3 (cholecalciferol) is an important nutrient for calcium homeostasis and bone health, that has also been recognized as a potent modulator of the immune response. Methicillin-resistant Staphylococcus aureus (MRSA) is the most important causative agent of both nosocomial and community-acquired infections. The aim of this study was to evaluate the inhibitory effect of alpha-tocopherol and cholecalciferol on both S. aureus and multidrug resistant S. aureus efflux pumps. The RN4220 strain has the plasmid pUL5054 that is the carrier of gene that encodes the macrolide resistance protein (an efflux pump) MsrA; the IS-58 strain possesses the TetK tetracycline efflux protein in its genome and the 1199B strain resists to hydrophilic fluoroquinolones via a NorA-mediated mechanism. The antibacterial activity was evaluated by determining the Minimal Inhibitory Concentration (MIC) and a possible inhibition of efflux pumps was associated to a reduction of the MIC. In this work we observed that in the presence of the treatments there was a decrease in the MIC for the RN4220 and IS-58 strains, suggesting that the substances presented an inhibitory effect on the efflux pumps of these strains. Significant efforts have been done to identify efflux pump inhibitors (EPIs) from natural sources and, therefore, the antibacterial properties of cholecalciferol and alpha-tocopherol might be attributed to a direct effect on the bacterial cell depending on their amphipathic structure.

  16. Vasopressin induces dopamine release and cyclic AMP efflux from the brain of water-deprived rats: inhibitory effect of vasopressin V2 receptor-mediated phosphorylation.

    PubMed

    Tyagi, M G; Handa, R K; Stephen, P M; Bapna, J S

    1998-01-01

    The neurohypophyseal hormone vasopressin (AVP) is widely distributed throughout the central nervous system. It acts as an excitatory transmitter in the CNS and plays an important physiological role in water and electrolyte homeostasis. However, water deprivation has been shown to induce changes in the levels of monoamines, but there is little knowledge about the influence of AVP on monoamine levels after water deprivation. In this study, we investigated the effect of AVP and its receptor antagonists on alterations in dopamine (DA) release and cyclic adenosine 3',5' monophosphate (cAMP) efflux from rat brain slices following water deprivation. Striatal brain slices (500 microm thick) were incubated in a medium with or without AVP (0. 1-1.0 microM) for 30 min. After 2 h of washout in normal medium, high KCl (40 mM)-evoked DA release and cAMP efflux from the rat brain slices were examined. In the brain slices of euhydrated animals, treatment with AVP slightly altered DA release and cAMP efflux from the brain. This increase in DA release and cAMP efflux was not significantly affected by the addition of a calcium/calmodulin-dependent protein phosphatase, calcineurin (20 microM), to the incubation medium or either by a V1 or V2 AVP receptor antagonist. In contrast, AVP significantly increased the DA release and enhanced the cAMP efflux from the brain slices of water-deprived animals. The AVP-induced increase of brain response in the water-deprived animals was significantly attenuated by a V2 receptor antagonist, partially by calcineurin, but not by a V1 receptor antagonist. The present results suggest that AVP may play a role in water-deprivation-induced DA release and cAMP efflux, which is possibly mediated through the activation of the V2 receptor. The V2 receptor action is attenuated by calcium/calmodulin-dependent dephosphorlyation of some cellular proteins critical for signal transduction.

  17. Efflux-Mediated Drug Resistance in Bacteria: an Update

    PubMed Central

    Li, Xian-Zhi; Nikaido, Hiroshi

    2010-01-01

    Drug efflux pumps play a key role in drug resistance and also serve other functions in bacteria. There has been a growing list of multidrug and drug-specific efflux pumps characterized from bacteria of human, animal, plant and environmental origins. These pumps are mostly encoded on the chromosome although they can also be plasmid-encoded. A previous article (Li X-Z and Nikaido H, Drugs, 2004; 64[2]: 159–204) had provided a comprehensive review regarding efflux-mediated drug resistance in bacteria. In the past five years, significant progress has been achieved in further understanding of drug resistance-related efflux transporters and this review focuses on the latest studies in this field since 2003. This has been demonstrated in multiple aspects that include but are not limited to: further molecular and biochemical characterization of the known drug efflux pumps and identification of novel drug efflux pumps; structural elucidation of the transport mechanisms of drug transporters; regulatory mechanisms of drug efflux pumps; determining the role of the drug efflux pumps in other functions such as stress responses, virulence and cell communication; and development of efflux pump inhibitors. Overall, the multifaceted implications of drug efflux transporters warrant novel strategies to combat multidrug resistance in bacteria. PMID:19678712

  18. Pseudomonas aeruginosa PAO1 resistance to Zinc pyrithione: phenotypic changes suggest the involvement of efflux pumps.

    PubMed

    Abdel Malek, Suzanne M; Al-Adham, Ibrahim S; Matalka, Khalid Z; Collier, Philip J

    2009-08-01

    The aim of this study is to investigate the involvement of an efflux pump in the development of Pseudomonas aeruginosa resistance to zinc pyrithione (ZnPT). In the presence of efflux inhibitor carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), the minimum inhibitory concentration of ZnPT for P. aeruginosa resistant cells is reduced significantly (p < 0.05). In addition, the concentration of ZnPT excluded by the resistant bacteria was reduced significantly (p < 0.01). However, the above reductions did not reach the levels measured for P. aeruginosa PAO1 sensitive strain. Furthermore, such changes in P. aeruginosa resistant cells were correlated with the overexpression of outer membrane proteins, reduced sensitivity toward imipenem (p < 0.01) and increased sensitivity toward sulphatriad and chloramphenicol (p < 0.05). In a continuation to a previous study, we conclude that P. aeruginosa resistance to ZnPT is multifactorial and involves induced efflux systems.

  19. Leiurus quinquestriatus venom inhibits BRL 34915-induced /sup 86/Rb/sup +/ efflux from the rat portal vein

    SciTech Connect

    Quast, U.; Cook, N.S.

    1988-01-01

    The effect of the crude venom of the Israeli scorpion Leiurus quinquestriatus hebraeus on the /sup 86/Rb/sup +/ efflux stimulated by the K/sup +/ channel opener BRL 34915 in the rat portal vein was examined. Applied alone, the venom greatly increased the spontaneous mechanical activity of and the concomitant /sup 86/Rb/sup +/ efflux from the vessel. When the excitability of the vein was suppressed by the dihydropyridine calcium antagonist, PN 200-110, the /sup 86/Rb/sup +/ efflux stimulated by BRL 34915 could be shown to be inhibited by the venom. From the concentration dependence of this inhibition an IC/sub 50/ value of 0.17 +/- 0.01 mg/ml was estimated. This venom is thus the most potent blocker of BRL 34915-evoked /sup 86/Rb/sup +/ efflux reported so far. 17 references, 2 figures.

  20. In vivo selection of a target/efflux double mutant of Pseudomonas aeruginosa by ciprofloxacin therapy.

    PubMed

    Le Thomas, I; Couetdic, G; Clermont, O; Brahimi, N; Plésiat, P; Bingen, E

    2001-10-01

    We report the emergence after 4 days of ciprofloxacin monotherapy of a double mutant of Pseudomonas aeruginosa overexpressing the multidrug efflux system MexAB-OprM and harbouring a mutation in the gyrB gene. Compared with its initial susceptible counterpart, this mutant exhibited a significant increase in resistance to most of the beta-lactam antibiotics tested (16 x MIC of ticarcillin) and to ciprofloxacin (128 x MIC). Combined ceftazidime and amikacin therapy finally eradicated the resistant isolate and cured the patient of his infection. This case illustrates how strains of P. aeruginosa may develop high levels of fluoroquinolone resistance by combining efflux mechanisms and target alterations.

  1. Posttranslational modification and trafficking of PIN auxin efflux carriers.

    PubMed

    Löfke, Christian; Luschnig, Christian; Kleine-Vehn, Jürgen

    2013-01-01

    Cell-to-cell communication is absolutely essential for multicellular organisms. Both animals and plants use chemicals called hormones for intercellular signaling. However, multicellularity of plants and animals has evolved independently, which led to establishment of distinct strategies in order to cope with variations in an ever-changing environment. The phytohormone auxin is crucial to plant development and patterning. PIN auxin efflux carrier-driven polar auxin transport regulates plant development as it controls asymmetric auxin distribution (auxin gradients), which in turn modulates a wide range of developmental processes. Internal and external cues trigger a number of posttranslational PIN auxin carrier modifications that were demonstrated to decisively influence variations in adaptive growth responses. In this review, we highlight recent advances in the analysis of posttranslational modification of PIN auxin efflux carriers, such as phosphorylation and ubiquitylation, and discuss their eminent role in directional vesicle trafficking, PIN protein de-/stabilization and auxin transport activity. We conclude with updated models, in which we attempt to integrate the mechanistic relevance of posttranslational modifications of PIN auxin carriers for the dynamic nature of plant development.

  2. PKCβ Inhibitors Attenuate Amphetamine-Stimulated Dopamine Efflux.

    PubMed

    Zestos, Alexander G; Mikelman, Sarah R; Kennedy, Robert T; Gnegy, Margaret E

    2016-06-15

    Amphetamine abuse afflicts over 13 million people, and there is currently no universally accepted treatment for amphetamine addiction. Amphetamine serves as a substrate for the dopamine transporter and reverses the transporter to cause an increase in extracellular dopamine. Activation of the beta subunit of protein kinase C (PKCβ) enhances extracellular dopamine in the presence of amphetamine by facilitating the reverse transport of dopamine and internalizing the D2 autoreceptor. We previously demonstrated that PKCβ inhibitors block amphetamine-stimulated dopamine efflux in synaptosomes from rat striatum in vitro. In this study, we utilized in vivo microdialysis in live, behaving rats to assess the effect of the PKCβ inhibitors, enzastaurin and ruboxistaurin, on amphetamine-stimulated locomotion and increases in monoamines and their metabolites. A 30 min perfusion of the nucleus accumbens core with 1 μM enzastaurin or 1 μM ruboxistaurin reduced efflux of dopamine and its metabolite 3-methoxytyramine induced by amphetamine by approximately 50%. The inhibitors also significantly reduced amphetamine-stimulated extracellular levels of norepinephrine. The stimulation of locomotor behavior by amphetamine, measured simultaneously with the analytes, was comparably reduced by the PKCβ inhibitors. Using a stable isotope label retrodialysis procedure, we determined that ruboxistaurin had no effect on basal levels of dopamine, norepinephrine, glutamate, or GABA. In addition, normal uptake function through the dopamine transporter was unaltered by the PKCβ inhibitors, as measured in rat synaptosomes. Our results support the utility of using PKCβ inhibitors to reduce the effects of amphetamine.

  3. Role of the cell envelope stress regulators BaeR and CpxR in control of RND-type multidrug efflux pumps and transcriptional cross talk with exopolysaccharide synthesis in Erwinia amylovora.

    PubMed

    Pletzer, Daniel; Stahl, Antje; Oja, Anna Elisabeth; Weingart, Helge

    2015-08-01

    The purpose of this study was to identify the role of the cell envelope stress-sensing systems BaeSR and CpxARP in regulation of multidrug efflux and exopolysaccharide synthesis in Erwinia amylovora. We have previously reported that BaeR activates transcription of the RND-type efflux pumps AcrD and MdtABC. In this study, we found that a cpxR-deficient mutant was highly susceptible to β-lactams, aminoglycosides and lincomycin, whereas a baeR mutant showed no change in antimicrobial sensitivity. However, overexpression of BaeR in a mutant lacking the major RND pump AcrB increased resistance of E. amylovora to several compounds that are not substrates of AcrD or MdtABC. Furthermore, we observed that overexpression of BaeR significantly increased amylovoran production. Moreover, the expression of RND-type efflux pumps was changed in regulatory mutants of exopolysaccharide production. Our data suggest that BaeSR and CpxARP regulate additional mechanisms, beside efflux, which are responsible for antimicrobial resistance of E. amylovora.

  4. Crystal structure of the CusBA heavy-metal efflux complex of Escherichia coli.

    PubMed

    Su, Chih-Chia; Long, Feng; Zimmermann, Michael T; Rajashankar, Kanagalaghatta R; Jernigan, Robert L; Yu, Edward W

    2011-02-24

    Gram-negative bacteria, such as Escherichia coli, expel toxic chemicals through tripartite efflux pumps that span both the inner and outer membrane. The three parts are an inner membrane, substrate-binding transporter; a membrane fusion protein; and an outer-membrane-anchored channel. The fusion protein connects the transporter to the channel within the periplasmic space. A crystallographic model of this tripartite efflux complex has been unavailable because co-crystallization of the various components of the system has proven to be extremely difficult. We previously described the crystal structures of both the inner membrane transporter CusA and the membrane fusion protein CusB of the CusCBA efflux system of E. coli. Here we report the co-crystal structure of the CusBA efflux complex, showing that the transporter (or pump) CusA, which is present as a trimer, interacts with six CusB protomers and that the periplasmic domain of CusA is involved in these interactions. The six CusB molecules seem to form a continuous channel. The affinity of the CusA and CusB interaction was found to be in the micromolar range. Finally, we have predicted a three-dimensional structure for the trimeric CusC outer membrane channel and developed a model of the tripartite efflux assemblage. This CusC(3)-CusB(6)-CusA(3) model shows a 750-kilodalton efflux complex that spans the entire bacterial cell envelope and exports Cu I and Ag I ions.

  5. Pharmacological evidence for common mechanisms underlying the effects of neurotensin and neuroleptics on in vivo dopamine efflux in the rat nucleus accumbens.

    PubMed

    Blaha, C D; Phillips, A G

    1992-08-01

    The effects of the neuropeptide neurotensin and the typical neuroleptic haloperidol on dopamine efflux were compared in the posteromedial nucleus accumbens of the chloral hydrate-anesthetized rat using in vivo chronoamperometry. Both neurotensin and haloperidol administration elicited an immediate increase in dopamine efflux in the nucleus accumbens. Gamma-hydroxybutyric acid lactone, an agent known to block impulse flow in dopamine neurons, either prevented when given before neurotensin or reversed neurotensin-induced increases in accumbens dopamine efflux. Haloperidol-induced increases in accumbens dopamine efflux were similarly affected by gamma-hydroxybutyric acid lactone. The dopamine receptor agonist apomorphine reversed neurotensin- and haloperidol-induced increases in dopamine efflux. Amphetamine, administered during the peak dopamine stimulatory effects induced by neurotensin or haloperidol, resulted in increases above baseline which were significantly greater than the effects of amphetamine alone. These combined drug treatment effects on baseline dopamine efflux were additive, indicating that the effects of amphetamine were not potentiated by neurotensin or haloperidol pretreatments. These in vivo results suggest that neurotensin and haloperidol may augment dopamine efflux in the nucleus accumbens via common mechanisms of action which may involve activation of mesotelencephalic dopamine neuronal firing. The inability of neurotensin to block amphetamine-induced efflux in the nucleus accumbens further suggests that neurotensin blockade of amphetamine-elicited locomotor activity is mediated by an action of neurotensin postsynaptic to dopamine nerve terminals in the nucleus accumbens.

  6. Active Shimmy Control System

    DTIC Science & Technology

    1975-12-01

    reviewed by thoe nformation Offite (01) end Is reslesuabe to the National Technical Wnrdstleftiv Oervico (WI2B). At N13..S it iuil be, avail-able th the...Figure 2, - are used only for the passive system. BH and BL are hydraulic (velocity squared) and linear shimmy damper constants, and KALP in the...NOTES KPH i.63E6 1.403E6 x KrI 11.20 5000 .. X &T, ~ ipl, , x KOC 77270 - X KALP 18000 -X IPH 69.7 83.9 X ITH .68 x "ITI, .03 - x ITII2 3.h9 - xIA .o

  7. Active Tensor Magnetic Gradiometer System

    DTIC Science & Technology

    2007-11-01

    Modify Forward Computer Models .............................................................................................2 Modify TMGS Simulator...active magnetic gradient measurement system are based upon the existing tensor magnetic gradiometer system ( TMGS ) developed under project MM-1328...Magnetic Gradiometer System ( TMGS ) for UXO Detection, Imaging, and Discrimination.” The TMGS developed under MM-1328 was successfully tested at the

  8. An Arg-Gly-Asp peptide stimulates Ca2+ efflux from osteoclast precursors through a novel mechanism

    NASA Technical Reports Server (NTRS)

    Yamakawa, K.; Duncan, R.; Hruska, K. A.

    1994-01-01

    We examined the effect of a peptide containing the Arg-Gly-Asp (RGD) sequence on 45Ca2+ efflux from osteoclast precursors. 45Ca(2+)-loaded osteoclast precursors were treated with GRGDSP (170 microM) for 10 min after 30 min of basal perfusion with a bicarbonate-containing buffer. GRGDSP significantly increased fractional efflux of Ca2+ from treated cells compared with vehicle-treated cells (P < 0.01) or cells treated with up to 200 micrograms/ml of a control peptide containing GRGESP. The effect of RGD was sustained for 15 min after the peptide was removed from the perfusate, but control levels of Ca2+ efflux returned by 1 h. The Ca2+ efflux effect of GRGDSP was most likely due to activation of the plasma membrane Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase) pump, as indicated by its inhibition with vanadate and a calmodulin antagonist, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, and the absence of an effect of Na+/Ca2+ exchange inhibition. An inhibitor of cyclic nucleotide-dependent protein kinases, N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (0.1 mM), failed to inhibit GRGDSP-stimulated Ca2+ efflux. However, genistein and herbimycin A, inhibitors of protein-tyrosine kinases, blocked Ca2+ efflux stimulated by GRGDSP. The results indicate that RGD sequences of matrix proteins may stimulate Ca2+ efflux from osteoclasts through activation of protein-tyrosine kinases and suggest that GRGDSP-stimulated Ca2+ efflux is mediated via the plasma membrane Ca(2+)-ATPase.

  9. Engineering microbial biofuel tolerance and export using efflux pumps

    PubMed Central

    Dunlop, Mary J; Dossani, Zain Y; Szmidt, Heather L; Chu, Hou Cheng; Lee, Taek Soon; Keasling, Jay D; Hadi, Masood Z; Mukhopadhyay, Aindrila

    2011-01-01

    Many compounds being considered as candidates for advanced biofuels are toxic to microorganisms. This introduces an undesirable trade-off when engineering metabolic pathways for biofuel production because the engineered microbes must balance production against survival. Cellular export systems, such as efflux pumps, provide a direct mechanism for reducing biofuel toxicity. To identify novel biofuel pumps, we used bioinformatics to generate a list of all efflux pumps from sequenced bacterial genomes and prioritized a subset of targets for cloning. The resulting library of 43 pumps was heterologously expressed in Escherichia coli, where we tested it against seven representative biofuels. By using a competitive growth assay, we efficiently distinguished pumps that improved survival. For two of the fuels (n-butanol and isopentanol), none of the pumps improved tolerance. For all other fuels, we identified pumps that restored growth in the presence of biofuel. We then tested a beneficial pump directly in a production strain and demonstrated that it improved biofuel yields. Our findings introduce new tools for engineering production strains and utilize the increasingly large database of sequenced genomes. PMID:21556065

  10. Engineering microbial biofuel tolerance and export using efflux pumps.

    PubMed

    Dunlop, Mary J; Dossani, Zain Y; Szmidt, Heather L; Chu, Hou Cheng; Lee, Taek Soon; Keasling, Jay D; Hadi, Masood Z; Mukhopadhyay, Aindrila

    2011-05-10

    Many compounds being considered as candidates for advanced biofuels are toxic to microorganisms. This introduces an undesirable trade-off when engineering metabolic pathways for biofuel production because the engineered microbes must balance production against survival. Cellular export systems, such as efflux pumps, provide a direct mechanism for reducing biofuel toxicity. To identify novel biofuel pumps, we used bioinformatics to generate a list of all efflux pumps from sequenced bacterial genomes and prioritized a subset of targets for cloning. The resulting library of 43 pumps was heterologously expressed in Escherichia coli, where we tested it against seven representative biofuels. By using a competitive growth assay, we efficiently distinguished pumps that improved survival. For two of the fuels (n-butanol and isopentanol), none of the pumps improved tolerance. For all other fuels, we identified pumps that restored growth in the presence of biofuel. We then tested a beneficial pump directly in a production strain and demonstrated that it improved biofuel yields. Our findings introduce new tools for engineering production strains and utilize the increasingly large database of sequenced genomes.

  11. Association between cholesterol efflux capacity and coronary restenosis after successful stent implantation.

    PubMed

    Imaizumi, Satoshi; Miura, Shin-Ichiro; Takata, Kohei; Takamiya, Yosuke; Kuwano, Takashi; Sugihara, Makoto; Ike, Amane; Iwata, Atsushi; Nishikawa, Hiroaki; Saku, Keijiro

    2016-08-01

    The measurement of high-density lipoprotein (HDL) functionality could be useful for identifying patients who have an increased risk of coronary restenosis after stent implantation. In the present study, we elucidates whether HDL functionality can predict restenosis. The participants included 48 consecutive patients who had stable angina and were successfully implanted with a drug-eluting stent (DES) or bare-metal stent. Follow-up coronary angiography was performed after 6-8 months of stenting. Cholesterol efflux and the anti-inflammatory capacity of HDL were measured before stenting (at baseline) and at follow-up. The mean age was 64 ± 11 years and the body mass index was 24 ± 3 kg/m(2). While HDL cholesterol (HDL-C) significantly increased from baseline to follow-up, there was no significant association between HDL-C level at baseline and in-stent late loss. Cholesterol efflux capacity was significantly increased from baseline to follow-up. The efflux capacity at baseline was negatively correlated with in-stent late loss, whereas the anti-oxidative activity of HDL at baseline was not associated with in-stent late loss. We analyzed the predictors of in-stent late loss using independent variables (efflux capacity and anti-oxidative capacity at baseline in addition to age, gender, HDL-C and low-density lipoprotein cholesterol at baseline, hypertension, diabetes mellitus, smoking, lesion length and DES implantation, history of myocardial infarction and prior percutaneous coronary intervention) by a multiple regression analysis. The efflux capacity at baseline was only independently associated with in-stent late loss. In conclusion, cholesterol efflux capacity at baseline could predict coronary restenosis in patients with successful stent implantation.

  12. Roles of pollen-specific boron efflux transporter, OsBOR4, in the rice fertilization process.

    PubMed

    Tanaka, Nobuhiro; Uraguchi, Shimpei; Saito, Akihiro; Kajikawa, Masataka; Kasai, Koji; Sato, Yutaka; Nagamura, Yoshiaki; Fujiwara, Toru

    2013-12-01

    Arabidopsis thaliana BOR1 was the first boron (B) transporter identified in living systems. There are four AtBOR1-like genes, OsBOR1, 2, 3 and 4, present in the rice genome. We characterized the activity, expression and physiological function of OsBOR4. OsBOR4 is an active efflux transporter of B. Quantitative PCR analysis and OsBOR4 promoter-green fluorescent protein (GFP) fusion revealed that OsBOR4 was both highly and specifically expressed in pollen. We obtained five Tos17 insertion mutants of osbor4. The pollen grains were viable and development of floral organs was normal in the homozygous osbor4 mutants. We observed that in all Tos17 insertion lines tested, the frequency of osbor4 homozygous plants was lower than expected in the progeny of self-fertilized heterozygous plants. These results establish that OsBOR4 is essential for normal reproductive processes. Pollen from osbor4 homozygous plants elongated fewer tubes on wild-type stigmas, and tube elongation of mutant pollen was less efficient compared with the wild-type pollen, suggesting reduced competence of osbor4 mutant pollen. The reduced competence of mutant pollen was further supported by the crosses of independent Tos17-inserted alleles of OsBOR4. Our results suggest that OsBOR4, a boron efflux transporter, is required for normal pollen germination and/or tube elongation.

  13. Multidrug efflux pumps in Staphylococcus aureus and their clinical implications.

    PubMed

    Jang, Soojin

    2016-01-01

    Antibiotic resistance is rapidly spreading among bacteria such as Staphylococcus aureus, an opportunistic bacterial pathogen that causes a variety of diseases in humans. For the last two decades, bacterial multidrug efflux pumps have drawn attention due to their potential association with clinical multidrug resistance. Numerous researchers have demonstrated efflux-mediated resistance in vitro and in vivo and found novel multidrug transporters using advanced genomic information about bacteria. This article aims to provide a concise summary of multidrug efflux pumps and their important clinical implications, focusing on recent findings concerning S. aureus efflux pumps.

  14. Evaluation of the tannic acid inhibitory effect against the NorA efflux pump of Staphylococcus aureus.

    PubMed

    Tintino, Saulo R; Oliveira-Tintino, Cícera D M; Campina, Fábia F; Silva, Raimundo L P; Costa, Maria do S; Menezes, Irwin R A; Calixto-Júnior, João T; Siqueira-Junior, José P; Coutinho, Henrique D M; Leal-Balbino, Tereza C; Balbino, Valdir Q

    2016-08-01

    During the early periods of antibiotic usage, bacterial infections were considered tamed. However, widespread antibiotic use has promoted the emergence of antibiotic-resistant pathogens, including multidrug resistant strains. Active efflux is a mechanism for bacterial resistance to inhibitory substances, known simply as drug efflux pumps. The bacterium Staphylococcus aureus is an important pathogenic bacterium responsible for an array of infections. The NorA efflux pump has been shown to be responsible for moderate fluoroquinolone resistance of S. aureus. The inhibition of the efflux pump was assayed using a sub-inhibitory concentration of standard efflux pump inhibitors and tannic acid (MIC/8), where its capacity to decrease the MIC of Ethidium bromide (EtBr) and antibiotics due to the possible inhibitory effect of these substances was observed. The MICs of EtBr and antibiotics were significantly reduced in the presence of tannic acid, indicating the inhibitory effect of this agent against the efflux pumps of both strains causing a three-fold reduction of the MIC when compared with the control. These results indicate the possible usage of tannic acid as an adjuvant in antibiotic therapy against multidrug resistant bacteria (MDR).

  15. 42K analysis of sodium-induced potassium efflux in barley: mechanism and relevance to salt tolerance.

    PubMed

    Britto, Dev T; Ebrahimi-Ardebili, Sasha; Hamam, Ahmed M; Coskun, Devrim; Kronzucker, Herbert J

    2010-04-01

    *Stimulation of potassium (K(+)) efflux by sodium (Na(+)) has been the subject of much recent attention, and its mechanism has been attributed to the activities of specific classes of ion channels. *The short-lived radiotracer (42)K(+) was used to test this attribution, via unidirectional K(+)-flux analysis at the root plasma membrane of intact barley (Hordeum vulgare), in response to NaCl, KCl, NH(4)Cl and mannitol, and to channel inhibitors. *Unidirectional K(+) efflux was strongly stimulated by NaCl, and K(+) influx strongly suppressed. Both effects were ameliorated by elevated calcium (Ca(2+)). As well, K(+) efflux was strongly stimulated by KCl, NH(4)Cl and mannitol , and NaCl also stimulated (13)NH(4)(+) efflux. The Na(+)-stimulated K(+) efflux was insensitive to cesium (Cs(+)) and pH 4.2, weakly sensitive to the K(+)-channel blocker tetraethylammonium (TEA(+)) and quinine, and moderately sensitive to zinc (Zn(2+)) and lanthanum (La(3+)). *We conclude that the stimulated efflux is: specific neither to Na(+) as effector nor K(+) as target; composed of fluxes from both cytosol and vacuole; mediated neither by outwardly-rectifying K(+) channels nor nonselective cation channels; attributable, alternatively, to membrane disintegration brought about by ionic and osmotic components; of limited long-term significance, unlike the suppression of K(+) influx by Na(+), which is a greater threat to K(+) homeostasis under salt stress.

  16. PKC phosphorylates residues in the N-terminal of the DA transporter to regulate amphetamine-induced DA efflux.

    PubMed

    Wang, Qiang; Bubula, Nancy; Brown, Jason; Wang, Yunliang; Kondev, Veronika; Vezina, Paul

    2016-05-27

    The DA transporter (DAT), a phosphoprotein, controls extracellular dopamine (DA) levels in the central nervous system through transport or reverse transport (efflux). Multiple lines of evidence support the claim that PKC significantly contributes to amphetamine-induced DA efflux. Other signaling pathways, involving CaMKII and ERK, have also been shown to regulate DAT mediated efflux. Here we assessed the contribution of putative PKC residues (S4, S7, S13) in the N-terminal of the DAT to amphetamine-induced DA efflux by transfecting DATs containing different serine to alanine (S-A) point mutations into DA pre-loaded HEK-293 cells and incubating these cells in amphetamine (2μM). The effects of a S-A mutation at the non-PKC residue S12 and a threonine to alanine (T-A) mutation at the ERK T53 residue were also assessed for comparison. WT-DATs were used as controls. In an initial experiment, we confirmed that inhibiting PKC with Go6976 (130nM) significantly reduced amphetamine-induced DA efflux. In subsequent experiments, cells transfected with the S4A, S12A, S13A, T53A and S4,7,13A mutants showed a reduction in amphetamine-induced DA efflux similar to that observed with Go6976. Interestingly, cells transfected with the S7A mutant, identified by some as a PKC-PKA residue, showed unperturbed WT-DAT levels of amphetamine-induced DA efflux. These results indicate that phosphorylation by PKC of select residues in the DAT N-terminal can regulate amphetamine-induced efflux. PKC can act either independently or in concert with other kinases such as ERK to produce this effect.

  17. Intestinal permeability and P-glycoprotein-mediated efflux transport of ticagrelor in Caco-2 monolayer cells.

    PubMed

    Marsousi, Niloufar; Doffey-Lazeyras, Fabienne; Rudaz, Serge; Desmeules, Jules A; Daali, Youssef

    2016-12-01

    Ticagrelor is the unique reversible oral antiplatelet drug commercialized today. During this study, the intestinal permeability of ticagrelor and its potential P-glycoprotein (P-gp)-mediated active transport were assessed. To this end, bidirectional transport of ticagrelor was performed across Caco-2 (human epithelial colorectal adenocarcinoma) monolayer model in the presence and absence of potent P-gp inhibitor valspodar. Ticagrelor presented an apical-basolateral apparent permeability coefficient (Papp ) of 6.0 × 10(-6) cm/s. On the other hand, mean efflux ratio (ER) of 2.71 was observed for ticagrelor describing a higher efflux permeability compared to the influx component. Valspodar showed a significant inhibitory effect on the efflux of ticagrelor suggesting involvement of P-gp in its oral disposition. Co-incubation of the P-gp inhibitor decreased the efflux Papp of ticagrelor from 1.60 × 10(-5) to 1.13 × 10(-5) cm/s and decreased its ER by 70%. Results suggest a modest active transport of ticagrelor by P-gp across the Caco-2 cell monolayer. The co-administration of ticagrelor with a P-gp inhibitor seems altogether unlikely to have an extended impact on pharmacokinetics of ticagrelor and cause bleeding events in patients.

  18. IP/sub 3/ stimulates CA/sup + +/ efflux from fusogenic carrot protoplasts

    SciTech Connect

    Rincon, M.; Boss, W.F.

    1986-04-01

    Polyphosphoinositide breakdown plays an important role in signal transduction in animal cells (Berridge and Irvine, 1984, Nature, 312:315). Upon stimulation, phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate (IP/sub 3/) and diacylglycerol both of which act as cellular second messengers. IP/sub 3/ mobilizes Ca/sup + +/ from internal stores, hence the cytosolic free Ca/sup + +/ concentration increases and those physiological activities regulated by Ca/sup + +/ are stimulated. To test if plant cells also responded to IP/sub 3/, Ca/sup + +/ efflux studies were done with fusogenic carrot protoplasts released in EGTA. The protoplasts were preloaded with /sup 45/Ca/sup + +/ placed in a Ca/sup + +/-free medium, and efflux determined as /sup 45/Ca/sup + +/ loss from the protoplasts. IP/sub 3/ (10-20..mu..M) caused enhanced /sup 45/Ca/sup + +/ efflux and the response was sustained for at least 15 min. In plants, as in animals, the observed IP/sub 3/-enhanced /sup 45/Ca/sup + +/ efflux suggested that IP/sub 3/ released Ca/sup + +/ from internal stores, and the increased free cytosolic Ca/sup + +/ activated Ca/sup + +/ pumping mechanisms which restored the Ca/sup + +/ concentration in the cytosol to the normal level.

  19. Characterization of zebrafish Abcc4 as an efflux transporter of organochlorine pesticides.

    PubMed

    Lu, Xing; Long, Yong; Lin, Li; Sun, Rongze; Zhong, Shan; Cui, Zongbin

    2014-01-01

    DDT and lindane are highly toxic organochlorine pesticides and posing adverse effects on the environment and public health due to their frequent usage in developing countries. ABCC4/MRP4 is an organic anion transporter that mediates cellular efflux of a wide range of exogenous and endogenous compounds such as cyclic nucleotides and anti-cancer drugs; however, it remains unclear whether ABCC4 and its orthologs function in the detoxification of organochlorine pesticides. Here, we demonstrated the roles of zebrafish Abcc4 in cellular efflux of DDT and lindane. Zebrafish abcc4 was maternally expressed in the oocytes and its transcripts were detected in the lens, pancreas, gills, liver, intestine and bladder of developing embryos and in adult tissues examined. DDT and lindane were able to induce the expression of abcc4 gene and overexpression of Abcc4 significantly decreased the cytotoxicity and accumulation of DDT and lindane in LLC-PK1 cells and developing embryos. In contrast, overexpression of an Abcc4-G1188D mutant abolished its transporter function without effects on its substrate binding activity, and sensitized LLC-PK1 cells and developing embryos to toxic pesticides. Moreover, glutathione (GSH) was involved in the efflux of cellular pesticides and ATPase activity in developing embryos can be induced by DDT or lindane. Thus, zebrafish Abcc4 plays crucial roles in cellular efflux of organochlorine pesticides and can be used a potential molecular marker for the monitor of DDT and lindane contamination in the aquatic environment.

  20. The ABCG2 efflux transporter from rabbit placenta: Cloning and functional characterization.

    PubMed

    Halwachs, Sandra; Kneuer, Carsten; Gohlsch, Katrin; Müller, Marian; Ritz, Vera; Honscha, Walther

    2016-02-01

    In human placenta, the ATP-binding cassette efflux transporter ABCG2 is highly expressed in syncytiotrophoblast cells and mediates cellular excretion of various drugs and toxins. Hence, physiological ABCG2 activity substantially contributes to the fetoprotective placenta barrier function during gestation. Developmental toxicity studies are often performed in rabbit. However, despite its toxicological relevance, there is no data so far on functional ABCG2 expression in this species. Therefore, we cloned ABCG2 from placenta tissues of chinchilla rabbit. Sequencing showed 84-86% amino acid sequence identity to the orthologues from man, rat and mouse. We transduced the rabbit ABCG2 clone (rbABCG2) in MDCKII cells and stable rbABCG2 gene and protein expression was shown by RT-PCR and Western blot analysis. The rbABCG2 efflux activity was demonstrated with the Hoechst H33342 assay using the specific ABCG2 inhibitor Ko143. We further tested the effect of established human ABCG2 (hABCG2) drug substrates including the antibiotic danofloxacin or the histamine H2-receptor antagonist cimetidine on H33342 accumulation in MDCKII-rbABCG2 or -hABCG2 cells. Human therapeutic plasma concentrations of all tested drugs caused a comparable competitive inhibition of H33342 excretion in both ABCG2 clones. Altogether, we first showed functional expression of the ABCG2 efflux transporter in rabbit placenta. Moreover, our data suggest a similar drug substrate spectrum of the rabbit and the human ABCG2 efflux transporter.

  1. Medial septum-diagonal band of Broca (MSDB) GABAergic regulation of hippocampal acetylcholine efflux is dependent on cognitive demands.

    PubMed

    Roland, Jessica J; Stewart, Amanda L; Janke, Kellie L; Gielow, Matthew R; Kostek, John A; Savage, Lisa M; Servatius, Richard J; Pang, Kevin C H

    2014-01-08

    The septohippocampal pathway contains cholinergic, GABAergic, and glutamatergic projections and has an established role in learning, memory, and hippocampal theta rhythm. Both GABAergic and cholinergic neurons in the medial septum-diagonal band of Broca (MSDB) have been associated with spatial memory, but the relationship between the two neuronal populations is not fully understood. The present study investigated the effect of selective GABAergic MSDB lesions on hippocampal acetylcholine (ACh) efflux and spatial memory during tasks that varied in memory demand. Male Sprague Dawley rats were given GABAergic lesions of the MSDB using GAT1-saporin (GAT1-SAP) and examined on spontaneous exploration (Experiment 1) and non-matching to position without (NMTP; Experiment 2) and with a delay (DNMTP; Experiment 3), while concurrently using in vivo microdialysis to measure hippocampal ACh efflux. Intraseptal GAT1-SAP treatment did not alter baseline or behaviorally stimulated hippocampal ACh efflux or maze exploration (Experiment 1). Moreover, GAT1-SAP did not alter evoked hippocampal ACh efflux related to NMTP nor did it impair working memory in NMTP (Experiment 2). In contrast, both ACh efflux and performance in DNMTP were impaired by intraseptal GAT1-SAP. Thus, GABAergic MSDB neurons are important for spatial working memory and modulate hippocampal ACh efflux under conditions of high memory load. The relationship between the septohippocampal cholinergic and GABAergic systems and working memory will be discussed.

  2. Active Response Gravity Offload System

    NASA Technical Reports Server (NTRS)

    Valle, Paul; Dungan, Larry; Cunningham, Thomas; Lieberman, Asher; Poncia, Dina

    2011-01-01

    The Active Response Gravity Offload System (ARGOS) provides the ability to simulate with one system the gravity effect of planets, moons, comets, asteroids, and microgravity, where the gravity is less than Earth fs gravity. The system works by providing a constant force offload through an overhead hoist system and horizontal motion through a rail and trolley system. The facility covers a 20 by 40-ft (approximately equals 6.1 by 12.2m) horizontal area with 15 ft (approximately equals4.6 m) of lifting vertical range.

  3. Mathematical modelling of in situ and in vitro efflux of ciprofloxacin and grepafloxacin.

    PubMed

    Rodríguez-Ibáñez, M; Sánchez-Castaño, G; Montalar-Montero, M; Garrigues, T M; Bermejo, M; Merino, V

    2006-01-03

    The efflux process due to p-glycoprotein-like mechanisms of ciprofloxacin (CIP) and grepafloxacin (GRX) has been studied "in situ" in rats and "in vitro" in Caco-2 cells. The results were modelled by a curve fitting procedure which allowed the characterization of the passive (Pd) and carrier mediated parameters (Vm and Km) from the raw data without initial velocities estimation. CIP absorption in rat was characterized as a passive diffusion at the assayed concentrations. Although the involvement of an efflux transporter cannot be ruled out, its relevance in the transport of the fluoroquinolone is negligible. In GRX absorption, an efflux process is implicated and it is detected in both absorption models. GRX permeability depends on the intestinal segment, reflecting the previously reported different expression level of the efflux transporters along the gut in rat. A first attempt to correlate the "in vitro" and the "in situ" data has been done. The mathematical model has been constructed using very simplistic assumptions and it will require further refinement but, nevertheless, the results are promising and demonstrate that a good modelling approach helps to identify the system critical parameters and how the system behaviour change when the parameters are modified as it happens when we move from the "in vitro" to the "in situ" level. Predicted versus experimental permeability values show a good correlation, demonstrating that the relevance of the secretion process "in situ" in rat can be predicted from the "in vitro" cell results.

  4. In situ kinetic modelling of intestinal efflux in rats: functional characterization of segmental differences and correlation with in vitro results.

    PubMed

    González-Alvarez, Isabel; Fernández-Teruel, Carlos; Casabó-Alós, Vicente G; Garrigues, Teresa M; Polli, James E; Ruiz-García, Ana; Bermejo, Marival

    2007-07-01

    The objective was to devise and apply a novel modelling approach to combine segmental in situ rat perfusion data and in vitro cell culture data, in order to elucidate the contribution of efflux in drug absorption kinetics. The fluoroquinolone CNV97100 was used as a model P-gp substrate. In situ intestinal perfusion was performed in rat duodenum, jejunum, ileum and colon to measure the influence of P-gp expression on efflux. Inhibition studies of CNV97100 were performed in the presence of verapamil, quinidine, cyclosporin A and p-aminohippuric acid. Absorption/efflux parameters were modelled simultaneously, using data from both in situ studies as well as in vitro studies. The maximal efflux velocity was modelled as a baseline value, corrected for each segment based on the expression level. CNV97100 passive diffusional permeability (P(diff)) and its affinity for the efflux carrier (K(m)) were assumed to be the same in all segments. The results indicate the new approach to combine in situ data and in vitro data succeed in yielding a unified, quantitative model for absorption/efflux. The model incorporated a quantitative relationship between P-gp expression level and the efflux functionality, both across in situ and in vitro systems, as well across different intestinal segments in the in situ studies. Permeability values decreased from duodenum to ileum in accordance with the increasing P-gp expression levels in rat intestine. The developed model reflects a strong correlation between in vitro and in situ results, including intrinsic differences in surface area. The successful application of a model approach to combine absorption data from two different experimental systems holds promise for future efforts to predict absorption results from one system to a second system.

  5. Sodium efflux in plant roots: what do we really know?

    PubMed

    Britto, D T; Kronzucker, H J

    2015-08-15

    The efflux of sodium (Na(+)) ions across the plasma membrane of plant root cells into the external medium is surprisingly poorly understood. Nevertheless, Na(+) efflux is widely regarded as a major mechanism by which plants restrain the rise of Na(+) concentrations in the cytosolic compartments of root cells and, thus, achieve a degree of tolerance to saline environments. In this review, several key ideas and bodies of evidence concerning root Na(+) efflux are summarized with a critical eye. Findings from decades past are brought to bear on current thinking, and pivotal studies are discussed, both "purely physiological", and also with regard to the SOS1 protein, the only major Na(+) efflux transporter that has, to date, been genetically characterized. We find that the current model of rapid transmembrane sodium cycling (RTSC), across the plasma membrane of root cells, is not adequately supported by evidence from the majority of efflux studies. An alternative hypothesis cannot be ruled out, that most Na(+) tracer efflux from the root in the salinity range does not proceed across the plasma membrane, but through the apoplast. Support for this idea comes from studies showing that Na(+) efflux, when measured with tracers, is rarely affected by the presence of inhibitors or the ionic composition in saline rooting media. We conclude that the actual efflux of Na(+) across the plasma membrane of root cells may be much more modest than what is often reported in studies using tracers, and may predominantly occur in the root tips, where SOS1 expression has been localized.

  6. Comparison of surficial CO2 efflux to other measures of subsurface crude oil degradation

    USGS Publications Warehouse

    Warren, Ean; Sihota, Natasha J.; Hostettler, Frances D.; Bekins, Barbara A.

    2014-01-01

    At a spill site near Bemidji, Minnesota, crude oil at the water table has been undergoing anaerobic biodegradation for over 30 years. Previous work at this site has shown that methane produced from biodegradation of the oil migrates upward and is oxidized in a methanotrophic zone midway between the water table and the surface. To compare microbial activity measurement methods from multiple locations in the oil body, surficial carbon dioxide efflux, methanogen and methanotroph concentrations, and oil degradation state were collected. Carbon dioxide effluxes over the oil body averaged more than four times those at the background site. Methanotrophic bacteria concentrations measured using pmoA were over 105 times higher above the oil-contaminated sediments compared with the background site. Methanogenic archaea measured using mcrA ranged from 105 to over 107 in the oil and were below detection in the background. Methanogens correlated very well with methanotroph concentrations (r = 0.99), n-alkylcyclohexane losses as a proxy for degradation state (r = − 0.96), and somewhat less well with carbon dioxide efflux (r = 0.92). Carbon dioxide efflux similarly correlated to methanotroph concentrations (r = 0.90) and n-alkylcyclohexane losses (r = − 0.91).

  7. Human paraoxonase 1 overexpression in mice stimulates HDL cholesterol efflux and reverse cholesterol transport

    PubMed Central

    Ikhlef, Souade; Berrougui, Hicham; Kamtchueng Simo, Olivier; Zerif, Echarki

    2017-01-01

    This study was aimed to investigate the effect of human PON1 overexpression in mice on cholesterol efflux and reverse cholesterol transport. PON1 overexpression in PON1-Tg mice induced a significant 3-fold (p<0.0001) increase in plasma paraoxonase activity and a significant ~30% (p<0.0001) increase in the capacity of HDL to mediate cholesterol efflux from J774 macrophages compared to wild-type mice. It also caused a significant 4-fold increase (p<0.0001) in the capacity of macrophages to transfer cholesterol to apoA-1, a significant 2-fold (p<0.0003) increase in ABCA1 mRNA and protein expression, and a significant increase in the expression of PPARγ (p<0.0003 and p<0.04, respectively) and LXRα (p<0.0001 and p<0.01, respectively) mRNA and protein compared to macrophages from wild-type mice. Moreover, transfection of J774 macrophages with human PON1 also increased ABCA1, PPARγ and LXRα protein expression and stimulates macrophages cholesterol efflux to apo A1. In vivo measurements showed that the overexpression of PON1 significantly increases the fecal elimination of macrophage-derived cholesterol in PON1-Tg mice. Overall, our results suggested that the overexpression of PON1 in mice may contribute to the regulation of the cholesterol homeostasis by improving the capacity of HDL to mediate cholesterol efflux and by stimulating reverse cholesterol transport. PMID:28278274

  8. Insight into determinants of substrate binding and transport in a multidrug efflux protein.

    PubMed

    Alegre, Kamela O; Paul, Stephanie; Labarbuta, Paola; Law, Christopher J

    2016-03-10

    Multidrug resistance arising from the activity of integral membrane transporter proteins presents a global public health threat. In bacteria such as Escherichia coli, transporter proteins belonging to the major facilitator superfamily make a considerable contribution to multidrug resistance by catalysing efflux of myriad structurally and chemically different antimicrobial compounds. Despite their clinical relevance, questions pertaining to mechanistic details of how these promiscuous proteins function remain outstanding, and the role(s) played by individual amino acid residues in recognition, binding and subsequent transport of different antimicrobial substrates by multidrug efflux members of the major facilitator superfamily requires illumination. Using in silico homology modelling, molecular docking and mutagenesis studies in combination with substrate binding and transport assays, we identified several amino acid residues that play important roles in antimicrobial substrate recognition, binding and transport by Escherichia coli MdtM, a representative multidrug efflux protein of the major facilitator superfamily. Furthermore, our studies suggested that 'aromatic clamps' formed by tyrosine and phenylalanine residues located within the substrate binding pocket of MdtM may be important for antimicrobial substrate recognition and transport by the protein. Such 'clamps' may be a structurally and functionally important feature of all major facilitator multidrug efflux proteins.

  9. Pregnancy Represses Induction of Efflux Transporters in Livers of Type I Diabetic Mice

    PubMed Central

    Aleksunes, Lauren M.; Xu, Jialin; Lin, Eugenia; Wen, Xia; Goedken, Michael J.; Slitt, Angela L.

    2013-01-01

    Purpose The purpose of this study was to determine whether down-regulation of transcription factor signaling during pregnancy disrupts the induction of efflux transporters in type I diabetic mice. Methods Type I diabetes was induced in female C57BL/6 mice with multiple low dose intraperitoneal injections of streptozotocin (STZ) at least 2 weeks prior to mating with normoglycemic male mice. On gestation day 14, livers were collected from vehicle- and STZ-treated non-pregnant and pregnant mice for quantification of efflux transporter and transcription factor signaling. Results STZ treatment up-regulated expression of Mrp1–5, Mdr1, Abcg5, Abcg8, Bcrp, and Bsep mRNA and/or protein in the livers of non-pregnant mice. Interestingly, little to no change in transporter expression was observed in STZ-treated mice that became pregnant. Enhanced PPARγ, Nrf2, and FXR transcription factor signaling, as quantified by nuclear binding and target gene regulation, was also observed in non-pregnant mice treated with STZ. Similar to efflux transporter expression, activation of these transcriptional pathways was diminished by pregnancy in STZ-treated mice. Conclusions This study demonstrates the opposing regulation of hepatobiliary efflux transporters in response to diabetes and pregnancy and points to PPARγ, Nrf2, and FXR as candidate pathways underlying the differential expression of transporters. PMID:23319174

  10. Efflux of hydraulically lifted water from mycorrhizal fungal hyphae during imposed drought

    PubMed Central

    Querejeta, José Ignacio; Allen, Michael F

    2008-01-01

    Apart from improving plant and soil water status during drought, it has been suggested that hydraulic lift (HL) could enhance plant nutrient capture through the flow of mineral nutrients directly from the soil to plant roots, or by maintaining the functioning of mycorrhizal fungi. We evaluated the extent to which the diel cycle of water availability created by HL covaries with the efflux of HL water from the tips of extramatrical (external) mycorrhizal hyphae, and the possible effects on biogeochemical processes. Phenotypic mycorrhizal fungal variables, such as total and live hyphal lengths, were positively correlated with HL efflux from hyphae, soil water potential (dawn), and plant response variables (foliar 15N). The efflux of HL water from hyphae was also correlated with bacterial abundance and soil enzyme activity (P), and the moistening of soil organic matter. Such findings indicate that the efflux of HL water from the external mycorrhizal mycelia may be a complementary explanation for plant nutrient acquisition and survival during drought. PMID:19704776

  11. Matrix metalloproteinase 8 degrades apolipoprotein A-I and reduces its cholesterol efflux capacity.

    PubMed

    Salminen, Aino; Åström, Pirjo; Metso, Jari; Soliymani, Rabah; Salo, Tuula; Jauhiainen, Matti; Pussinen, Pirkko J; Sorsa, Timo

    2015-04-01

    Various cell types in atherosclerotic lesions express matrix metalloproteinase (MMP)-8. We investigated whether MMP-8 affects the structure and antiatherogenic function of apolipoprotein (apo) A-I, the main protein component of HDL particles. Furthermore, we studied serum lipid profiles and cholesterol efflux capacity in MMP-8-deficient mouse model. Incubation of apoA-I (28 kDa) with activated MMP-8 yielded 22 kDa and 25 kDa apoA-I fragments. Mass spectrometric analyses revealed that apoA-I was cleaved at its carboxyl-terminal part. Treatment of apoA-I and HDL with MMP-8 resulted in significant reduction (up to 84%, P < 0.001) in their ability to facilitate cholesterol efflux from cholesterol-loaded THP-1 macrophages. The cleavage of apoA-I by MMP-8 and the reduction in its cholesterol efflux capacity was inhibited by doxycycline. MMP-8-deficient mice had significantly lower serum triglyceride (TG) levels (P = 0.003) and larger HDL particles compared with wild-type (WT) mice. However, no differences were observed in the apoA-I levels or serum cholesterol efflux capacities between the mouse groups. Proteolytic modification of apoA-I by MMP-8 may impair the first steps of reverse cholesterol transport, leading to increased accumulation of cholesterol in the vessel walls. Eventually, inhibition of MMPs by doxycycline may reduce the risk for atherosclerotic vascular diseases.

  12. Comparison of surficial CO2 efflux to other measures of subsurface crude oil degradation.

    PubMed

    Warren, Ean; Sihota, Natasha J; Hostettler, Frances D; Bekins, Barbara A

    2014-08-01

    At a spill site near Bemidji, Minnesota, crude oil at the water table has been undergoing anaerobic biodegradation for over 30years. Previous work at this site has shown that methane produced from biodegradation of the oil migrates upward and is oxidized in a methanotrophic zone midway between the water table and the surface. To compare microbial activity measurement methods from multiple locations in the oil body, surficial carbon dioxide efflux, methanogen and methanotroph concentrations, and oil degradation state were collected. Carbon dioxide effluxes over the oil body averaged more than four times those at the background site. Methanotrophic bacteria concentrations measured using pmoA were over 10(5) times higher above the oil-contaminated sediments compared with the background site. Methanogenic archaea measured using mcrA ranged from 10(5) to over 10(7) in the oil and were below detection in the background. Methanogens correlated very well with methanotroph concentrations (r=0.99), n-alkylcyclohexane losses as a proxy for degradation state (r=-0.96), and somewhat less well with carbon dioxide efflux (r=0.92). Carbon dioxide efflux similarly correlated to methanotroph concentrations (r=0.90) and n-alkylcyclohexane losses (r=-0.91).

  13. Comparison of surficial CO2 efflux to other measures of subsurface crude oil degradation

    NASA Astrophysics Data System (ADS)

    Warren, Ean; Sihota, Natasha J.; Hostettler, Frances D.; Bekins, Barbara A.

    2014-08-01

    At a spill site near Bemidji, Minnesota, crude oil at the water table has been undergoing anaerobic biodegradation for over 30 years. Previous work at this site has shown that methane produced from biodegradation of the oil migrates upward and is oxidized in a methanotrophic zone midway between the water table and the surface. To compare microbial activity measurement methods from multiple locations in the oil body, surficial carbon dioxide efflux, methanogen and methanotroph concentrations, and oil degradation state were collected. Carbon dioxide effluxes over the oil body averaged more than four times those at the background site. Methanotrophic bacteria concentrations measured using pmoA were over 105 times higher above the oil-contaminated sediments compared with the background site. Methanogenic archaea measured using mcrA ranged from 105 to over 107 in the oil and were below detection in the background. Methanogens correlated very well with methanotroph concentrations (r = 0.99), n-alkylcyclohexane losses as a proxy for degradation state (r = - 0.96), and somewhat less well with carbon dioxide efflux (r = 0.92). Carbon dioxide efflux similarly correlated to methanotroph concentrations (r = 0.90) and n-alkylcyclohexane losses (r = - 0.91).

  14. Compartmental models for apical efflux by P-glycoprotein. Part 1. Evaluation of model complexity

    PubMed Central

    Nagar, Swati; Tucker, Jalia; Weiskircher, Erica A.; Bhoopathy, Siddhartha; Hidalgo, Ismael J.; Korzekwa, Ken

    2013-01-01

    Purpose With the goal of quantifying P-gp transport kinetics, Part 1 of these manuscripts evaluates different compartmental models and Part 2 applies these models to kinetic data. Methods Models were developed to simulate the effect of apical efflux transporters on intracellular concentrations of six drugs. The effect of experimental variability on model predictions was evaluated. Several models were evaluated, and characteristics including membrane configuration, lipid content, and apical surface area (asa) were varied. Results Passive permeabilities from MDCK-MDR1 cells in the presence of cyclosporine gave lower model errors than from MDCK control cells. Consistent with the results in Part 2, model configuration had little impact on calculated model errors. The 5-compartment model was the simplest model that reproduced experimental lag times. Lipid content and asa had minimal effect on model errors, predicted lag times, and intracellular concentrations. Including endogenous basolateral uptake activity can decrease model errors. Models with and without explicit membrane barriers differed markedly in their predicted intracellular concentrations for basolateral drug exposure. Single point data resulted in clearances similar to time course data. Conclusions Compartmental models are useful to evaluate the impact of efflux transporters on intracellular concentrations. Whereas a 3-compartment model may be sufficient to predict the impact of transporters that efflux drugs from the cell, a 5-compartment model with explicit membranes may be required to predict intracellular concentrations when efflux occurs from the membrane. More complex models including additional compartments may be unnecessary. PMID:24019023

  15. Adaptive Resistance in Bacteria Requires Epigenetic Inheritance, Genetic Noise, and Cost of Efflux Pumps

    PubMed Central

    Motta, Santiago Sandoval; Cluzel, Philippe; Aldana, Maximino

    2015-01-01

    Adaptive resistance emerges when populations of bacteria are subjected to gradual increases of antibiotics. It is characterized by a rapid emergence of resistance and fast reversibility to the non-resistant phenotype when the antibiotic is removed from the medium. Recent work shows that adaptive resistance requires epigenetic inheritance and heterogeneity of gene expression patterns that are, in particular, associated with the production of porins and efflux pumps. However, the precise mechanisms by which inheritance and variability govern adaptive resistance, and what processes cause its reversibility remain unclear. Here, using an efflux pump regulatory network (EPRN) model, we show that the following three mechanisms are essential to obtain adaptive resistance in a bacterial population: 1) intrinsic variability in the expression of the EPRN transcription factors; 2) epigenetic inheritance of the transcription rate of EPRN associated genes; and 3) energetic cost of the efflux pumps activity that slows down cell growth. While the first two mechanisms acting together are responsible for the emergence and gradual increase of the resistance, the third one accounts for its reversibility. In contrast with the standard assumption, our model predicts that adaptive resistance cannot be explained by increased mutation rates. Our results identify the molecular mechanism of epigenetic inheritance as the main target for therapeutic treatments against the emergence of adaptive resistance. Finally, our theoretical framework unifies known and newly identified determinants such as the burden of efflux pumps that underlie bacterial adaptive resistance to antibiotics. PMID:25781931

  16. Adaptive resistance in bacteria requires epigenetic inheritance, genetic noise, and cost of efflux pumps.

    PubMed

    Motta, Santiago Sandoval; Cluzel, Philippe; Aldana, Maximino

    2015-01-01

    Adaptive resistance emerges when populations of bacteria are subjected to gradual increases of antibiotics. It is characterized by a rapid emergence of resistance and fast reversibility to the non-resistant phenotype when the antibiotic is removed from the medium. Recent work shows that adaptive resistance requires epigenetic inheritance and heterogeneity of gene expression patterns that are, in particular, associated with the production of porins and efflux pumps. However, the precise mechanisms by which inheritance and variability govern adaptive resistance, and what processes cause its reversibility remain unclear. Here, using an efflux pump regulatory network (EPRN) model, we show that the following three mechanisms are essential to obtain adaptive resistance in a bacterial population: 1) intrinsic variability in the expression of the EPRN transcription factors; 2) epigenetic inheritance of the transcription rate of EPRN associated genes; and 3) energetic cost of the efflux pumps activity that slows down cell growth. While the first two mechanisms acting together are responsible for the emergence and gradual increase of the resistance, the third one accounts for its reversibility. In contrast with the standard assumption, our model predicts that adaptive resistance cannot be explained by increased mutation rates. Our results identify the molecular mechanism of epigenetic inheritance as the main target for therapeutic treatments against the emergence of adaptive resistance. Finally, our theoretical framework unifies known and newly identified determinants such as the burden of efflux pumps that underlie bacterial adaptive resistance to antibiotics.

  17. The Role of Efflux Pumps in Schistosoma mansoni Praziquantel Resistant Phenotype

    PubMed Central

    Armada, Ana; Belo, Silvana; Carrilho, Emanuel; Viveiros, Miguel; Afonso, Ana

    2015-01-01

    Background Schistosomiasis is a neglected disease caused by a trematode of the genus Schistosoma that is second only to malaria in public health significance in Africa, South America, and Asia. Praziquantel (PZQ) is the drug of choice to treat this disease due to its high cure rates and no significant side effects. However, in the last years increasingly cases of tolerance to PZQ have been reported, which has caused growing concerns regarding the emergency of resistance to this drug. Methodology/Principal Findings Here we describe the selection of a parasitic strain that has a stable resistance phenotype to PZQ. It has been reported that drug resistance in helminths might involve efflux pumps such as members of ATP-binding cassette transport proteins, including P-glycoprotein and multidrug resistance-associated protein families. Here we evaluate the role of efflux pumps in Schistosoma mansoni resistance to PZQ, by comparing the efflux pumps activity in susceptible and resistant strains. The evaluation of the efflux activity was performed by an ethidium bromide accumulation assay in presence and absence of Verapamil. The role of efflux pumps in resistance to PZQ was further investigated comparing the response of susceptible and resistant parasites in the absence and presence of different doses of Verapamil, in an ex vivo assay, and these results were further reinforced through the comparison of the expression levels of SmMDR2 RNA by RT-PCR. Conclusions/Significance This work strongly suggests the involvement of Pgp-like transporters SMDR2 in Praziquantel drug resistance in S. mansoni. Low doses of Verapamil successfully reverted drug resistance. Our results might give an indication that a combination therapy with PZQ and natural or synthetic Pgp modulators can be an effective strategy for the treatment of confirmed cases of resistance to PZQ in S. mansoni. PMID:26445012

  18. Dopamine and noradrenaline efflux in the prefrontal cortex in the light and dark period: effects of novelty and handling and comparison to the nucleus accumbens.

    PubMed

    Feenstra, M G; Botterblom, M H; Mastenbroek, S

    2000-01-01

    We used on-line microdialysis measurements of dopamine and noradrenaline extracellular concentrations in the medial prefrontal cortex of awake, freely moving rats during the dark and the light period of the day to study whether (i) basal efflux would be higher in the active, dark period than in the inactive, light period; (ii) the activation induced by environmental stimuli would be dependent on these conditions. When determined one day after cannula placement, noradrenaline and dopamine levels were higher during the dark. Maximal relative increases induced by novelty and handling were 150% and 175-200%, respectively, and were very similar in the light and the dark, but the net increases were higher in the dark. Separate groups were tested one week after cannula placement to ensure recovery of possibly disturbed circadian rhythms. While basal levels in the dark were now approximately twice those in the light, the maximal relative and net increases after both novelty and handling were very similar. Basal levels of dopamine in the nucleus accumbens (one day after cannula placement) were not different in the light or dark, but were increased by novelty and handling to about 130% only in the light period, not in the dark. Thus, in the prefrontal cortex, dopamine strongly resembles noradrenaline, in that basal efflux was state dependent, whereas activation by stimuli was not. In the nucleus accumbens, basal dopamine efflux was not state dependent, but activation by stimuli was. These results suggest that there are differential effects of circadian phase on basal activity and responsiveness of the mesolimbic vs the mesocortical dopamine system.

  19. Endothelium-dependent relaxation of the pig aorta: relationship to stimulation of 86Rb efflux from isolated endothelial cells.

    PubMed Central

    Gordon, J. L.; Martin, W.

    1983-01-01

    Bradykinin, adenosine triphosphate (ATP) and acetylcholine each relaxed histamine-contracted strips of pig aorta in a dose-dependent manner. These relaxations were abolished when the endothelium was removed. Relaxation induced by ATP was mimicked by adenosine diphosphate (ADP) but adenosine monophosphate (AMP) and adenosine were about 120 times less potent. Relaxation induced by acetylcholine was antagonized by atropine in a competitive manner, and carbachol induced the same degree of relaxation as acetylcholine, but was about 10 times less potent. The calcium ionophore, A23187, also induced a dose-dependent relaxation of pig aortic strips provided the endothelium was present, suggesting that a rise in the level of ionized calcium within the endothelial cells is one means by which vascular smooth muscle relaxation can be triggered. Bradykinin, ATP, ADP, AMP, adenosine and A23187 each induced a dose-dependent increase in 86Rb efflux from preloaded pig aortic endothelial cells. The dose-response curves for stimulation of 86Rb efflux and for endothelium-dependent relaxation were similar for each individual compound. ADP was equipotent with ATP, but AMP and adenosine were about 120 times less potent. Neither acetylcholine nor carbachol, in concentrations that induce endothelium-dependent relaxation, had any effect on 86Rb efflux from isolated aortic endothelial cells. Lanthanum, which blocks calcium influx, abolished the increases in 86Rb efflux induced by bradykinin and ATP, and the calcium ionophore A23187 was the most effective stimulant of 86Rb efflux, suggesting that the potassium transport induced by these agents is calcium-activated. It is concluded that endothelial responses to bradykinin and ATP can be assessed by monitoring 86Rb efflux, which probably reflects a calcium-activated efflux of potassium associated with the endothelium-dependent vascular relaxation induced by these agents. This pathway is apparently not involved in endothelial responses to

  20. Identification and Characterization of Inhibitors of Multidrug Resistance Efflux Pumps in Pseudomonas aeruginosa: Novel Agents for Combination Therapy

    PubMed Central

    Lomovskaya, Olga; Warren, Mark S.; Lee, Angela; Galazzo, Jorge; Fronko, Richard; Lee, May; Blais, Johanne; Cho, Deidre; Chamberland, Suzanne; Renau, Tom; Leger, Roger; Hecker, Scott; Watkins, Will; Hoshino, Kazuki; Ishida, Hiroko; Lee, Ving J.

    2001-01-01

    Whole-cell assays were implemented to search for efflux pump inhibitors (EPIs) of the three multidrug resistance efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN) that contribute to fluoroquinolone resistance in clinical isolates of Pseudomonas aeruginosa. Secondary assays were developed to identify lead compounds with exquisite activities as inhibitors. A broad-spectrum EPI which is active against all three known Mex efflux pumps from P. aeruginosa and their close Escherichia coli efflux pump homolog (AcrAB-TolC) was discovered. When this compound, MC-207,110, was used, the intrinsic resistance of P. aeruginosa to fluoroquinolones was decreased significantly (eightfold for levofloxacin). Acquired resistance due to the overexpression of efflux pumps was also decreased (32- to 64-fold reduction in the MIC of levofloxacin). Similarly, 32- to 64-fold reductions in MICs in the presence of MC-207,110 were observed for strains with overexpressed efflux pumps and various target mutations that confer resistance to levofloxacin (e.g., gyrA and parC). We also compared the frequencies of emergence of levofloxacin-resistant variants in the wild-type strain at four times the MIC of levofloxacin (1 μg/ml) when it was used either alone or in combination with EPI. In the case of levofloxacin alone, the frequency was ∼10−7 CFU/ml. In contrast, with an EPI, the frequency was below the level of detection (<10−11). In summary, we have demonstrated that inhibition of efflux pumps (i) decreased the level of intrinsic resistance significantly, (ii) reversed acquired resistance, and (iii) resulted in a decreased frequency of emergence of P. aeruginosa strains that are highly resistant to fluoroquinolones. PMID:11120952

  1. HDL Cholesterol Efflux Predicts Graft Failure in Renal Transplant Recipients

    PubMed Central

    Annema, Wijtske; Dikkers, Arne; Freark de Boer, Jan; Dullaart, Robin P. F.; Sanders, Jan-Stephan F.; Bakker, Stephan J. L.

    2016-01-01

    High-density lipoprotein (HDL) particles are involved in the protection against cardiovascular disease by promoting cholesterol efflux, in which accumulated cholesterol is removed from macrophage foam cells. We investigated whether HDL cholesterol efflux capacity is associated with cardiovascular mortality, all-cause mortality, and graft failure in a cohort of renal transplant recipients (n=495, median follow-up 7.0 years). Cholesterol efflux capacity at baseline was quantified using incubation of human macrophage foam cells with apolipoprotein B–depleted plasma. Baseline efflux capacity was not different in deceased patients and survivors (P=0.60 or P=0.50 for cardiovascular or all-cause mortality, respectively), whereas recipients developing graft failure had lower efflux capacity than those with functioning grafts (P<0.001). Kaplan–Meier analysis demonstrated a lower risk for graft failure (P=0.004) but not cardiovascular (P=0.30) or all-cause mortality (P=0.31) with increasing gender-stratified tertiles of efflux capacity. Cox regression analyses adjusted for age and gender showed that efflux capacity was not associated with cardiovascular mortality (hazard ratio [HR], 0.89; 95% confidence interval [95% CI], 0.67 to 1.19; P=0.43). Furthermore, the association between efflux capacity and all-cause mortality (HR, .79; 95% CI, 0.63 to 0.98; P=0.031) disappeared after further adjustment for potential confounders. However, efflux capacity at baseline significantly predicted graft failure (HR, 0.43; 95% CI, 0.29 to 0.64; P<0.001) independent of apolipoprotein A-I, HDL cholesterol, or creatinine clearance. In conclusion, this prospective study shows that cholesterol efflux capacity from macrophage foam cells is not associated with cardiovascular or all-cause mortality but is a strong predictor of graft failure independent of plasma HDL cholesterol levels in renal transplant recipients. PMID:26319244

  2. Energy Metabolism and Drug Efflux in Mycobacterium tuberculosis

    PubMed Central

    Black, Philippa A.; Warren, Robin M.; Louw, Gail E.; van Helden, Paul D.; Victor, Thomas C.

    2014-01-01

    The inherent drug susceptibility of microorganisms is determined by multiple factors, including growth state, the rate of drug diffusion into and out of the cell, and the intrinsic vulnerability of drug targets with regard to the corresponding antimicrobial agent. Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant source of global morbidity and mortality, further exacerbated by its ability to readily evolve drug resistance. It is well accepted that drug resistance in M. tuberculosis is driven by the acquisition of chromosomal mutations in genes encoding drug targets/promoter regions; however, a comprehensive description of the molecular mechanisms that fuel drug resistance in the clinical setting is currently lacking. In this context, there is a growing body of evidence suggesting that active extrusion of drugs from the cell is critical for drug tolerance. M. tuberculosis encodes representatives of a diverse range of multidrug transporters, many of which are dependent on the proton motive force (PMF) or the availability of ATP. This suggests that energy metabolism and ATP production through the PMF, which is established by the electron transport chain (ETC), are critical in determining the drug susceptibility of M. tuberculosis. In this review, we detail advances in the study of the mycobacterial ETC and highlight drugs that target various components of the ETC. We provide an overview of some of the efflux pumps present in M. tuberculosis and their association, if any, with drug transport and concomitant effects on drug resistance. The implications of inhibiting drug extrusion, through the use of efflux pump inhibitors, are also discussed. PMID:24614376

  3. Spectinamides: A New Class of Semisynthetic Anti-Tuberculosis Agents that Overcome Native Drug Efflux

    PubMed Central

    Vaddady, Pavan K; Zheng, Zhong; Qi, Jianjun; Akbergenov, Rashid; Das, Sourav; Madhura, Dora B.; Rathi, Chetan; Trivedi, Ashit; Villellas, Cristina; Lee, Robin. B.; Rakesh; Waidyarachchi, Samanthi L.; Sun, Dianqing; McNeil, Michael R.; Ainsa, Jose A.; Boshoff, Helena I.; Gonzalez-Juarrero, Mercedes

    2014-01-01

    Although the classical antibiotic spectinomycin is a potent bacterial protein synthesis inhibitor, poor antimycobacterial activity limits its clinical application for treating tuberculosis. Using structure-based design, a novel semisynthetic series of spectinomycin analogs was generated with selective ribosomal inhibition and excellent narrow-spectrum antitubercular activity. In multiple murine infection models, these spectinamides were well tolerated, significantly reduced lung mycobacterial burden and increased survival. In vitro studies demonstrated a lack of cross-resistance with existing tuberculosis therapeutics, activity against MDR/XDR-tuberculosis, and an excellent pharmacological profile. Key to their potent antitubercular properties was their structural modification to evade the Rv1258c efflux pump, which is upregulated in MDR strains and is implicated in macrophage induced drug tolerance. The antitubercular efficacy of spectinamides demonstrates that synthetic modifications to classical antibiotics can overcome the challenge of intrinsic efflux pump-mediated resistance and expands opportunities for target based tuberculosis drug discovery. PMID:24464186

  4. Inhibition of the Human ABC Efflux Transporters P-gp and BCRP by the BDE-47 Hydroxylated Metabolite 6-OH-BDE-47: Considerations for Human Exposure.

    PubMed

    Marchitti, Satori A; Mazur, Christopher S; Dillingham, Caleb M; Rawat, Swati; Sharma, Anshika; Zastre, Jason; Kenneke, John F

    2017-01-01

    High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [((3)H)-paclitaxel and ((3)H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity. These findings suggest that some effects previously attributed to BDE-47 in biological systems may actually be due to 6-OH-BDE-47. Considerations for human exposure are discussed.

  5. Amphipathic polyproline peptides stimulate cholesterol efflux by the ABCA1 transporter.

    PubMed

    Sviridov, D O; Drake, S K; Freeman, L A; Remaley, A T

    2016-03-18

    ApoA-I mimetics are short synthetic peptides that contain an amphipathic α-helix and stimulate cholesterol efflux by the ABCA1 transporter in a detergent-like extraction mechanism. We investigated the use of amphipathic peptides with a polypro helix for stimulating cholesterol efflux by ABCA1. Polypro peptides were synthesized with modified prolines, containing either a hydrophobic phenyl group (Prop) or a polar N-acetylgalactosamine (Prog) attached to the pyrrolidine ring and were designated as either PP-2, 3, 4, or 5, depending on the number of 3 amino acid repeat units (Prop-Prog-Prop). Based on molecular modeling, these peptides were predicted to be relatively rigid and to bind to a phospholipid bilayer. By CD spectroscopy, PP peptides formed a Type-II polypro helix in an aqueous solution. PP-2 was inactive in promoting cholesterol efflux, but peptides with more than 2 repeat units were active. PP-4 showed a similar Vmax as a much longer amphipathic α-helical peptide, containing 37 amino acids, but had a Km that was approximately 20-fold lower. PP peptides were specific in that they did not stimulate cholesterol efflux from cells not expressing ABCA1 and were also non-cytotoxic. Addition of PP-3, 4 and 5 to serum promoted the formation of smaller size HDL species (7 nM) and increased its capacity for ABCA1-dependent cholesterol efflux by approximately 20-35% (p < 0.05). Because of their relatively small size and increased potency, amphipathic peptides with a polypro helix may represent an alternative structural motif for the development of apoA-I mimetic peptides.

  6. Genomic potential for arsenic efflux and methylation varies among global Prochlorococcus populations

    PubMed Central

    Saunders, Jaclyn K; Rocap, Gabrielle

    2016-01-01

    The globally significant picocyanobacterium Prochlorococcus is the main primary producer in oligotrophic subtropical gyres. When phosphate concentrations are very low in the marine environment, the mol:mol availability of phosphate relative to the chemically similar arsenate molecule is reduced, potentially resulting in increased cellular arsenic exposure. To mediate accidental arsenate uptake, some Prochlorococcus isolates contain genes encoding a full or partial efflux detoxification pathway, consisting of an arsenate reductase (arsC), an arsenite-specific efflux pump (acr3) and an arsenic-related repressive regulator (arsR). This efflux pathway was the only previously known arsenic detox pathway in Prochlorococcus. We have identified an additional putative arsenic mediation strategy in Prochlorococcus driven by the enzyme arsenite S-adenosylmethionine methyltransferase (ArsM) which can convert inorganic arsenic into more innocuous organic forms and appears to be a more widespread mode of detoxification. We used a phylogenetically informed approach to identify Prochlorococcus linked arsenic genes from both pathways in the Global Ocean Sampling survey. The putative arsenic methylation pathway is nearly ubiquitously present in global Prochlorococcus populations. In contrast, the complete efflux pathway is only maintained in populations which experience extremely low PO4:AsO4, such as regions in the tropical and subtropical Atlantic. Thus, environmental exposure to arsenic appears to select for maintenance of the efflux detoxification pathway in Prochlorococcus. The differential distribution of these two pathways has implications for global arsenic cycling, as their associated end products, arsenite or organoarsenicals, have differing biochemical activities and residence times. PMID:26151644

  7. A fluorescent microplate assay quantifies bacterial efflux and demonstrates two distinct compound binding sites in AcrB.

    PubMed

    Iyer, Ramkumar; Ferrari, Annette; Rijnbrand, R; Erwin, Alice L

    2015-04-01

    A direct assay of efflux by Escherichia coli AcrAB-TolC and related multidrug pumps would have great value in discovery of new Gram-negative antibiotics. The current understanding of how efflux is affected by the chemical structure and physical properties of molecules is extremely limited, derived from antibacterial data for compounds that inhibit growth of wild-type E. coli. We adapted a previously described fluorescent efflux assay to a 96-well microplate format that measured the ability of test compounds to compete for efflux with Nile Red (an environment-sensitive fluor), independent of antibacterial activity. We show that Nile Red and the lipid-sensitive probe DiBAC4-(3) [bis-(1,3-dibutylbarbituric acid)-trimethine oxonol] can quantify efflux competition in E. coli. We extend the previous findings that the tetracyclines compete with Nile Red and show that DiBAC4-(3) competes with macrolides. The extent of the competition shows a modest correlation with the effect of the acrB deletion on MICs within the compound sets for both dyes. Crystallographic studies identified at least two substrate binding sites in AcrB, the proximal and distal pockets. High-molecular-mass substrates bound the proximal pocket, while low-mass substrates occupied the distal pocket. As DiBAC4-(3) competes with macrolides but not with Nile Red, we propose that DiBAC4-(3) binds the proximal pocket and Nile Red likely binds the distal site. In conclusion, competition with fluorescent probes can be used to study the efflux process for diverse chemical structures and may provide information as to the site of binding and, in some cases, enable rank-ordering a series of related compounds by efflux.

  8. A Fluorescent Microplate Assay Quantifies Bacterial Efflux and Demonstrates Two Distinct Compound Binding Sites in AcrB

    PubMed Central

    Ferrari, Annette; Rijnbrand, R.; Erwin, Alice L.

    2015-01-01

    A direct assay of efflux by Escherichia coli AcrAB-TolC and related multidrug pumps would have great value in discovery of new Gram-negative antibiotics. The current understanding of how efflux is affected by the chemical structure and physical properties of molecules is extremely limited, derived from antibacterial data for compounds that inhibit growth of wild-type E. coli. We adapted a previously described fluorescent efflux assay to a 96-well microplate format that measured the ability of test compounds to compete for efflux with Nile Red (an environment-sensitive fluor), independent of antibacterial activity. We show that Nile Red and the lipid-sensitive probe DiBAC4-(3) [bis-(1,3-dibutylbarbituric acid)-trimethine oxonol] can quantify efflux competition in E. coli. We extend the previous findings that the tetracyclines compete with Nile Red and show that DiBAC4-(3) competes with macrolides. The extent of the competition shows a modest correlation with the effect of the acrB deletion on MICs within the compound sets for both dyes. Crystallographic studies identified at least two substrate binding sites in AcrB, the proximal and distal pockets. High-molecular-mass substrates bound the proximal pocket, while low-mass substrates occupied the distal pocket. As DiBAC4-(3) competes with macrolides but not with Nile Red, we propose that DiBAC4-(3) binds the proximal pocket and Nile Red likely binds the distal site. In conclusion, competition with fluorescent probes can be used to study the efflux process for diverse chemical structures and may provide information as to the site of binding and, in some cases, enable rank-ordering a series of related compounds by efflux. PMID:25645845

  9. Bacterial Multidrug Efflux Pumps: Much More Than Antibiotic Resistance Determinants

    PubMed Central

    Blanco, Paula; Hernando-Amado, Sara; Reales-Calderon, Jose Antonio; Corona, Fernando; Lira, Felipe; Alcalde-Rico, Manuel; Bernardini, Alejandra; Sanchez, Maria Blanca; Martinez, Jose Luis

    2016-01-01

    Bacterial multidrug efflux pumps are antibiotic resistance determinants present in all microorganisms. With few exceptions, they are chromosomally encoded and present a conserved organization both at the genetic and at the protein levels. In addition, most, if not all, strains of a given bacterial species present the same chromosomally-encoded efflux pumps. Altogether this indicates that multidrug efflux pumps are ancient elements encoded in bacterial genomes long before the recent use of antibiotics for human and animal therapy. In this regard, it is worth mentioning that efflux pumps can extrude a wide range of substrates that include, besides antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals or bacterial metabolites, among others. In the current review, we present information on the different functions that multidrug efflux pumps may have for the bacterial behaviour in different habitats as well as on their regulation by specific signals. Since, in addition to their function in non-clinical ecosystems, multidrug efflux pumps contribute to intrinsic, acquired, and phenotypic resistance of bacterial pathogens, the review also presents information on the search for inhibitors of multidrug efflux pumps, which are currently under development, in the aim of increasing the susceptibility of bacterial pathogens to antibiotics. PMID:27681908

  10. Bacterial multidrug efflux pumps: mechanisms, physiology and pharmacological exploitations.

    PubMed

    Sun, Jingjing; Deng, Ziqing; Yan, Aixin

    2014-10-17

    Multidrug resistance (MDR) refers to the capability of bacterial pathogens to withstand lethal doses of structurally diverse drugs which are capable of eradicating non-resistant strains. MDR has been identified as a major threat to the public health of human being by the World Health Organization (WHO). Among the four general mechanisms that cause antibiotic resistance including target alteration, drug inactivation, decreased permeability and increased efflux, drug extrusion by the multidrug efflux pumps serves as an important mechanism of MDR. Efflux pumps not only can expel a broad range of antibiotics owing to their poly-substrate specificity, but also drive the acquisition of additional resistance mechanisms by lowering intracellular antibiotic concentration and promoting mutation accumulation. Over-expression of multidrug efflux pumps have been increasingly found to be associated with clinically relevant drug resistance. On the other hand, accumulating evidence has suggested that efflux pumps also have physiological functions in bacteria and their expression is subject tight regulation in response to various of environmental and physiological signals. A comprehensive understanding of the mechanisms of drug extrusion, and regulation and physiological functions of efflux pumps is essential for the development of anti-resistance interventions. In this review, we summarize the development of these research areas in the recent decades and present the pharmacological exploitation of efflux pump inhibitors as a promising anti-drug resistance intervention.

  11. Bacterial Multidrug Efflux Pumps: Much More Than Antibiotic Resistance Determinants.

    PubMed

    Blanco, Paula; Hernando-Amado, Sara; Reales-Calderon, Jose Antonio; Corona, Fernando; Lira, Felipe; Alcalde-Rico, Manuel; Bernardini, Alejandra; Sanchez, Maria Blanca; Martinez, Jose Luis

    2016-02-16

    Bacterial multidrug efflux pumps are antibiotic resistance determinants present in all microorganisms. With few exceptions, they are chromosomally encoded and present a conserved organization both at the genetic and at the protein levels. In addition, most, if not all, strains of a given bacterial species present the same chromosomally-encoded efflux pumps. Altogether this indicates that multidrug efflux pumps are ancient elements encoded in bacterial genomes long before the recent use of antibiotics for human and animal therapy. In this regard, it is worth mentioning that efflux pumps can extrude a wide range of substrates that include, besides antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals or bacterial metabolites, among others. In the current review, we present information on the different functions that multidrug efflux pumps may have for the bacterial behaviour in different habitats as well as on their regulation by specific signals. Since, in addition to their function in non-clinical ecosystems, multidrug efflux pumps contribute to intrinsic, acquired, and phenotypic resistance of bacterial pathogens, the review also presents information on the search for inhibitors of multidrug efflux pumps, which are currently under development, in the aim of increasing the susceptibility of bacterial pathogens to antibiotics.

  12. Plasma cholesterol efflux capacity from human THP-1 macrophages is reduced in HIV-infected patients: impact of HAART[S

    PubMed Central

    El Khoury, Petra; Ghislain, Mathilde; Villard, Elise F.; Le Goff, Wilfried; Lascoux-Combe, Caroline; Yeni, Patrick; Meyer, Laurence; Vigouroux, Corinne; Goujard, Cécile; Guerin, Maryse

    2015-01-01

    The capacity of HDL to remove cholesterol from macrophages is inversely associated with the severity of angiographic coronary artery disease. The effect of human immunodeficiency virus (HIV) infection or its treatment on the ability of HDL particles to stimulate cholesterol efflux from human macrophages has never been studied. We evaluated the capacity of whole plasma and isolated HDL particles from HIV-infected subjects (n = 231) and uninfected controls (n = 200), as well as in a subset of 41 HIV subjects receiving highly active antiretroviral therapy (HAART) to mediate cholesterol efflux from human macrophages. Plasma cholesterol efflux capacity was reduced (−12%; P = 0.001) in HIV patients as compared with controls. HIV infection reduced by 27% (P < 0.05) the capacity of HDL subfractions to promote cholesterol efflux from macrophages. We observed a reduced ABCA1-dependent efflux capacity of plasma (−27%; P < 0.0001) from HIV-infected subjects as a result of a reduction in the efflux capacity of HDL3 particles. HAART administration restored the capacity of plasma from HIV patients to stimulate cholesterol efflux from human macrophages (9.4%; P = 0.04). During HIV infection, the capacity of whole plasma to remove cholesterol from macrophages is reduced, thus potentially contributing to the increased coronary heart disease in the HIV population. HAART administration restored the removal of cholesterol from macrophages by increasing HDL functionality. PMID:25573889

  13. Rubidium efflux as a tool for the pharmacological characterisation of compounds with BK channel opening properties.

    PubMed

    McKay, Neil G; Kirby, Robert W; Lawson, Kim

    2008-01-01

    This chapter describes a method of assaying rubidium (Rb(+)) efflux as a measure of potassium channel activity. In this assay, rubidium acts as a tracer for potassium movement across the cell membrane. HEK 293 cells expressing the alpha subunit of the human brain large-conductance, voltage-activated, calcium-sensitive potassium channel (BK channel) are loaded with Rb(+), washed, and then incubated under experimental conditions. The cell supernatant is removed, and the remaining cell monolayer lysed. These two samples contain Rb(+) that has moved out of the cell and Rb(+) that remains in the cell, respectively. Measurement of the Rb(+) content of these samples by flame atomic absorption spectrometry allows calculation of the percentage Rb(+) efflux and, depending on the experimental design, provides pharmacological data about the control and test compounds used. In this chapter, we describe the protocol and steps for optimisation and illustrate this with data obtained using NS1619, a well-characterised BK channel opener.

  14. Co-Regulations of Spartina alterniflora Invasion and Exogenous Nitrogen Loading on Soil N2O Efflux in Subtropical Mangrove Mesocosms.

    PubMed

    Jia, Dai; Qi, Fei; Xu, Xia; Feng, Jianxiang; Wu, Hao; Guo, Jiemin; Lu, Weizhi; Peng, Ronghao; Zhu, Xiaoshan; Luo, Yiqi; Lin, Guanghui

    2016-01-01

    Both plant invasion and nitrogen (N) enrichment should have significant impact on mangrove ecosystems in coastal regions around the world. However, how N2O efflux in mangrove wetlands responds to these environmental changes has not been well studied. Here, we conducted a mesocosm experiment with native mangrove species Kandelia obovata, invasive salt marsh species Spartina alterniflora, and their mixture in a simulated tide rotation system with or without nitrogen addition. In the treatments without N addition, the N2O effluxes were relatively low and there were no significant variations among the three vegetation types. A pulse loading of exogenous ammonium nitrogen increased N2O effluxes from soils but the stimulatory effect gradually diminished over time, suggesting that frequent measurements are necessary to accurately understand the behavior of N-induced response of N2O emissions. With the N addition, the N2O effluxes from the invasive S. alterniflora were lower than that from native K. obovata mesocosms. This result may be attributed to higher growth of S. alterniflora consuming most of the available nitrogen in soils, and thus inhibiting N2O production. We concluded that N loading significantly increased N2O effluxes, while the invasion of S. alterniflora reduced N2O effluxes response to N loading in this simulated mangrove ecosystem. Thus, both plant invasion and excessive N loading can co-regulate soil N2O emissions from mangrove wetlands, which should be considered when projecting future N2O effluxes from this type of coastal wetland.

  15. Differences in trans-stimulated chloroquine efflux kinetics are linked to PfCRT in Plasmodium falciparum

    PubMed Central

    Sanchez, Cecilia P.; Rohrbach, Petra; McLean, Jeremy E.; Fidock, David A.; Stein, Wilfred D.; Lanzer, Michael

    2010-01-01

    Summary The mechanism underpinning chloroquine drug resistance in the human malarial parasite Plasmodium falciparum has remained controversial. Currently discussed models include a carrier or a channel for chloroquine, the former actively expelling the drug, the latter facilitating its passive diffusion, out of the parasite’s food vacuole, where chloroquine accumulates and inhibits haem detoxification. Here we have challenged both models using an established trans-stimulation efflux protocol. While carriers may demonstrate trans-stimulation, channels do not. Our data reveal that extracellular chloroquine stimulates chloroquine efflux in the presence and absence of metabolic energy in both chloroquine-sensitive and -resistant parasites, resulting in a hyperbolic increase in the apparent initial efflux rates as the concentration of external chloroquine increases. In the absence of metabolic energy, the apparent initial efflux rates were comparable in both parasites. Significant differences were only observed in the presence of metabolic energy, where consistently higher apparent initial efflux rates were found in chloroquine-resistant parasites. As trans-stimulation is characteristic of a carrier, and not a channel, we interpret our data in favour of a carrier for chloroquine being present in both chloroquine-sensitive and -resistant parasites, however, with different transport modalities. PMID:17493125

  16. The diffusive influx and carrier efflux have a strong effect on the bistability of the lac operon in Escherichia coli.

    PubMed

    Noel, Jason T; Pilyugin, Sergei S; Narang, Atul

    2009-01-07

    In the presence of gratuitous inducers, the lac operon of Escherichia coli exhibits bistability. Most models in the literature assume that the inducer enters the cell via the carrier (permease), and exits by a diffusion-like process. The diffusive influx and carrier efflux are neglected. However, analysis of the data shows that in non-induced cells, the diffusive influx is comparable to the carrier influx, and in induced cells, the carrier efflux is comparable to the diffusive efflux. Since bistability entails the coexistence of steady states corresponding to both non-induced and induced cells, neither one of these fluxes can be ignored. We present a model accounting for both fluxes, and show that: (1) The thresholds (i.e., the extracellular inducer levels at which transcription turns on or off) are profoundly affected by both fluxes. The diffusive influx reduces the on threshold, and eliminates irreversible bistability, a phenomenon that is inconsistent with data. The carrier efflux increases the off threshold, and abolishes bistability at large permease activities, a conclusion that can be tested experimentally. (2) The thresholds are well approximated by simple analytical expressions obtained by considering two limiting cases (no carrier efflux and no diffusive influx). (3) The simulations are in good agreement with the data for isopropyl thiogalactoside (IPTG), but somewhat discrepant with respect to the data for thiomethyl galactoside (TMG). We discuss the potential sources of the discrepancy.

  17. Hyperoxia decreases muscle glycogenolysis, lactate production, and lactate efflux during steady-state exercise.

    PubMed

    Stellingwerff, Trent; Leblanc, Paul J; Hollidge, Melanie G; Heigenhauser, George J F; Spriet, Lawrence L

    2006-06-01

    The aim of this study was to determine whether the decreased muscle and blood lactate during exercise with hyperoxia (60% inspired O2) vs. room air is due to decreased muscle glycogenolysis, leading to decreased pyruvate and lactate production and efflux. We measured pyruvate oxidation via PDH, muscle pyruvate and lactate accumulation, and lactate and pyruvate efflux to estimate total pyruvate and lactate production during exercise. We hypothesized that 60% O2 would decrease muscle glycogenolysis, resulting in decreased pyruvate and lactate contents, leading to decreased muscle pyruvate and lactate release with no change in PDH activity. Seven active male subjects cycled for 40 min at 70% VO2 peak on two occasions when breathing 21 or 60% O2. Arterial and femoral venous blood samples and blood flow measurements were obtained throughout exercise, and muscle biopsies were taken at rest and after 10, 20, and 40 min of exercise. Hyperoxia had no effect on leg O2 delivery, O2 uptake, or RQ during exercise. Muscle glycogenolysis was reduced by 16% with hyperoxia (267 +/- 19 vs. 317 +/- 21 mmol/kg dry wt), translating into a significant, 15% reduction in total pyruvate production over the 40-min exercise period. Decreased pyruvate production during hyperoxia had no effect on PDH activity (pyruvate oxidation) but significantly decreased lactate accumulation (60%: 22.6 +/- 6.4 vs. 21%: 31.3 +/- 8.7 mmol/kg dry wt), lactate efflux, and total lactate production over 40 min of cycling. Decreased glycogenolysis in hyperoxia was related to an approximately 44% lower epinephrine concentration and an attenuated accumulation of potent phosphorylase activators ADPf and AMPf during exercise. Greater phosphorylation potential during hyperoxia was related to a significantly diminished rate of PCr utilization. The tighter metabolic match between pyruvate production and oxidation resulted in a decrease in total lactate production and efflux over 40 min of exercise during hyperoxia.

  18. Solar active region display system

    NASA Astrophysics Data System (ADS)

    Golightly, M.; Raben, V.; Weyland, M.

    2003-04-01

    The Solar Active Region Display System (SARDS) is a client-server application that automatically collects a wide range of solar data and displays it in a format easy for users to assimilate and interpret. Users can rapidly identify active regions of interest or concern from color-coded indicators that visually summarize each region's size, magnetic configuration, recent growth history, and recent flare and CME production. The active region information can be overlaid onto solar maps, multiple solar images, and solar difference images in orthographic, Mercator or cylindrical equidistant projections. Near real-time graphs display the GOES soft and hard x-ray flux, flare events, and daily F10.7 value as a function of time; color-coded indicators show current trends in soft x-ray flux, flare temperature, daily F10.7 flux, and x-ray flare occurrence. Through a separate window up to 4 real-time or static graphs can simultaneously display values of KP, AP, daily F10.7 flux, GOES soft and hard x-ray flux, GOES >10 and >100 MeV proton flux, and Thule neutron monitor count rate. Climatologic displays use color-valued cells to show F10.7 and AP values as a function of Carrington/Bartel's rotation sequences - this format allows users to detect recurrent patterns in solar and geomagnetic activity as well as variations in activity levels over multiple solar cycles. Users can customize many of the display and graph features; all displays can be printed or copied to the system's clipboard for "pasting" into other applications. The system obtains and stores space weather data and images from sources such as the NOAA Space Environment Center, NOAA National Geophysical Data Center, the joint ESA/NASA SOHO spacecraft, and the Kitt Peak National Solar Observatory, and can be extended to include other data series and image sources. Data and images retrieved from the system's database are converted to XML and transported from a central server using HTTP and SOAP protocols, allowing

  19. Efficiency of N use by wheat as a function of influx and efflux of NO sub 3

    NASA Technical Reports Server (NTRS)

    Huffaker, R. C.; Aslam, M.; Ward, M. R.

    1989-01-01

    Since N assimilation is one of the most costly functions of a plant, its efflux before assimilation results in a serious energy cost and loss in efficiency which could decrease yields. Efficient crop production is critical to the Controlled Ecological Life-Support System (CELSS). The objective is to determine the extent of efflux of the N species NO3(-), NH4(+), NO2(-), and urea after uptake, and possible means of regulation. It was found that NO3(-) efflux became serious as its substrate level increased. Efflux/Influx (E/I) of 3NO3(-) was greater in darkness (35 pct) than in light (14 pct) and the ratio greatly increased with increased substrate NO3(-), (up to 45 pct at 10 mM). It seems advantageous to use the lowest possible nutrient concentration of NO3(-). The feasibility of using ClO3(-) as a trapping agent (competitive inhibitor of NO3(-) uptake) for effluxed NO3(-) was assessed and its toxicity determined.

  20. Use and engineering of efflux pumps for the export of olefins in microbes

    SciTech Connect

    Mukhopadhyay, Aindrila

    2016-07-14

    The scope of the project is to investigate efflux pump systems in engineered host microorganisms, such as E. coli, and develop a pump engineered to export a target compound. To initiate the project in coordination with other TOTAL driven projects, the first target compound to be studied was 1-hexene. However, we were investigating other chemicals as Styrene. The main goal of the project was to generate a set of optimized efflux pump systems for microorganisms (E. coli and Streptomyces or other host) engineered to contain biosynthetic pathways to export large titers of target compounds that are toxic (or accumulate and push back biosynthesis) to the host cell. An optimized microbial host will utilize specific and efficient cell wall located pumps to extrude harmful target compounds and enable greater production of these compounds.

  1. Multidrug efflux pumps in Gram-negative bacteria and their role in antibiotic resistance.

    PubMed

    Blair, Jessica M A; Richmond, Grace E; Piddock, Laura J V

    2014-01-01

    Gram-negative bacteria express a plethora of efflux pumps that are capable of transporting structurally varied molecules, including antibiotics, out of the bacterial cell. This efflux lowers the intracellular antibiotic concentration, allowing bacteria to survive at higher antibiotic concentrations. Overexpression of some efflux pumps can cause clinically relevant levels of antibiotic resistance in Gram-negative pathogens. This review discusses the role of efflux in resistance of clinical isolates of Gram-negative bacteria, the regulatory mechanisms that control efflux pump expression, the recent advances in our understanding of efflux pump structure and how inhibition of efflux is a promising future strategy for tackling multidrug resistance in Gram-negative pathogens.

  2. Experimental investigation of charged liquid jet efflux from a capillary

    NASA Astrophysics Data System (ADS)

    Zhakin, A. I.; Belov, P. A.; Kuz'ko, A. E.

    2013-03-01

    The shapes and electrical characteristics of charged liquid (water, ethanol, glycerol, castor oil) jets emitted from a metal capillary have been experimentally studied depending on the applied high voltage. A map of efflux regimes in the flow velocity-applied voltage coordinates is constructed for water. The effects of medium viscosity, surface tension, and charge relaxation time on the laws of jet efflux are analyzed.

  3. Multidrug Resistance-associated Protein-1 (MRP-1)-dependent Glutathione Disulfide (GSSG) Efflux as a Critical Survival Factor for Oxidant-enriched Tumorigenic Endothelial Cells.

    PubMed

    Gordillo, Gayle M; Biswas, Ayan; Khanna, Savita; Spieldenner, James M; Pan, Xueliang; Sen, Chandan K

    2016-05-06

    Endothelial cell tumors are the most common soft tissue tumors in infants. Tumor-forming endothelial (EOMA) cells are able to escape cell death fate despite excessive nuclear oxidant burden. Our previous work recognized perinuclear Nox-4 as a key contributor to EOMA growth. The objective of this work was to characterize the mechanisms by which EOMA cells evade oxidant toxicity and thrive. In EOMA cells, compared with in the cytosol, the nuclear GSSG/GSH ratio was 5-fold higher. Compared to the ratio observed in healthy murine aortic endothelial (MAE) cells, GSSG/GSH was over twice as high in EOMA cells. Multidrug resistance-associated protein-1 (MRP-1), an active GSSG efflux mechanism, showed 2-fold increased activity in EOMA compared with MAE cells. Hyperactive YB-1 and Ape/Ref-1 were responsible for high MRP-1 expression in EOMA. Proximity ligand assay demonstrated MRP-1 and YB-1 binding. Such binding enabled the nuclear targeting of MRP-1 in EOMA in a leptomycin-B-sensitive manner. MRP-1 inhibition as well as knockdown trapped nuclear GSSG, causing cell death of EOMA. Disulfide loading of cells by inhibition of GSSG reductase (bischoloronitrosourea) or thioredoxin reductase (auranofin) was effective in causing EOMA death as well. In sum, EOMA cells survive a heavy oxidant burden by rapid efflux of GSSG, which is lethal if trapped within the cell. A hyperactive MRP-1 system for GSSG efflux acts as a critical survival factor for these cells, making it a potential target for EOMA therapeutics.

  4. Calcium induces tobramycin resistance in Pseudomonas aeruginosa by regulating RND efflux pumps.

    PubMed

    Khanam, Sharmily; Guragain, Manita; Lenaburg, Dirk L; Kubat, Ryan; Patrauchan, Marianna A

    2017-01-01

    Pseudomonas aeruginosa is an opportunistic multidrug resistant pathogen causing severe chronic infections. Our previous studies showed that elevated calcium (Ca(2+)) enhances production of several virulence factors and plant infectivity of the pathogen. Here we show that Ca(2+) increases resistance of P. aeruginosa PAO1 to tobramycin, antibiotic commonly used to treat Pseudomonas infections. LC-MS/MS-based comparative analysis of the membrane proteomes of P aeruginosa grown at elevated versus not added Ca(2+), determined that the abundances of two RND (resistance-nodulation-cell division) efflux pumps, MexAB-OprM and MexVW-OprM, were increased in the presence of elevated Ca(2+). Analysis of twelve transposon mutants with disrupted RND efflux pumps showed that six of them (mexB, muxC, mexY, mexJ, czcB, and mexE) contribute to Ca(2+)-induced tobramycin resistance. Transcriptional analyses by promoter activity and RT-qPCR showed that the expression of mexAB, muxABC, mexXY, mexJK, czcCBA, and mexVW is increased by elevated Ca(2+). Disruption of mexJ, mexC, mexI, and triA significantly decreased Ca(2+)-induced plant infectivity of the pathogen. Earlier, our group showed that PAO1 maintains intracellular Ca(2+) (Ca(2+)in) homeostasis, which mediates Ca(2+) regulation of P. aeruginosa virulence, and identified four putative Ca(2+) transporters involved in this process (Guragain et al., 2013). Here we show that three of these transporters (PA2435, PA2092, PA4614) play role in Ca(2+)-induced tobramycin resistance and one of them (PA2435) contributes to Ca(2+) regulation of mexAB-oprM promoter activity. Furthermore, mexJ, czcB, and mexE contribute to the maintenance of Ca(2+)in homeostasis. This provides the first evidence that Ca(2+)in homeostasis mediates Ca(2+) regulation of RND transport systems, which contribute to Ca(2+)-enhanced tobramycin resistance and plant infectivity in P. aeruginosa.

  5. Control of Angiogenesis by AIBP-mediated Cholesterol Efflux

    PubMed Central

    Fang, Longhou; Choi, Soo-Ho; Baek, Ji Sun; Liu, Chao; Almazan, Felicidad; Ulrich, Florian; Wiesner, Philipp; Taleb, Adam; Deer, Elena; Pattison, Jennifer; Torres-Vázquez, Jesús; Li, Andrew C.; Miller, Yury I.

    2013-01-01

    Cholesterol is a structural component of the cell, indispensable for normal cellular function, but its excess often leads to abnormal proliferation, migration, inflammatory responses and/or cell death. To prevent cholesterol overload, ATP-binding cassette (ABC) transporters mediate cholesterol efflux from the cells to apolipoprotein A-I (ApoA-I) and to the ApoA-I-containing high-density lipoprotein (HDL)1-3. Maintaining efficient cholesterol efflux is essential for normal cellular function4-6. However, the role of cholesterol efflux in angiogenesis and the identity of its local regulators are poorly understood. Here we show that ApoA-I binding protein (AIBP) accelerates cholesterol efflux from endothelial cells (EC) to HDL and thereby regulates angiogenesis. AIBP/HDL-mediated cholesterol depletion reduces lipid rafts, interferes with VEGFR2 dimerization and signaling, and inhibits VEGF-induced angiogenesis in vitro and mouse aortic neovascularization ex vivo. Remarkably, Aibp regulates the membrane lipid order in embryonic zebrafish vasculature and functions as a non-cell autonomous regulator of zebrafish angiogenesis. Aibp knockdown results in dysregulated sprouting/branching angiogenesis, while forced Aibp expression inhibits angiogenesis. Dysregulated angiogenesis is phenocopied in Abca1/Abcg1-deficient embryos, and cholesterol levels are increased in Aibp-deficient and Abca1/Abcg1-deficient embryos. Our findings demonstrate that secreted AIBP positively regulates cholesterol efflux from EC and that effective cholesterol efflux is critical for proper angiogenesis. PMID:23719382

  6. The PerR-regulated P1B-4 type ATPase (PmtA) acts as a ferrous iron efflux pump in Streptococcus pyogenes.

    PubMed

    Turner, Andrew G; Ong, Cheryl-Lynn Y; Djoko, Karrera Y; West, Nicholas P; Davies, Mark R; McEwan, Alastair G; Walker, Mark J

    2017-04-03

    Streptococcus pyogenes (group A Streptococcus; GAS) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and as such, tightly modulates metal uptake and efflux during infection. Metal acquisition systems are required to combat metal sequestration by the host, while metal efflux systems are essential to protect against metal overload poisoning. Here, we investigated the function of PmtA (PerR-regulated metal transporter A), a P1B-4 type ATPase efflux pump, in the invasive GAS M1T1 strain 5448. We reveal that PmtA functions as a ferrous iron [Fe(II)] efflux system. In the presence of high Fe(II) concentrations, the 5448ΔpmtA deletion mutant exhibited diminished growth and accumulated 5-fold higher intracellular Fe(II) compared to the wild-type and complemented mutant. The 5448ΔpmtA deletion mutant also showed enhanced susceptibility to killing by the Fe-dependent antibiotic, streptonigrin, as well as increased sensitivity to hydrogen peroxide and superoxide. We suggest that the PerR-mediated control of Fe(II) efflux by PmtA is important for bacterial defense against oxidative stress. PmtA represents an exemplar for a Fe(II) efflux system in a host-adapted Gram-positive bacterial pathogen.

  7. Staphylococcus aureus MnhF Mediates Cholate Efflux and Facilitates Survival under Human Colonic Conditions

    PubMed Central

    Sannasiddappa, Thippeswamy H.; Hood, Graham A.; Hanson, Kevan J.; Costabile, Adele; Gibson, Glenn R.

    2015-01-01

    Resistance to the innate defenses of the intestine is crucial for the survival and carriage of Staphylococcus aureus, a common colonizer of the human gut. Bile salts produced by the liver and secreted into the intestines are one such group of molecules with potent antimicrobial activity. The mechanisms by which S. aureus is able to resist such defenses in order to colonize and survive in the human gut are unknown. Here we show that mnhF confers resistance to bile salts, which can be abrogated by efflux pump inhibitors. MnhF mediates the efflux of radiolabeled cholic acid both in S. aureus and when heterologously expressed in Escherichia coli, rendering them resistant. Deletion of mnhF attenuated the survival of S. aureus in an anaerobic three-stage continuous-culture model of the human colon (gut model), which represents different anatomical areas of the large intestine. PMID:25824834

  8. Resistance-Nodulation-Division Multidrug Efflux Pumps in Gram-Negative Bacteria: Role in Virulence.

    PubMed

    Fernando, Dinesh M; Kumar, Ayush

    2013-03-18

    Resistance-Nodulation-Division (RND) efflux pumps are one of the most important determinants of multidrug resistance (MDR) in Gram-negative bacteria. With an ever increasing number of Gram-negative clinical isolates exhibiting MDR phenotypes as a result of the activity of RND pumps, it is clear that the design of novel effective clinical strategies against such pathogens must be grounded in a better understanding of these pumps, including their physiological roles. To this end, recent evidence suggests that RND pumps play an important role in the virulence of Gram-negative pathogens. In this review, we discuss the important role RND efflux pumps play in different facets of virulence including colonization, evasion of host defense mechanisms, and biofilm formation. These studies provide key insights that may ultimately be applied towards strategies used in the design of effective therapeutics against MDR Gram negative bacterial pathogens.

  9. Volume-dependent osmolyte efflux from neural tissues

    PubMed Central

    Fisher, Stephen K.; Cheema, Tooba A.; Foster, Daniel J.; Heacock, Anne M.

    2008-01-01

    The CNS is particularly vulnerable to reductions in plasma osmolarity, such as occurr during hyponatremia, the most commonly encountered electrolyte disorder in clinical practice. In response to a lowered plasma osmolarity, neural cells initially swell but then are able to restore their original volume through the release of osmolytes, both inorganic and organic, and the exit of osmotically obligated water. Given the importance of the maintenance of cell volume within the CNS, mechanisms underlying the release of osmolytes assume major significance. In this context, we review recent evidence obtained from our laboratory and others that indicates that the activation of specific G-protein-coupled receptors can markedly enhance the volume-dependent release of osmolytes from neural cells. Of particular significance is the observation that receptor activation significantly lowers the osmotic threshold at which osmolyte release occurs, thereby facilitating the ability of the cells to respond to small, more physiologically relevant, reductions in osmolarity. The mechanisms underlying G-protein-coupled receptor-mediated osmolyte release and the possibility that this efflux can result in both physiologically beneficial and potentially harmful pathophysiological consequences are discussed. PMID:18518929

  10. Bypassing P-Glycoprotein Drug Efflux Mechanisms: Possible Applications in Pharmacoresistant Schizophrenia Therapy

    PubMed Central

    Hoosain, Famida G.; Choonara, Yahya E.; Tomar, Lomas K.; Kumar, Pradeep; Tyagi, Charu; du Toit, Lisa C.; Pillay, Viness

    2015-01-01

    The efficient noninvasive treatment of neurodegenerative disorders is often constrained by reduced permeation of therapeutic agents into the central nervous system (CNS). A vast majority of bioactive agents do not readily permeate into the brain tissue due to the existence of the blood-brain barrier (BBB) and the associated P-glycoprotein efflux transporter. The overexpression of the MDR1 P-glycoprotein has been related to the occurrence of multidrug resistance in CNS diseases. Various research outputs have focused on overcoming the P-glycoprotein drug efflux transporter, which mainly involve its inhibition or bypassing mechanisms. Studies into neurodegenerative disorders have shown that the P-glycoprotein efflux transporter plays a vital role in the progression of schizophrenia, with a noted increase in P-glycoprotein function among schizophrenic patients, thereby reducing therapeutic outcomes. In this review, we address the hypothesis that methods employed in overcoming P-glycoprotein in cancer and other disease states at the level of the BBB and intestine may be applied to schizophrenia drug delivery system design to improve clinical efficiency of drug therapies. In addition, the current review explores polymers and drug delivery systems capable of P-gp inhibition and modulation. PMID:26491671

  11. Functional cloning and expression of emeA, and characterization of EmeA, a multidrug efflux pump from Enterococcus faecalis.

    PubMed

    Lee, Eun-Woo; Chen, Jing; Huda, Md Nazmul; Kuroda, Teruo; Mizushima, Tohru; Tsuchiya, Tomofusa

    2003-02-01

    A fragment of chromosomal DNA from Enterococcus faecalis ATCC 29212 was cloned using Escherichia coli KAM32 host cells lacking major multidrug efflux pumps. E. coli KAM32 cells were sensitive to many antimicrobial agents, and the transformed cells harboring a recombinant plasmid became resistant to several structurally unrelated antimicrobial agents such as tetraphenylphosphonium chloride, 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33342, acriflavine, benzalkonium chloride, norfloxacin and ethidium bromide. This suggests that the cloned DNA fragment carries a gene(s) encoding a multidrug efflux pump. Determination of the nucleotide sequence of the cloned DNA revealed a gene designated as emeA. The transformed E. coli cells showed efflux activity of several antimicrobial agents such as DAPI, Hoechst 33342 and acriflavine. Efflux of DAPI via EmeA was strongly inhibited by reserpine.

  12. Identification and expression of nor efflux family genes in Staphylococcus epidermidis that act against gatifloxacin.

    PubMed

    Juárez-Verdayes, Marco A; Parra-Ortega, Berenice; Hernández-Rodríguez, César; Betanzos-Cabrera, Gabriel; Rodríguez-Martínez, Sandra; Cancino-Diaz, Mario E; Cancino-Diaz, Juan C

    2012-06-01

    NorA, NorB, and NorC are efflux proteins in the Nor family that regulate the secretion of fluoroquinolones, and MgrA/NorR is a transcription factor of the Nor family. Overexpression of Nor family proteins provides fluoroquinolone resistance in Staphylococcus aureus. However, in coagulase-negative staphylococci (CNS), members of the Nor family had not been identified. In this work, the presence of Nor family proteins in Staphylococcus spp. and the expression of Nor family in gatifloxacin resistant S. epidermidis strains obtained from ocular infections (OI) were identified and analyzed. S. epidermidis strains from OIs (n = 44) and healthy skin (HS; n = 52) were isolated. The nor family genes were identified in CNS using PCR, sequencing and phylogenetic approaches. Nor family expression was determined by RT-PCR. NorA efflux activity was determined using the automated ethidium bromide method. In-silico analysis showed that norA, mgrA/norR, and "norB-like" and "norC-like" (norB/norC) genes are present in CNS. The nor family genes were distributed and constitutively expressed in all S. epidermidis strains studied. In one gatifloxacin resistant strain isolated from the endophthalmitis, treatment with gatifloxacin induced overexpression of the norA gene and resulted in high activity of NorA efflux. These results indicate that the Nor family of proteins is present in CNS, and the NorA efflux mechanism for gatifloxacin response occurs in at least one strain of S. epidermidis, contributing to gatifloxacin resistance.

  13. Enhanced Corneal Absorption of Erythromycin by Modulating P-Glycoprotein and MRP Mediated Efflux with Corticosteroids

    PubMed Central

    Hariharan, Sudharshan; Gunda, Sriram; Mishra, Gyan P.; Pal, Dhananjay; Mitra, Ashim K.

    2015-01-01

    Purpose The objectives were (i) to test in vivo functional activity of MRP2 on rabbit corneal epithelium and (ii) to evaluate modulation of P-gp and MRP2 mediated efflux of erythromycin when co-administered with corticosteroids. Methods Cultured rabbit primary corneal epithelial cells (rPCECs) was employed as an in vitro model for rabbit cornea. Cellular accumulation and bi-directional transport studies were conducted across Madin-Darby Canine Kidney (MDCK) cells overexpressing MDR1 and MRP2 proteins to delineate transporter specific interaction of steroids. Ocular pharmacokinetic studies were conducted in rabbits following a single-dose infusion of erythromycin in the presence of specific inhibitors and steroids. Results Bi-directional transport of erythromycin across MDCK-MDR1 and MDCK-MRP2 cells showed significant difference between BL-AP and AP-BL permeability, suggesting that erythromycin is a substrate for P-gp and MRP2. Cellular accumulation of erythromycin in rPCEC was inhibited by steroids in a dose dependent manner. MK571, a specific MRP inhibitor, modulated the aqueous humor concentration of erythromycin in vivo. Even, steroids inhibited P-gp and MRP2 mediated efflux with maximum increase in ka, AUC0−∞, Cmax and Clast values of erythromycin, observed with 6α-methyl prednisolone. Conclusion MRP2 is functionally active along with P-gp in effluxing drug molecules out of corneal epithelium. Steroids were able to significantly inhibit both P-gp and MRP2 mediated efflux of erythromycin. PMID:18958406

  14. Significance of the percentage of cholesterol efflux capacity and total cholesterol efflux capacity in patients with or without coronary artery disease.

    PubMed

    Norimatsu, Kenji; Kuwano, Takashi; Miura, Shin-Ichiro; Shimizu, Tomohiko; Shiga, Yuhei; Suematsu, Yasunori; Miyase, Yuiko; Adachi, Sen; Nakamura, Ayumi; Imaizumi, Satoshi; Iwata, Atsushi; Nishikawa, Hiroaki; Uehara, Yoshinari; Saku, Keijiro

    2017-01-01

    We hypothesized that cholesterol efflux capacity is more useful than the lipid profile as a marker of the presence and the severity of coronary artery disease (CAD). Therefore, we investigated the associations between the presence and the severity of CAD and both the percentage of cholesterol efflux capacity and total cholesterol efflux capacity and the lipid profile including the high-density lipoprotein cholesterol (HDL-C) level in patients who underwent coronary computed tomography angiography (CTA). The subjects consisted of 204 patients who were clinically suspected to have CAD and underwent CTA. We isolated HDL from plasma by ultracentrifugation and measured the percentage of cholesterol efflux capacity using (3)H-cholesterol-labeled J774 macrophage cells and calculated total cholesterol efflux capacity as follows: the percentage of cholesterol efflux capacity/100× HDL-C levels. While the percentage of cholesterol efflux capacity was not associated with the presence or the severity of CAD, total cholesterol efflux capacity and HDL-C in patients with CAD were significantly lower than those in patients without CAD. In addition, total cholesterol efflux capacity and HDL-C, but not the percentage of cholesterol efflux capacity, significantly decreased as the number of coronary arteries with significant stenosis increased. Total cholesterol efflux capacity was positively correlated with HDL-C, whereas the percentage of cholesterol efflux capacity showed only weak association. In a logistic regression analysis, the presence of CAD was independently associated with total cholesterol efflux capacity, in addition to age and gender. Finally, a receiver-operating characteristic curve analysis indicated that the areas under the curves for total cholesterol efflux capacity and HDL-C were similar. In conclusion, the percentage of cholesterol efflux capacity using the fixed amount of isolated HDL was not associated with CAD. On the other hand, the calculated total

  15. Comparative Molecular Docking Studies with ABCC1 and Aquaporin 9 in the Arsenite Complex Efflux

    PubMed Central

    Poojan, Shiv; Dhasmana, Anupam; Jamal, Qazi Mohammad Sajid; Haneef, Mohd; Lohani, Mohtashim

    2014-01-01

    Arsenic is the most toxic metalloid present in the natural environment in both organic and inorganic arsenic forms. Inorganic arsenic is often more hazardous than the organic form. Arsenite and arsenate compounds are the major inorganic forms which are toxic causing severe human health dysfunction including cancer. Excretion of arsenic from the system is found elusive. Therefore, it is of interest to screen channel proteins with the arsenic complex in the different combination of arsenic, GSH (glutathione) and arsenic, selenium using docking methods. The mode of arsenic removal. The complex structure revealed the mode of arsenic binding efficiency with the receptor aquaporine 9 and ABCC1 channel protein. This provides insights to understand the mechanism of arsenic efflux. These inferences find application in the design, identification and development of novel nutracetucal or any other formulation useful in the balance of arsenic efflux. PMID:25258480

  16. Active thermal control system evolution

    NASA Technical Reports Server (NTRS)

    Petete, Patricia A.; Ames, Brian E.

    1991-01-01

    The 'restructured' baseline of the Space Station Freedom (SSF) has eliminated many of the growth options for the Active Thermal Control System (ATCS). Modular addition of baseline technology to increase heat rejection will be extremely difficult. The system design and the available real estate no longer accommodate this type of growth. As the station matures during its thirty years of operation, a demand of up to 165 kW of heat rejection can be expected. The baseline configuration will be able to provide 82.5 kW at Eight Manned Crew Capability (EMCC). The growth paths necessary to reach 165 kW have been identified. Doubling the heat rejection capability of SSF will require either the modification of existing radiator wings or the attachment of growth structure to the baseline truss for growth radiator wing placement. Radiator performance can be improved by enlarging the surface area or by boosting the operating temperature with a heat pump. The optimal solution will require both modifications. The addition of growth structure would permit the addition of a parallel ATCS using baseline technology. This growth system would simplify integration. The feasibility of incorporating these growth options to improve the heat rejection capacity of SSF is under evaluation.

  17. Resistance-nodulation-division efflux pump acrAB is modulated by florfenicol and contributes to drug resistance in the fish pathogen Piscirickettsia salmonis.

    PubMed

    Sandoval, Rodrigo; Oliver, Cristian; Valdivia, Sharin; Valenzuela, Karla; Haro, Ronie E; Sánchez, Patricio; Olavarría, Víctor H; Valenzuela, Paulina; Avendaño-Herrera, Rubén; Romero, Alex; Cárcamo, Juan G; Figueroa, Jaime E; Yáñez, Alejandro J

    2016-06-01

    Piscirickettsia salmonis is a fastidious intracellular pathogen responsible for high mortality rates in farmed salmonids, with serious economic consequences for the Chilean aquaculture industry. Oxytetracycline and florfenicol are the most frequently used antibiotics against P. salmonis, but routine use could contribute to drug resistance. This study identified differentiated florfenicol susceptibilities in two P. salmonis strains, LF-89 and AUSTRAL-005. The less susceptible isolate, AUSTRAL-005, also showed a high ethidium bromide efflux rate, indicating a higher activity of general efflux pump genes than LF-89. The P. salmonis genome presented resistance nodulation division (RND) family members, a family containing typical multidrug resistance-related efflux pumps in Gram-negative bacteria. Additionally, efflux pump acrAB genes were overexpressed in AUSTRAL-005 following exposure to the tolerated maximal concentration of florfenicol, in contrast to LF-89. These results indicate that tolerated maximum concentrations of florfenicol can modulate RND gene expression and increase efflux pump activity. We propose that the acrAB efflux pump is essential for P. salmonis survival at critical florfenicol concentrations and for the generation of antibiotic-resistant bacterial strains.

  18. The Effects of Nitrogen Enrichment and a Simulated Rainfall Event on Soil Carbon Dioxide Efflux in an Annual California Grassland

    NASA Astrophysics Data System (ADS)

    Johnson, T. P.; Strong, A. L.; Chiariello, N.; Field, C. B.

    2013-12-01

    Soils contain the largest pool of carbon in terrestrial ecosystems and play a critical role in the global carbon cycle. Previous studies have shown that enhanced precipitation (projected by climate models) and human activities (such as increased fertilizer use) may alter this cycle by enhancing soil microbial activity, although effects are often variable. Soils in semi-arid grasslands play a vital role in the global carbon cycle and may be responsive to environmental perturbations. Previous studies have demonstrated that wet-up treatments positively influence soil carbon dioxide efflux rates, which are otherwise low during dry summers. A preliminary study performed in a semi-arid annual grassland has shown that long-term nitrogen enrichment (equivalent to 70kg N per hectare) positively influences soil carbon dioxide efflux during peak biomass in the wet season. However, the combined effect and seasonal dynamics of these environmental changes is poorly understood. In order to assess this interaction, we explore the short-term response of soil carbon dioxide efflux rates in a semi-arid grassland to a combination of long-term nitrogen enrichment and a simulated 20-mm rainfall event in the Jasper Ridge Global Change Experiment (JRCGE), a long-term, multi-factorial experiment in a semi-arid annual grassland located in the foothills of the Santa Cruz mountains in central California. We measured soil carbon dioxide efflux rates from pre-installed soil respiration collars for forty-eight hours after a simulated rainfall event (20mm) during the dry season in late July 2013. Both the enhanced and non-enhanced nitrogen treatments had an immediate pronounced response to the wet-up stimulation in which efflux rates increased by an average of more than six-fold. In contrast with previous studies of soil carbon dioxide efflux at JRGCE during the wet season in which N enrichment elevated efflux rates relative to controls, however, the soil carbon dioxide efflux rates in response

  19. Alpinetin enhances cholesterol efflux and inhibits lipid accumulation in oxidized low-density lipoprotein-loaded human macrophages.

    PubMed

    Jiang, Zhengming; Sang, Haiqiang; Fu, Xin; Liang, Ying; Li, Ling

    2015-01-01

    Alpinetin is a natural flavonoid abundantly present in the ginger family. Here, we investigated the effect of alpinetin on cholesterol efflux and lipid accumulation in oxidized low-density lipoprotein (ox-LDL)-treated THP-1 macrophages and human peripheral blood monocyte-derived macrophages (HMDMs). After exposing THP-1 macrophages to alpinetin, cholesterol efflux was determined by liquid scintillator. The mRNA and protein levels of peroxisome proliferator-activated receptor gamma (PPAR-γ), liver X receptor alpha (LXR-α), ATP-binding cassette transporter A1 (ABCA1), and ABCG1 and scavenger receptor class B member 1 were determined by reverse-transcriptase PCR (RT-PCR) and Western blot analysis, respectively. Alpinetin promoted apolipoprotein A-I- and high-density-lipoprotein-mediated cholesterol efflux and elevated PPAR-γ and LXR-α mRNA and protein expression in a dose-dependent fashion in ox-LDL-treated THP-1 macrophages and HMDMs. Small interfering RNA-mediated silencing of PPAR-γ or LXR-α dose dependently reversed alpinetin-increased cholesterol efflux in THP-1 macrophages, indicating the involvement of PPAR-γ and LXR-α in alpinetin-promoted cholesterol efflux. Alpinetin inhibited ox-LDL-induced lipid accumulation and enhanced the expression of ABCA1 and ABCG1 mRNA and protein, which was reversed by specific knockdown of PPAR-γ or LXR-α. Taken together, our results reveal that alpinetin exhibits positive effects on cholesterol efflux and inhibits ox-LDL-induced lipid accumulation, which might be through PPAR-γ/LXR-α/ABCA1/ABCG1 pathway.

  20. Group X Secretory Phospholipase A2 Negatively Regulates ABCA1 and ABCG1 expression and cholesterol efflux in macrophages

    PubMed Central

    Shridas, Preetha; Bailey, William M; Gizard, Florence; Oslund, Rob C; Gelb, Michael H; Bruemmer, Dennis; Webb, Nancy R

    2010-01-01

    Objectives Group X secretory phospholipase A2 (GX sPLA2) potently hydrolyzes plasma membranes to generate lysophospholipids and free fatty acids and has been implicated in inflammatory diseases including atherosclerosis. Here we identify a novel role for GX sPLA2 in modulating ABCA1 and ABCG1 expression and hence macrophage cholesterol efflux. Methods and Results Overexpression or exogenous addition of GX sPLA2 significantly reduced ABCA1 and ABCG1 expression in J774 macrophage-like cells, whereas GX sPLA2 deficiency in mouse peritoneal macrophages (MPMs) was associated with enhanced expression. Altered ABC transporter expression led to reduced cholesterol efflux in GX sPLA2 overexpressing J774 cells, and increased efflux in GX sPLA2-deficient MPMs. Gene regulation was dependent on GX sPLA2 catalytic activity, mimicked by arachidonic acid, abrogated when LXRα/β expression was suppressed, and partially reversed by the LXR agonist T0901317. Reporter assays indicated that GX sPLA2 suppresses the ability of LXR to trans-activate its promoters through a mechanism involving the C-terminal portion of LXR spanning the ligand binding domain. Conclusions GX sPLA2 modulates gene expression in macrophages by generating lipolytic products that suppress LXR activation. GX sPLA2 may play a previously unrecognized role in atherosclerotic lipid accumulation by negatively regulating genes critical for cellular cholesterol efflux. PMID:20844270

  1. MexEF-OprN efflux pump exports the Pseudomonas quinolone signal (PQS) precursor HHQ (4-hydroxy-2-heptylquinoline).

    PubMed

    Lamarche, Martin G; Déziel, Eric

    2011-01-01

    Bacterial cells have evolved the capacity to communicate between each other via small diffusible chemical signals termed autoinducers. Pseudomonas aeruginosa is an opportunistic pathogen involved, among others, in cystic fibrosis complications. Virulence of P. aeruginosa relies on its ability to produce a number of autoinducers, including 4-hydroxy-2-alkylquinolines (HAQ). In a cell density-dependent manner, accumulated signals induce the expression of multiple targets, especially virulence factors. This phenomenon, called quorum sensing, promotes bacterial capacity to cause disease. Furthermore, P. aeruginosa possesses many multidrug efflux pumps conferring adaptive resistance to antibiotics. Activity of some of these efflux pumps also influences quorum sensing. The present study demonstrates that the MexEF-OprN efflux pump modulates quorum sensing through secretion of a signalling molecule belonging to the HAQ family. Moreover, activation of MexEF-OprN reduces virulence factor expression and swarming motility. Since MexEF-OprN can be activated in infected hosts even in the absence of antibiotic selective pressure, it could promote establishment of chronic infections in the lungs of people suffering from cystic fibrosis, thus diminishing the immune response to virulence factors. Therapeutic drugs that affect multidrug efflux pumps and HAQ-mediated quorum sensing would be valuable tools to shut down bacterial virulence.

  2. Inhibition of uncoupled respiration in tumor cells. A possible role of mitochondrial Ca2+ efflux.

    PubMed

    Gabai, V L

    1993-08-23

    Uncouplers CCCP (2-4 microM) or DNP (200-400 microM) when added to EL-4 thymoma or Ehrlich carcinoma ascites cells initially stimulated endogenous respiration about 2-fold but then inhibited it to a first-order rate 20-25% of controls. This inhibition was accelerated by intracellular acidification or by A23187, a Ca2+/H(+)-antiporter (i.e. when mitochondrial Ca2+ efflux was stimulated) whereas Ruthenium red, an inhibitor of uniporter-driven Ca2+ efflux, significantly slowed down the effect of uncouplers. The respiratory inhibition was associated with NAD(P)H oxidation and was partially reversed by exogenous substrates (glutamine or glucose). In the permeabilized cells, endogenous and glutamine-supported respiration was inhibited by EGTA, while succinate-supported respiration was Ca2+ independent. It is suggested that mitochondrial Ca2+ is necessary for NADH-dependent respiration of tumor cells, and uncouplers inhibit it by activation of mitochondrial Ca2+ efflux.

  3. Identification of Transport Proteins Involved in Free Fatty Acid Efflux in Escherichia coli

    PubMed Central

    Lennen, Rebecca M.; Politz, Mark G.; Kruziki, Max A.

    2013-01-01

    Escherichia coli has been used as a platform host for studying the production of free fatty acids (FFA) and other energy-dense compounds useful in biofuel applications. Most of the FFA produced by E. coli are found extracellularly. This finding suggests that a mechanism for transport across the cell envelope exists, yet knowledge of proteins that may be responsible for export remains incomplete. Production of FFA has been shown to cause cell lysis, induce stress responses, and impair basic physiological processes. These phenotypes could potentially be diminished if efflux rates were increased. Here, a total of 15 genes and operons were deleted and screened for their impact on cell viability and titer in FFA-producing E. coli. Deletions of acrAB and rob and, to a lower degree of statistical confidence, emrAB, mdtEF, and mdtABCD reduced multiple measures of viability, while deletion of tolC nearly abolished FFA production. An acrAB emrAB deletion strain exhibited greatly reduced FFA titers approaching the tolC deletion phenotype. Expression of efflux pumps on multicopy plasmids did not improve endogenous FFA production in an acrAB+ strain, but plasmid-based expression of acrAB, mdtEF, and an mdtEF-tolC artificial operon improved the MIC of exogenously added decanoate for an acrAB mutant strain. The findings suggest that AcrAB-TolC is responsible for most of the FFA efflux in E. coli, with residual activity provided by other resistance-nodulation-cell division superfamily-type efflux pumps, including EmrAB-TolC and MdtEF-TolC. While the expression of these proteins on multicopy plasmids did not improve production over the basal level, their identification enables future engineering efforts. PMID:23104810

  4. Plasmid-determined inducible efflux is responsible for resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus.

    PubMed Central

    Nies, D H; Silver, S

    1989-01-01

    In Alcaligenes eutrophus CH34, resistance to chromate is plasmid determined, inducible, and based on decreased net accumulation of the metal anion. Plasmid-encoded resistances to zinc, cadmium, cobalt, and nickel are resulting from inducible, energy-dependent cation efflux systems. PMID:2914875

  5. Interaction of Ocular Hypotensive Agents (PGF2α Analogs—Bimatoprost, Latanoprost, and Travoprost) With MDR Efflux Pumps on the Rabbit Cornea

    PubMed Central

    Mitra, Ashim K.

    2009-01-01

    Purpose: The objectives of this work were (i) to screen ocular hypotensive prostaglandin (PGF2α) analogs—bimatoprost, latanoprost, and travoprost as well as their free acid forms—for interaction with efflux pumps on the cornea and (ii) to assess the modulation of efflux upon co-administration of these prostaglandin analogs. Methods: Cultured rabbit primary corneal epithelial cells (rPCEC) were employed as an in vitro model for rabbit cornea. Transporter-specific interaction studies were carried out using Madin-Darby canine kidney (MDCK) cells overexpressing MDR1, MRP1, MRP2, MRP5, and BCRP. Freshly excised rabbit cornea was used as an ex vivo model to determine transcorneal permeability. Results: Cellular accumulation studies clearly showed that all prostaglandin analogs and their free acid forms are substrates of MRP1, MRP2, and MRP5. Bimatoprost was the only prostaglandin analog in this study to interact with P-gp. In addition, none of these molecules showed any affinity for BCRP. Ki values of these prostaglandin analogs obtained from dose-dependent inhibition of erythromycin efflux in rPCEC showed bimatoprost (82.54 μM) and travoprost (94.77 μM) to have similar but higher affinity to efflux pumps than latanoprost (163.20 μM). Ex vivo studies showed that the permeation of these molecules across cornea was significantly elevated in the presence of specific efflux modulators. Finally, both in vitro and ex vivo experiments demonstrated that the efflux of these prostaglandin analogs could be modulated by co-administering them together. Conclusion: Bimatoprost, latanoprost, travoprost, and their free acid forms are substrates of multiple drug efflux pumps on the cornea. Co-administration of these molecules together is a viable strategy to overcome efflux, which could simultaneously elicit a synergistic pharmacological effect, since these molecules have been shown to activate different receptor population for the reduction of intraocular pressure (IOP). PMID

  6. Multidrug Efflux Pumps in Staphylococcus aureus: an Update

    PubMed Central

    Costa, Sofia Santos; Viveiros, Miguel; Amaral, Leonard; Couto, Isabel

    2013-01-01

    The emergence of infections caused by multi- or pan-resistant bacteria in the hospital or in the community settings is an increasing health concern. Albeit there is no single resistance mechanism behind multiresistance, multidrug efflux pumps, proteins that cells use to detoxify from noxious compounds, seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria. During the last decades, experimental data has established their contribution to low level resistance to antimicrobials in bacteria and their potential role in the appearance of MDR phenotypes, by the extrusion of multiple, unrelated compounds. Recent studies suggest that efflux pumps may be used by the cell as a first-line defense mechanism, avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration occurs, that allows survival in the presence of that agent. In this paper we review the current knowledge on MDR efflux pumps and their intricate regulatory network in Staphylococcus aureus, a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that S. aureus MDR efflux pumps, either chromosomal or plasmid-encoded, have on resistance towards different antimicrobial agents and on the selection of drug - resistant strains. We will also discuss the many questions that still remain on the role of each specific efflux pump and the need to establish appropriate methodological approaches to address all these questions. PMID:23569469

  7. Multidrug Efflux Pumps in Staphylococcus aureus: an Update.

    PubMed

    Costa, Sofia Santos; Viveiros, Miguel; Amaral, Leonard; Couto, Isabel

    2013-01-01

    The emergence of infections caused by multi- or pan-resistant bacteria in the hospital or in the community settings is an increasing health concern. Albeit there is no single resistance mechanism behind multiresistance, multidrug efflux pumps, proteins that cells use to detoxify from noxious compounds, seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria. During the last decades, experimental data has established their contribution to low level resistance to antimicrobials in bacteria and their potential role in the appearance of MDR phenotypes, by the extrusion of multiple, unrelated compounds. Recent studies suggest that efflux pumps may be used by the cell as a first-line defense mechanism, avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration occurs, that allows survival in the presence of that agent. In this paper we review the current knowledge on MDR efflux pumps and their intricate regulatory network in Staphylococcus aureus, a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that S. aureus MDR efflux pumps, either chromosomal or plasmid-encoded, have on resistance towards different antimicrobial agents and on the selection of drug - resistant strains. We will also discuss the many questions that still remain on the role of each specific efflux pump and the need to establish appropriate methodological approaches to address all these questions.

  8. Structure and function of efflux pumps that confer resistance to drugs.

    PubMed Central

    Borges-Walmsley, M Ines; McKeegan, Kenneth S; Walmsley, Adrian R

    2003-01-01

    Resistance to therapeutic drugs encompasses a diverse range of biological systems, which all have a human impact. From the relative simplicity of bacterial cells, fungi and protozoa to the complexity of human cancer cells, resistance has become problematic. Stated in its simplest terms, drug resistance decreases the chance of providing successful treatment against a plethora of diseases. Worryingly, it is a problem that is increasing, and consequently there is a pressing need to develop new and effective classes of drugs. This has provided a powerful stimulus in promoting research on drug resistance and, ultimately, it is hoped that this research will provide novel approaches that will allow the deliberate circumvention of well understood resistance mechanisms. A major mechanism of resistance in both microbes and cancer cells is the membrane protein-catalysed extrusion of drugs from the cell. Resistant cells exploit proton-driven antiporters and/or ATP-driven ABC (ATP-binding cassette) transporters to extrude cytotoxic drugs that usually enter the cell by passive diffusion. Although some of these drug efflux pumps transport specific substrates, many are transporters of multiple substrates. These multidrug pumps can often transport a variety of structurally unrelated hydrophobic compounds, ranging from dyes to lipids. If we are to nullify the effects of efflux-mediated drug resistance, we must first of all understand how these efflux pumps can accommodate a diverse range of compounds and, secondly, how conformational changes in these proteins are coupled to substrate translocation. These are key questions that must be addressed. In this review we report on the advances that have been made in understanding the structure and function of drug efflux pumps. PMID:13678421

  9. Evading P-glycoprotein mediated-efflux chemoresistance using Solid Lipid Nanoparticles.

    PubMed

    Cavaco, Marco C; Pereira, Carolina; Kreutzer, Bruna; Gouveia, Luis F; Silva-Lima, Beatriz; Brito, Alexandra M; Videira, Mafalda

    2017-01-01

    Multidrug resistance (MDR), whereby cancer cells become resistant to the cytotoxic effects of various structurally and mechanistically unrelated chemotherapeutic agents, is a major problem in the clinical treatment of cancer. P-glycoprotein (P-gp) is a transmembrane protein responsible for drug efflux, which decreases drug intracellular bioavailability, consequently decreasing their efficacy against cancer. Solid Lipid Nanoparticles (SLNs) have not only the ability to protect the entrapped drug against proteolytic degradation, but also allow a selective intracellular targeting. Hypothetically, the entrapped drug enter the target cells by different uptake mechanisms, "nanocitose", as compared to the free drug and may evade efflux-transporters, like P-gp. The functional role of P-gp in limiting the permeability of the anticancer drug paclitaxel (Ptx) was assessed in MDA-MB-436 cells. The observed increase in the pharmacologic efficacy of drug entrapped in SLN relatively to the free drug indicates that this system is shielding the drug. Therefore, "blinding" the nanoparticle from the efflux transporters. The effect was confirmed by the decrease expression of P-gp with loaded-SLNs and through the impact on cellular MDR1 expression. Besides the ability to prevent MDR events, functionalization of SLN with a specific antibody against membrane receptors (anti-CD44v6) improves the nanoparticle capability to target selectively malignant cells. This results allow to anticipate that poor clinical outcomes related to tumour P-gp overexpression might be overcome in a near future.

  10. Efflux pump gene hefA of Helicobacter pylori plays an important role in multidrug resistance

    PubMed Central

    Liu, Zhi-Qiang; Zheng, Peng-Yuan; Yang, Ping-Chang

    2008-01-01

    AIM: To determine whether efflux systems contribute to multidrug resistance of H pylori. METHODS: A chloramphenicol-induced multidrug resistance model of six susceptible H pylori strains (5 isolates and H pylori NCTC11637) was developed. Multidrug-resistant (MDR) strains were selected and the minimal inhibitory concentration (MIC) of erythromycin, metronidazole, penicillin G, tetracycline, and ciprofloxacin in multidrug resistant strains and their parent strains was determined by agar dilution tests. The level of mRNA expression of hefA was assessed by fluorescence real-time quantitative PCR. A H pylori LZ1026 knockout mutant (ΔH pylori LZ1026) for (putative) efflux protein was constructed by inserting the kanamycin resistance cassette from pEGFP-N2 into hefA, and its susceptibility profiles to 10 antibiotics were evaluated. RESULTS: The MIC of six multidrug-resistant strains (including 5 clinical isolates and H pylori NCTC11637) increased significantly (≥ 4-fold) compared with their parent strains. The expression level of hefA gene was significantly higher in the MDR strains than in their parent strains (P = 0.033). A H pylori LZ1026 mutant was successfully constructed and the ΔH pylori LZ1026 was more susceptible to four of the 10 antibiotics. All the 20 strains displayed transcripts for hefA that confirmed the in vitro expression of these genes. CONCLUSION: The efflux pump gene hefA plays an important role in multidrug resistance of H pylori. PMID:18777600

  11. Elevational change in woody tissue CO2 efflux in a tropical mountain rain forest in southern Ecuador.

    PubMed

    Zach, Alexandra; Horna, Viviana; Leuschner, Christoph

    2008-01-01

    Much uncertainty exists about the magnitude of woody tissue respiration and its environmental control in highly diverse tropical moist forests. In a tropical mountain rain forest in southern Ecuador, we measured the apparent diurnal gas exchange of stems and coarse roots (diameter 1-4 cm) of trees from representative families along an elevational transect with plots at 1050, 1890 and 3050 m a.s.l. Mean air temperatures were 20.8, 17.2 and 10.6 degrees C, respectively. Stem and root CO(2) efflux of 13 to 21 trees per stand from dominant families were investigated with an open gas exchange system while stand microclimate was continuously monitored. Substantial variation in respiratory activity among and within species was found at all sites. Mean daily CO(2) release rates from stems declined 6.6-fold from 1.38 micromol m(-2) s(-1) at 1050 m to 0.21 micromol m(-2) s(-1) at 3050 m. Mean daily CO(2) release from coarse roots decreased from 0.35 to 0.20 micromol m(-2) s(-1) with altitude, but the differences were not significant. There was, thus, a remarkable shift from a high ratio of stem to coarse root respiration rates at the lowest elevation to an apparent equivalence of stem and coarse root CO(2) efflux rates at the highest elevation. We conclude that stem respiration, but not root respiration, greatly decreases with elevation in this transect, coinciding with a substantial decrease in relative stem diameter increment and a large increase in fine and coarse root biomass production with elevation.

  12. Rapid efflux of Ca2+ from heart mitochondria in the presence of inorganic pyrophosphate.

    PubMed

    Vercesi, A; Lehninger, A L

    1984-01-13

    Inorganic pyrophosphate (PPi) in the intracellular concentration range causes rapid efflux of Ca2+ from rat heart mitochondria oxidizing pyruvate + malate in a low Na+ medium. Half-maximal rates of Ca2+ efflux were given by 20 microM PPi. During and after PPi-stimulated Ca2+ efflux the mitochondria retain their structural integrity and complete respiratory control. Carboxyatractyloside inhibits PPi-stimulated Ca2+ efflux, indicating PPi must enter the matrix in order to promote Ca2+ efflux. Heart mitochondria have a much higher affinity for PPi uptake and PPi-induced Ca2+ efflux than liver mitochondria.

  13. Contribution of efflux pumps, porins, and β-lactamases to multidrug resistance in clinical isolates of Acinetobacter baumannii.

    PubMed

    Rumbo, C; Gato, E; López, M; Ruiz de Alegría, C; Fernández-Cuenca, F; Martínez-Martínez, L; Vila, J; Pachón, J; Cisneros, J M; Rodríguez-Baño, J; Pascual, A; Bou, G; Tomás, M

    2013-11-01

    We investigated the mechanisms of resistance to carbapenems, aminoglycosides, glycylcyclines, tetracyclines, and quinolones in 90 multiresistant clinical strains of Acinetobacter baumannii isolated from two genetically unrelated A. baumannii clones: clone PFGE-ROC-1 (53 strains producing the OXA-58 β-lactamase enzyme and 18 strains with the OXA-24 β-lactamase) and clone PFGE-HUI-1 (19 strains susceptible to carbapenems). We used real-time reverse transcriptase PCR to correlate antimicrobial resistance (MICs) with expression of genes encoding chromosomal β-lactamases (AmpC and OXA-51), porins (OmpA, CarO, Omp33, Dcap-like, OprB, Omp25, OprC, OprD, and OmpW), and proteins integral to six efflux systems (AdeABC, AdeIJK, AdeFGH, CraA, AbeM, and AmvA). Overexpression of the AdeABC system (level of expression relative to that by A. baumannii ATCC 17978, 30- to 45-fold) was significantly associated with resistance to tigecycline, minocycline, and gentamicin and other biological functions. However, hyperexpression of the AdeIJK efflux pump (level of expression relative to that by A. baumannii ATCC 17978, 8- to 10-fold) was significantly associated only with resistance to tigecycline and minocycline (to which the TetB efflux system also contributed). TetB and TetA(39) efflux pumps were detected in clinical strains and were associated with resistance to tetracyclines and doxycycline. The absence of the AdeABC system and the lack of expression of other mechanisms suggest that tigecycline-resistant strains of the PFGE-HUI-1 clone may be associated with a novel resistance-nodulation-cell efflux pump (decreased MICs in the presence of the inhibitor Phe-Arg β-naphthylamide dihydrochloride) and the TetA(39) system.

  14. Contribution of Efflux Pumps, Porins, and β-Lactamases to Multidrug Resistance in Clinical Isolates of Acinetobacter baumannii

    PubMed Central

    Rumbo, C.; Gato, E.; López, M.; Ruiz de Alegría, C.; Fernández-Cuenca, F.; Martínez-Martínez, L.; Vila, J.; Pachón, J.; Cisneros, J. M.; Rodríguez-Baño, J.; Pascual, A.

    2013-01-01

    We investigated the mechanisms of resistance to carbapenems, aminoglycosides, glycylcyclines, tetracyclines, and quinolones in 90 multiresistant clinical strains of Acinetobacter baumannii isolated from two genetically unrelated A. baumannii clones: clone PFGE-ROC-1 (53 strains producing the OXA-58 β-lactamase enzyme and 18 strains with the OXA-24 β-lactamase) and clone PFGE-HUI-1 (19 strains susceptible to carbapenems). We used real-time reverse transcriptase PCR to correlate antimicrobial resistance (MICs) with expression of genes encoding chromosomal β-lactamases (AmpC and OXA-51), porins (OmpA, CarO, Omp33, Dcap-like, OprB, Omp25, OprC, OprD, and OmpW), and proteins integral to six efflux systems (AdeABC, AdeIJK, AdeFGH, CraA, AbeM, and AmvA). Overexpression of the AdeABC system (level of expression relative to that by A. baumannii ATCC 17978, 30- to 45-fold) was significantly associated with resistance to tigecycline, minocycline, and gentamicin and other biological functions. However, hyperexpression of the AdeIJK efflux pump (level of expression relative to that by A. baumannii ATCC 17978, 8- to 10-fold) was significantly associated only with resistance to tigecycline and minocycline (to which the TetB efflux system also contributed). TetB and TetA(39) efflux pumps were detected in clinical strains and were associated with resistance to tetracyclines and doxycycline. The absence of the AdeABC system and the lack of expression of other mechanisms suggest that tigecycline-resistant strains of the PFGE-HUI-1 clone may be associated with a novel resistance-nodulation-cell efflux pump (decreased MICs in the presence of the inhibitor Phe-Arg β-naphthylamide dihydrochloride) and the TetA(39) system. PMID:23939894

  15. RND multidrug efflux pumps: what are they good for?

    PubMed

    Alvarez-Ortega, Carolina; Olivares, Jorge; Martínez, José L

    2013-01-01

    Multidrug efflux pumps are chromosomally encoded genetic elements capable of mediating resistance to toxic compounds in several life forms. In bacteria, these elements are involved in intrinsic and acquired resistance to antibiotics. Unlike other well-known horizontally acquired antibiotic resistance determinants, genes encoding for multidrug efflux pumps belong to the core of bacterial genomes and thus have evolved over millions of years. The selective pressure stemming from the use of antibiotics to treat bacterial infections is relatively recent in evolutionary terms. Therefore, it is unlikely that these elements have evolved in response to antibiotics. In the last years, several studies have identified numerous functions for efflux pumps that go beyond antibiotic extrusion. In this review we present some examples of these functions that range from bacterial interactions with plant or animal hosts, to the detoxification of metabolic intermediates or the maintenance of cellular homeostasis.

  16. RND multidrug efflux pumps: what are they good for?

    PubMed Central

    Alvarez-Ortega, Carolina; Olivares, Jorge; Martínez, José L.

    2013-01-01

    Multidrug efflux pumps are chromosomally encoded genetic elements capable of mediating resistance to toxic compounds in several life forms. In bacteria, these elements are involved in intrinsic and acquired resistance to antibiotics. Unlike other well-known horizontally acquired antibiotic resistance determinants, genes encoding for multidrug efflux pumps belong to the core of bacterial genomes and thus have evolved over millions of years. The selective pressure stemming from the use of antibiotics to treat bacterial infections is relatively recent in evolutionary terms. Therefore, it is unlikely that these elements have evolved in response to antibiotics. In the last years, several studies have identified numerous functions for efflux pumps that go beyond antibiotic extrusion. In this review we present some examples of these functions that range from bacterial interactions with plant or animal hosts, to the detoxification of metabolic intermediates or the maintenance of cellular homeostasis. PMID:23386844

  17. Vertical variations in wood CO2 efflux for live emergent trees in a Bornean tropical rainforest.

    PubMed

    Katayama, Ayumi; Kume, Tomonori; Komatsu, Hikaru; Ohashi, Mizue; Matsumoto, Kazuho; Ichihashi, Ryuji; Kumagai, Tomo'omi; Otsuki, Kyoichi

    2014-05-01

    Difficult access to 40-m-tall emergent trees in tropical rainforests has resulted in a lack of data related to vertical variations in wood CO2 efflux, even though significant variations in wood CO2 efflux are an important source of errors when estimating whole-tree total wood CO2 efflux. This study aimed to clarify vertical variations in wood CO2 efflux for emergent trees and to document the impact of the variations on the whole-tree estimates of stem and branch CO2 efflux. First, we measured wood CO2 efflux and factors related to tree morphology and environment for seven live emergent trees of two dipterocarp species at four to seven heights of up to ∼ 40 m for each tree using ladders and a crane. No systematic tendencies in vertical variations were observed for all the trees. Wood CO2 efflux was not affected by stem and air temperature, stem diameter, stem height or stem growth. The ratios of wood CO2 efflux at the treetop to that at breast height were larger in emergent trees with relatively smaller diameters at breast height. Second, we compared whole-tree stem CO2 efflux estimates using vertical measurements with those based on solely breast height measurements. We found similar whole-tree stem CO2 efflux estimates regardless of the patterns of vertical variations in CO2 efflux because the surface area in the canopy, where wood CO2 efflux often differed from that at breast height, was very small compared with that at low stem heights, resulting in little effect of the vertical variations on the estimate. Additionally, whole-tree branch CO2 efflux estimates using measured wood CO2 efflux in the canopy were considerably different from those measured using only breast height measurements. Uncertainties in wood CO2 efflux in the canopy did not cause any bias in stem CO2 efflux scaling, but affected branch CO2 efflux.

  18. Molecular Dynamics Computer Simulations of Multidrug RND Efflux Pumps.

    PubMed

    Ruggerone, Paolo; Vargiu, Attilio V; Collu, Francesca; Fischer, Nadine; Kandt, Christian

    2013-01-01

    Over-expression of multidrug efflux pumps of the Resistance Nodulation Division (RND) protein super family counts among the main causes for microbial resistance against pharmaceuticals. Understanding the molecular basis of this process is one of the major challenges of modern biomedical research, involving a broad range of experimental and computational techniques. Here we review the current state of RND transporter investigation employing molecular dynamics simulations providing conformational samples of transporter components to obtain insights into the functional mechanism underlying efflux pump-mediated antibiotics resistance in Escherichia coli and Pseudomonas aeruginosa.

  19. Molecular Dynamics Computer Simulations of Multidrug RND Efflux Pumps

    PubMed Central

    Ruggerone, Paolo; Vargiu, Attilio V.; Collu, Francesca; Fischer, Nadine; Kandt, Christian

    2013-01-01

    Over-expression of multidrug efflux pumps of the Resistance Nodulation Division (RND) protein super family counts among the main causes for microbial resistance against pharmaceuticals. Understanding the molecular basis of this process is one of the major challenges of modern biomedical research, involving a broad range of experimental and computational techniques. Here we review the current state of RND transporter investigation employing molecular dynamics simulations providing conformational samples of transporter components to obtain insights into the functional mechanism underlying efflux pump-mediated antibiotics resistance in Escherichia coli and Pseudomonas aeruginosa. PMID:24688701

  20. Incubation temperature, osmolarity, and salicylate affect the expression of resistance-nodulation-division efflux pumps and outer membrane porins in Acinetobacter baumannii ATCC19606T.

    PubMed

    Bazyleu, Andrei; Kumar, Ayush

    2014-08-01

    In this study, we examined the impact of various environmental conditions on the expression of resistance-nodulation-division (RND) efflux pumps and outer membrane (OM) porins, two key determinants of Acinetobacter baumannii's intrinsic resistance, an organism known to cause various multidrug resistant infections in immunocompromised individuals. Quantitative RT-PCR was used to analyze the expression of adeB, adeG, and adeJ (genes encoding RND pumps) and 33 kDa, carO, and oprD (genes encoding OM porins) of A. baumannii ATCC19606(T) under different incubation temperatures (30, 37, and 42 °C) and in the presence of high osmolarity and salicylate. Downregulation of all three RND pumps was observed at 30 °C, while downregulation of all three porins tested was observed at increased osmolarity. Downregulation of RND efflux pumps, particularly AdeABC, was consistent with increased susceptibility to antibiotics that are substrates of this pump. Expression of the adeR response regulator gene of the AdeRS system, the activator of the AdeABC pump, was also analyzed. Our work shows that various environmental stress conditions can influence the expression of RND pumps and porins in A. baumannii ATCC19606(T) and thus may play a role in the modulation of its antibiotic resistance.

  1. Efflux-mediated fluoroquinolone resistance in the multidrug-resistant Pseudomonas aeruginosa clinical isolate PA7: identification of a novel MexS variant involved in upregulation of the mexEF-oprN multidrug efflux operon

    PubMed Central

    Morita, Yuji; Tomida, Junko; Kawamura, Yoshiaki

    2014-01-01

    The emergence of multidrug-resistant Pseudomonas aeruginosa has become a serious problem in medical settings. P. aeruginosa clinical isolate PA7 is resistant to fluoroquinolones, aminoglycosides, and most β-lactams but not imipenem. In this study, enhanced efflux-mediated fluoroquinolone resistance of PA7 was shown to reflect increased expression of two resistance nodulation cell division (RND) -type multidrug efflux operons, mexEF-oprN and mexXY-oprA. Such a clinical isolate has rarely been reported because MexEF-OprN-overproducing mutants often increase susceptibility to aminoglycosides apparently owing to impairment of the MexXY system. A mutant of PA7 lacking three RND-type multidrug efflux operons (mexAB-oprM, mexEF-oprN, and mexXY-oprA) was susceptible to all anti-pseudomonas agents we tested, supporting an idea that these RND-type multidrug efflux transporters are molecular targets to overcome multidrug resistance in P. aeruginosa. mexEF-oprN-upregulation in P. aeruginosa PA7 was shown due to a MexS variant harboring the Valine-155 amino acid residue. This is the first genetic evidence shown that a MexS variant causes mexEF-oprN-upregulation in P. aeruginosa clinical isolates. PMID:25653649

  2. Effect of biofilm formation by clinical isolates of Helicobacter pylori on the efflux-mediated resistance to commonly used antibiotics

    PubMed Central

    Attaran, Bahareh; Falsafi, Tahereh; Ghorbanmehr, Nassim

    2017-01-01

    AIM To evaluate the role of biofilm formation on the resistance of Helicobacter pylori (H. pylori) to commonly prescribed antibiotics, the expression rates of resistance genes in biofilm-forming and planktonic cells were compared. METHODS A collection of 33 H. pylori isolates from children and adult patients with chronic infection were taken for the present study. The isolates were screened for biofilm formation ability, as well as for polymerase chain reaction (PCR) reaction with HP1165 and hp1165 efflux pump genes. Susceptibilities of the selected strains to antibiotic and differences between susceptibilities of planktonic and biofilm-forming cell populations were determined. Quantitative real-time PCR (qPCR) analysis was performed using 16S rRNA gene as a H. pylori-specific primer, and two efflux pumps-specific primers, hp1165 and hefA. RESULTS The strains were resistant to amoxicillin, metronidazole, and erythromycin, except for one strain, but they were all susceptible to tetracycline. Minimum bactericidal concentrations of antibiotics in the biofilm-forming cells were significantly higher than those of planktonic cells. qPCR demonstrated that the expression of efflux pump genes was significantly higher in the biofilm-forming cells as compared to the planktonic ones. CONCLUSION The present work demonstrated an association between H. pylori biofilm formation and decreased susceptibility to all the antibiotics tested. This decreased susceptibility to antibiotics was associated with enhanced functional activity of two efflux pumps: hp1165 and hefA. PMID:28275296

  3. Recent Advances in Multi-Drug Resistance (MDR) Efflux Pump Inhibitors of Gram-Positive Bacteria S. aureus

    PubMed Central

    Handzlik, Jadwiga; Matys, Anna; Kieć-Kononowicz, Katarzyna

    2013-01-01

    The paper focuses on recent achievements in the search for new chemical compounds able to inhibit multidrug resistance (MDR) mechanisms in Gram-positive pathogens. An analysis of the results of the search for new efflux pump inhibitors (EPIs) for Gram-positive bacteria, which have been performed over the last decade, indicates that almost all efforts are focused on the NorA (MFS) efflux pump in S. aureus. Considering the chemical structures of the NorA EPIs that have been identified, it can be observed that the most active agents belong to the families of compounds possessing conjugated double bonds, e.g., chalcones, piperine-like compounds, N-cinnamoylphenalkylamides or citral amide derivatives. Indole-, dihydronaphthyl-, 2-chloro-5-bromo-phenyl- or piperidine moieties seem to be profitable for the EPI properties, as well. These results, together with an increasing knowledge about a variety of efflux pumps that are involved in MDR of Gram-positive pathogens underline that further search for new EPIs should pay more attention to develop MDR efflux protein targets, including SMR, MATE, ABC or other members of the MFS family. PMID:27029290

  4. Recent Advances in Multi-Drug Resistance (MDR) Efflux Pump Inhibitors of Gram-Positive Bacteria S. aureus.

    PubMed

    Handzlik, Jadwiga; Matys, Anna; Kieć-Kononowicz, Katarzyna

    2013-02-05

    The paper focuses on recent achievements in the search for new chemical compounds able to inhibit multidrug resistance (MDR) mechanisms in Gram-positive pathogens. An analysis of the results of the search for new efflux pump inhibitors (EPIs) for Gram-positive bacteria, which have been performed over the last decade, indicates that almost all efforts are focused on the NorA (MFS) efflux pump in S. aureus. Considering the chemical structures of the NorA EPIs that have been identified, it can be observed that the most active agents belong to the families of compounds possessing conjugated double bonds, e.g., chalcones, piperine-like compounds, N-cinnamoylphenalkylamides or citral amide derivatives. Indole-, dihydronaphthyl-, 2-chloro-5-bromo-phenyl- or piperidine moieties seem to be profitable for the EPI properties, as well. These results, together with an increasing knowledge about a variety of efflux pumps that are involved in MDR of Gram-positive pathogens underline that further search for new EPIs should pay more attention to develop MDR efflux protein targets, including SMR, MATE, ABC or other members of the MFS family.

  5. Interplay between Cellular Methyl Metabolism and Adaptive Efflux during Oncogenic Transformation from Chronic Arsenic Exposure in Human Cells*S⃞

    PubMed Central

    Coppin, Jean-François; Qu, Wei; Waalkes, Michael P.

    2008-01-01

    After protracted low level arsenic exposure, the normal human prostate epithelial cell line RWPE-1 acquires a malignant phenotype with DNA hypomethylation, indicative of disrupted methyl metabolism, and shows arsenic adaptation involving glutathione overproduction and enhanced arsenic efflux. Thus, the interplay between methyl and glutathione metabolism during this progressive arsenic adaptation was studied. Arsenic-treated cells showed a time-dependent increase in LC50 and a marked increase in homocysteine (Hcy) levels. A marked suppression of S-adenosylmethionine (SAM) levels occurred with decreased methionine adenosyltransferase 2A (converts methionine to SAM) expression and increased negative regulator methionine adenosyltransferase B, suggesting reduced conversion of Hcy to SAM. Consistent with Hcy overproduction, activity and expression of S-adenosylhomocysteine hydrolase (converts S-adenosylhomocysteine to Hcy) were both increased. Expression of cystathionine β-synthase, a key gene in the transsulfuration pathway, and various glutathione production genes were increased, resulting in a 5-fold increase in glutathione. Arsenic efflux increased along with expression of ATP-binding cassette protein C1, which effluxes arsenic as a glutathione conjugate. Evidence of genomic DNA hypomethylation was observed during early arsenic exposure, indicating that the disruption in methyl metabolism had a potential impact related to oncogenesis. Thus, cellular arsenic adaptation is a dynamic, progressive process that involves decreased SAM recycling and concurrent accumulation of Hcy, which is channeled via transsulfuration to increase glutathione and enhance arsenic efflux but may also impact the carcinogenic process. PMID:18487201

  6. Proton motive force-driven and ATP-dependent drug extrusion systems in multidrug-resistant Lactococcus lactis.

    PubMed Central

    Bolhuis, H; Molenaar, D; Poelarends, G; van Veen, H W; Poolman, B; Driessen, A J; Konings, W N

    1994-01-01

    Three mutants of Lactococcus lactis subsp. lactis MG1363, termed EthR, DauR, and RhoR, were selected for resistance to high concentrations of ethidium bromide, daunomycin, and rhodamine 6G, respectively. These mutants were found to be cross resistant to a number of structurally and functionally unrelated drugs, among which were typical substrates of the mammalian multidrug transporter (P-glycoprotein) such as daunomycin, quinine, actinomycin D, gramicidin D, and rhodamine 6G. The three multidrug-resistant strains showed an increased rate of energy-dependent ethidium and daunomycin efflux compared with that of the wild-type strain. This suggests that resistance to these toxic compounds is at least partly due to active efflux. Efflux of ethidium from the EthR strain could occur against a 37-fold inwardly directed concentration gradient. In all strains, ethidium efflux was inhibited by reserpine, a well-known inhibitor of P-glycoprotein. Ionophores which selectively dissipate the membrane potential or the pH gradient across the membrane inhibited ethidium and daunomycin efflux in the wild-type strain, corresponding with a proton motive force-driven efflux system. The ethidium efflux system in the EthR strain, on the other hand, was inhibited by ortho-vanadate and not upon dissipation of the proton motive force, which suggests the involvement of ATP in the energization of transport. The partial inhibition of ethidium efflux by ortho-vanadate and nigericin in the DauR and RhoR strains suggest that a proton motive force-dependent and an ATP-dependent system are expressed simultaneously. This is the first report of an ATP-dependent transport system in prokaryotes which confers multidrug resistance to the organism. PMID:7961458

  7. Content Analysis in Systems Engineering Acquisition Activities

    DTIC Science & Technology

    2016-04-30

    shape requirements definitions for system upgrade or modification contracts and new baseline contracts. Finally, content analysis training and skill...back to the system designers, this information can then be used to shape requirements definition for system upgrade or modification contracts and new...Activity System Requirements Definition Ensuring the system requirements adequately reflect the stakeholder requirements Negotiating modifications to

  8. EEPD1 Is a Novel LXR Target Gene in Macrophages Which Regulates ABCA1 Abundance and Cholesterol Efflux

    PubMed Central

    Nelson, Jessica Kristine; Koenis, Duco Steven; Scheij, Saskia; Cook, Emma Clare Laura; Moeton, Martina; Santos, Ana; Lobaccaro, Jean-Marc Adolphe; Baron, Silvere

    2017-01-01

    Objective— The sterol-responsive nuclear receptors, liver X receptors α (LXRα, NR1H3) and β (LXRβ, NR1H2), are key determinants of cellular cholesterol homeostasis. LXRs are activated under conditions of high cellular sterol load and induce expression of the cholesterol efflux transporters ABCA1 and ABCG1 to promote efflux of excess cellular cholesterol. However, the full set of genes that contribute to LXR-stimulated cholesterol efflux is unknown, and their identification is the objective of this study. Approach and Results— We systematically compared the global transcriptional response of macrophages to distinct classes of LXR ligands. This allowed us to identify both common and ligand-specific transcriptional responses in macrophages. Among these, we identified endonuclease–exonuclease–phosphatase family domain containing 1 (EEPD1/KIAA1706) as a direct transcriptional target of LXRs in human and murine macrophages. EEPD1 specifically localizes to the plasma membrane owing to the presence of a myristoylation site in its N terminus. Accordingly, the first 10 amino acids of EEPD1 are sufficient to confer plasma membrane localization in the context of a chimeric protein with GFP. Functionally, we report that silencing expression of EEPD1 blunts maximal LXR-stimulated Apo AI-dependent efflux and demonstrate that this is the result of reduced abundance of ABCA1 protein in human and murine macrophages. Conclusions— In this study, we identify EEPD1 as a novel LXR-regulated gene in macrophages and propose that it promotes cellular cholesterol efflux by controlling cellular levels and activity of ABCA1. PMID:28082258

  9. The decrease of paclitaxel efflux by pretreatment of interferon-γ and tumor necrosis factor-α after intracerebral microinjection.

    PubMed

    Lee, Na-Young; Kang, Young-Sook

    2013-03-07

    Paclitaxel is highly efficacious in the treatment of patients suffering from a broad spectrum of neoplastic diseases. However, its efficacy against malignant glioma is very moderate. Paclitaxel is known to be a substrate for P-glycoprotein (P-gp), so this transporter may be due to insufficient access of paclitaxel to the brain. First, we investigated the brain-to-blood transport of paclitaxel across the blood-brain barrier (BBB) using the brain efflux index method. [(3)H]Paclitaxel was eliminated from rat brain with an efflux transport rate of 1.87×10(-2)±0.16×10(-2)min(-1). The elimination of [(3)H]paclitaxel was inhibited by unlabeled paclitaxel and verapamil, suggesting a carrier-mediated transport process via P-gp. Furthermore, TNF-α and IFN-γ induced significant decrease of paclitaxel efflux 1 and 24h pre-treatment. These results suggest that P-gp efflux function at the BBB is reduced by TNF-α and IFN-γ. Therefore, the distribution of P-gp dependant drugs including paclitaxel in the central nervous system can be modulated by neurological diseases.

  10. Radiocarbon measurements of soil organic matter (SOM) and soil CO2 efflux provide unique insights into the SOM dynamics of managed loblolly pine plantations

    NASA Astrophysics Data System (ADS)

    Vogel, J. G.; Schuur, E. A.; Bracho, R.; Jokela, E. J.

    2011-12-01

    37-45%, respectively. Nearly 82% of all soil CO2 efflux measurements fell between the root and microbial values, and in two cases, soil CO2 efflux was estimated to be several hundred years old. The soil CO2 efflux values outside the root and microbial end-members prevented a partitioning of soil CO2 efflux, but suggested that the decomposition of very old carbon was contributing significant amounts of CO2 to efflux. Overall, the insights gained from the radiocarbon measurements suggest that very old SOM may be actively decomposed in these forests and that intensive fertilization will result in a decrease in fine root inputs of C to the soil.

  11. Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance

    PubMed Central

    2009-01-01

    Background Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear. Results To investigate the contribution of efflux pumps to intrinsic drug resistance of B. cenocepacia J2315, we deleted 3 operons encoding the putative RND transporters RND-1, RND-3, and RND-4 containing the genes BCAS0591-BCAS0593, BCAL1674-BCAL1676, and BCAL2822-BCAL2820. Each deletion included the genes encoding the RND transporter itself and those encoding predicted periplasmic proteins and outer membrane pores. In addition, the deletion of rnd-3 also included BCAL1672, encoding a putative TetR regulator. The B. cenocepacia rnd-3 and rnd-4 mutants demonstrated increased sensitivity to inhibitory compounds, suggesting an involvement of these proteins in drug resistance. Moreover, the rnd-3 and rnd-4 mutants demonstrated reduced accumulation of N-acyl homoserine lactones in the growth medium. In contrast, deletion of the rnd-1 operon had no detectable phenotypes under the conditions assayed. Conclusion Two of the three inactivated RND efflux pumps in B. cenocepacia J2315 contribute to the high level of intrinsic resistance of this strain to some antibiotics and other inhibitory compounds. Furthermore, these efflux systems also mediate accumulation in the growth medium of quorum sensing molecules that have been shown to contribute to infection. A systematic study of RND efflux systems in B. cenocepacia is required to provide a full picture of intrinsic antibiotic resistance in this opportunistic bacterium. PMID:19761586

  12. Data base management systems activities

    NASA Technical Reports Server (NTRS)

    1983-01-01

    The Data Management System-1100 is designed to operate in conjunction with the UNIVAC 1100 Series Operating System on any 1100 Series computer. DMS-1100 is divided into the following four major software components: (1) Data Definition Languages (DDL); (2) Data Management Routine (DMR); (3) Data Manipulation Languages (DML); and (4) Data Base Utilities (DBU). These software components are described in detail.

  13. Orbiter active thermal control system description

    NASA Technical Reports Server (NTRS)

    Laubach, G. E.

    1975-01-01

    A brief description of the Orbiter Active Thermal Control System (ATCS) including (1) major functional requirements of heat load, temperature control and heat sink utilization, (2) the overall system arrangement, and (3) detailed description of the elements of the ATCS.

  14. Salvianolic acid B accelerated ABCA1-dependent cholesterol efflux by targeting PPAR-γ and LXRα

    SciTech Connect

    Yue, Jianmei; Li, Bo; Jing, Qingping; Guan, Qingbo

    2015-07-03

    Objectives: Cholesterol efflux has been thought to be the main and basic mechanism by which free cholesterol is transferred from extra hepatic cells to the liver or intestine for excretion. Salvianolic acid B (Sal B) has been widely used for the prevention and treatment of atherosclerotic diseases. Here, we sought to investigate the effects of Sal B on the cholesterol efflux in THP-1 macrophages. Methods: After PMA-stimulated THP-1 cells were exposed to 50 mg/L of oxLDL and [{sup 3}H] cholesterol (1.0 μCi/mL) for another 24 h, the effect of Sal B on cholesterol efflux was evaluated in the presence of apoA-1, HDL{sub 2} or HDL{sub 3}. The expression of ATP binding cassette transporter A1 (ABCA1), peroxisome proliferator-activated receptor-gamma (PPAR-γ), and liver X receptor-alpha (LXRα) was detected both at protein and mRNA levels in THP-1 cells after the stimulation of Sal B. Meanwhile, specific inhibition of PPAR-γ and LXRα were performed to investigate the mechanism. Results: The results showed that Sal B significantly accelerated apoA-I- and HDL-mediated cholesterol efflux in both dose- and time-dependent manners. Meanwhile, Sal B treatment also enhanced the expression of ABCA1 at both mRNA and protein levels. Then the data demonstrated that Sal B increased the expression of PPAR-γ and LXRα. And the application of specific agonists and inhibitors of further confirmed that Sal exert the function through PPAR-γ and LXRα. Conclusion: These results demonstrate that Sal B promotes cholesterol efflux in THP-1 macrophages through ABCA1/PPAR-γ/LXRα pathway. - Highlights: • Sal B promotes the expression of ABCA1. • Sal B promotes cholesterol efflux in macrophages. • Sal B promotes the expression of ABCA1 and cholesterol efflux through PPAR-γ/LXRα signaling pathway.

  15. Cholesterol efflux from THP-1 macrophages is impaired by the fatty acid component from lipoprotein hydrolysis by lipoprotein lipase

    SciTech Connect

    Yang, Yanbo; Thyagarajan, Narmadaa; Coady, Breanne M.; Brown, Robert J.

    2014-09-05

    Highlights: • Lipoprotein hydrolysis products were produced by lipoprotein lipase. • Hydrolysis products lowers expression of macrophage cholesterol transporters. • Hydrolysis products reduces expression of select nuclear receptors. • Fatty acid products lowers cholesterol transporters and select nuclear receptors. • Fatty acid products reduces cholesterol efflux from macrophages. - Abstract: Lipoprotein lipase (LPL) is an extracellular lipase that primarily hydrolyzes triglycerides within circulating lipoproteins. Macrophage LPL contributes to atherogenesis, but the mechanisms behind it are poorly understood. We hypothesized that the products of lipoprotein hydrolysis generated by LPL promote atherogenesis by inhibiting the cholesterol efflux ability by macrophages. To test this hypothesis, we treated human THP-1 macrophages with total lipoproteins that were hydrolyzed by LPL and we found significantly reduced transcript levels for the cholesterol transporters ATP binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor BI. These decreases were likely due to significant reductions for the nuclear receptors liver-X-receptor-α, peroxisome proliferator activated receptor (PPAR)-α, and PPAR-γ. We prepared a mixture of free fatty acids (FFA) that represented the ratios of FFA species within lipoprotein hydrolysis products, and we found that the FFA mixture also significantly reduced cholesterol transporters and nuclear receptors. Finally, we tested the efflux of cholesterol from THP-1 macrophages to apolipoprotein A-I, and we found that the treatment of THP-1 macrophages with the FFA mixture significantly attenuated cholesterol efflux. Overall, these data show that the FFA component of lipoprotein hydrolysis products generated by LPL may promote atherogenesis by inhibiting cholesterol efflux, which partially explains the pro-atherogenic role of macrophage LPL.

  16. Molecular Mechanism of MBX2319 Inhibition of Escherichia coli AcrB Multidrug Efflux Pump and Comparison with Other Inhibitors

    PubMed Central

    Vargiu, Attilio V.; Ruggerone, Paolo; Opperman, Timothy J.; Nguyen, Son T.

    2014-01-01

    Efflux pumps of the resistance nodulation division (RND) superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. The development of inhibitors of the RND pumps would improve the efficacy of current and next-generation antibiotics. To date, however, only one inhibitor has been cocrystallized with AcrB. Thus, in silico structure-based analysis is essential for elucidating the interaction between other inhibitors and the efflux pumps. In this work, we used computer docking and molecular dynamics simulations to study the interaction between AcrB and the compound MBX2319, a novel pyranopyridine efflux pump inhibitor with potent activity against RND efflux pumps of Enterobacteriaceae species, as well as other known inhibitors (D13-9001, 1-[1-naphthylmethyl]-piperazine, and phenylalanylarginine-β-naphthylamide) and the binding of doxorubicin to the efflux-defective F610A variant of AcrB. We also analyzed the binding of a substrate, minocycline, for comparison. Our results show that MBX2319 binds very tightly to the lower part of the distal pocket in the B protomer of AcrB, strongly interacting with the phenylalanines lining the hydrophobic trap, where the hydrophobic portion of D13-9001 was found to bind by X-ray crystallography. Additionally, MBX2319 binds to AcrB in a manner that is similar to the way in which doxorubicin binds to the F610A variant of AcrB. In contrast, 1-(1-naphthylmethyl)-piperazine and phenylalanylarginine-β-naphthylamide appear to bind to somewhat different areas of the distal pocket in the B protomer of AcrB than does MBX2319. However, all inhibitors (except D13-9001) appear to distort the structure of the distal pocket, impairing the proper binding of substrates. PMID:25114133

  17. Tigecycline Susceptibility and the Role of Efflux Pumps in Tigecycline Resistance in KPC-Producing Klebsiella pneumoniae

    PubMed Central

    Chen, Qiong; Ruan, Zhi; Hua, Xiaoting; Zhou, Hua; Yu, Yunsong

    2015-01-01

    KPC-producing Klebsiella pneumoniae isolates have emerged as important pathogens of nosocomial infections, and tigecycline is one of the antibiotics recommended for severe infections caused by KPC-producing K. pneumoniae. To identify the susceptibility profile of KPC-producing K. pneumoniae to tigecycline and investigate the role of efflux pumps in tigecycline resistance, a total of 215 KPC-producing K. pneumoniae isolates were collected. The minimum inhibitory concentration (MIC) of tigecycline was determined by standard broth microdilution tests. Isolates showing resistance to tigecycline underwent susceptibility test with efflux pump inhibitors. Expression levels of efflux pump genes (acrB and oqxB) and their regulators (ramA, marA, soxS and rarA) were examined by real-time PCR, and the correlation between tigecycline MICs and gene expression levels were analysed. Our results show that the tigecycline resistance rate in these isolates was 11.2%. Exposure of the tigecycline-resistant isolates to the efflux pump inhibitor NMP resulted in an obvious decrease in MICs and restored susceptibility to tigecycline in 91.7% of the isolates. A statistically significant association between acrB expression and tigecycline MICs was observed, and overexpression of ramA was found in three tigecycline-resistant isolates, further analysis confirmed ramR mutations existed in these isolates. Transformation of one mutant with wild-type ramR restored susceptibility to tigecycline and repressed overexpression of ramA and acrB. These data indicate that efflux pump AcrAB, which can be up-regulated by ramR mutations and subsequent ramA activation, contributed to tigecycline resistance in K. pneumoniae clinical isolates. PMID:25734903

  18. Tigecycline susceptibility and the role of efflux pumps in tigecycline resistance in KPC-producing Klebsiella pneumoniae.

    PubMed

    He, Fang; Fu, Ying; Chen, Qiong; Ruan, Zhi; Hua, Xiaoting; Zhou, Hua; Yu, Yunsong

    2015-01-01

    KPC-producing Klebsiella pneumoniae isolates have emerged as important pathogens of nosocomial infections, and tigecycline is one of the antibiotics recommended for severe infections caused by KPC-producing K. pneumoniae. To identify the susceptibility profile of KPC-producing K. pneumoniae to tigecycline and investigate the role of efflux pumps in tigecycline resistance, a total of 215 KPC-producing K. pneumoniae isolates were collected. The minimum inhibitory concentration (MIC) of tigecycline was determined by standard broth microdilution tests. Isolates showing resistance to tigecycline underwent susceptibility test with efflux pump inhibitors. Expression levels of efflux pump genes (acrB and oqxB) and their regulators (ramA, marA, soxS and rarA) were examined by real-time PCR, and the correlation between tigecycline MICs and gene expression levels were analysed. Our results show that the tigecycline resistance rate in these isolates was 11.2%. Exposure of the tigecycline-resistant isolates to the efflux pump inhibitor NMP resulted in an obvious decrease in MICs and restored susceptibility to tigecycline in 91.7% of the isolates. A statistically significant association between acrB expression and tigecycline MICs was observed, and overexpression of ramA was found in three tigecycline-resistant isolates, further analysis confirmed ramR mutations existed in these isolates. Transformation of one mutant with wild-type ramR restored susceptibility to tigecycline and repressed overexpression of ramA and acrB. These data indicate that efflux pump AcrAB, which can be up-regulated by ramR mutations and subsequent ramA activation, contributed to tigecycline resistance in K. pneumoniae clinical isolates.

  19. Berry anthocyanins and anthocyanidins exhibit distinct affinities for the efflux transporters BCRP and MDR1

    PubMed Central

    Dreiseitel, A; Oosterhuis, B; Vukman, KV; Schreier, P; Oehme, A; Locher, S; Hajak, G; Sand, PG

    2009-01-01

    Background and purpose: Dietary anthocyanins hold great promise in the prevention of chronic disease but factors affecting their bioavailability remain poorly defined. Specifically, the role played by transport mechanisms at the intestinal and blood–brain barriers (BBB) is currently unknown. Experimental approach: In the present study, 16 anthocyanins and anthocyanidins were exposed to the human efflux transporters multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP), using dye efflux, ATPase and, for BCRP, vesicular transport assays. Key results: All test compounds interacted with the BCRP transporter in vitro. Of these, seven emerged as potential BCRP substrates (malvidin, petunidin, malvidin-3-galactoside, malvidin-3,5-diglucoside, cyanidin-3-galactoside, peonidin-3-glucoside, cyanidin-3-glucoside) and 12 as potential inhibitors of BCRP (cyanidin, peonidin, cyanidin-3,5-diglucoside, malvidin, pelargonidin, delphinidin, petunidin, delphinidin-3-glucoside, cyanidin-3-rutinoside, malvidin-3-glucoside, pelargonidin-3,5-diglucoside, malvidin-3-galactoside). Malvidin, malvidin-3-galactoside and petunidin exhibited bimodal activities serving as BCRP substrates at low concentrations and, at higher concentrations, as BCRP inhibitors. Effects on MDR1, in contrast, were weak. Only aglycones exerted mild inhibitory activity. Conclusions and implications: Although the anthocyanidins under study may alter pharmacokinetics of drugs that are BCRP substrates, they are less likely to interfere with activities of MDR1 substrates. The present data suggest that several anthocyanins and anthocyanidins may be actively transported out of intestinal tissues and endothelia, limiting their bioavailability in plasma and brain. PMID:19922539

  20. Quantitative investigation of the brain-to-cerebrospinal fluid unbound drug concentration ratio under steady-state conditions in rats using a pharmacokinetic model and scaling factors for active efflux transporters.

    PubMed

    Kodaira, Hiroshi; Kusuhara, Hiroyuki; Fuse, Eiichi; Ushiki, Junko; Sugiyama, Yuichi

    2014-06-01

    A pharmacokinetic model was constructed to explain the difference in brain- and cerebrospinal fluid (CSF)-to-plasma and brain-to-CSF unbound drug concentration ratios (Kp,uu,brain, Kp,uu,CSF, and Kp,uu,CSF/brain, respectively) of drugs under steady-state conditions in rats. The passive permeability across the blood-brain barrier (BBB), PS1, was predicted by two methods using log(D/molecular weight(0.5)) for PS1(1) or the partition coefficient in octanol/water at pH 7.4 (LogD), topologic van der Waals polar surface area, and van der Waals surface area of the basic atoms for PS1(2). The coefficients of each parameter were determined using previously reported in situ rat BBB permeability. Active transport of drugs by P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) measured in P-gp- and Bcrp-overexpressing cells was extrapolated to in vivo by introducing scaling factors. Brain- and CSF-to-plasma unbound concentration ratios (Kp,uu,brain and Kp,uu,CSF, respectively) of 19 compounds, including P-gp and Bcrp substrates (daidzein, dantrolene, flavopiridol, genistein, loperamide, quinidine, and verapamil), were simultaneously fitted to the equations in a three-compartment model comprising blood, brain, and CSF compartments. The calculated Kp,uu,brain and Kp,uu,CSF of 17 compounds were within a factor of three of experimental values. Kp,uu,CSF values of genistein and loperamide were outliers of the prediction, and Kp,uu,brain of dantrolene also became an outlier when PS1(2) was used. Kp,uu,CSF/brain of the 19 compounds was within a factor of three of experimental values. In conclusion, the Kp,uu,CSF/brain of drugs, including P-gp and Bcrp substrates, could be successfully explained by a kinetic model using scaling factors combined with in vitro evaluation of P-gp and Bcrp activities.

  1. Modeling Cytoskeletal Active Matter Systems

    NASA Astrophysics Data System (ADS)

    Blackwell, Robert

    Active networks of filamentous proteins and crosslinking motor proteins play a critical role in many important cellular processes. One of the most important microtubule-motor protein assemblies is the mitotic spindle, a self-organized active liquid-crystalline structure that forms during cell division and that ultimately separates chromosomes into two daughter cells. Although the spindle has been intensively studied for decades, the physical principles that govern its self-organization and function remain mysterious. To evolve a better understanding of spindle formation, structure, and dynamics, I investigate course-grained models of active liquid-crystalline networks composed of microtubules, modeled as hard spherocylinders, in diffusive equilibrium with a reservoir of active crosslinks, modeled as hookean springs that can adsorb to microtubules and and translocate at finite velocity along the microtubule axis. This model is investigated using a combination of brownian dynamics and kinetic monte carlo simulation. I have further refined this model to simulate spindle formation and kinetochore capture in the fission yeast S. pombe. I then make predictions for experimentally realizable perturbations in motor protein presence and function in S. pombe.

  2. Hydrogen peroxide efflux from muscle mitochondria underestimates matrix superoxide production: a correction using glutathione depletion

    PubMed Central

    TREBERG, Jason R.; QUINLAN, Casey L.; BRAND, Martin D.

    2010-01-01

    Summary The production of H2O2 by isolated mitochondria is frequently used as a measure of mitochondrial superoxide formation. Matrix superoxide dismutase quantitatively converts matrix superoxide to H2O2. However, matrix enzymes such as the glutathione peroxidases can consume H2O2 and compete with efflux of H2O2, causing an underestimate of superoxide production. To assess this underestimate we depleted matrix glutathione in rat skeletal muscle mitochondria by more than 90% by pretreatment with 1-chloro-2,4-dintrobenzene (CDNB). The pretreatment protocol strongly diminished the mitochondrial capacity to consume exogenous H2O2, consistent with decreased peroxidase capacity, but avoided direct stimulation of superoxide production from complex I. It elevated the observed rates of H2O2 formation from matrix-directed superoxide up to two-fold from several sites of production, defined by substrates and electron transport inhibitors, over a wide range of control rates, from 0.2 to 2.5 nmol H2O2 • min−1 • mg protein−1. Similar results were obtained when glutathione was depleted using monochlorobimane or when soluble matrix peroxidase activity was removed by preparation of submitochondrial particles. The data indicate that the increased H2O2 efflux observed with CDNB pretreatment was a result of glutathione depletion and compromised peroxidase activity. A hyperbolic correction curve was constructed, making H2O2 efflux a more quantitative measure of matrix superoxide production. For rat muscle mitochondria, the correction equation was: [CDNB pretreated rate = control rate + (1.43*(control rate))/(0.55+control rate)]. These results have significant ramifications for the rates and topology of superoxide production by isolated mitochondria. PMID:20491900

  3. Role of acid pH and deficient efflux of pyrazinoic acid in unique susceptibility of Mycobacterium tuberculosis to pyrazinamide.

    PubMed

    Zhang, Y; Scorpio, A; Nikaido, H; Sun, Z

    1999-04-01

    Pyrazinamide (PZA) is an important antituberculosis drug. Unlike most antibacterial agents, PZA, despite its remarkable in vivo activity, has no activity against Mycobacterium tuberculosis in vitro except at an acidic pH. M. tuberculosis is uniquely susceptible to PZA, but other mycobacteria as well as nonmycobacteria are intrinsically resistant. The role of acidic pH in PZA action and the basis for the unique PZA susceptibility of M. tuberculosis are unknown. We found that in M. tuberculosis, acidic pH enhanced the intracellular accumulation of pyrazinoic acid (POA), the active derivative of PZA, after conversion of PZA by pyrazinamidase. In contrast, at neutral or alkaline pH, POA was mainly found outside M. tuberculosis cells. PZA-resistant M. tuberculosis complex organisms did not convert PZA into POA. Unlike M. tuberculosis, intrinsically PZA-resistant M. smegmatis converted PZA into POA, but it did not accumulate POA even at an acidic pH, due to a very active POA efflux mechanism. We propose that a deficient POA efflux mechanism underlies the unique susceptibility of M. tuberculosis to PZA and that the natural PZA resistance of M. smegmatis is due to a highly active efflux pump. These findings may have implications with regard to the design of new antimycobacterial drugs.

  4. Environmental effects on CO sub 2 efflux from water track and tussock tundra in arctic Alaska, U. S. A

    SciTech Connect

    Oberbauer, S.F. ); Tenhunen, J.D.; Reynolds, J.F. )

    1991-05-01

    CO{sub 2} efflux and variation in soil environmental characteristics were examined in two tundra vegetation communities, water track (a small drainage of intermittent water flow) and tussock tundra, in the northern foothills of the Philip Smith Mountains in arctic Alaska. Correlation analyses were performed on the observations made at six times during the growing season in order to evaluate the relationships between system CO{sub 2} loss and soil moisture, soil temperature, depth of thaw, and depth to the water table. The two sites differed significantly in terms of soil moisture, soil temperature, depth of thaw, and water table depth on several of the sampling dates. During four of the six measurement periods, the rate of CO{sub 2} efflux differed significantly between sites. Early in the season, respiration was greater in tussock tundra than at the water track, but later in the season, rates at the water track exceeded those at the tussock site. Highest rates were measured at the water track near mid-season. Efflux of CO{sub 2} at both sites was positively correlated with soil temperature. Soil surface (0-5 cm depth) environmental conditions were better predictors of CO{sub 2} efflux than were conditions measured at greater depth (5-10 cm). Soil moisture appeared to increase respiration between 100 and 700% of soil dry weight and decrease soil respiration at higher water contents. The effects of soil moisture were stronger in tussock tundra than in the water track community. These data suggest that both soil temperature and soil moisture limit CO{sub 2} efflux in water track and tussock tundra communities and that the relative importance of these factors changes throughout the growing season.

  5. A robust activity marking system for exploring active neuronal ensembles.

    PubMed

    Sørensen, Andreas T; Cooper, Yonatan A; Baratta, Michael V; Weng, Feng-Ju; Zhang, Yuxiang; Ramamoorthi, Kartik; Fropf, Robin; LaVerriere, Emily; Xue, Jian; Young, Andrew; Schneider, Colleen; Gøtzsche, Casper René; Hemberg, Martin; Yin, Jerry Cp; Maier, Steven F; Lin, Yingxi

    2016-09-23

    Understanding how the brain captures transient experience and converts it into long lasting changes in neural circuits requires the identification and investigation of the specific ensembles of neurons that are responsible for the encoding of each experience. We have developed a Robust Activity Marking (RAM) system that allows for the identification and interrogation of ensembles of neurons. The RAM system provides unprecedented high sensitivity and selectivity through the use of an optimized synthetic activity-regulated promoter that is strongly induced by neuronal activity and a modified Tet-Off system that achieves improved temporal control. Due to its compact design, RAM can be packaged into a single adeno-associated virus (AAV), providing great versatility and ease of use, including application to mice, rats, flies, and potentially many other species. Cre-dependent RAM, CRAM, allows for the study of active ensembles of a specific cell type and anatomical connectivity, further expanding the RAM system's versatility.

  6. Co-Regulations of Spartina alterniflora Invasion and Exogenous Nitrogen Loading on Soil N2O Efflux in Subtropical Mangrove Mesocosms

    PubMed Central

    Jia, Dai; Qi, Fei; Xu, Xia; Feng, Jianxiang; Wu, Hao; Guo, Jiemin; Lu, Weizhi; Peng, Ronghao; Zhu, Xiaoshan; Luo, Yiqi; Lin, Guanghui

    2016-01-01

    Both plant invasion and nitrogen (N) enrichment should have significant impact on mangrove ecosystems in coastal regions around the world. However, how N2O efflux in mangrove wetlands responds to these environmental changes has not been well studied. Here, we conducted a mesocosm experiment with native mangrove species Kandelia obovata, invasive salt marsh species Spartina alterniflora, and their mixture in a simulated tide rotation system with or without nitrogen addition. In the treatments without N addition, the N2O effluxes were relatively low and there were no significant variations among the three vegetation types. A pulse loading of exogenous ammonium nitrogen increased N2O effluxes from soils but the stimulatory effect gradually diminished over time, suggesting that frequent measurements are necessary to accurately understand the behavior of N-induced response of N2O emissions. With the N addition, the N2O effluxes from the invasive S. alterniflora were lower than that from native K. obovata mesocosms. This result may be attributed to higher growth of S. alterniflora consuming most of the available nitrogen in soils, and thus inhibiting N2O production. We concluded that N loading significantly increased N2O effluxes, while the invasion of S. alterniflora reduced N2O effluxes response to N loading in this simulated mangrove ecosystem. Thus, both plant invasion and excessive N loading can co-regulate soil N2O emissions from mangrove wetlands, which should be considered when projecting future N2O effluxes from this type of coastal wetland. PMID:26727205

  7. Two Distinct Major Facilitator Superfamily Drug Efflux Pumps Mediate Chloramphenicol Resistance in Streptomyces coelicolor▿

    PubMed Central

    Vecchione, James J.; Alexander, Blair; Sello, Jason K.

    2009-01-01

    Chloramphenicol, florfenicol, and thiamphenicol are used as antibacterial drugs in clinical and veterinary medicine. Two efflux pumps of the major facilitator superfamily encoded by the cmlR1 and cmlR2 genes mediate resistance to these antibiotics in Streptomyces coelicolor, a close relative of Mycobacterium tuberculosis. The transcription of both genes was observed by reverse transcription-PCR. Disruption of cmlR1 decreased the chloramphenicol MIC 1.6-fold, while disruption of cmlR2 lowered the MIC 16-fold. The chloramphenicol MIC of wild-type S. coelicolor decreased fourfold and eightfold in the presence of reserpine and Phe-Arg-β-naphthylamide, respectively. These compounds are known to potentiate the activity of some antibacterial drugs via efflux pump inhibition. While reserpine is known to potentiate drug activity against gram-positive bacteria, this is the first time that Phe-Arg-β-naphthylamide has been shown to potentiate drug activity against a gram-positive bacterium. PMID:19687245

  8. Lactose uptake driven by galactose efflux in Streptococcus thermophilus: Evidence for a galactose-lactose antiporter

    SciTech Connect

    Hutkins, R.W.; Ponne, C. )

    1991-04-01

    Galactose-nonfermenting (Gal{sup {minus}}) Streptococcus thermophilus TS2 releases galactose into the extracellular medium when grown in medium containing excess lactose. Starved and de-energized Gal{sup {minus}} cells, however, could be loaded with galactose to levels approximately equal to the extracellular concentration (0 to 50 mM). When loaded cells were separated from the medium and resuspended in fresh broth containing 5 mM lactose, galactose efflux occurred. De-energized, galactose-loaded cells, resuspended in buffer or medium, accumulated ({sup 14}C)lactose at a greater rate and to significantly higher intracellular concentrations than unloaded cells. Uptake of lactose by loaded cells was inhibited more than that by unloaded cells in the presence of extracellular galactose, indicating that a galactose gradient was involved in the exchange system. When de-energized, galactose-loaded cells were resuspended in carbohydrate-free medium at pH 6.7, a proton motive force ({Delta}p) of 86 to 90 mV was formed, whereas de-energized, nonloaded cells maintained a {Delta}p of about 56 mV. However, uptake of lactose by loaded cells occurred when the proton motive force was abolished by the addition of an uncoupler or in the presence of a proto