Science.gov

Sample records for active efflux system

  1. Substrate-Dependent Activation of the Vibrio cholerae vexAB RND Efflux System Requires vexR

    PubMed Central

    Taylor, Dawn L.; Ante, Vanessa M.; Bina, X. Renee; Howard, Mondraya F.; Bina, James E.

    2015-01-01

    Vibrio cholerae encodes six resistance-nodulation-division (RND) efflux systems which function in antimicrobial resistance, virulence factor production, and intestinal colonization. Among the six RND efflux systems, VexAB exhibited broad substrate specificity and played a predominant role in intrinsic antimicrobial resistance. The VexAB system was encoded in an apparent three gene operon that included vexR; which encodes an uncharacterized TetR family regulator. In this work we examined the role of vexR in vexRAB expression. We found that VexR bound to the vexRAB promoter and vexR deletion resulted in decreased vexRAB expression and increased susceptibility to VexAB antimicrobial substrates. Substrate-dependent induction of vexRAB was dependent on vexR and episomal vexR expression provided a growth advantage in the presence of the VexAB substrate deoxycholate. The expression of vexRAB increased, in a vexR-dependent manner, in response to the loss of RND efflux activity. This suggested that VexAB may function to export intracellular metabolites. Support for this hypothesis was provided by data showing that vexRAB was upregulated in several metabolic mutants including tryptophan biosynthetic mutants that were predicted to accumulate indole. In addition, vexRAB was found to be upregulated in response to exogenous indole and to contribute to indole resistance. The collective results indicate that vexR is required for vexRAB expression in response to VexAB substrates and that the VexAB RND efflux system modulates the intracellular levels of metabolites that could otherwise accumulate to toxic levels. PMID:25695834

  2. Substrate-dependent activation of the Vibrio cholerae vexAB RND efflux system requires vexR.

    PubMed

    Taylor, Dawn L; Ante, Vanessa M; Bina, X Renee; Howard, Mondraya F; Bina, James E

    2015-01-01

    Vibrio cholerae encodes six resistance-nodulation-division (RND) efflux systems which function in antimicrobial resistance, virulence factor production, and intestinal colonization. Among the six RND efflux systems, VexAB exhibited broad substrate specificity and played a predominant role in intrinsic antimicrobial resistance. The VexAB system was encoded in an apparent three gene operon that included vexR; which encodes an uncharacterized TetR family regulator. In this work we examined the role of vexR in vexRAB expression. We found that VexR bound to the vexRAB promoter and vexR deletion resulted in decreased vexRAB expression and increased susceptibility to VexAB antimicrobial substrates. Substrate-dependent induction of vexRAB was dependent on vexR and episomal vexR expression provided a growth advantage in the presence of the VexAB substrate deoxycholate. The expression of vexRAB increased, in a vexR-dependent manner, in response to the loss of RND efflux activity. This suggested that VexAB may function to export intracellular metabolites. Support for this hypothesis was provided by data showing that vexRAB was upregulated in several metabolic mutants including tryptophan biosynthetic mutants that were predicted to accumulate indole. In addition, vexRAB was found to be upregulated in response to exogenous indole and to contribute to indole resistance. The collective results indicate that vexR is required for vexRAB expression in response to VexAB substrates and that the VexAB RND efflux system modulates the intracellular levels of metabolites that could otherwise accumulate to toxic levels. PMID:25695834

  3. Metal-induced conformational changes in ZneB suggest an active role of membrane fusion proteins in efflux resistance systems.

    PubMed

    De Angelis, Fabien; Lee, John K; O'Connell, Joseph D; Miercke, Larry J W; Verschueren, Koen H; Srinivasan, Vasundara; Bauvois, Cédric; Govaerts, Cédric; Robbins, Rebecca A; Ruysschaert, Jean-Marie; Stroud, Robert M; Vandenbussche, Guy

    2010-06-15

    Resistance nodulation cell division (RND)-based efflux complexes mediate multidrug and heavy-metal resistance in many Gram-negative bacteria. Efflux of toxic compounds is driven by membrane proton/substrate antiporters (RND protein) in the plasma membrane, linked by a membrane fusion protein (MFP) to an outer-membrane protein. The three-component complex forms an efflux system that spans the entire cell envelope. The MFP is required for the assembly of this complex and is proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we chose ZneB, the MFP component of the ZneCAB heavy-metal efflux system from Cupriavidus metallidurans CH34. ZneB is shown to be highly specific for Zn(2+) alone. The crystal structure of ZneB to 2.8 A resolution defines the basis for metal ion binding in the coordination site at a flexible interface between the beta-barrel and membrane proximal domains. The conformational differences observed between the crystal structures of metal-bound and apo forms are monitored in solution by spectroscopy and chromatography. The structural rearrangements between the two states suggest an active role in substrate efflux through metal binding and release. PMID:20534468

  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. Carbapenem Activities against Pseudomonas aeruginosa: Respective Contributions of OprD and Efflux Systems

    PubMed Central

    Köhler, Thilo; Michea-Hamzehpour, Mehri; Epp, Simone F.; Pechere, Jean-Claude

    1999-01-01

    While meropenem MICs were strongly influenced by the presence or absence of the MexAB-OprM efflux pump in both OprD-proficient and -deficient strain backgrounds, MICs of imipenem and of ER-35786 remained unchanged, demonstrating that meropenem is a substrate of MexAB-OprM but not imipenem and ER-35786. In vitro, all three carbapenems selected loss of OprD as a first mechanism of resistance. However, in an OprD-deficient background, meropenem was able to select MexAB-OprM overproducers as a secondary resistance mechanism, while ER-35786 selected a mutant cross-resistant to sparfloxacin and cefpirome. PMID:9925552

  6. 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

  7. Brain Efflux Index To Investigate the Influence of Active Efflux on Brain Distribution of Pemetrexed and Methotrexate

    PubMed Central

    Li, Li; Agarwal, Sagar

    2013-01-01

    Antifolates, in particular methotrexate (MTX), have been widely used in the treatment of primary and secondary tumors of the central nervous system (CNS). Pemetrexed (PMX) is a novel antifolate that also exhibits potent antitumor activity against CNS malignancies. Studies have shown that brain distribution of both antifolates is significantly restricted, possible due to active efflux transport at the blood-brain barrier (BBB). This study characterizes the brain-to-blood transport of PMX and MTX and examines the role of several efflux transporters in brain distribution of the antifolates by use of the intracerebral microinjection technique (brain efflux index). The results from this study show that both PMX and MTX undergo saturable efflux transport across the BBB, with elimination half-lives of approximately 39 minutes and 29 minutes, respectively. Of the various efflux transporters this study investigated, multidrug resistance-associated protein 2 (Mrp2) does not play an important role in the brain distribution of the two antifolate drugs. Interestingly, breast-cancer resistance protein (Bcrp) makes a significant contribution to the brain elimination of MTX but not PMX. In addition, the brain-to-blood transport of both antifolates was inhibited by probenecid and benzylpenicillin, suggesting the involvement of organic anion transporters in the efflux of these compounds from the brain, with organic anion transporter 3 (Oat3) being a possibility. Our results suggest that one of the underlying mechanisms behind the limited brain distribution of PMX and MTX is active efflux transport processes at the BBB, including a benzylpenicillin-sensitive transport system and/or the active transporter Bcrp. PMID:23297298

  8. Zidovudine and ursodeoxycholic acid conjugation: design of a new prodrug potentially able to bypass the active efflux transport systems of the central nervous system.

    PubMed

    Dalpiaz, Alessandro; Paganetto, Guglielmo; Pavan, Barbara; Fogagnolo, Marco; Medici, Alessandro; Beggiato, Sarah; Perrone, Daniela

    2012-04-01

    We have synthesized a new prodrug obtained by the 5'-ester conjugation of zidovudine (AZT), an antiviral agent substrate of active efflux transport systems (AET), with ursodeoxycholic acid (UDCA), a bile acid able to permeate into the central nervous system (CNS). We have demonstrated, by HPLC analysis, that UDCA-AZT is quickly hydrolyzed in rat plasma and whole blood (half-life <10 s). The same compound was hydrolyzed with slower rates in human plasma (half-life =7.53 ± 0.44 h) and whole blood (half-life =3.71 ± 0.16 h), allowing to control the AZT release. UDCA-AZT appeared hydrolyzed also in rat brain (half-life = 7.24 ± 0.45 min) and liver homogenates (half-life = 2.70 ± 0.14 min). In the aim to study the permeation properties of the UDCA-AZT across physiological barriers, we have used an established human retinal pigment epithelium (HRPE) cell line to obtain a polarized cell monolayer showing epithelial features. The bidirectional permeation of 30 μM AZT across this monolayer was regulated by apparent permeability coefficients (P(E)) higher from the apical to basolateral compartments (P(E) = 209 ± 4 × 10⁻⁵ cm/min) than in the opposite way (P(E) = 133 ± 8 × 10⁻⁵ cm/min), in conformity with the in vivo behavior of AZT, actively effluxed from the CNS. The influx (P(E) = 39.1 ± 1.2 × 10⁻⁵ cm/min) and efflux (P(E) = 31.3 ± 3.6 × 10⁻⁵ cm/min) permeability coefficients of 30 μM UDCA-AZT were instead the same, suggesting the ability of the prodrug to avoid the AET systems and, potentially, to allow its accumulation in the CNS. The relatively low P(E) values of UDCA-AZT were associated with a partial hydrolysis during its permeation across the cell monolayer. PMID:22356133

  9. Efflux pump inhibitors: targeting mycobacterial efflux systems to enhance TB therapy.

    PubMed

    Pule, Caroline M; Sampson, Samantha L; Warren, Robin M; Black, Philippa A; van Helden, Paul D; Victor, Tommie C; Louw, Gail E

    2016-01-01

    The emergence of drug resistance continues to plague TB control, with a global increase in the prevalence of MDR-TB. This acts as a gateway to XDR-TB and thus emphasizes the urgency for drug development and optimal treatment options. Bedaquiline is the first new anti-TB drug approved by the FDA in 40 years and has been shown to be an effective treatment option for MDR Mycobacterium tuberculosis infection. Bedaquiline has also recently been included in clinical trials for new regimens with the aim of improving and shortening treatment periods. Alarmingly, efflux-mediated bedaquiline resistance, as well as efflux-mediated cross-resistance to clofazimine, has been identified in treatment failures. This mechanism of resistance results in efflux of a variety of anti-TB drugs from the bacterial cell, thereby decreasing the intracellular drug concentration. In doing so, the bacillus is able to render the antibiotic treatment ineffective. Recent studies have explored strategies to reverse the resistance phenotype conferred by efflux pump activation. It was observed that the addition of efflux pump inhibitors partially restored drug susceptibility in vitro and in vivo. This has significant clinical implications, especially in MDR-TB management where treatment options are extremely limited. This review aims to highlight the current efflux pump inhibitors effective against M. tuberculosis, the effect of efflux pump inhibitors on mycobacterial growth and the clinical promise of treatment with efflux pump inhibitors and standard anti-TB therapy. PMID:26472768

  10. Efflux systems in bacterial pathogens: an opportunity for therapeutic intervention? An industry view.

    PubMed

    Lynch, A Simon

    2006-03-30

    The efflux systems of bacteria protect cells from antibiotics and biocides by actively transporting compounds out of the cytoplasm and/or periplasm and thereby limit their steady-state accumulation at their site(s) of action. The impact of efflux systems on the efficacy of antibiotics used in human medicine and animal husbandry is becoming increasingly apparent from the characterization of drug-resistant strains with altered drug efflux properties. In most instances, efflux-mediated antibiotic resistance arises from mutational events that result in their elevated expression and, in the case of efflux pumps with broad substrate specificity, can confer multi-drug resistance (MDR) to structurally unrelated antibiotics. Knowledge of the role of efflux systems in conferring antibiotic resistance has now been successfully exploited in the pharmaceutical industry and contributed, in part, to the development of new members of the macrolide and tetracycline classes of antibiotics that circumvent the efflux-based resistance mechanisms that have limited the clinical utility of their progenitors. The therapeutic utility of compounds that inhibit bacterial drug efflux pumps and therein potentiate the activity of a co-administered antibiotic agent remains to be validated in the clinical setting, but the approach holds promise for the future in improving the efficacy and/or extending the clinical utility of existing antibiotics. This review discusses the potential of further exploiting the knowledge of efflux-mediated antibiotic resistance in bacteria toward the discovery and development of new chemotherapeutic agents. PMID:16290174

  11. Active efflux of fluoroquinolones in Mycobacterium smegmatis mediated by LfrA, a multidrug efflux pump.

    PubMed Central

    Liu, J; Takiff, H E; Nikaido, H

    1996-01-01

    The lfrA gene cloned from chromosomal DNA of quinolone-resistant Mycobacterium smegmatis mc2-552 conferred low-level resistance to fluoroquinolones when present on multicopy plasmids. Sequence analysis suggested that lfrA encodes a membrane efflux pump of the major facilitator family (H. E. Takiff, M. Cimino, M. C. Musso, T. Weisbrod, R. Martinez, M. B. Delgado, L Salazar, B. R. Bloom, and W. R. Jacbos, Jr., Proc. Natl. Acad. Sci. USA 93:362-366, 1996). In this work, we studied the role of LfrA in the accumulation of fluoroquinolones by M. smegmatis. The steady-state accumulation level of a hydrophilic quinolone, norfloxacin, by M. smegmatis harboring a plasmid carrying the lfrA gene was about 50% of that by the parent strain but was increased to the same level as that of the parent strain by addition of a proton conductor, carbonyl cyanide m-chorophenylhydrazone. Norfloxacin efflux mediated by LfrA was competed for strongly by ciprofloxacin but not by nalidixic acid. Furthermore, we showed that portions of norfloxacin accumulated by starved cells were pumped out upon reenergization of the cells, and the rates of this efflux showed evidence of saturation at higher intracellular concentrations of the drug. These results suggest that the LfrA polypeptide catalyzes the active efflux of several quinolones. PMID:8682782

  12. 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

  13. Ethidium Bromide MIC Screening for Enhanced Efflux Pump Gene Expression or Efflux Activity in Staphylococcus aureus▿

    PubMed Central

    Patel, Diixa; Kosmidis, Christos; Seo, Susan M.; Kaatz, Glenn W.

    2010-01-01

    Multidrug resistance efflux pumps contribute to antimicrobial and biocide resistance in Staphylococcus aureus. The detection of strains capable of efflux is time-consuming and labor-intensive using currently available techniques. A simple and inexpensive method to identify such strains is needed. Ethidium bromide is a substrate for all but one of the characterized S. aureus multidrug-resistant (MDR) efflux pumps (NorC), leading us to examine the utility of simple broth microtiter MIC determinations using this compound in identifying efflux-proficient strains. Quantitative reverse transcription-PCR identified the increased expression of one or more MDR efflux pump genes in 151/309 clinical strains (49%). Ethidium bromide MIC testing was insensitive (48%) but specific (92%) in identifying strains with gene overexpression, but it was highly sensitive (95%) and specific (99%) in identifying strains capable of ethidium efflux. The increased expression of norA with or without other genes was most commonly associated with efflux, and in the majority of cases that efflux was inhibited by reserpine. Ethidium bromide MIC testing is a simple and straightforward method to identify effluxing strains and can provide accurate predictions of efflux prevalence in large strain sets in a short period of time. PMID:20855743

  14. Involvement of Antibiotic Efflux Machinery in Glutathione-Mediated Decreased Ciprofloxacin Activity in Escherichia coli.

    PubMed

    Goswami, Manish; Subramanian, Mahesh; Kumar, Ranjeet; Jass, Jana; Jawali, Narendra

    2016-07-01

    We have analyzed the contribution of different efflux components to glutathione-mediated abrogation of ciprofloxacin's activity in Escherichia coli and the underlying potential mechanism(s) behind this phenomenon. The results indicated that glutathione increased the total active efflux, thereby partially contributing to glutathione-mediated neutralization of ciprofloxacin's antibacterial action in E. coli However, the role of glutathione-mediated increased efflux becomes evident in the absence of a functional TolC-AcrAB efflux pump. PMID:27139480

  15. 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

  16. Identification and molecular characterization of an efflux system involved in Pseudomonas putida S12 multidrug resistance.

    PubMed

    Kieboom, J; de Bont, J

    2001-01-01

    The authors previously described srpABC, an operon involved in proton-dependent solvent efflux in the solvent-tolerant Pseudomonas putida S12. Recently, it was shown that organic solvents and not antibiotics induce this operon. In the present study, the authors characterize a new efflux pump, designated ArpABC, on the basis of two isolated chloramphenicol-sensitive transposon mutants. The arpABC operon is involved in the active efflux of multiple antibiotics, such as tetracycline, chloramphenicol, carbenicillin, streptomycin, erythromycin and novobiocin. The deduced amino acid sequences encoded by the three genes involved show a striking resemblance to proteins of the resistance/nodulation/cell division family, which are involved in both organic solvent and multiple drug efflux. These findings demonstrate that ArpABC is highly homologous to the MepABC and TtgABC efflux systems for organic solvents and multiple antibiotics. However, ArpABC does not contribute to organic solvent tolerance in P. putida S12 but is solely involved in multidrug resistance. PMID:11160799

  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. Correlation between AcrB trimer association affinity and efflux activity.

    PubMed

    Ye, Cui; Wang, Zhaoshuai; Lu, Wei; Zhong, Meng; Chai, Qian; Wei, Yinan

    2014-06-17

    The majority of membrane proteins function as oligomers. However, it remains largely unclear how the oligomer stability of protein complexes correlates with their function. Understanding the relationship between oligomer stability and activity is essential to protein research and to virtually all cellular processes that depend on the function of protein complexes. Proteins make lasting or transient interactions as they perform their functions. Obligate oligomeric proteins exist and function exclusively at a specific oligomeric state. Although oligomerization is clearly critical for such proteins to function, a direct correlation between oligomer affinity and biological activity has not yet been reported. Here, we used an obligate trimeric membrane transporter protein, AcrB, as a model to investigate the correlation between its relative trimer affinity and efflux activity. AcrB is a component of the major multidrug efflux system in Escherichia coli. We created six AcrB constructs with mutations at the transmembrane intersubunit interface, and we determined their activities using both a drug susceptibility assay and an ethidium bromide accumulation assay. The relative trimer affinities of these mutants in detergent micelles were obtained using blue native polyacrylamide gel electrophoresis. A correlation between the relative trimer affinity and substrate efflux activity was observed, in which a threshold trimer stability was required to maintain efflux activity. The trimer affinity of the wild-type protein was approximately 3 kcal/mol more stable than the threshold value. Once the threshold was reached, an additional increase of stability in the range observed had no observable effect on protein activity. PMID:24854514

  19. Reciprocal Regulation of Resistance-Nodulation-Division Efflux Systems and the Cpx Two-Component System in Vibrio cholerae

    PubMed Central

    Taylor, Dawn L.; Bina, X. Renee; Slamti, Leyla; Waldor, Matthew K.

    2014-01-01

    The Cpx two-component regulatory system has been shown in Escherichia coli to alleviate stress caused by misfolded cell envelope proteins. The Vibrio cholerae Cpx system was previously found to respond to cues distinct from those in the E. coli system, suggesting that this system fulfills a different physiological role in the cholera pathogen. Here, we used microarrays to identify genes that were regulated by the V. cholerae Cpx system. Our observations suggest that the activation of the V. cholerae Cpx system does not induce expression of genes involved in the mitigation of stress generated by misfolded cell envelope proteins but promotes expression of genes involved in antimicrobial resistance. In particular, activation of the Cpx system induced expression of the genes encoding the VexAB and VexGH resistance-nodulation-division (RND) efflux systems and their cognate outer membrane pore protein TolC. The promoters for these loci contained putative CpxR consensus binding sites, and ectopic cpxR expression activated transcription from the promoters for the RND efflux systems. CpxR was not required for intrinsic antimicrobial resistance, but CpxR activation enhanced resistance to antimicrobial substrates of VexAB and VexGH. Mutations that inactivated VexAB or VexGH efflux activity resulted in the activation of the Cpx response, suggesting that vexAB and vexGH and the cpxP-cpxRA system are reciprocally regulated. We speculate that the reciprocal regulation of the V. cholerae RND efflux systems and the Cpx two-component system is mediated by the intracellular accumulation of an endogenously produced metabolic by-product that is normally extruded from the cell by the RND efflux systems. PMID:24799626

  20. MexXY multidrug efflux system of Pseudomonas aeruginosa

    PubMed Central

    Morita, Yuji; Tomida, Junko; Kawamura, Yoshiaki

    2012-01-01

    Anti-pseudomonas aminoglycosides, such as amikacin and tobramycin, are used in the treatment of Pseudomonas aeruginosa infections. However, their use is linked to the development of resistance. During the last decade, the MexXY multidrug efflux system has been comprehensively studied, and numerous reports of laboratory and clinical isolates have been published. This system has been increasingly recognized as one of the primary determinants of aminoglycoside resistance in P. aeruginosa. In P. aeruginosa cystic fibrosis isolates, upregulation of the pump is considered the most common mechanism of aminoglycoside resistance. Non-fermentative Gram-negative pathogens possessing very close MexXY orthologs such as Achromobacter xylosoxidans and various Burkholderia species (e.g., Burkholderia pseudomallei and B. cepacia complexes), but not B. gladioli, are intrinsically resistant to aminoglycosides. Here, we summarize the properties (e.g., discovery, mechanism, gene expression, clinical significance) of the P. aeruginosa MexXY pump and other aminoglycoside efflux pumps such as AcrD of Escherichia coli, AmrAB-OprA of B. pseudomallei, and AdeABC of Acinetobacter baumannii. MexXY inducibility of the PA5471 gene product, which is dependent on ribosome inhibition or oxidative stress, is noteworthy. Moreover, the discovery of the cognate outer membrane component (OprA) of MexXY in the multidrug-resistant clinical isolate PA7, serotype O12 deserves special attention. PMID:23233851

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

    PubMed Central

    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. PMID:25972842

  2. Goldenseal (Hydrastis canadensis L.) extracts synergistically enhance the antibacterial activity of berberine via efflux pump inhibition.

    PubMed

    Ettefagh, Keivan A; Burns, Johnna T; Junio, Hiyas A; Kaatz, Glenn W; Cech, Nadja B

    2011-05-01

    Goldenseal (Hydrastis canadensis L.) is used to combat inflammation and infection. Its antibacterial activity in vitRO has been attributed to its alkaloids, the most abundant of which is berberine. The goal of these studies was to compare the composition, antibacterial activity, and efflux pump inhibitory activity of ethanolic extracts prepared from roots and aerial portions of H. canadensis. Ethanolic extracts were prepared separately from roots and aerial portions of six H. canadensis plants. Extracts were analyzed for alkaloid concentration using LC-MS and tested for antimicrobial activity against Staphylococcus aureus (NCTC 8325-4) and for inhibition of ethidium bromide efflux. Synergistic antibacterial activity was observed between the aerial extract (FIC 0.375) and to a lesser extent the root extract (FIC 0.750) and berberine. The aerial extract inhibited ethidium bromide efflux from wild-type S. aureus but had no effect on the expulsion of this compound from an isogenic derivative deleted for norA. Our studies indicate that the roots of H. canadensis contain higher levels of alkaloids than the aerial portions, but the aerial portions synergize with berberine more significantly than the roots. Furthermore, extracts from the aerial portions of H. canadensis contain efflux pump inhibitors, while efflux pump inhibitory activity was not observed for the root extract. The three most abundant H. canadensis alkaloids, berberine, hydrastine, and canadine, are not responsible for the efflux pump inhibitory activity of the extracts from H. canadensis aerial portions. PMID:21157683

  3. Correlation of Ciprofloxacin Resistance with the AdeABC Efflux System in Acinetobacter baumannii Clinical Isolates

    PubMed Central

    Ardebili, Abdollah; Talebi, Malihe

    2014-01-01

    Background Acinetobacter baumannii is one of the most important pathogens capable of colonization in burn patients, leading to drug-resistant wound infections. This study evaluated the distribution of the AdeABC efflux system genes and their relationship to ciprofloxacin resistance in A. baumannii isolates collected from burn patients. Methods A total of 68 A. baumannii clinical strains were isolated from patients hospitalized in Motahari Burns Center in Tehran, Iran. Ciprofloxacin susceptibility was tested by the disk diffusion and agar dilution methods. PCR amplification of the adeRS-adeB drug efflux genes was performed for all resistant and susceptible isolates. To assess the role of the drug efflux pump in ciprofloxacin susceptibility, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as an efflux pump inhibitor (EPI). Results Approximately 95.6% of the Acinetobacter isolates were resistant to ciprofloxacin, with minimum inhibitory concentration (MIC) values ranging from 4 to ≥128 µg/mL. The susceptibility of 86.1% of the resistant isolates increased by factors of 2 to 64 in the presence of CCCP. All resistant isolates were positive for the adeRS-adeB genes, and 73.2% of them had mutations in the AdeRS regulatory system. Conclusions The results showed that AdeABC genes are common in A. baumannii, which might be associated with ciprofloxacin non-susceptibility, as indicated by the observed linkage to the presence of the genes essential for the activity of the AdeABC, several single mutations occurring in the adeRS regulatory system, and an increase of ciprofloxacin susceptibility in the presence of a CCCP EPI. PMID:25368818

  4. Regulation of retinoid mediated cholesterol efflux involves liver X receptor activation in mouse macrophages.

    PubMed

    Manna, Pulak R; Sennoune, Souad R; Martinez-Zaguilan, Raul; Slominski, Andrzej T; Pruitt, Kevin

    2015-08-14

    Removal of cholesterol from macrophage-derived foam cells is a critical step to the prevention of atherosclerotic lesions. We have recently demonstrated the functional importance of retinoids in the regulation of the steroidogenic acute regulatory (StAR) protein that predominantly mediates the intramitochondrial transport of cholesterol in target tissues. In the present study, treatment of mouse macrophages with retinoids, particularly all-trans retinoic acid (atRA) and 9-cis RA, resulted in increases in cholesterol efflux to apolipoprotein AI (Apo-A1). Activation of the PKA pathway by a cAMP analog, (Bu)2cAMP, markedly augmented retinoid mediated cholesterol efflux. Macrophages overexpressing hormone-sensitive lipase increased the hydrolysis of cholesteryl esters and concomitantly enhanced the efficacy of retinoic acid receptor and liver X receptor (LXR) ligands on StAR and ATP-binding cassette transporter A1 (ABCA1) protein levels. RAs elevated StAR promoter activity in macrophages, and an increase in StAR levels augmented cholesterol efflux to Apo-A1, suggesting retinoid-mediated efflux of cholesterol involves enhanced oxysterol production. Further studies revealed that retinoids activate the LXR regulated genes, sterol receptor-element binding protein-1c and ABCA1. These findings provide insights into the regulatory events in which retinoid signaling effectively enhances macrophage cholesterol efflux and indicate that retinoid therapy may have important implications in limiting and/or regressing atherosclerotic cardiovascular disease. PMID:26119689

  5. 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

  6. Ferroportin and Exocytoplasmic Ferroxidase Activity Are Required for Brain Microvascular Endothelial Cell Iron Efflux*

    PubMed Central

    McCarthy, Ryan C.; Kosman, Daniel J.

    2013-01-01

    The mechanism(s) of iron flux across the brain microvasculature endothelial cells (BMVEC) of the blood-brain barrier remains unknown. Although both hephaestin (Hp) and the ferrous iron permease ferroportin (Fpn) have been identified in BMVEC, their roles in iron efflux have not been examined. Using a human BMVEC line (hBMVEC), we have demonstrated that these proteins are required for iron efflux from these cells. Expression of both Hp and Fpn protein was confirmed in hBMVEC by immunoblot and indirect immunofluorescence; we show that hBMVEC express soluble ceruloplasmin (Cp) transcript as well. Depletion of endogenous Hp and Cp via copper chelation leads to the reduction of hBMVEC Fpn protein levels as well as a complete inhibition of 59Fe efflux. Both hBMVEC Fpn protein and 59Fe efflux activity are restored upon incubation with 6.6 nm soluble plasma Cp. These results are independent of the source of cell iron, whether delivered as transferrin- or non-transferrin-bound 59Fe. Our results demonstrate that iron efflux from hBMVEC Fpn requires the action of an exocytoplasmic ferroxidase, which can be either endogenous Hp or extracellular Cp. PMID:23640881

  7. Solute carrier 41A3 encodes for a mitochondrial Mg(2+) efflux system.

    PubMed

    Mastrototaro, Lucia; Smorodchenko, Alina; Aschenbach, Jörg R; Kolisek, Martin; Sponder, Gerhard

    2016-01-01

    The important role of magnesium (Mg(2+)) in normal cellular physiology requires flexible, yet tightly regulated, intracellular Mg(2+) homeostasis (IMH). However, only little is known about Mg(2+) transporters of subcellular compartments such as mitochondria, despite their obvious importance for the deposition and reposition of intracellular Mg(2+) pools. In particular, knowledge about mechanisms responsible for extrusion of Mg(2+) from mitochondria is lacking. Based on circumstantial evidence, two possible mechanisms of Mg(2+) release from mitochondria were predicted: (1) Mg(2+) efflux coupled to ATP translocation via the ATP-Mg/Pi carrier, and (2) Mg(2+) efflux via a H(+)/Mg(2+) exchanger. Regardless, the identity of the H(+)-coupled Mg(2+) efflux system is unknown. We demonstrate here that member A3 of solute carrier (SLC) family 41 is a mitochondrial Mg(2+) efflux system. Mitochondria of HEK293 cells overexpressing SLC41A3 exhibit a 60% increase in the extrusion of Mg(2+) compared with control cells. This efflux mechanism is Na(+)-dependent and temperature sensitive. Our data identify SLC41A3 as the first mammalian mitochondrial Mg(2+) efflux system, which greatly enhances our understanding of intracellular Mg(2+) homeostasis. PMID:27302215

  8. Solute carrier 41A3 encodes for a mitochondrial Mg2+ efflux system

    PubMed Central

    Mastrototaro, Lucia; Smorodchenko, Alina; Aschenbach, Jörg R.; Kolisek, Martin; Sponder, Gerhard

    2016-01-01

    The important role of magnesium (Mg2+) in normal cellular physiology requires flexible, yet tightly regulated, intracellular Mg2+ homeostasis (IMH). However, only little is known about Mg2+ transporters of subcellular compartments such as mitochondria, despite their obvious importance for the deposition and reposition of intracellular Mg2+ pools. In particular, knowledge about mechanisms responsible for extrusion of Mg2+ from mitochondria is lacking. Based on circumstantial evidence, two possible mechanisms of Mg2+ release from mitochondria were predicted: (1) Mg2+ efflux coupled to ATP translocation via the ATP-Mg/Pi carrier, and (2) Mg2+ efflux via a H+/Mg2+ exchanger. Regardless, the identity of the H+-coupled Mg2+ efflux system is unknown. We demonstrate here that member A3 of solute carrier (SLC) family 41 is a mitochondrial Mg2+ efflux system. Mitochondria of HEK293 cells overexpressing SLC41A3 exhibit a 60% increase in the extrusion of Mg2+ compared with control cells. This efflux mechanism is Na+-dependent and temperature sensitive. Our data identify SLC41A3 as the first mammalian mitochondrial Mg2+ efflux system, which greatly enhances our understanding of intracellular Mg2+ homeostasis. PMID:27302215

  9. Heavy metal transport by the CusCFBA efflux system.

    PubMed

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

    2015-11-01

    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

    2016-06-01

    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. PMID:26554512

  11. MexCD-OprJ Multidrug Efflux System of Pseudomonas aeruginosa: Involvement in Chlorhexidine Resistance and Induction by Membrane-Damaging Agents Dependent upon the AlgU Stress Response Sigma Factor▿

    PubMed Central

    Fraud, Sebastien; Campigotto, Aaron J.; Chen, Zhilin; Poole, Keith

    2008-01-01

    The biocide chlorhexidine (CHX) as well as additional membrane-active agents were shown to induce expression of the mexCD-oprJ multidrug efflux operon, dependent upon the AlgU stress response sigma factor. Hyperexpression of this efflux system in nfxB mutants was also substantially AlgU dependent. CHX resistance correlated with efflux gene expression in various mutants, consistent with MexCD-OprJ being a determinant of CHX resistance. PMID:18838593

  12. MexCD-OprJ multidrug efflux system of Pseudomonas aeruginosa: involvement in chlorhexidine resistance and induction by membrane-damaging agents dependent upon the AlgU stress response sigma factor.

    PubMed

    Fraud, Sebastien; Campigotto, Aaron J; Chen, Zhilin; Poole, Keith

    2008-12-01

    The biocide chlorhexidine (CHX) as well as additional membrane-active agents were shown to induce expression of the mexCD-oprJ multidrug efflux operon, dependent upon the AlgU stress response sigma factor. Hyperexpression of this efflux system in nfxB mutants was also substantially AlgU dependent. CHX resistance correlated with efflux gene expression in various mutants, consistent with MexCD-OprJ being a determinant of CHX resistance. PMID:18838593

  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. Identification and characterization of the emhABC efflux system for polycyclic aromatic hydrocarbons in Pseudomonas fluorescens cLP6a.

    PubMed

    Hearn, Elizabeth M; Dennis, Jonathan J; Gray, Murray R; Foght, Julia M

    2003-11-01

    The hydrocarbon-degrading environmental isolate Pseudomonas fluorescens LP6a possesses an active efflux mechanism for the polycyclic aromatic hydrocarbons phenanthrene, anthracene, and fluoranthene but not for naphthalene or toluene. PCR was used to detect efflux pump genes belonging to the resistance-nodulation-cell division (RND) superfamily in a plasmid-cured derivative, P. fluorescens cLP6a, which is unable to metabolize hydrocarbons. One RND pump, whose gene was identified in P. fluorescens cLP6a and was designated emhB, showed homology to the multidrug and solvent efflux pumps in Pseudomonas aeruginosa and Pseudomonas putida. The emhB gene is located in a gene cluster with the emhA and emhC genes, which encode the membrane fusion protein and outer membrane protein components of the efflux system, respectively. Disruption of emhB by insertion of an antibiotic resistance cassette demonstrated that the corresponding gene product was responsible for the efflux of polycyclic aromatic hydrocarbons. The emhB gene disruption did not affect the resistance of P. fluorescens cLP6a to tetracycline, erythromycin, trimethoprim, or streptomycin, but it did decrease resistance to chloramphenicol and nalidixic acid, indicating that the EmhABC system also functions in the efflux of these compounds and has an unusual selectivity. Phenanthrene efflux was observed in P. aeruginosa, P. putida, and Burkholderia cepacia but not in Azotobacter vinelandii. Polycyclic aromatic hydrocarbons represent a new class of nontoxic, highly hydrophobic compounds that are substrates of RND efflux systems, and the EmhABC system in P. fluorescens cLP6a has a narrow substrate range for these hydrocarbons and certain antibiotics. PMID:14563857

  16. Reversal of efflux mediated antifungal resistance underlies synergistic activity of two monoterpenes with fluconazole.

    PubMed

    Ahmad, Aijaz; Khan, Amber; Manzoor, Nikhat

    2013-01-23

    Thymol (THY) and carvacrol (CARV), the principal chemical components of thyme oil have long been known for their wide use in medicine due to antimicrobial and disinfectant properties. This study, however, draws attention to a possible synergistic antifungal effect of these monoterpenes with azole antimycotic-fluconazole. Resistance to azoles in Candida albicans involves over-expression of efflux-pump genes MDR1, CDR1, CDR2 or mutations and over-expression of target gene ERG11. The inhibition of drug efflux pumps is considered a feasible strategy to overcome clinical antifungal resistance. To put forward this approach, we investigated the combination effects of these monoterpenes and FLC against 38 clinically obtained FLC-sensitive, and eleven FLC-resistant Candida isolates. Synergism was observed with combinations of THY-FLC and CARV-FLC evaluated by checkerboard microdilution method and nature of the interactions was calculated by FICI. In addition, antifungal activity was assessed using agar-diffusion and time-kill curves. The drug efflux activity was determined using two dyes, Rhodamine6G (R6G) and fluorescent Hoechst 33342. No significant differences were observed in dye uptakes between FLC-susceptible and resistant isolates, incubated in glucose free buffer. However, a significantly higher efflux was recorded in FLC-resistant isolates when glucose was added. Both monoterpenes inhibited efflux by 70-90%, showing their high potency to block drug transporter pumps. Significant differences, in the expression levels of CDR1 and MDR1, induced by monoterpenes revealed reversal of FLC-resistance. The selectively fungicidal characteristics and ability to restore FLC susceptibility in resistant isolates signify a promising candidature of THY and CARV as antifungal agents in combinational treatments for candidiasis. PMID:23111348

  17. Nek7 is an essential mediator of NLRP3 activation downstream of potassium efflux

    PubMed Central

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

    2016-01-01

    Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases1. To date, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-182,3. The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases4,5 as well as Cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations6,7. Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by multiple stimuli8,9. Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. We report here the identification of Nek7, a member of the family of mammalian NIMA-related kinases (Neks)10, 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 inflammasome. NLRP3-activating stimuli promoted the NLRP3-Nek7 interaction in a process dependent on potassium efflux. NLRP3 associated with the catalytic domain of Nek7, but the catalytic activity of Nek7 was 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 were abrogated in the absence of Nek7. Nek7 was required for macrophages harboring the CAPS-associated NLRP3R258W activating mutation to activate caspase-1. Mouse chimeras reconstituted with wild-type, Nek7−/− or Nlrp3−/− hematopoietic cells revealed 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 mediate NLRP3 inflammasome

  18. 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

  19. 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. PMID:23886790

  20. Hydraulics are a first-order control on CO2 efflux from fluvial systems

    NASA Astrophysics Data System (ADS)

    Long, Hazel; Vihermaa, Leena; Waldron, Susan; Hoey, Trevor; Quemin, Simon; Newton, Jason

    2015-10-01

    Evasion of carbon dioxide (CO2) from fluvial systems is now recognized as a significant component of the global carbon cycle. However, the magnitude of, and controls on, this flux remains uncertain, and improved understanding of both is required to refine global estimates of fluvial CO2 efflux. CO2 efflux data show no pattern with latitude suggesting that catchment biological productivity is not a primary control and that an alternative explanation for intersite variability is required. It has been suggested that increased flow velocity and turbulence enhance CO2 efflux, but this is not confirmed. Here using contemporaneous measurements of efflux (range: 0.07-107 µmol CO2 m-2 s-1), flow hydraulics (mean velocity range: 0.03-1.39 m s-1), and pCO2 (range: 174-10712 µatm) at six sites, we find that flow intensity is a primary control on efflux across two climatically different locations (where pH is not a limiting factor) and that the relationship is refined by incorporating the partial pressure of CO2 (pCO2) of the water. A remaining challenge is how to upscale from point to reach or river basin level. Remote imaging or river surface may be worth exploring if subjectivity in interpreting surface state can be overcome.

  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. PMID:27332787

  3. NorA efflux pump inhibitory activity of coumarins from Mesua ferrea.

    PubMed

    Roy, Somendu K; Kumari, Neela; Pahwa, Sonika; Agrahari, Udai C; Bhutani, Kamlesh K; Jachak, Sanjay M; Nandanwar, Hemraj

    2013-10-01

    The purpose of this investigation was to study the modulator and efflux pump inhibitor activity of coumarins isolated from Mesua ferrea against clinical strains as well as NorA-over expressed strain of Staphylococcus aureus 1199B. Seven coumarins were tested for modulator activity using ethidium bromide (EtBr) as a substrate. Compounds 1, 4-7 modulated the MIC of EtBr by ≥ 2 fold against wild type clinical strains of S. aureus 1199 and S. aureus 1199B, whereas compounds 4-7 modulated the MIC of EtBr by ≥ 16 fold against MRSA 831. Compounds 1, 4-7 also reduced the MIC of norfloxacin by ≥ 8 fold against S. aureus 1199B, and 4-6 reduced the MIC of norfloxacin by ≥ 8 fold against MRSA 831 at half of their MICs. Inhibition of EtBr efflux by NorA-overproducing S. aureus 1199B and MRSA 831 confirmed the role of compounds 4-6 as NorA efflux pump inhibitors (EPI). Dose-dependent activity at sub-inhibitory concentration (6.25 μg/mL) suggested that compounds 4 and 5 are promising EPI compared to verapamil against 1199B and MRSA 831 strains. PMID:23892000

  4. Influence of multidrug efflux systems on methylene blue-mediated photodynamic inactivation of Candida albicans

    PubMed Central

    Prates, Renato A.; Kato, Ilka T.; Ribeiro, Martha S.; Tegos, George P.; Hamblin, Michael R.

    2011-01-01

    Objectives To investigate whether the major fungal multidrug efflux systems (MESs) affect the efficiency of methylene blue (MB)-mediated antimicrobial photodynamic inactivation (APDI) in pathogenic fungi and test specific inhibitors of these efflux systems to potentiate APDI. Methods Candida albicans wild-type and mutants that overexpressed two classes of MESs [ATP-binding cassette (ABC) and major facilitator superfamily (MFS)] were tested for APDI using MB as the photosensitizer with and without addition of MES inhibitors. The uptake and cytoplasm localization of photosensitizer were achieved using laser confocal microscopy. Results ABC MES overexpression reduced MB accumulation and APDI killing more than MFS MES overexpression. Furthermore, by combining MB APDI with the ABC inhibitor verapamil, fungal killing and MB uptake were potentiated, while by combining MB APDI with the MFS inhibitor INF271, fungal killing and MB uptake were inhibited. This latter surprising finding may be explained by the hypothesis that the MFS channel can also serve as an uptake mechanism for MB. Conclusions The ABC pumps are directly implicated in MB efflux from the cell cytoplasm. Both the influx and efflux of MB may be regulated by MFS systems, and blocking this gate before incubation with MB can decrease the uptake and APDI effects. An ABC inhibitor could be usefully combined with MB APDI for treating C. albicans infections. PMID:21525022

  5. Auxin efflux carrier activity and auxin accumulation regulate cell division and polarity in tobacco cells.

    PubMed

    Petrásek, Jan; Elckner, Miroslav; Morris, David A; Zazímalová, Eva

    2002-12-01

    Division and growth of most types of in vitro-cultured plant cells require an external source of auxin. In such cultures, the ratio of external to internal auxin concentration is crucial for the regulation of the phases of the standard growth cycle. In this report the internal concentration of auxin in suspension-cultured cells of Nicotiana tabacum L., strain VBI-0, was manipulated either (i) by increasing 10-fold the normal concentration of 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid in the external medium; or (ii) by addition 1-N-naphthylphthalamic acid (NPA; an inhibitor of auxin efflux and of auxin efflux carrier traffic). Both treatments delayed the onset of cell division for 6-7 days without loss of cell viability. In both cases, cell division activity subsequently resumed coincident with a reduction in the ability of cells to accumulate [(3)H]NAA from an external medium. Following renewed cell division, a significant proportion of the NPA-treated cells but not those grown at high auxin concentration, exhibited changes in the orientation of new cell divisions and loss of polarity. We conclude that cell division, but not cell elongation, is prevented when the internal auxin concentration rises above a critical threshold value and that the directed traffic of auxin efflux carriers to the plasma membrane may regulate the orientation of cell divisions. PMID:12447544

  6. Ventral Tegmental Area Neurotensin Signaling Links the Lateral Hypothalamus to Locomotor Activity and Striatal Dopamine Efflux in Male Mice

    PubMed Central

    Patterson, Christa M.; Wong, Jenny-Marie T.; Leinninger, Gina M.; Allison, Margaret B.; Mabrouk, Omar S.; Kasper, Chelsea L.; Gonzalez, Ian E.; Mackenzie, Alexander; Jones, Justin C.

    2015-01-01

    Projections from the lateral hypothalamic area (LHA) innervate components of the mesolimbic dopamine (MLDA) system, including the ventral tegmental area (VTA) and nucleus accumbens (NAc), to modulate motivation appropriately for physiologic state. Neurotensin (NT)-containing LHA neurons respond to multiple homeostatic challenges and project to the VTA, suggesting that these neurons could link such signals to MLDA function. Indeed, we found that pharmacogenetic activation of LHA NT neurons promoted prolonged DA-dependent locomotor activity and NAc DA efflux, suggesting the importance of VTA neurotransmitter release by LHA NT neurons for the control of MLDA function. Using a microdialysis-mass spectrometry technique that we developed to detect endogenous NT in extracellular fluid in the mouse brain, we found that activation of LHA NT cells acutely increased the extracellular concentration of NT (a known activator of VTA DA cells) in the VTA. In contrast to the prolonged elevation of extracellular NAc DA, however, VTA NT concentrations rapidly returned to baseline. Intra-VTA infusion of NT receptor antagonist abrogated the ability of LHA NT cells to increase extracellular DA in the NAc, demonstrating that VTA NT promotes NAc DA release. Thus, transient LHA-derived NT release in the VTA couples LHA signaling to prolonged changes in DA efflux and MLDA function. PMID:25734363

  7. Potentiation of antibacterial activity of the MB-1 siderophore-monobactam conjugate using an efflux pump inhibitor.

    PubMed

    Tomaras, Andrew P; Crandon, Jared L; McPherson, Craig J; Nicolau, David P

    2015-04-01

    Preliminary enthusiasm over the encouraging spectrum and in vitro activities of siderophore conjugates, such as MB-1, was recently tempered by unexpected variability in in vivo efficacy. The need for these conjugates to compete for iron with endogenously produced siderophores has exposed a significant liability for this novel antibacterial strategy. Here, we have exploited dependence on efflux for siderophore secretion in Pseudomonas aeruginosa and provide evidence that efflux inhibition may circumvent this in vivo-relevant resistance liability. PMID:25605364

  8. Potentiation of Antibacterial Activity of the MB-1 Siderophore-Monobactam Conjugate Using an Efflux Pump Inhibitor

    PubMed Central

    Crandon, Jared L.; McPherson, Craig J.; Nicolau, David P.

    2015-01-01

    Preliminary enthusiasm over the encouraging spectrum and in vitro activities of siderophore conjugates, such as MB-1, was recently tempered by unexpected variability in in vivo efficacy. The need for these conjugates to compete for iron with endogenously produced siderophores has exposed a significant liability for this novel antibacterial strategy. Here, we have exploited dependence on efflux for siderophore secretion in Pseudomonas aeruginosa and provide evidence that efflux inhibition may circumvent this in vivo-relevant resistance liability. PMID:25605364

  9. 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

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

    PubMed Central

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inês CR; 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

    2014-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.02860.001 PMID:24948515

  11. 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

    2014-01-01

    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. PMID:24948515

  12. Inhibition of topoisomerase I activity and efflux drug transporters' expression by xanthohumol. from hops.

    PubMed

    Lee, Sung Ho; Kim, Hyun Jung; Lee, Jung Sun; Lee, Ik-Soo; Kang, Bok Yun

    2007-11-01

    Xanthohumol (XN) and its related compounds were evaluated for their cytotoxicity against four different human cancer cell lines, A549 (lung), SK-OV-3 (ovarian), SK-MEL-2 (melanoma), and HCT-15 (colon) using a sulforhodamine B assay. XN showed the most active cytotoxicity against the human cancer cell lines. Isoxanthohumol, 8-prenylnaringenin, and xanthohumol 4'-O-beta-D-glucopyranoside showed comparable cytotoxicity and (2S)-5-methoxy-8-prenylnaringenin 7-O-beta-D-glucopyranoside was the least cytotoxic compound. The anticancer properties of XN, the most active cytotoxic compound, were further investigated. XN showed an inhibitory effect on the activity of DNA topoisomerase I (topo I), which was measured from the relaxation of supercoiled DNA. The inhibition of topo I by XN might explain the cytotoxicity against the human cancer cell lines. Moreover, the expression of the drug efflux genes was investigated to predict the drug resistance. XN clearly decreased the mRNA levels of ABCB1 (MDR1), ABCC1 (MRP1), ABCC2 (MRP2), and ABCC3 (MRP3). These results suggest that XN has anticancer properties by inhibiting the topo I activity and it might be used in conjunction with other anticancer chemotherapeutic agents to reduce the drug resistance inhibiting the efflux drug transporters. PMID:18087812

  13. Effects of aging on agonist-activated sup 86 Rb efflux in arteries of Fischer 344 rats

    SciTech Connect

    Cox, R.H.; Tulenko, T.N. )

    1989-08-01

    Segments of thoracic aorta (DTA), tail artery (TA), and mesenteric artery branches (MAB) were obtained from male Fischer 344 rats at ages of 1, 2, 6, 12, 24, and 30 mo and were used to determine the effects of aging on agonist-activated {sup 86}Rb (and {sup 42}K) efflux. At all three arterial sites, basal efflux decreased during development (1-6 mo), but no further changes were observed with aging (6-30 mo). The initial efflux response to 10 microM norepinephrine (NE) in the presence of 1 microM propranolol exhibited either no change (DTA) or an increase (TA and MAB) during development (1-6 mo), but all three sites showed a large decrease during aging (6-30 mo). Changes in the steady-state response to NE paralleled changes in the basal efflux at all ages and arterial sites. The initial efflux response to 75 mM K+-physiological salt solution (PSS) for the DTA in the presence of 1 microM phentolamine and 1 microM propranolol decreased during development followed by an increase during aging, whereas for the TA and MAB, there were no significant changes with age. The steady-state efflux response to K+ decreased during development at all three sites but was increased only for the DTA during aging. The steady-state efflux response to K+ was not altered for the TA and MAB during aging. Efflux responses using {sup 42}K were qualitatively similar, but rate constants were quantitatively larger than those with {sup 86}Rb at all three arterial sites and at all ages.

  14. 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 ...

  15. 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

  16. 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.

  17. Comparison of effects of cromakalim and pinacidil on mechanical activity and 86Rb efflux in dog coronary arteries

    SciTech Connect

    Masuzawa, K.; Asano, M.; Matsuda, T.; Imaizumi, Y.; Watanabe, M. )

    1990-05-01

    Effects of two K+ channel openers, cromakalim and pinacidil, on mechanical activity and on 86Rb efflux were compared in strips of dog coronary arteries. Cromakalim and pinacidil produced the relaxation in 20.9 mM K(+)-contracted strips with a pD2 of 6.53 and 5.95, respectively. In 65.9 mM K(+)-contracted strips, high concentrations of pinacidil, but not cromakalim, produced relaxation. Ca+(+)-induced contractions in 80 mM K(+)-depolarized strips were also inhibited by pinacidil but not by cromakalim. Glibenclamide, a blocker of ATP-regulated K+ (KATP) channels, competitively antagonized the relaxant responses to cromakalim with a pA2 value of 7.62. However, the antagonism by glibenclamide of the relaxant responses to pinacidil was not a typical competitive type, suggesting the contribution of other effects than the KATP channel opening activity to the relaxant effects of pinacidil. In resting strips preloaded with 86Rb, cromakalim and pinacidil increased the basal 86Rb efflux in a dose-dependent manner. The increase in the 86Rb efflux induced by cromakalim was greater than that by pinacidil. When the effects of cromakalim and pinacidil on the 86Rb efflux were determined in the 20.9 or 65.9 mM K(+)-contracted strips, both drugs increased the 86Rb efflux. Under the same conditions nifedipine, a Ca(+)+ channel blocker, produced the relaxation that is accompanied by the decrease in 86Rb efflux. The increase in the 86Rb efflux induced by cromakalim was much greater than that by pinacidil.

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

    PubMed

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

    2016-08-01

    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

  19. Novel Resistance-Nodulation-Cell Division Efflux System AdeDE in Acinetobacter Genomic DNA Group 3

    PubMed Central

    Chau, Sze-Lok; Chu, Yiu-Wai; Houang, Elizabeth T. S.

    2004-01-01

    Resistance-nodulation-cell division type efflux pump AdeDE was identified in acinetobacters belonging to genomic DNA group 3. Inactivation of adeE showed that it may be responsible for reduced susceptibility to amikacin, ceftazidime, chloramphenicol, ciprofloxacin, erythromycin, ethidium bromide, meropenem, rifampin, and tetracycline. However, unlike what was found for other similar efflux systems, the open reading frame for the outer membrane component was not found downstream of the adeDE gene cluster. PMID:15388479

  20. Altered agonist-activated sup 86 Rb+ efflux from arteries in canine renal hypertension

    SciTech Connect

    Cox, R.H.; Bagshaw, R.J. )

    1989-07-01

    Basal rate constants for {sup 86}Rb+ efflux from renal arteries of renal hypertensive dogs were lower than those of control animals whereas no differences were found for coronary arteries. Norepinephrine produced parallel increases in efflux rate constants for hypertensive and control renal arteries, but serotonin produced smaller responses in hypertensive compared to control coronary arteries.

  1. Identification of a Cyanobacterial RND-Type Efflux System Involved in Export of Free Fatty Acids.

    PubMed

    Kato, Akihiro; Takatani, Nobuyuki; Use, Kazuhide; Uesaka, Kazuma; Ikeda, Kazutaka; Chang, Yajun; Kojima, Kouji; Aichi, Makiko; Ihara, Kunio; Nakahigashi, Kenji; Maeda, Shin-Ichi; Omata, Tatsuo

    2015-12-01

    An RND (resistance-nodulation-division)-type transporter having the capacity to export free fatty acids (FFAs) was identified in the cyanobacterium Synechococcus elongatus strain PCC 7942 during characterization of a mutant strain engineered to produce FFAs. The basic strategy for construction of the FFA-producing mutant was a commonly used one, involving inactivation of the endogenous acyl-acyl carrier protein synthetase gene (aas) and introduction of a foreign thioesterase gene ('tesA), but a nitrate transport mutant NA3 was used as the parental strain to achieve slow, nitrate-limited growth in batch cultures. Also, a nitrogen-regulated promoter PnirA was used to drive 'tesA to maximize thioesterase expression during the nitrate-limited growth. The resulting mutant (dAS2T) was, however, incapable of growth under the conditions of nitrate limitation, presumably due to toxicity associated with FFA overproduction. Incubation of the mutant culture under the non-permissive conditions allowed for isolation of a pseudorevertant (dAS2T-pr1) capable of growth on nitrate. Genome sequence and gene expression analyses of this strain suggested that expression of an RND-type efflux system had rescued growth on nitrate. Targeted inactivation of the RND-type transporter genes in the wild-type strain resulted in loss of tolerance to exogenously added FFAs including capric, lauric, myristic, oleic and linolenic acids. Overexpression of the genes in dAS2T, on the other hand, enhanced FFA excretion and cell growth in nitrate-containing medium, verifying that the genes encode an efflux pump for FFAs. These results demonstrate the importance of the efflux system in efficient FFA production using genetically engineered cyanobacteria. PMID:26468506

  2. Methane efflux from marine sediments in passive and active margins: Estimations from bioenergetic reaction-transport simulations

    NASA Astrophysics Data System (ADS)

    Dale, A. W.; Van Cappellen, P.; Aguilera, D. R.; Regnier, P.

    2008-01-01

    A simplified version of a kinetic-bioenergetic reaction model for anaerobic oxidation of methane (AOM) in marine sediments [Dale, A.W., Regnier, P., Van Cappellen, P., 2006. Bioenergetic controls on anaerobic oxidation of methane (AOM) in coastal marine sediments: a theoretical analysis. Am. J. Sci. 306, 246-294.] is used to assess the impact of transport processes on biomass distributions, AOM rates and methane release fluxes from the sea floor. The model explicitly represents the functional microbial groups and the kinetic and bioenergetic limitations of the microbial metabolic pathways involved in AOM. Model simulations illustrate the dominant control exerted by the transport regime on the activity and abundance of AOM communities. Upward fluid flow at active seep systems restricts AOM to a narrow subsurface reaction zone and sustains high rates of methane oxidation. In contrast, pore-water transport dominated by molecular diffusion leads to deeper and broader zones of AOM, characterized by much lower rates and biomasses. Under steady-state conditions, less than 1% of the upward dissolved methane flux reaches the water column, irrespective of the transport regime. However, a sudden increase in the advective flux of dissolved methane, for example as a result of the destabilization of methane hydrates, causes a transient efflux of methane from the sediment. The benthic efflux of dissolved methane is due to the slow growth kinetics of the AOM community and lasts on the order of 60 years. This time window is likely too short to allow for a significant escape of pore-water methane following a large scale gas hydrate dissolution event such as the one that may have accompanied the Paleocene/Eocene Thermal Maximum (PETM).

  3. Identification and characterization of maize and barley Lsi2-like silicon efflux transporters reveals a distinct silicon uptake system from that in rice.

    PubMed

    Mitani, Namiki; Chiba, Yukako; Yamaji, Naoki; Ma, Jian Feng

    2009-07-01

    Silicon (Si) uptake has been extensively examined in rice (Oryza sativa), but it is poorly understood in other gramineous crops. We identified Low Silicon Rice 2 (Lsi2)-like Si efflux transporters from two important gramineous crops: maize (Zea mays) and barley (Hordeum vulgare). Both maize and barley Lsi2 expressed in Xenopus laevis oocytes showed Si efflux transport activity. Furthermore, barley Lsi2 was able to recover Si uptake in a rice mutant defective in Si efflux. Maize and barley Lsi2 were only expressed in the roots. Expression of maize and barley Lsi2 was downregulated in response to exogenously applied Si. Moreover, there was a significant positive correlation between the ability of roots to absorb Si and the expression levels of Lsi2 in eight barley cultivars, suggesting that Lsi2 is a key Si transporter in barley. Immunostaining showed that maize and barley Lsi2 localized only at the endodermis, with no polarity. Protein gel blot analysis indicated that maize and barley Lsi2 localized on the plasma membrane. The unique features of maize and barley Si influx and efflux transporters, including their cell-type specificity and the lack of polarity of their localization in Lsi2, indicate that these crops have a different Si uptake system from that in rice. PMID:19574435

  4. The effects of the putative potassium channel activator WAY-120,491 on 86Rb efflux from the rabbit aorta

    SciTech Connect

    Lodge, N.J.; Cohen, R.B.; Havens, C.N.; Colatsky, T.J. )

    1991-02-01

    WAY-120,491 ((-)-(3S-trans)-2-(3,4-dihydro-3-hydroxy-2,2-dimethyl-6-(trifluoromet hox y)- 2H-1-benzopyran-4-yl)-2,3-dihydro-1H-isoindol-1-one) is a novel antihypertensive agent. We have investigated the effects of this compound on contractile force and 86Rb efflux, using the rabbit aorta, in order to assess its K channel activator properties. K channel blockers and ionic conditions thought to modulate specific K channel types have been used to provide insight into the K channel(s) affected by this compound. WAY-120,491 evoked relaxation of precontracted rabbit aortic rings and increased the rate of 86Rb efflux from strips of rabbit aorta; both effects occurring in a concentration-dependent manner. The WAY-120,491 (1 microM)-induced 86Rb efflux was inhibited by tetraethylammonium (IC50 = 0.38 mM), indicating that the increased efflux was mediated by K channels. Glyburide completely blocked the WAY-120,491 (1 microM)-evoked 86Rb efflux with 50% block occurring at a concentration of 0.48 microM. Glyburide also antagonized the WAY-120,491-induced relaxation of aortic rings. Omission of Ca from the solution bathing the aorta did not inhibit the WAY-120,491 induced 86Rb efflux but rather caused an augmentation of the response. It is concluded that WAY-120,491 may be classified as a K channel opener. Furthermore, the K channel upon which WAY-120,491 acts exhibits some characteristics normally associated with the ATP regulated K channel although the involvement of other K channel types has not been ruled out.

  5. Intra-annual dynamics of stem CO2 efflux in relation to cambial activity and xylem development in Pinus cembra

    PubMed Central

    GRUBER, A.; WIESER, G.; OBERHUBER, W.

    2010-01-01

    Summary The relationship between stem CO2 efflux (ES), cambial activity and xylem production in Pinus cembra was determined at the timberline (1950 m a.s.l.) of the Central Austrian Alps, throughout one year. ES was measured continuously from June 2006 to August 2007 using an infrared gas-analysis system. Cambial activity and xylem production was determined by repeated microcore sampling of the developing tree ring and radial increment was monitored using automated point dendrometers. Aside of temperature, the number of living tracheids and cambial cells was predominantly responsible for ES: ES normalized to 10°C (ES10) was significantly correlated to number of living cells throughout the year (r2 = 0,574; p < 0,001). However, elevated ES and missing correlation between ES10 and xylem production was detected during cambial reactivation in April and during transition from active phase to rest, which occurred in August and lasted until early September. Results of this study indicate that (i) during seasonal variations in cambial activity non-linearity between ES and xylem production occurs and (ii) elevated metabolic activity during transition stages in the cambial activity-dormancy cycle influence the carbon budget of Pinus cembra. Daily radial stem increment was primarily influenced by the number of enlarging cells and was not correlated to ES. PMID:19203979

  6. Bioluminescent imaging of ABCG2 efflux activity at the blood-placenta barrier.

    PubMed

    Kumar, Jeyan S; Wei, Bih-Rong; Madigan, James P; Simpson, R Mark; Hall, Matthew D; Gottesman, Michael M

    2016-01-01

    Physiologic barriers such as the blood placenta barrier (BPB) and the blood brain barrier protect the underlying parenchyma from pathogens and toxins. ATP-binding cassette (ABC) transporters are transmembrane proteins found at these barriers, and function to efflux xenobiotics and maintain chemical homeostasis. Despite the plethora of ex vivo and in vitro data showing the function and expression of ABC transporters, no imaging modality exists to study ABC transporter activity in vivo at the BPB. In the present study, we show that in vitro models of the placenta possess ABCG2 activity and can specifically transport D-luciferin, the endogenous substrate of firefly luciferase. To test ABCG2 transport activity at the BPB, we devised a breeding strategy to generate a bioluminescent pregnant mouse model to demonstrate transporter function in vivo. We found that coadministering the ABCG2 inhibitors Ko143 and gefitinib with D-luciferin increased bioluminescent signal from fetuses and placentae, whereas the control P-gp inhibitor DCPQ had no effect. We believe that our bioluminescent pregnant mouse model will facilitate greater understanding of the BPB and ABCG2 activity in health and disease. PMID:26853103

  7. Bioluminescent imaging of ABCG2 efflux activity at the blood-placenta barrier

    PubMed Central

    Kumar, Jeyan S.; Wei, Bih-Rong; Madigan, James P.; Simpson, R. Mark; Hall, Matthew D.; Gottesman, Michael M.

    2016-01-01

    Physiologic barriers such as the blood placenta barrier (BPB) and the blood brain barrier protect the underlying parenchyma from pathogens and toxins. ATP-binding cassette (ABC) transporters are transmembrane proteins found at these barriers, and function to efflux xenobiotics and maintain chemical homeostasis. Despite the plethora of ex vivo and in vitro data showing the function and expression of ABC transporters, no imaging modality exists to study ABC transporter activity in vivo at the BPB. In the present study, we show that in vitro models of the placenta possess ABCG2 activity and can specifically transport D-luciferin, the endogenous substrate of firefly luciferase. To test ABCG2 transport activity at the BPB, we devised a breeding strategy to generate a bioluminescent pregnant mouse model to demonstrate transporter function in vivo. We found that coadministering the ABCG2 inhibitors Ko143 and gefitinib with D-luciferin increased bioluminescent signal from fetuses and placentae, whereas the control P-gp inhibitor DCPQ had no effect. We believe that our bioluminescent pregnant mouse model will facilitate greater understanding of the BPB and ABCG2 activity in health and disease. PMID:26853103

  8. Multidrug efflux transporter activity in sea urchin embryos:Does localization provide a diffusive advantage?

    NASA Astrophysics Data System (ADS)

    Song, Xianfeng; Setayeshgar, Sima; Cole, Bryan; Hamdoun, Amro; Epel, David

    2008-03-01

    Experiments have shown upregulation of multidrug efflux transporter activity approximately 30 min after fertilization in the sea urchin embryo [1]. These ATP-hydrolyzing transporter proteins pump moderately hydrophobic molecules out of the cell and represent the cell's first line of defense againstexogenous toxins. It has also been shown that transporters are moved in vesicles along microfilaments and localized to tips of microvilli prior to activation. We have constructed a geometrically realistic model of the embryo, including microvilli, to explore the functional role of this localization in the efficient elimination of toxins from the standpoint of diffusion. We compute diffusion of toxins in extracellular, membrane and intracellular spaces coupled with transporter activity, using experimentally derived values for physical parameters. For transporters uniformly distributed along microvilli and tip-localized transporters we compare regions in parameter space where each distribution provides diffusive advantage, and comment on the physically expected conditions. [1] A. M. Hamdoun, G. N. Cherr, T. A. Roepke and D. Epel, Developmental Biology 276 452 (2004).

  9. Development of a novel antimicrobial screening system targeting the pyoverdine-mediated iron acquisition system and xenobiotic efflux pumps.

    PubMed

    Sato, Kazuki; Ushioda, Kenichi; Akiba, Keiji; Matsumoto, Yoshimi; Maseda, Hideaki; Ando, Tasuke; Isogai, Emiko; Nakae, Taiji; Yoneyama, Hiroshi

    2015-01-01

    The iron acquisition systems in Pseudomonas aeruginosa are inducible in response to low-iron conditions and important for growth of this organism under iron limitation. OprM is the essential outer membrane subunit of the MexAB-OprM xenobiotic efflux pump. We designed and constructed a new model antimicrobial screening system targeting both the iron-uptake system and xenobiotic efflux pumps. The oprM gene was placed immediately downstream of the ferri-pyoverdine receptor gene, fpvA, in the host lacking chromosomal oprM and the expression of oprM was monitored by an antibiotic susceptibility test under iron depleted and replete conditions. The recombinant cells showed wild-type susceptibility to pump substrate antibiotics, e.g., aztreonam, under iron limitation and became supersusceptible to them under iron repletion, suggesting that expression of oprM is under control of the iron acquisition system. Upon screening of a chemical library comprising 2952 compounds using this strain, a compound-ethyl 2-(1-acetylpiperidine-4-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate-was found to enhance the efficacy of aztreonam under iron limitation, suggesting that the compound inhibits either the iron acquisition system or the MexAB-OprM efflux pump. This compound was subsequently found to inhibit the growth of wild-type cells in the presence of sublethal amounts of aztreonam, regardless of the presence or absence of dipyridyl, an iron-chelator. The compound was eventually identified to block the function of the MexAB-OprM efflux pump, showing the validity of this new method. PMID:25939068

  10. 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

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

    PubMed

    Machado, Diana; Pires, David; 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

  12. Rapid activation of catalase followed by citrate efflux effectively improves aluminum tolerance in the roots of chick pea (Cicer arietinum).

    PubMed

    Sharma, Manorma; Sharma, Vinay; Tripathi, Bhumi Nath

    2016-05-01

    The present study demonstrates the comparative response of two contrasting genotypes (aluminum (Al) tolerant and Al sensitive) of chick pea (Cicer arietinum) against Al stress. The Al-tolerant genotype (RSG 974) showed lesser inhibition of root growth as well as lower oxidative damages, measured in terms of the accumulation of H2O2 and lipid peroxidation compared to the Al-sensitive genotype (RSG 945). The accumulation of Al by roots of both genotypes was almost equal at 96 and 144 h after Al treatment; however, it was higher in Al-tolerant than Al-sensitive genotype at 48 h after Al treatment. Further, the Al-mediated induction of superoxide dismutase (SOD) activity was significantly higher in Al-tolerant than Al-sensitive genotype. Ascorbate peroxidase (APX) activity was almost similar in both genotypes. Al treatment promptly activated catalase activity in Al-tolerant genotype, and it was remarkably higher than that of Al-sensitive genotype. As another important Al detoxification mechanism, citrate efflux was almost equal in both genotypes except at 1000 μM Al treatment for 96 and 144 h. Further, citrate carrier and anion channel inhibitor experiment confirmed the contribution of citrate efflux in conferring Al tolerance in Al-tolerant genotype. Based on the available data, the present study concludes that rapid activation of catalase (also SOD) activity followed by citrate efflux effectively improves Al tolerance in chick pea. PMID:26615604

  13. 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

  14. Both STAT3 activation and cholesterol efflux contribute to the anti-inflammatory effect of apoA-I/ABCA1 interaction in macrophages.

    PubMed

    Tang, Chongren; Houston, Barbara A; Storey, Carl; LeBoeuf, Renee C

    2016-05-01

    ABCA1 exports excess cholesterol from cells to apoA-I and is essential for HDL synthesis. Genetic studies have shown that ABCA1 protects against cardiovascular disease. We have previously shown that the interaction of apoA-I with ABCA1 activates signaling molecule Janus kinase 2 (JAK2), which optimizes the cholesterol efflux activity of ABCA1. ABCA1-mediated activation of JAK2 also activates signal transducer and activator of transcription 3 (STAT3), which significantly attenuates proinflammatory cytokine expression in macrophages. To determine the mechanisms of the anti-inflammatory effects of apoA-I/ABCA1 interaction, we identified two special ABCA1 mutants, one with normal STAT3-activating capacity but lacking cholesterol efflux ability and the other with normal cholesterol efflux ability but lacking STAT3-activating capacity. We showed that activation of STAT3 by the interaction of apoA-I/ABCA1 without cholesterol efflux could significantly decrease proinflammatory cytokine expression in macrophages. Mechanistic studies showed that the anti-inflammatory effect of the apoA-I/ABCA1/STAT3 pathway is suppressor of cytokine signaling 3 dependent. Moreover, we showed that apoA-I/ABCA1-mediated cholesterol efflux without STAT3 activation can also reduce proinflammatory cytokine expression in macrophages. These findings suggest that the interaction of apoA-I/ABCA1 activates cholesterol efflux and STAT3 branch pathways to synergistically suppress inflammation in macrophages. PMID:26989082

  15. Motesanib (AMG706), a potent multikinase inhibitor, antagonizes multidrug resistance by inhibiting the efflux activity of the ABCB1

    PubMed Central

    Wang, Yi-Jun; Kathawala, Rishil J.; Zhang, Yun-Kai; Patel, Atish; Kumar, Priyank; Shukla, Suneet; Fung, King Leung; Ambudkar, Suresh V.; Talele, Tanaji T.; Chen, Zhe-Sheng

    2014-01-01

    Cancer cells often become resistant to chemotherapy through a phenomenon known as multidrug resistance (MDR). Several factors are responsible for the development of MDR, preeminent among them being the accelerated drug efflux mediated by overexpression of ATP binding cassette (ABC) transporters. Some small molecule tyrosine kinase inhibitors (TKIs) were recently reported to modulate the activity of ABC transporters. Therefore, the purpose of this study was to determine if motesanib, a multikinase inhibitor, could reverse ABCB1-mediated MDR. The results showed that motesanib significantly sensitized both ABCB1-transfected and drug-selected cell lines overexpressing this transporter to its substrate anticancer drugs. Motesanib significantly increased the accumulation of [3H]-paclitaxel in ABCB1 overexpressing cells by blocking the efflux function of ABCB1 transporter. In contrast, no significant change in the expression levels and localization pattern of ABCB1 was observed when ABCB1 overexpressing cells were exposed to 3 µM motesanib for 72 h. Moreover, motesanib stimulated the ATPase activity of ABCB1 in a concentration-dependent manner, indicating a direct interaction with the transporter. Consistent with these findings, the docking studies indicated favorable binding of motesanib within the transmembrane region of homology modeled human ABCB1. Here, we report for the first time, motesanib, at clinically achievable plasma concentrations, antagonizes MDR by inhibiting the efflux activity of the ABCB1 transporter. These findings may be useful for cancer combination therapy with TKIs in the clinic. PMID:24937702

  16. Motesanib (AMG706), a potent multikinase inhibitor, antagonizes multidrug resistance by inhibiting the efflux activity of the ABCB1.

    PubMed

    Wang, Yi-Jun; Kathawala, Rishil J; Zhang, Yun-Kai; Patel, Atish; Kumar, Priyank; Shukla, Suneet; Fung, King Leung; Ambudkar, Suresh V; Talele, Tanaji T; Chen, Zhe-Sheng

    2014-08-15

    Cancer cells often become resistant to chemotherapy through a phenomenon known as multidrug resistance (MDR). Several factors are responsible for the development of MDR, preeminent among them being the accelerated drug efflux mediated by overexpression of ATP binding cassette (ABC) transporters. Some small molecule tyrosine kinase inhibitors (TKIs) were recently reported to modulate the activity of ABC transporters. Therefore, the purpose of this study was to determine if motesanib, a multikinase inhibitor, could reverse ABCB1-mediated MDR. The results showed that motesanib significantly sensitized both ABCB1-transfected and drug-selected cell lines overexpressing this transporter to its substrate anticancer drugs. Motesanib significantly increased the accumulation of [(3)H]-paclitaxel in ABCB1 overexpressing cells by blocking the efflux function of ABCB1 transporter. In contrast, no significant change in the expression levels and localization pattern of ABCB1 was observed when ABCB1 overexpressing cells were exposed to 3μM motesanib for 72h. Moreover, motesanib stimulated the ATPase activity of ABCB1 in a concentration-dependent manner, indicating a direct interaction with the transporter. Consistent with these findings, the docking studies indicated favorable binding of motesanib within the transmembrane region of homology modeled human ABCB1. Here, we report for the first time, motesanib, at clinically achievable plasma concentrations, antagonizes MDR by inhibiting the efflux activity of the ABCB1 transporter. These findings may be useful for cancer combination therapy with TKIs in the clinic. PMID:24937702

  17. 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

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

    PubMed Central

    Lau, Calvin Ho-Fung; Gilmour, Christie; Hao, Youai; Lam, Joseph S.

    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

  19. Brain distribution of 6-mercaptopurine is regulated by the efflux transport system in the blood-brain barrier.

    PubMed

    Deguchi, Y; Yokoyama, Y; Sakamoto, T; Hayashi, H; Naito, T; Yamada, S; Kimura, R

    2000-01-01

    6-mercaptopurine (6-MP) has been used clinically for 40 years to maintain remission in patients with acute lymphoblastic leukemia (ALL). However, central nervous system (CNS) relapses frequently occur in patients with ALL who continuously receive anticancer drugs, including 6-MP, during remission maintenance therapy. The cause of such CNS relapse is not well understood. One possible reason may involve the restricted distribution of 6-MP in the brain. This study, therefore, investigates the blood-brain barrier (BBB) transport which largely regulates 6-MP distribution in the brain using a quantitative microdialysis technique and centers on the efflux transport of 6-MP across the BBB. The brain tissue, cerebrospinal fluid (CSF), or hippocampal interstitial fluid (ISF) concentration of 6-MP was very low compared with the unbound plasma concentration, suggesting that 6-MP distribution in the brain is highly restricted. Kinetic analyses of this BBB transport showed that the efflux clearance from brain ISF to plasma across the BBB (CLout) is approximately 20-times greater than the influx clearance from plasma to brain (CLin). The CLout was significantly reduced by 1mM N-ethylmaleimide (NEM), a sulfhydryl-modifying agent, suggesting the participation of transport protein in the efflux of 6-MP across the BBB. In addition, efflux transport was inhibited by an intracerebral infusion of probenecid (1.5 mM), p-aminohippuric acid (PAH, 3.0 mM), benzoate (3.6 mM), or salicylate (3.7 mM) administered through a microdialysis probe, but neither choline (0.8 mM) nor tetraethylammonium (TEA, 0.7 mM) had any effect. These data suggest that the restricted 6-MP brain distribution may be ascribed to efficient efflux from the brain, possibly via both the organic anion transport system, shared with probenecid and PAH, and the monocarboxylic acid transport system, shared with benzoate and salicylate. PMID:10794520

  20. Co-treatment with grapefruit juice inhibits while chronic administration activates intestinal P-glycoprotein-mediated drug efflux.

    PubMed

    Panchagnula, R; Bansal, T; Varma, M V S; Kaul, C L

    2005-12-01

    P-Glycoprotein (P-gp) mediated efflux is recognized as a significant biochemical barrier affecting oral absorption for a number of drugs. Various conflicting reports have been published regarding the effects of grapefruit juice (GFJ) on P-gp-mediated drug efflux, in which GFJ has been shown both to inhibit and activate it. Hence, the present study adopted a two-way approach, involving both co-treatment and chronic administration. Bi-directional transport of paclitaxel (PCL) was carried out in the absence and presence of GFJ extract, in rat everted ileum sac. Further, the effect of chronic administration of GFJ to rats was characterized by permeability studies with indinavir (INDI). Co-treatment of GFJ extract at 100% concentration reduced the asymmetric transport of PCL (efflux ratio = 20.8) by increasing absorptive (A --> B) transport by 921% and reducing secretory (B --> A) transport by 41%. Further, GFJ showed a concentration dependent effect on PCL permeability. Imipramine, a passive permeability marker with absorptive permeability of 15.33 +/- 4.26 x 10(-6) cm/s showed no asymmetric transport and also no significant (P < 0.05) change in permeability in the presence of GFJ. Chronic administration of GFJ resulted in a significant decrease in absorptive transport of indinavir, which was even greater than that produced by rifampicin pretreatment. No change in permeability of propranolol, a passive permeability marker, was observed. Further, the decrease in absorptive transport of INDI was reversed by the P-gp inhibitor verapamil. In conclusion, GFJ extract inhibited P-gp-mediated efflux in co-treatment, whereas chronic administration led to increased levels of P-gp expression, thus having a profound effect on intestinal absorption and GFJ-drug interactions in vivo. PMID:16398269

  1. The "racemic approach" in the evaluation of the enantiomeric NorA efflux pump inhibition activity of 2-phenylquinoline derivatives.

    PubMed

    Carotti, Andrea; Ianni, Federica; Sabatini, Stefano; Di Michele, Alessandro; Sardella, Roccaldo; Kaatz, Glenn W; Lindner, Wolfgang; Cecchetti, Violetta; Natalini, Benedetto

    2016-09-10

    Among the mechanisms adopted by bacteria, efflux pumps (EPs) have been recognized as being significantly involved in contributing to resistance to commonly used antibacterial agents. However, little is known about their three-dimensional structures or the steric requirements for their inhibition. Lack of such knowledge includes NorA, one of the most studied Staphylococcus aureus EPs. In the present study, the use of two commercialized Cinchona alkaloid-based zwitterionic chiral stationary phases allowed the enantioseparation of four 2-((2-(4-propoxyphenyl)quinolin-4-yl)oxy)alkylamines 1-4 previously found to be potent S. aureus NorA efflux pump inhibitors when tested as racemates. In the identified optimal polar-ionic conditions (MeOH/THF/H2O-49/49/2 (v/v/v)+25mM formic acid, 12.5mM diethylamine), repeated consecutive injections of 1 allowed the isolation of sufficient amount of its enantiomers (2.6mg and 2.8mg, for (R)-1 and (S)-1, respectively) and then to evaluate their ability to inhibit the S. aureus NorA efflux pump. The biological evaluation highlighted the main contribution of the (R)-1 enantiomer to both the EtBr efflux inhibition and synergistic effect with against SA-1199B (norA+/A116E GrlA) respect to the racemate activity. The comparison between the experimental electronic circular dichroism and the time-dependent density functional theory calculations spectra of the two isolated enantiomeric fractions allowed for all compounds a clear and easy assignment of the enantiomeric elution order. PMID:27429367

  2. The Peptide Toxin Amylosin of Bacillus amyloliquefaciens from Moisture-Damaged Buildings Is Immunotoxic, Induces Potassium Efflux from Mammalian Cells, and Has Antimicrobial Activity

    PubMed Central

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

    2015-01-01

    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. PMID:25681192

  3. Bacterial multidrug efflux transporters.

    PubMed

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

    2014-01-01

    Infections caused by bacteria are a leading cause of death worldwide. Although antibiotics remain a key clinical therapy, their effectiveness has been severely compromised by the development of drug resistance in bacterial pathogens. Multidrug efflux transporters--a common and powerful resistance mechanism--are capable of extruding a number of structurally unrelated antimicrobials from the bacterial cell, including antibiotics and toxic heavy metal ions, facilitating their survival in noxious environments. Transporters of the resistance-nodulation-cell division (RND) superfamily typically assemble as tripartite efflux complexes spanning the inner and outer membranes of the cell envelope. In Escherichia coli, the CusCFBA complex, which mediates resistance to copper(I) and silver(I) ions, is the only known RND transporter specific to heavy metals. Here, we describe the current knowledge of individual pump components of the Cus system, a paradigm for efflux machinery, and speculate on how RND pumps assemble to fight diverse antimicrobials. PMID:24702006

  4. Exploring the HME and HAE1 efflux systems in the genus Burkholderia

    PubMed Central

    2010-01-01

    Background The genus Burkholderia includes a variety of species with opportunistic human pathogenic strains, whose increasing global resistance to antibiotics has become a public health problem. In this context a major role could be played by multidrug efflux pumps belonging to Resistance Nodulation Cell-Division (RND) family, which allow bacterial cells to extrude a wide range of different substrates, including antibiotics. This study aims to i) identify rnd genes in the 21 available completely sequenced Burkholderia genomes, ii) analyze their phylogenetic distribution, iii) define the putative function(s) that RND proteins perform within the Burkholderia genus and iv) try tracing the evolutionary history of some of these genes in Burkholderia. Results BLAST analysis of the 21 Burkholderia sequenced genomes, using experimentally characterized ceoB sequence (one of the RND family counterpart in the genus Burkholderia) as probe, allowed the assembly of a dataset comprising 254 putative RND proteins. An extensive phylogenetic analysis revealed the occurrence of several independent events of gene loss and duplication across the different lineages of the genus Burkholderia, leading to notable differences in the number of paralogs between different genomes. A putative substrate [antibiotics (HAE1 proteins)/heavy-metal (HME proteins)] was also assigned to the majority of these proteins. No correlation was found between the ecological niche and the lifestyle of Burkholderia strains and the number/type of efflux pumps they possessed, while a relation can be found with genome size and taxonomy. Remarkably, we observed that only HAE1 proteins are mainly responsible for the different number of proteins observed in strains of the same species. Data concerning both the distribution and the phylogenetic analysis of the HAE1 and HME in the Burkholderia genus allowed depicting a likely evolutionary model accounting for the evolution and spreading of HME and HAE1 systems in the

  5. 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

  6. 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

  7. Efflux pump activity in fluoroquinolone and tetracycline resistant Salmonella and E. coli implicated in reduced susceptibility to household antimicrobial cleaning agents.

    PubMed

    Thorrold, C A; Letsoalo, M E; Dusé, A G; Marais, E

    2007-02-15

    It has been shown that the inappropriate use of antimicrobial household agents selects for organisms with resistance mechanisms (e.g. efflux pumps), which could lead to the development of antibiotic resistance. The reverse hypothesis, that antibiotic-resistant organisms become tolerant to other antibacterial agents (e.g. disinfectants) due to the action of efflux pumps, has however not been extensively examined. The objective of this study was to establish whether there is a link between antibiotic resistance in potential gastrointestinal pathogens and reduced sensitivity of these organisms to commonly used household antimicrobial agents. In this study, tetracycline and ofloxacin sensitive and resistant Escherichia coli (9 strains) and Salmonella spp. (8 strains) were isolated from poultry and clinical samples. In order to assess whether these bacteria had active efflux pumps, ethidium bromide accumulation assays were performed. Extrusion of the active components of three commercial household agents (triclosan, sodium salicylate, and ortho-phenylphenol) by efflux pumps was tested using spectrophotometric accumulation assays. In order to simulate the kitchen environment, in-use disinfectant testing using the commercial household agents was performed to determine changes in their efficacy due to antibiotic resistance. Active efflux pump activity and extrusion of all three active ingredients was observed only in the antibiotic resistant organisms. The antibiotic sensitive bacteria were also more susceptible than the resistant isolates to the household antimicrobial agents at concentrations below that recommended by the manufacturer. These resistant bacteria could potentially be selected for and result in hard to treat infections. PMID:17126442

  8. Comparison of models for determining soil-surface carbon dioxide effluxes in different agricultural systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil-surface CO2 efflux (SCE) models are appealing due to expense and labor of fine temporal- and spatial-resolution field measurements. However, several simple SCE models are reported in the literature. Our objective was to compare and validate selected soil temperature (Ts)- and water content ('v)...

  9. 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

  10. Kinetic analysis of receptor-controlled tracer efflux from sealed membrane fragments

    PubMed Central

    Bernhardt, Julius; Neumann, Eberhard

    1978-01-01

    A detailed kinetic analysis is presented for activator-receptor-mediated efflux of tracer substances from vesicular membrane systems in general and from sealed fragments of excitable membranes in particular. Rate constants and amplitudes, as the primary measurable quantities of the efflux kinetics, are expressed in terms of fundamental properties of vesicular membrane systems containing receptors of chemical gating systems. The experimental determination and theoretical analysis of single contributions to a complex receptor-controlled efflux has been treated for the acetylcholine receptor system; also the effect of “pharmacological densensitization” on efflux is explicitly formulated. The dependence of the measured efflux parameters on the concentration of activators can be used to derive the kinetic and thermodynamic constants for receptor activation and inactivation processes; a general kinetic scheme and two limiting cases are analyzed. The efflux of 22Na from “excitable microsacs” of Torpedo marmorata is treated as an example, and the power of the rigorous analytical method is demonstrated. In particular, the analysis of efflux amplitudes from only a few data points offers an alternative to the longer lasting measurements for obtaining efflux curves when a safety factor is involved, as in the case of tracer ions like 22Na. PMID:16592553

  11. Use of a combined effect model approach for discriminating between ABCB1- and ABCC1-type efflux activities in native bivalve gill tissue.

    PubMed

    Faria, Melissa; Pavlichenko, Vasiliy; Burkhardt-Medicke, Kathleen; Soares, Amadeu M V M; Altenburger, Rolf; Barata, Carlos; Luckenbach, Till

    2016-04-15

    Aquatic organisms, such as bivalves, employ ATP binding cassette (ABC) transporters for efflux of potentially toxic chemicals. Anthropogenic water contaminants can, as chemosensitizers, disrupt efflux transporter function enabling other, putatively toxic compounds to enter the organism. Applying rapid amplification of cDNA ends (RACE) PCR we identified complete cDNAs encoding ABCB1- and ABCC1-type transporter homologs from zebra mussel providing the molecular basis for expression of both transporter types in zebra mussel gills. Further, efflux activities of both transporter types in gills were indicated with dye accumulation assays where efflux of the dye calcein-am was sensitive to both ABCB1- (reversin 205, verapamil) and ABCC1- (MK571) type specific inhibitors. The assumption that different inhibitors targeted different efflux pump types was confirmed when comparing measured effects of binary inhibitor compound mixtures in dye accumulation assays with predictions from mixture effect models. Effects by the MK571/reversin 205 mixture corresponded better with independent action, whereas reversin 205/verapamil joint effects were better predicted by the concentration addition model indicating different and equal targets, respectively. The binary mixture approach was further applied to identify the efflux pump type targeted by environmentally relevant chemosensitizing compounds. Pentachlorophenol and musk ketone, which were selected after a pre-screen of twelve compounds that previously had been identified as chemosensitizers, showed mixture effects that corresponded better with concentration addition when combined with reversine 205 but with independent action predictions when combined with MK571 indicating targeting of an ABCB1-type efflux pump by these compounds. PMID:26929997

  12. Association of hp1181 and hp1184 Genes With the Active Efflux Phenotype in Multidrug-Resistant Isolates of Helicobacter pylori

    PubMed Central

    Falsafi, Tahereh; Ehsani, Azadeh; Attaran, Bahareh; Niknam, Vahid

    2016-01-01

    Background During the last decades the rate of multidrug resistance among clinical Helicobacter pylori isolates has increased. Active pumping out of the drugs may be an important mechanism for multidrug resistance in H. pylori strains. Objectives The aim of this study was to evaluate the association of two H. pylori efflux-genes, hp1181 and hp1184 with the active-efflux phenotype in MDR clinical-strains of H. pylori. Materials and Methods Minimal inhibitory concentration (MIC) and drug accumulation for β-lactames, Tetracycline (TET), Erythromycin (ERY), Metronidazole (MTZ), Ciprofloxacin (CIP) and Ethidium Bromide (EtBr) was performed in the presence and absence of carbonyl cyanide M-Chlorophenyl Hydrazone (CCCP). Presence of hp1181 and hp1184 genes was detected by the polymerase chain reaction (PCR). RT-PCR was performed to compare expression of efflux genes by MDR strains, demonstrating active efflux with the strains without active efflux. Results Two- to four-fold decrease in minimum inhibitory concentration (MIC) and two-fold increase in accumulation were observed for EtBr in the presence of CCCP for 67% (8) of 12 MDR strains. With CCCP, two- to four-fold decrease in MIC and 1.4- to 1.8-fold increase in the accumulation of β-lactames, TET, CIP and MTZ were obtained for 42% (5) of the MDR strains. Six, five and three of the 12 MDR strains amplified hp1184, hp1181, and both of them, respectively. The RT-PCR product for expression of hp1181 by MDR strains was approximately 100 bp shorter than that of the 26695 susceptible standard strain. Conclusions Expression of the genes hp1184 and hp1181 are associated with the specific active efflux of EtBr and non-related antibiotics, respectively. For displaying these phenotypes, a post-transcriptional regulation step may be required. PMID:27303615

  13. Impairment of blood-cerebrospinal fluid barrier properties by retrovirus-activated T lymphocytes: reduction in cerebrospinal fluid-to-blood efflux of prostaglandin E2.

    PubMed

    Khuth, Seng Thuon; Strazielle, Nathalie; Giraudon, Pascale; Belin, Marie-Françoise; Ghersi-Egea, Jean-François

    2005-09-01

    The choroid plexus epithelium forms the interface between the blood and the CSF. In conjunction with the tight junctions restricting the paracellular pathway, polarized specific transport systems in the choroidal epithelium allow a fine regulation of CSF-borne biologically active mediators. The highly vascularized stroma delimited by the choroidal epithelium can be a reservoir for retrovirus-infected or activated immune cells. In this work, new insight in the implication of the blood-CSF barrier in neuroinfectious and inflammatory diseases is provided by using a differentiated cellular model of the choroidal epithelium, exposed to infected T lymphocytes. We demonstrate that T cells activated by a retroviral infection, but not non-infected cells, reduce the transporter-mediated CSF-to-blood efflux of organic anions, in particular that of the potent pro-inflammatory prostaglandin PGE2, via the release of soluble factors. A moderate alteration of the paracellular permeability also occurs. We identified the viral protein Tax, oxygenated free radicals, matrix-metalloproteinases and pro-inflammatory cytokines as active molecules released during the exposure of the epithelium to infected T cells. Among them, tumour necrosis factor and interleukin 1 are directly involved in the mechanism underlying the decrease in some choroidal organic anion efflux. Given the strong involvement of CSF-borne PGE2 in sickness behaviour syndrome, these data suggest that the blood-CSF barrier plays an important role in the pathophysiology of neuroinflammation and neuroinfection, via changes in the transport processes controlling the CSF biodisposition of PGE2. PMID:16026393

  14. Antitumor activity of 7-aminocarboxycoumarin derivatives, a new class of potent inhibitors of lactate influx but not efflux.

    PubMed

    Draoui, Nihed; Schicke, Olivier; Seront, Emmanuel; Bouzin, Caroline; Sonveaux, Pierre; Riant, Olivier; Feron, Olivier

    2014-06-01

    High lactate concentration in tumors is associated with bad prognosis. Lactate is released by glycolytic cells in tumors and recaptured by oxidative cancer cells to feed the tricarboxylic acid (TCA) cycle after conversion into pyruvate. Monocarboxylate transporters (MCT) mediate these fluxes of proton-linked lactate and represent attractive targets to interrupt lactate shuttle and to inhibit tumor growth. Here, we investigated the properties of 7-aminocarboxycoumarins (7ACC) developed to selectively interfere with lactate fluxes in the lactate-rich tumor microenvironment. The pharmacologic properties of two compounds of this family, including their effects on lactate influx and efflux and antitumor activity, were investigated using human cancer cell lines and mouse xenograft models. Contrary to the reference MCT1 inhibitor AR-C155858, 7ACC unexpectedly inhibited lactate influx but not efflux in tumor cells expressing MCT1 and MCT4 transporters. 7ACC delayed the growth of cervix SiHa tumors, colorectal HCT116 tumors, and orthoptopic MCF-7 breast tumors. MCT target engagement was confirmed by the lack of activity of 7ACC on bladder UM-UC-3 carcinoma that does not express functional MCT. 7ACC also inhibited SiHa tumor relapse after treatment with cisplatin. Finally, we found that contrary to AR-C155858, 7ACC did not prevent the cell entry of the substrate-mimetic drug 3-bromopyruvate (3BP) through MCT1, and contributed to the inhibition of tumor relapse after 3BP treatment. In conclusion, our results indicate that 7ACC selectively affects a single part of the MCT symporter translocation cycle, leading to strict inhibition of lactate influx. This singular activity is associated with antitumor effects less prone to resistance and side effects. PMID:24672058

  15. 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.

  16. 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. PMID:22167946

  17. 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. PMID:25697558

  18. Dissociation between the induction of potassium efflux and cytostatic activity of polyene macrolides in mammalian cells.

    PubMed Central

    Malewicz, B; Jenkin, H M; Borowski, E

    1980-01-01

    The paper contains data on the induction of K+ efflux and viability of baby hamster kidney (BHK-21) cells after their treatment with macrolide antibiotics inducing specific pores in membrane. New water-soluble semisynthetic derivatives of amphotericin B and aureofacin (N-glycosyl and trimethylammonium methyl ester derivatives) as well as the parent compounds were used to compare the concentration of antibiotics inducing permeabilizing and cytostatic effects. We found that a two- to eight-times-higher concentration of polyene antibiotic was required to observe a cytostatic effect than for release of 50% of the cellular potassium (K50 concentration) from BHK-21 cells. These differences were larger for water-soluble derivatives than for the parent compounds. The amount of intracellular potassium in treated cells incubated under optimal growth conditions was higher than in cells which had been further washed with K+-free maintenance medium. The membrane permeability changes induced by low concentrations of specific polyenes were observed to be reversible. BHK-21 cells were able to repair polyene-induced membrane permeability within 3 to 12 h under optimal growth conditions, after cell treatment with K50 concentration of specific macrolide antibiotics. The repair phenomenon is postulated as an explanation for the dissociation observed between permeabilizing and cytostatic effect of specific polyenes in BHK-21 cells. PMID:7396461

  19. Regulation of the MtrC-MtrD-MtrE efflux-pump system modulates the in vivo fitness of Neisseria gonorrhoeae.

    PubMed

    Warner, Douglas M; Folster, Jason P; Shafer, William M; Jerse, Ann E

    2007-12-15

    The Neisseria gonorrhoeae MtrC-MtrD-MtrE multidrug-resistance efflux pump expels macrolide antibiotics, penicillin, and antimicrobial effectors of the innate defense. Mutation of the mtrR locus, which encodes a transcriptional repressor of the mtrCDE operon, increases gonococcal resistance to these agents. Here we report that, in a mouse infection model, an mtrR mutant is more fit than the wild-type bacteria. Consistent with derepression of the mtrCDE operon as the primary reason for the fitness benefit, an mtrR,mtrE double mutant and an mtrE mutant showed no difference in survival phenotype. Gonococcal mutants deficient in MtrA, an activator of the mtrCDE operon, exhibited significantly reduced fitness in vivo, and mtrA mutants with spontaneous compensatory mtrR mutations were selected during infection. These results confirm the importance of the MtrC-MtrD-MtrE efflux-pump system during experimental gonococcal genital-tract infection and also illustrate an antibiotic-resistance mechanism that is accompanied by a fitness benefit rather than a fitness cost. PMID:18190261

  20. Increased Systemic Exposure of Methotrexate by a Polyphenol-Rich Herb via Modulation on Efflux Transporters Multidrug Resistance-Associated Protein 2 and Breast Cancer Resistance Protein.

    PubMed

    Yu, Chung-Ping; Hsieh, Yun-Chung; Shia, Chi-Sheng; Hsu, Pei-Wen; Chen, Jen-Yuan; Hou, Yu-Chi; Hsieh, Yo-Wen

    2016-01-01

    Scutellariae radix (SR, roots of Scutellaria baicalensis Georgi), a popular Chinese medicine, contains plenty of flavonoids such as baicalin, wogonoside, baicalein, and wogonin. Methotrexate (MTX), an important immunosuppressant with a narrow therapeutic index, is a substrate of multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein (BCRP). This study investigated the effect of SR on MTX pharmacokinetics and the underlying mechanisms. Rats were orally administered MTX alone and with 1.0 or 2.0 g/kg of SR. The serum concentrations of MTX were determined by a fluorescence polarization immunoassay. Cell models were used to explore the involvement of MRP2 and BCRP in the interaction. The results showed that 1.0 g/kg of SR significantly increased Cmax, AUC(0-30), AUC(0-2880), and mean residence time (MRT) of MTX by 50%, 45%, 501%, and 347%, respectively, and 2.0 g/kg of SR significantly enhanced the AUC(0-2880) and MRT by 242% and 293%, respectively, but decreased AUC(0-30) by 41%. Cell line studies indicated that SR activated the BCRP-mediated efflux transport, whereas the serum metabolites of SR inhibited both the BCRP- and MRP2-mediated efflux transports. In conclusion, SR ingestion increased the systemic exposure and MRT of MTX via modulation on MRP2 and BCRP. PMID:26852865

  1. In vivo evaluation of anionic thiolated polymers as oral delivery systems for efflux pump inhibition.

    PubMed

    Palmberger, Thomas F; Laffleur, Flavia; Greindl, Melanie; Bernkop-Schnürch, Andreas

    2015-08-01

    Recently, the cationic polymer thiolated chitosan has been reported to modulate drug absorption by inhibition of intestinal efflux pumps. The objective of this study was to evaluate in vitro and in vivo whether thiolated anionic biopolymers also show an efflux pump inhibitory effect in order to improve intestinal transcellular drug uptake. Therefore, three thiomers have been synthesized due covalent attachment of cysteine to various polymer backbones: pectin-cysteine (pect-cys), carboxymethylcellulose-cysteine (CMC-cys) and alginate-cysteine (alg-cys). In vitro, the permeation enhancing properties of these thiomers and their corresponding unmodified polymers have been evaluated on rat small intestine in Ussing-type chambers, using sulforhodamine 101 (SR-101) as MRP2 model substrate. In comparison to buffer only, SR-101 transport in presence of pect-cys, CMC-cys and alg-cys was improved 1.5-fold, 1.8-fold and 3.0-fold, respectively. Due to the comparatively best in vitro performance of thiolated alginate, it has been chosen for in vivo studies: a SR-101 solution containing 4% (w/v) alg-cys led to an AUC0 ≥ 12 of SR-101 of 109 ng ml(-1)h in rats representing a 3.8-fold improvement in comparison to a SR-101 buffer solution. Unmodified alginate improved the AUC0 ≥ 12 of SR-101 by a factor of 1.9. These findings suggest thiolated alginate as promising auxiliary agent for drugs being anionic efflux pump substrates, since the oral bioavailability of a MRP2 substrate could be significantly improved. PMID:26095915

  2. Characterisation of SalRAB a Salicylic Acid Inducible Positively Regulated Efflux System of Rhizobium leguminosarum bv viciae 3841

    PubMed Central

    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. PMID:25133394

  3. Involvement of a Novel Efflux System in Biofilm-Specific Resistance to Antibiotics▿

    PubMed Central

    Zhang, Li; Mah, Thien-Fah

    2008-01-01

    Bacteria growing in biofilms are more resistant to antibiotics than their planktonic counterparts. How this transition occurs is unclear, but it is likely there are multiple mechanisms of resistance that act together in order to provide an increased overall level of resistance to the biofilm. We have identified a novel efflux pump in Pseudomonas aeruginosa that is important for biofilm-specific resistance to a subset of antibiotics. Complete deletion of the genes encoding this pump, PA1874 to PA1877 (PA1874-1877) genes, in an P. aeruginosa PA14 background results in an increase in sensitivity to tobramycin, gentamicin, and ciprofloxacin, specifically when this mutant strain is growing in a biofilm. This efflux pump is more highly expressed in biofilm cells than in planktonic cells, providing an explanation for why these genes are important for biofilm but not planktonic resistance to antibiotics. Furthermore, expression of these genes in planktonic cells increases their resistance to antibiotics. We have previously shown that ndvB is important for biofilm-specific resistance (T. F. Mah, B. Pitts, B. Pellock, G. C. Walker, P. S. Stewart, and G. A. O'Toole, Nature 426:306-310, 2003). Our discovery that combining the ndvB mutation with the PA1874-1877 gene deletion results in a mutant strain that is more sensitive to antibiotics than either single mutant strain suggests that ndvB and PA1874-1877 contribute to two different mechanisms of biofilm-specific resistance to antibiotics. PMID:18469108

  4. 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

  5. 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. PMID:25652088

  6. Conditioning of Roots with Hypoxia Increases Aluminum and Acid Stress Tolerance by Mitigating Activation of K+ Efflux Channels by ROS in Barley: Insights into Cross-Tolerance Mechanisms.

    PubMed

    Ma, Yanling; Zhu, Min; Shabala, Lana; Zhou, Meixue; Shabala, Sergey

    2016-01-01

    Aluminum (Al) is prevalent in soils, but Al toxicity is manifested only under acid conditions. It causes severe damages to the root system. Short-term waterlogging stress can occur simultaneously with Al toxicity in areas with high rainfall or an inappropriate irrigation pattern. Barley (Hordeum vulgare L.) is one of the most Al-sensitive small-grained cereals. In this work, we have investigated effects of short-term treatments with hypoxia and phenolic acid (two major constraints in waterlogged soils) on root sensitivity to low-pH and Al stresses. We showed that hypoxia-primed roots maintained higher cell viability when exposed to low-pH/Al stress, in both elongation and mature root zones, and possessed superior ability to retain K(+) in response to low-pH/Al stresses. These priming effects were not related to higher H(+)-ATPase activity and better membrane potential maintenance, and could not be explained by the increased expression levels of HvHAK1, which mediates high-affinity K(+) uptake in roots. Instead, hypoxia-conditioned roots were significantly less sensitive to H2O2 treatment, indicated by the 10-fold reduction in the magnitude of K(+) efflux changes. This suggested that roots pre-treated with hypoxia desensitized reactive oxygen species (ROS)-inducible K(+) efflux channels in root epidermis, most probably via enhanced antioxidative capacity. A possible role for Ca(2+) in stress-induced ROS signaling pathways is also discussed. Overall, our results report, for the first time, the phenomenon of cross-protection between hypoxia and low-pH/Al stresses, and causally link it to the cell's ability to maintain cytosolic K(+) homeostasis. PMID:26581863

  7. Nrf2 activation ameliorates cytotoxic effects of arsenic trioxide in acute promyelocytic leukemia cells through increased glutathione levels and arsenic efflux from cells.

    PubMed

    Nishimoto, Shoichi; Suzuki, Toshihiro; Koike, Shin; Yuan, Bo; Takagi, Norio; Ogasawara, Yuki

    2016-08-15

    Carnosic acid (CA), a phenolic diterpene isolated from Rosmarinus officinalis, has been shown to activate nuclear transcription factor E2-related factor 2 (Nrf2), which plays a central role in cytoprotective responses to oxidative and electrophilic stress. Recently, the Nrf2-Kelch ECH associating protein 1 (Keap1) pathway has been associated with cancer drug resistance attributable to modulation of the expression and activation of antioxidant and detoxification enzymes. However, the exact mechanisms by which Nrf2 activation results in chemoresistance are insufficiently understood to date. This study investigated the mechanisms by which the cytotoxic effects of arsenic trioxide (ATO), an anticancer drug, were decreased in acute promyelocytic leukemia cells treated with CA, a typical activator of Nrf2 used to stimulate the Nrf2/Keap1 system. Our findings suggest that arsenic is non-enzymatically incorporated into NB4 cells and forms complexes that are dependent on intracellular glutathione (GSH) concentrations. In addition, the arsenic complexes are recognized as substrates by multidrug resistance proteins and subsequently excreted from the cells. Therefore, Nrf2-associated activation of the GSH biosynthetic pathway, followed by increased levels of intracellular GSH, are key mechanisms underlying accelerated arsenic efflux and attenuation of the cytotoxic effects of ATO. PMID:27317373

  8. 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

  9. 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. PMID:18396538

  10. 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

  11. Impairment of the ABCA1 and SR-BI-mediated cholesterol efflux pathways and HDL anti-inflammatory activity in Alzheimer's disease.

    PubMed

    Khalil, Abdelouahed; Berrougui, Hicham; Pawelec, Graham; Fulop, Tamas

    2012-01-01

    The aim of our study was to investigate the effect of Alzheimer's disease (AD) on the cholesterol efflux capacity and anti-inflammatory activity of HDL. HDL and apoA-I were isolated from 20 healthy subjects and from 39 AD patients. Our results showed that serum- and HDL-mediated cholesterol efflux is significantly impaired in AD patients. This impairment of serum and HDL cholesterol efflux capacity was significantly inversely correlated to the AD severity as evaluated by MMSE scores. Results obtained from SR-BI-enriched Fu5AH and ABCA1-enriched J774 cells revealed that AD impaired the interaction of HDL and apoA-I with both the ABCA1 transporter and SR-BI receptor. Purified apoA-I from AD patients also failed to remove free excess cholesterol from ABCA1-enriched J774 macrophages. Interestingly, the decrease in plasma α-tocopherol content and the increase in MDA formation and HDL relative electrophoretic mobility indicated that AD patients had higher levels of oxidative stress. The anti-inflammatory activity of HDL was also significantly lower in AD patients as measured by the level of ICAM-1 expression. In conclusion, our study provides evidence for the first time that the functionality of HDL is impaired in AD and that this alteration might be caused by AD-associated oxidative stress and inflammation. PMID:22178419

  12. Structural Elements in the Transmembrane and Cytoplasmic Domains of the Metal Transporter SLC30A10 Are Required for Its Manganese Efflux Activity.

    PubMed

    Zogzas, Charles E; Aschner, Michael; Mukhopadhyay, Somshuvra

    2016-07-29

    Homozygous mutations in SLC30A10 lead to the development of familial manganese-induced parkinsonism. We previously demonstrated that SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism-causing mutations block its trafficking and efflux activity. Interestingly, other transporters in the SLC30 family mediate zinc efflux. Determining the mechanisms that allow SLC30A10 to transport manganese, which are unclear, is essential to understand its role in parkinsonism. Here, we generated a predicted structure of SLC30A10, based on the structure of the bacterial zinc transporter YiiP, and performed functional studies. In YiiP, side chains of residues Asp-45 and Asp-49 in the second and His-153 and Asp-157 in the fifth transmembrane segments coordinate zinc and are required for transport. In SLC30A10, the corresponding residues are Asn-43 and Asp-47 in the second and His-244 and Asp-248 in the fifth transmembrane segments. Surprisingly, although alanine substitution of Asp-248 abolished manganese efflux, that of Asn-43 and Asp-47 did not. Instead, side chains of charged or polar residues adjacent to Asp-248 in the first (Glu-25) or fourth (Asn-127) transmembrane segments were required. Further analyses revealed that residues His-333 and His-350 in the cytoplasmic C-terminal domain were required for full activity. However, the C-terminal domain failed to transfer manganese transport capability to a related zinc transporter. Overall, our results indicate that residues in the transmembrane and C-terminal domains together confer optimal manganese transport capability to SLC30A10 and suggest that the mechanism of ion coordination in the transmembrane domain of SLC30A10 may be substantially different from that in YiiP/other SLC30 proteins. PMID:27307044

  13. Efflux-Mediated Antifungal Drug Resistance†

    PubMed Central

    Cannon, Richard D.; Lamping, Erwin; Holmes, Ann R.; Niimi, Kyoko; Baret, Philippe V.; Keniya, Mikhail V.; Tanabe, Koichi; Niimi, Masakazu; Goffeau, Andre; Monk, Brian C.

    2009-01-01

    Summary: Fungi cause serious infections in the immunocompromised and debilitated, and the incidence of invasive mycoses has increased significantly over the last 3 decades. Slow diagnosis and the relatively few classes of antifungal drugs result in high attributable mortality for systemic fungal infections. Azole antifungals are commonly used for fungal infections, but azole resistance can be a problem for some patient groups. High-level, clinically significant azole resistance usually involves overexpression of plasma membrane efflux pumps belonging to the ATP-binding cassette (ABC) or the major facilitator superfamily class of transporters. The heterologous expression of efflux pumps in model systems, such Saccharomyces cerevisiae, has enabled the functional analysis of efflux pumps from a variety of fungi. Phylogenetic analysis of the ABC pleiotropic drug resistance family has provided a new view of the evolution of this important class of efflux pumps. There are several ways in which the clinical significance of efflux-mediated antifungal drug resistance can be mitigated. Alternative antifungal drugs, such as the echinocandins, that are not efflux pump substrates provide one option. Potential therapeutic approaches that could overcome azole resistance include targeting efflux pump transcriptional regulators and fungal stress response pathways, blockade of energy supply, and direct inhibition of efflux pumps. PMID:19366916

  14. Biochemistry of Bacterial Multidrug Efflux Pumps

    PubMed Central

    Kumar, Sanath; Varela, Manuel F.

    2012-01-01

    Bacterial pathogens that are multi-drug resistant compromise the effectiveness of treatment when they are the causative agents of infectious disease. These multi-drug resistance mechanisms allow bacteria to survive in the presence of clinically useful antimicrobial agents, thus reducing the efficacy of chemotherapy towards infectious disease. Importantly, active multi-drug efflux is a major mechanism for bacterial pathogen drug resistance. Therefore, because of their overwhelming presence in bacterial pathogens, these active multi-drug efflux mechanisms remain a major area of intense study, so that ultimately measures may be discovered to inhibit these active multi-drug efflux pumps. PMID:22605991

  15. 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

  16. 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

  17. The multi-xenobiotic resistance (MXR) efflux activity in hemocytes of Mytilus edulis is mediated by an ATP binding cassette transporter of class C (ABCC) principally inducible in eosinophilic granulocytes.

    PubMed

    Rioult, Damien; Pasquier, Jennifer; Boulangé-Lecomte, Céline; Poret, Agnès; Abbas, Imane; Marin, Matthieu; Minier, Christophe; Le Foll, Frank

    2014-08-01

    In marine and estuarine species, immunotoxic and/or immunomodulatory mechanisms are the crossroad of interactions between xenobiotics, microorganisms and physicochemical variations of the environment. In mussels, immunity relies exclusively on innate responses carried out by cells collectively called hemocytes and found in the open hemolymphatic circulatory system of these organisms. However, hemocytes do not form a homogenous population of immune cells since distinct subtypes of mussel blood cells can be distinguished by cytochemistry, flow cytometry or cell motility analysis. Previous studies have also shown that these cells are able to efflux xenobiotics by means of ATP binding cassette (ABC) transporter activities conferring a multixenobiotic resistance (MXR) phenotype. ABC transporters corresponding to vertebrate class B/P-glycoprotein (P-gp) and to class C/multidrug resistance related protein (MRP) are characterized in Mytilidae. Herein, we have investigated the relative contributions of ABCB- and ABCC-mediated efflux within the different hemocyte subpopulations of Mytilus edulis mussels, collected from areas differentially impacted by chemical contaminants in Normandy (France). RT-PCR analyses provide evidence for the presence of ABCB and ABCC transporters transcripts in hemocytes. Immunodetection of ABCB/P-gp with the monoclonal antibody UIC2 in living hemocytes revealed that expression was restricted to granular structures of spread cells. Efflux transporter activities, with calcein-AM as fluorescent probe, were measured by combining flow cytometry to accurate Coulter cell size measurements in order to get a cell-volume normalized fluorescence concentration. In these conditions, basal fluorescence levels were higher in hemocytes originating from Yport (control site) than in cells collected from the harbor of Le Havre, where mussels are more exposed to with persistent pollutants. By using specific ABCB/P-gp (verapamil, PSC833, zosuquidar) and ABCC/MRP (MK

  18. BBA, a Synthetic Derivative of 23-hydroxybutulinic Acid, Reverses Multidrug Resistance by Inhibiting the Efflux Activity of MRP7 (ABCC10)

    PubMed Central

    Chen, Jun-Jiang; Patel, Atish; Sodani, Kamlesh; Xiao, Zhi-Jie; Tiwari, Amit K.; Zhang, Dong-Mei; Li, Ying-Jie; Yang, Dong-Hua; Ye, Wen-Cai; Chen, Si-Dong; Chen, Zhe-Sheng

    2013-01-01

    Natural products are frequently used for adjuvant chemotherapy in cancer treatment. 23-O-(1,4'-bipiperidine-1-carbonyl) betulinic acid (BBA) is a synthetic derivative of 23-hydroxybutulinic acid (23-HBA), which is a natural pentacyclic triterpene and the major active constituent of the root of Pulsatillachinensis. We previously reported that BBA could reverse P-glycoprotein (P-gp/ABCB1)-mediated multidrug resistance (MDR). In the present study, we investigated whether BBA has the potential to reverse multidrug resistance protein 7 (MRP7/ABCC10)-mediated MDR. We found that BBA concentration-dependently enhanced the sensitivity of MRP7-transfected HEK293 cells to paclitaxel, docetaxel and vinblastine. Accumulation and efflux experiments demonstrated that BBA increased the intracellular accumulation of [3H]-paclitaxel by inhibiting the efflux of [3H]-paclitaxel from HEK293/MRP7 cells. In addition, immunoblotting and immunofluorescence analyses indicated no significant alteration of MRP7 protein expression and localization in plasma membranes after treatment with BBA. These results demonstrate that BBA reverses MRP7-mediated MDR through blocking the drug efflux function of MRP7 without affecting the intracellular ATP levels. Our findings suggest that BBA has the potential to be used in combination with conventional chemotherapeutic agents to augment the response to chemotherapy. PMID:24069321

  19. Expression of homologous RND efflux pump genes is dependent upon AcrB expression: implications for efflux and virulence inhibitor design

    PubMed Central

    Blair, Jessica M. A.; Smith, Helen E.; Ricci, Vito; Lawler, Amelia J.; Thompson, Louisa J.; Piddock, Laura J. V.

    2015-01-01

    Objectives Enterobacteriaceae have multiple efflux pumps that confer intrinsic resistance to antibiotics. AcrB mediates clinically relevant multidrug resistance and is required for virulence and biofilm formation, making it an attractive target for the design of inhibitors. The aim of this study was to assess the viability of single transporters as a target for efflux inhibition using Salmonella Typhimurium as the model pathogen. Methods The expression of resistance–nodulation–division (RND) efflux pump genes in response to the inactivation of single or multiple homologues was measured using real-time RT–PCR. Phenotypes of mutants were characterized by measuring antimicrobial susceptibility, dye accumulation and the ability to cause infection in vitro. Results The expression of all RND efflux pump genes was increased when single or multiple acr genes were inactivated, suggesting a feedback mechanism that activates the transcription of homologous efflux pump genes. When two or three acr genes were inactivated, the mutants had further reduced efflux, altered susceptibility to antimicrobials (including increased susceptibility to some, but conversely and counterintuitively, decreased susceptibility to some others) and were more attenuated in the tissue culture model than mutants lacking single pumps were. Conclusions These data indicate that it is critical to understand which pumps an inhibitor is active against and the effect of this on the expression of homologous systems. For some antimicrobials, an inhibitor with activity against multiple pumps will have a greater impact on susceptibility, but an unintended consequence of this may be decreased susceptibility to other drugs, such as aminoglycosides. PMID:25288678

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

    PubMed

    Blair, Jessica M A; Piddock, Laura J V

    2016-01-01

    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

  1. 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

  2. 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

  3. [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. PMID:26084076

  4. An RND-Type Efflux System in Borrelia burgdorferi Is Involved in Virulence and Resistance to Antimicrobial Compounds

    PubMed Central

    Bunikis, Ignas; Denker, Katrin; Östberg, Yngve; Andersen, Christian; Benz, Roland; Bergström, Sven

    2008-01-01

    Borrelia burgdorferi is remarkable for its ability to thrive in widely different environments due to its ability to infect various organisms. In comparison to enteric Gram-negative bacteria, these spirochetes have only a few transmembrane proteins some of which are thought to play a role in solute and nutrient uptake and excretion of toxic substances. Here, we have identified an outer membrane protein, BesC, which is part of a putative export system comprising the components BesA, BesB and BesC. We show that BesC, a TolC homolog, forms channels in planar lipid bilayers and is involved in antibiotic resistance. A besC knockout was unable to establish infection in mice, signifying the importance of this outer membrane channel in the mammalian host. The biophysical properties of BesC could be explained by a model based on the channel-tunnel structure. We have also generated a structural model of the efflux apparatus showing the putative spatial orientation of BesC with respect to the AcrAB homologs BesAB. We believe that our findings will be helpful in unraveling the pathogenic mechanisms of borreliae as well as in developing novel therapeutic agents aiming to block the function of this secretion apparatus. PMID:18389081

  5. Reversal of efflux of an anticancer drug in human drug-resistant breast cancer cells by inhibition of protein kinase Cα (PKCα) activity.

    PubMed

    Kim, Chan Woo; Asai, Daisuke; Kang, Jeong-Hun; Kishimura, Akihiro; Mori, Takeshi; Katayama, Yoshiki

    2016-02-01

    P-glycoprotein (Pgp) is a 170-kDa transmembrane protein that mediates the efflux of anticancer drugs from cells. Pgp overexpression has a distinct role in cells exhibiting multidrug resistance (MDR). We examined reversal of drug resistance in human MDR breast cancer cells by inhibition of protein kinase Cα (PKCα) activity, which is associated with Pgp-mediated efflux of anticancer drugs. PKCα activity was confirmed by measurement of phosphorylation levels of a PKCα-specific peptide substrate (FKKQGSFAKKK-NH2), showing relatively higher basal activity in drug-resistant MCF-7/ADR cells (84 %) than that in drug-sensitive MCF-7 cells (63 %). PKCα activity was effectively suppressed by the PKC inhibitor, Ro-31-7549, and reversal of intracellular accumulation of doxorubicin was observed by inhibition of PKCα activity in MCF-7/ADR cells compared with their intrinsic drug resistance. Importantly, increased accumulation of doxorubicin could enhance the therapeutic efficacy of doxorubicin in MDR cells significantly. These results suggest a potential for overcoming MDR via inhibition of PKCα activity with conventional anticancer drugs. PMID:26323260

  6. 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 Central

    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

  7. Carbon Dioxide Efflux From Poultry Litter Applied Soils under Conservation Tillage Systems in North Alabama

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increased carbon dioxide release from soils resulting from agricultural practices such as tillage and application of poultry litter in cotton production systems has generated concerns about contributions to global warming which negatively impacts natural ecosystems, weather patterns, and food produc...

  8. Neutrophil IL-1β processing induced by pneumolysin is mediated by the NLRP3/ASC inflammasome and caspase-1 activation, and is dependent on K+ efflux

    PubMed Central

    Karmakar, Mausita; Katsnelson, Michael; Malak, Hesham A.; Greene, Neil G.; Howell, Scott J.; Hise, Amy G.; Camilli, Andrew; Kadioglu, Aras; Dubyak, George R.; Pearlman, Eric

    2014-01-01

    Although neutrophils are the most abundant cells in acute infection and inflammation, relatively little attention has been paid to their role in inflammasome formation and IL-1β processing. In the current study, we investigated the mechanism by which neutrophils process IL-1β in response to Streptococcus pneumoniae. Using a murine model of S. pneumoniae corneal infection, we demonstrated a requirement for IL-1β in bacterial clearance, and showed that NLRP3, ASC and caspase-1 are essential for IL-1β production and bacterial killing in the cornea. Neutrophils in infected corneas had multiple specks with enzymatically active caspase-1 (FLICA-660+), and bone marrow neutrophils stimulated with heat killed S. pneumoniae (signal 1) and pneumolysin (signal 2) exhibited multiple specks after staining with FLICA-660, NLRP3 or ASC. High molecular weight ASC complexes were also detected, consistent with oligomer formation. Pneumolysin induced K+ efflux in neutrophils, and blocking K+ efflux inhibited caspase-1 activation and IL-1β processing; however, neutrophils did not undergo pyroptosis, indicating that K+ efflux and IL-1β processing is not a consequence of cell death. There was also no role for lysosomal destabilization or neutrophil elastase in pneumolysin mediated IL-1β processing in neutrophils. Together, these findings demonstrate an essential role for neutrophil derived IL-1β in S. pneumoniae infection, and elucidate the role of the NLRP3 inflammasome in neutrophil cleavage and secretion of mature IL-1β. Given the ubiquitous presence of neutrophils in acute bacterial and fungal infections, these findings will have implications for other microbial diseases. PMID:25609842

  9. 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

  10. 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

  11. 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.

  12. 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

  13. A subsurface, closed-loop system for soil carbon dioxide and its application to the gradient efflux approach

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Carbon dioxide concentrations in the soil can vary both temporally and spatially. The subsurface gradient approach is commonly used to estimate CO2 efflux but spatial integration has been limited. Methodology was developed to semi-continuously measure subsurface concentrations of CO2 using porous T...

  14. 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. PMID:22033226

  15. 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.

  16. K+ efflux is the Common Trigger of NLRP3 inflammasome Activation by Bacterial Toxins and Particulate Matter

    PubMed Central

    Muñoz-Planillo, Raúl; Kuffa, Peter; Martínez-Colón, Giovanny; Smith, Brenna L.; Rajendiran, Thekkelnaycke M.; Núñez, Gabriel

    2013-01-01

    Summary The NLRP3 inflammasome is an important component of the innate immune system. However, its mechanism of activation remains largely unknown. We show that NLRP3 activators including bacterial pore-forming toxins, nigericin, ATP and particulate matter caused mitochondrial perturbation or the opening of a large membrane pore; but this was not required for NLRP3 activation. Furthermore, reactive oxygen species generation or a change in cell volume was not necessary for NLRP3 activation. Instead, the only common activity induced by all NLRP3 agonists was the permeation of the cell membrane to K+ and Na+. Notably, reduction of the intracellular K+ concentration was sufficient to activate NLRP3 whereas an increase in intracellular Na+ modulated, but was not strictly required for inflammasome activation. These results provide a unifying model for the activation of the NLRP3 inflammasome in which a drop in cytosolic K+ is the common step that is necessary and sufficient for caspase-1 activation. PMID:23809161

  17. 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. PMID:27190152

  18. TLR2/MyD88/NF-κB Pathway, Reactive Oxygen Species, Potassium Efflux Activates NLRP3/ASC Inflammasome during Respiratory Syncytial Virus Infection

    PubMed Central

    Mgbemena, Victoria; Tsai, Su-Yu; Chang, Te-Hung; Berton, Michael T.; Morris, Ian R.; Allen, Irving C.; Ting, Jenny P.-Y.; Bose, Santanu

    2012-01-01

    Human respiratory syncytial virus (RSV) constitute highly pathogenic virus that cause severe respiratory diseases in newborn, children, elderly and immuno-compromised individuals. Airway inflammation is a critical regulator of disease outcome in RSV infected hosts. Although “controlled” inflammation is required for virus clearance, aberrant and exaggerated inflammation during RSV infection results in development of inflammatory diseases like pneumonia and bronchiolitis. Interleukin-1β (IL-1β) plays an important role in inflammation by orchestrating the pro-inflammatory response. IL-1β is synthesized as an immature pro-IL-1β form. It is cleaved by activated caspase-1 to yield mature IL-1β that is secreted extracellularly. Activation of caspase-1 is mediated by a multi-protein complex known as the inflammasome. Although RSV infection results in IL-1β release, the mechanism is unknown. Here in, we have characterized the mechanism of IL-1β secretion following RSV infection. Our study revealed that NLRP3/ASC inflammasome activation is crucial for IL-1β production during RSV infection. Further studies illustrated that prior to inflammasome formation; the “first signal” constitutes activation of toll-like receptor-2 (TLR2)/MyD88/NF-κB pathway. TLR2/MyD88/NF-κB signaling is required for pro-IL-1β and NLRP3 gene expression during RSV infection. Following expression of these genes, two “second signals” are essential for triggering inflammasome activation. Intracellular reactive oxygen species (ROS) and potassium (K+) efflux due to stimulation of ATP-sensitive ion channel promote inflammasome activation following RSV infection. Thus, our studies have underscored the requirement of TLR2/MyD88/NF-κB pathway (first signal) and ROS/potassium efflux (second signal) for NLRP3/ASC inflammasome formation, leading to caspase-1 activation and subsequent IL-1β release during RSV infection. PMID:22295065

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

    PubMed

    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

  20. Commensal Bacteria-Induced Inflammasome Activation in Mouse and Human Macrophages Is Dependent on Potassium Efflux but Does Not Require Phagocytosis or Bacterial Viability.

    PubMed

    Chen, Kejie; Shanmugam, Nanda Kumar N; Pazos, Michael A; Hurley, Bryan P; Cherayil, Bobby J

    2016-01-01

    Gut commensal bacteria contribute to the pathogenesis of inflammatory bowel disease, in part by activating the inflammasome and inducing secretion of interleukin-1ß (IL-1ß). Although much has been learned about inflammasome activation by bacterial pathogens, little is known about how commensals carry out this process. Accordingly, we investigated the mechanism of inflammasome activation by representative commensal bacteria, the Gram-positive Bifidobacterium longum subspecies infantis and the Gram-negative Bacteroides fragilis. B. infantis and B. fragilis induced IL-1ß secretion by primary mouse bone marrow-derived macrophages after overnight incubation. IL-1ß secretion also occurred in response to heat-killed bacteria and was only partly reduced when phagocytosis was inhibited with cytochalasin D. Similar results were obtained with a wild-type immortalized mouse macrophage cell line but neither B. infantis nor B. fragilis induced IL-1ß secretion in a mouse macrophage line lacking the nucleotide-binding/leucine-rich repeat pyrin domain containing 3 (NLRP3) inflammasome. IL-1ß secretion in response to B. infantis and B. fragilis was significantly reduced when the wild-type macrophage line was treated with inhibitors of potassium efflux, either increased extracellular potassium concentrations or the channel blocker ruthenium red. Both live and heat-killed B. infantis and B. fragilis also induced IL-1ß secretion by human macrophages (differentiated THP-1 cells or primary monocyte-derived macrophages) after 4 hours of infection, and the secretion was inhibited by raised extracellular potassium and ruthenium red but not by cytochalasin D. Taken together, our findings indicate that the commensal bacteria B. infantis and B. fragilis activate the NLRP3 inflammasome in both mouse and human macrophages by a mechanism that involves potassium efflux and that does not require bacterial viability or phagocytosis. PMID:27505062

  1. Commensal Bacteria-Induced Inflammasome Activation in Mouse and Human Macrophages Is Dependent on Potassium Efflux but Does Not Require Phagocytosis or Bacterial Viability

    PubMed Central

    Chen, Kejie; Shanmugam, Nanda Kumar N.; Pazos, Michael A.; Hurley, Bryan P.; Cherayil, Bobby J.

    2016-01-01

    Gut commensal bacteria contribute to the pathogenesis of inflammatory bowel disease, in part by activating the inflammasome and inducing secretion of interleukin-1ß (IL-1ß). Although much has been learned about inflammasome activation by bacterial pathogens, little is known about how commensals carry out this process. Accordingly, we investigated the mechanism of inflammasome activation by representative commensal bacteria, the Gram-positive Bifidobacterium longum subspecies infantis and the Gram-negative Bacteroides fragilis. B. infantis and B. fragilis induced IL-1ß secretion by primary mouse bone marrow-derived macrophages after overnight incubation. IL-1ß secretion also occurred in response to heat-killed bacteria and was only partly reduced when phagocytosis was inhibited with cytochalasin D. Similar results were obtained with a wild-type immortalized mouse macrophage cell line but neither B. infantis nor B. fragilis induced IL-1ß secretion in a mouse macrophage line lacking the nucleotide-binding/leucine-rich repeat pyrin domain containing 3 (NLRP3) inflammasome. IL-1ß secretion in response to B. infantis and B. fragilis was significantly reduced when the wild-type macrophage line was treated with inhibitors of potassium efflux, either increased extracellular potassium concentrations or the channel blocker ruthenium red. Both live and heat-killed B. infantis and B. fragilis also induced IL-1ß secretion by human macrophages (differentiated THP-1 cells or primary monocyte-derived macrophages) after 4 hours of infection, and the secretion was inhibited by raised extracellular potassium and ruthenium red but not by cytochalasin D. Taken together, our findings indicate that the commensal bacteria B. infantis and B. fragilis activate the NLRP3 inflammasome in both mouse and human macrophages by a mechanism that involves potassium efflux and that does not require bacterial viability or phagocytosis. PMID:27505062

  2. Quantitative evaluation of brain distribution and blood-brain barrier efflux transport of probenecid in rats by microdialysis: possible involvement of the monocarboxylic acid transport system.

    PubMed

    Deguchi, Y; Nozawa, K; Yamada, S; Yokoyama, Y; Kimura, R

    1997-02-01

    This study was performed to evaluate quantitatively the brain distribution and the efflux transport across the blood-brain barrier of probenecid, using in vivo microdialysis and in situ brain perfusion techniques. The brain interstitial fluid (ISF)-to-plasma cerebrospinal fluid (CSF)-to-plasma and brain tissue-to-plasma unbound concentration ratios of probenecid at steady state were less than unity, which suggests restricted distribution in the brain. An uphill concentration gradient from ISF to plasma and a downhill concentration gradient from CSF to ISF were observed. Kinetic analysis revealed that the efflux clearance from brain ISF to plasma (0.0373 ml/min/g brain) was significantly greater than the influx clearance from plasma to brain (0.00733 ml/min/g brain). The ratio of the ISF concentration (Cisf) to the plasma unbound concentration (Cp,f) of probenecid was increased 2- to 3-fold by salicylate (3.7 mM) and benzoate (3.6 mM), which are accepted as substrates of the monocarboxylic acid transport system, compared with the same ratio for the control. In addition, the ratio Cisf/Cp,f was increased by treatment with N-ethylmaleimide, a sulfhydryl-modifying agent, whereas p-aminohippuric acid and choline did not produce increasing effects on Cisf/Cp,f. These data suggest that the restricted distribution of probenecid in the brain may be ascribed to efficient efflux from the brain ISF, which may be regulated by the monocarboxylic acid transport system at a relatively high ISF concentration. PMID:9023263

  3. 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. PMID:27392698

  4. 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

  5. AcrS/EnvR Represses Expression of the acrAB Multidrug Efflux Genes in Escherichia coli▿

    PubMed Central

    Hirakawa, Hidetada; Takumi-Kobayashi, Asuka; Theisen, Ulrike; Hirata, Takahiro; Nishino, Kunihiko; Yamaguchi, Akihito

    2008-01-01

    The acrS regulatory gene is located upstream of the acrEF multidrug efflux system genes. However, the roles of AcrS in regulation of drug efflux pumps have not been clearly understood. Here we show that AcrS represses other multidrug efflux genes, acrAB, which encode a major efflux system in Escherichia coli. PMID:18567659

  6. Mitochondria Influence CDR1 Efflux Pump Activity, Hog1-Mediated Oxidative Stress Pathway, Iron Homeostasis, and Ergosterol Levels in Candida albicans

    PubMed Central

    Thomas, Edwina; Roman, Elvira; Claypool, Steven; Manzoor, Nikhat; Pla, Jesús

    2013-01-01

    Mitochondrial dysfunction in Candida albicans is known to be associated with drug susceptibility, cell wall integrity, phospholipid homeostasis, and virulence. In this study, we deleted CaFZO1, a key component required during biogenesis of functional mitochondria. Cells with FZO1 deleted displayed fragmented mitochondria, mitochondrial genome loss, and reduced mitochondrial membrane potential and were rendered sensitive to azoles and peroxide. In order to understand the cellular response to dysfunctional mitochondria, genome-wide expression profiling of fzo1Δ/Δ cells was performed. Our results show that the increased susceptibility to azoles was likely due to reduced efflux activity of CDR efflux pumps, caused by the missorting of Cdr1p into the vacuole. In addition, fzo1Δ/Δ cells showed upregulation of genes involved in iron assimilation, in iron-sufficient conditions, characteristic of iron-starved cells. One of the consequent effects was downregulation of genes of the ergosterol biosynthesis pathway with a commensurate decrease in cellular ergosterol levels. We therefore connect deregulated iron metabolism to ergosterol biosynthesis pathway in response to dysfunctional mitochondria. Impaired activation of the Hog1 pathway in the mutant was the basis for increased susceptibility to peroxide and increase in reactive oxygen species, indicating the importance of functional mitochondria in controlling Hog1-mediated oxidative stress response. Mitochondrial phospholipid levels were also altered as indicated by an increase in phosphatidylserine and phosphatidylethanolamine and decrease in phosphatidylcholine in fzo1Δ/Δ cells. Collectively, these findings reinforce the connection between functional mitochondria and azole tolerance, oxidant-mediated stress, and iron homeostasis in C. albicans. PMID:23979757

  7. 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

  8. Role of outer membrane barrier in efflux-mediated tetracycline resistance of Escherichia coli.

    PubMed Central

    Thanassi, D G; Suh, G S; Nikaido, H

    1995-01-01

    Accumulation of tetracycline in Escherichia coli was studied to determine its permeation pathway and to provide a basis for understanding efflux-mediated resistance. Passage of tetracycline across the outer membrane appeared to occur preferentially via the porin OmpF, with tetracycline in its magnesium-bound form. Rapid efflux of magnesium-chelated tetracycline from the periplasm was observed. In E. coli cells that do not contain exogenous tetracycline resistance genes, the steady-state level of tetracycline accumulation was decreased when porins were absent or when the fraction of Mg(2+)-chelated tetracycline was small. This is best explained by assuming the presence of a low-level endogenous active efflux system that bypasses the outer membrane barrier. When influx of tetracycline is slowed, this efflux is able to reduce the accumulation of tetracycline in the cytoplasm. In contrast, we found no evidence of a special outer membrane bypass mechanism for high-level efflux via the Tet protein, which is an inner membrane efflux pump coded for by exogenous tetA genes. Fractionation and equilibrium density gradient centrifugation experiments showed that the Tet protein is not localized to regions of inner and outer membrane adhesion. Furthermore, a high concentration of tetracycline was found in the compartment that rapidly equilibrated with the medium, most probably the periplasm, of Tet-containing E. coli cells, and the level of tetracycline accumulation in Tet-containing cells was not diminished by the mutational loss of the OmpF porin. These results suggest that the Tet protein, in contrast to the endogenous efflux system(s), pumps magnesium-chelated tetracycline into the periplasm. A quantitative model of tetracycline fluxes in E. coli cells of various types is presented. PMID:7860612

  9. Characterization of a MexAB-OprM efflux system necessary for productive metabolism of Pseudomonas azelaica HBP1 on 2-hydroxybiphenyl

    PubMed Central

    Czechowska, K.; Reimmann, C.; van der Meer, J. R.

    2013-01-01

    Pseudomonas azelaica HBP1 is one of the few bacteria known to completely mineralize the biocide and toxic compound 2-hydroxybiphenyl (2-HBP), but the mechanisms of its tolerance to the toxicity are unknown. By transposon mutant analysis and screening for absence of growth on water saturating concentrations of 2-HBP (2.7 mM) we preferentially found insertions in three genes with high homology to the mexA, mexB, and oprM efflux system. Mutants could grow at 2-HBP concentrations below 100 μM but at lower growth rates than the wild-type. Exposure of the wild-type to increasing 2-HBP concentrations resulted in acute cell growth arrest and loss of membrane potential, to which the cells adapt after a few hours. By using ethidium bromide (EB) as proxy we could show that the mutants are unable to expel EB effectively. Inclusion of a 2-HBP reporter plasmid revealed that the wild-type combines efflux with metabolism at all 2-HBP concentrations, whereas the mutants cannot remove the compound and arrest metabolism at concentrations above 24 μM. The analysis thus showed the importance of the MexAB-OprM system for productive metabolism of 2-HBP. PMID:23882265

  10. 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

  11. 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

  12. Vectorial transport of fexofenadine across Caco-2 cells: involvement of apical uptake and basolateral efflux transporters.

    PubMed

    Ming, Xin; Knight, Beverly M; Thakker, Dhiren R

    2011-10-01

    Fexofenadine is a nonsedative antihistamine that exhibits good oral bioavailability despite its zwitterionic chemical structure and efflux by P-gp. Evidence exists that multiple uptake and efflux transporters play a role in hepatic disposition of fexofenadine. However, the roles of specific transporters and their interrelationship in intestinal absorption of this drug are unclear. This study was designed to elucidate vectorial absorptive transport of fexofenadine across Caco-2 cells involving specific apical uptake and efflux transporters as well as basolateral efflux transporters. Studies with cellular models expressing single transporters showed that OATP2B1 expression stimulated uptake of fexofenadine at pH 6.0. Apical uptake of fexofenadine into Caco-2 cells was decreased by 45% by pretreatment with estrone 3-sulfate, an OATP inhibitor, at pH 6.0 but not at pH 7.4, indicating that OATP2B1 mediates apical uptake of fexofenadine into these cells. Examination of fexofenadine efflux from preloaded Caco-2 cells in the presence or absence of (i) the MRP inhibitor MK-571 and (ii) the P-gp inhibitor GW918 showed that apical efflux is predominantly mediated by P-gp, with a small contribution by MRP2, whereas basolateral efflux is predominantly mediated by MRP3. These results also showed that while OSTαβ is functionally active in the basolateral membrane of Caco-2 cells, it does not play a role in the export of fexofenadine. MK-571 decreased the absorptive transport of fexofenadine by 17%. However, the decrease in absorptive transport by MK-571 was 42% when P-gp was inhibited by GW918. The results provide a novel insight into a vectorial transport system mainly consisting of apical OATP2B1 and basolateral MRP3 that may play an important role in delivering hydrophilic anionic and zwitterionic drugs such as pravastatin and fexofenadine into systemic circulation upon oral administration. PMID:21780830

  13. 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

  14. 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. PMID:25085218

  15. 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

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

    PubMed

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

    2016-02-01

    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. PMID:26824720

  17. 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

  18. Oral and inhaled corticosteroids: differences in P-glycoprotein (ABCB1) mediated efflux.

    PubMed

    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(-6) cm/s) compared to the inhaled corticosteroids (>5×10(-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. PMID:22464980

  19. Tigecycline Efflux as a Mechanism for Nonsusceptibility in Acinetobacter baumannii.

    PubMed

    Peleg, Anton Y; Adams, Jennifer; Paterson, David L

    2007-06-01

    Tigecycline has an extended spectrum of in vitro antimicrobial activities, including that against multidrug-resistant Acinetobacter. After identifying bloodstream isolates of Acinetobacter with reduced susceptibilities to tigecycline, we performed a study to assess tigecycline efflux mediated by the resistance-nodulation-division-type transporter AdeABC. After exposure of two tigecycline-nonsusceptible isolates to the efflux pump inhibitor phenyl-arginine-beta-naphthylamide (PABN), a fourfold reduction in the tigecycline MIC was observed. Both tigecycline-susceptible and -nonsusceptible isolates were found to carry the gene coding for the transmembrane component of the AdeABC pump, adeB, and the two-component regulatory system comprising adeS and adeR. Previously unreported point mutations were identified in the regulatory system in tigecycline-nonsusceptible isolates. Real-time PCR identified 40-fold and 54-fold increases in adeB expression in the two tigecycline-nonsusceptible isolates compared to that in a tigecycline-susceptible isolate. In vitro exposure of a tigecycline-susceptible clinical strain to tigecycline caused a rapid rise in the MIC of tigecycline from 2 microg/ml to 24 microg/ml, which was reversible with PABN. A 25-fold increase in adeB expression was observed in a comparison between this tigecycline-susceptible isolate and its isogenic tigecycline-nonsusceptible mutant. These results indicate that an efflux-based mechanism plays a role in reduced tigecycline susceptibility in Acinetobacter. PMID:17420217

  20. Tigecycline Efflux as a Mechanism for Nonsusceptibility in Acinetobacter baumannii▿

    PubMed Central

    Peleg, Anton Y.; Adams, Jennifer; Paterson, David L.

    2007-01-01

    Tigecycline has an extended spectrum of in vitro antimicrobial activities, including that against multidrug-resistant Acinetobacter. After identifying bloodstream isolates of Acinetobacter with reduced susceptibilities to tigecycline, we performed a study to assess tigecycline efflux mediated by the resistance-nodulation-division-type transporter AdeABC. After exposure of two tigecycline-nonsusceptible isolates to the efflux pump inhibitor phenyl-arginine-β-naphthylamide (PABN), a fourfold reduction in the tigecycline MIC was observed. Both tigecycline-susceptible and -nonsusceptible isolates were found to carry the gene coding for the transmembrane component of the AdeABC pump, adeB, and the two-component regulatory system comprising adeS and adeR. Previously unreported point mutations were identified in the regulatory system in tigecycline-nonsusceptible isolates. Real-time PCR identified 40-fold and 54-fold increases in adeB expression in the two tigecycline-nonsusceptible isolates compared to that in a tigecycline-susceptible isolate. In vitro exposure of a tigecycline-susceptible clinical strain to tigecycline caused a rapid rise in the MIC of tigecycline from 2 μg/ml to 24 μg/ml, which was reversible with PABN. A 25-fold increase in adeB expression was observed in a comparison between this tigecycline-susceptible isolate and its isogenic tigecycline-nonsusceptible mutant. These results indicate that an efflux-based mechanism plays a role in reduced tigecycline susceptibility in Acinetobacter. PMID:17420217

  1. Calcium efflux activity of plasma membrane Ca2+ ATPase-4 (PMCA4) mediates cell cycle progression in vascular smooth muscle cells.

    PubMed

    Afroze, Talat; Yang, Ge; Khoshbin, Amir; Tanwir, Mansoor; Tabish, Taha; Momen, Abdul; Husain, Mansoor

    2014-03-01

    We explored the role played by plasma membrane calcium ATPase-4 (PMCA4) and its alternative splice variants in the cell cycle of vascular smooth muscle cells (VSMC). A novel variant (PMCA4e) was discovered. Quantitative real-time-PCR-quantified PMCA4 splice variant proportions differed in specific organs. The PMCA4a:4b ratio in uninjured carotid arteries (∼1:1) was significantly reduced by wire denudation injury (to ∼1:3) by modulation of alternative splicing, as confirmed by novel antibodies against PMCA4a/e and PMCA4b. Laser capture microdissection localized this shift to the media and adventitia. Primary carotid VSMC from PMCA4 knock-out (P4KO) mice showed impaired [(3)H]thymidine incorporation and G1 phase arrest as compared with wild type (P4WT). Electroporation of expression constructs encoding PMCA4a, PMCA4b, and a PMCA4b mutant lacking PDZ binding rescued this phenotype of P4KO cells, whereas a mutant with only 10% of normal Ca(2+) efflux activity could not. Microarray of early G1-synchronized VSMC showed 39-fold higher Rgs16 (NFAT (nuclear factor of activated T-cells) target; MAPK inhibitor) and 69-fold higher Decorin (G1 arrest marker) expression in P4KO versus P4WT. Validation by Western blot also revealed decreased levels of Cyclin D1 and NFATc3 in P4KO. Microarrays of P4KO VSMC rescued by PMCA4a or PMCA4b expression showed reversal of perturbed Rgs16, Decorin, and NFATc3 expression levels. However, PMCA4a rescue caused a 44-fold reduction in AP-2β, a known anti-proliferative transcription factor, whereas PMCA4b rescue resulted in a 50-fold reduction in p15 (Cyclin D1/Cdk4 inhibitor). We conclude that Ca(2+) efflux activity of PMCA4 underlies G1 progression in VSMC and that PMCA4a and PMCA4b differentially regulate specific downstream mediators. PMID:24448801

  2. 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. PMID:27129224

  3. Bacterial multi-drug efflux transporters

    PubMed Central

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

    2016-01-01

    Infections caused by bacteria remain a leading cause of death worldwide. While antibiotics remain a key clinical therapy, their effectiveness has been severely compromised by the development of drug resistance in these pathogens. A common and powerful resistance mechanism, multi-drug efflux transporters are capable of extruding a number of structurally unrelated antimicrobials from the bacterial cell, including antibiotics and toxic heavy metal ions, facilitating their survival in noxious environments. Those transporters belonging to the resistance-nodulation-cell division (RND) superfamily typically assemble as tripartite efflux complexes, spanning the inner and outer membranes of the cell envelope. In Escherichia coli, the CusCFBA complex, which mediates resistance to copper(I) and silver(I) ions, is the only known RND transporter with a specificity for heavy metals. Herein, we describe the current knowledge of individual pump components of the Cus system, a paradigm for efflux machinery, and speculate on how RND pumps assemble to fight diverse antimicrobials. PMID:24702006

  4. Vertical profile of branch CO2 efflux in a Norway spruce tree: a case study

    NASA Astrophysics Data System (ADS)

    Acosta, M.; Pavelka, M.

    2012-04-01

    Despite woody-tissue CO2 effluxes having been recognized as an important component of forest carbon budget due to the fraction of assimilates used and the dramatic increase in woody with stand development, there is limited research to determine the CO2 efflux vertical variability of woody-tissue components. For a better understanding and quantification of branch woody-tissue CO2 efflux in forest ecosystems, it is necessary to identify the environmental factors influencing it and the role of the branch distribution within the canopy. The proper assessment of this forest component will improve the knowledge of the ratio between ecosystem respiration and gross primary production at forest ecosystem. In order to achieve this goal, branch CO2 efflux of Norway spruce tree was measured in ten branches at five different whorls during the growing season 2004 (from June till October) in campaigns of 3-4 times per month at the Beskydy Mts., the Czech Republic, using a portable infrared gas analyzer operating as a closed system. Branch woody tissue temperature was measured continuously in ten minutes intervals for each sample position during the whole experiment period. On the basis of relation between CO2 efflux rate and woody tissue temperature a value of Q10 and normalized CO2 efflux rate (E10 - CO2 efflux rate at 10° C) were calculated for each sampled position. Estimated Q10 values ranged from 2.12 to 2.89 and E10 ranged from 0.41 to 1.19 ?molCO2m-2 s-1. Differences in branch CO2 efflux were found between orientations; East side branches presented higher efflux rate than west side branches. The highest branch CO2 efflux rate values were measured in August and the lowest in October, which were connected with woody tissue temperature and ontogenetic processes during these periods. Branch CO2 efflux was significantly and positively correlated with branch position within canopy and woody tissue temperature. Branches from the upper whorls showed higher respiration activity

  5. 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.

  6. NLRP3 inflammasome signaling is activated by low-level lysosome disruption but inhibited by extensive lysosome disruption: roles for K+ efflux and Ca2+ influx.

    PubMed

    Katsnelson, Michael A; Lozada-Soto, Kristen M; Russo, Hana M; Miller, Barbara A; Dubyak, George R

    2016-07-01

    Nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) is a cytosolic protein that nucleates assembly of inflammasome signaling platforms, which facilitate caspase-1-mediated IL-1β release and other inflammatory responses in myeloid leukocytes. NLRP3 inflammasomes are assembled in response to multiple pathogen- or environmental stress-induced changes in basic cell physiology, including the destabilization of lysosome integrity and activation of K(+)-permeable channels/transporters in the plasma membrane (PM). However, the quantitative relationships between lysosome membrane permeabilization (LMP), induction of increased PM K(+) permeability, and activation of NLRP3 signaling are incompletely characterized. We used Leu-Leu-O-methyl ester (LLME), a soluble lysosomotropic agent, to quantitatively track the kinetics and extent of LMP in relation to NLRP3 inflammasome signaling responses (ASC oligomerization, caspase-1 activation, IL-1β release) and PM cation fluxes in murine bone marrow-derived dendritic cells (BMDCs). Treatment of BMDCs with submillimolar (≤1 mM) LLME induced slower and partial increases in LMP that correlated with robust NLRP3 inflammasome activation and K(+) efflux. In contrast, supramillimolar (≥2 mM) LLME elicited extremely rapid and complete collapse of lysosome integrity that was correlated with suppression of inflammasome signaling. Supramillimolar LLME also induced dominant negative effects on inflammasome activation by the canonical NLRP3 agonist nigericin; this inhibition correlated with an increase in NLRP3 ubiquitination. LMP elicited rapid BMDC death by both inflammasome-dependent pyroptosis and inflammasome-independent necrosis. LMP also triggered Ca(2+) influx, which attenuated LLME-stimulated NLRP3 inflammasome signaling but potentiated LLME-induced necrosis. Taken together, these studies reveal a previously unappreciated signaling network that defines the coupling between LMP, changes

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

    PubMed

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

    2016-03-18

    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

  8. Hypoxic Stress Induced by Hydralazine Leads to a Loss of Blood-Brain Barrier Integrity and an Increase in Efflux Transporter Activity

    PubMed Central

    Chatard, Morgane; Puech, Clémentine

    2016-01-01

    Understanding cellular and molecular mechanisms induced by hypoxic stress is crucial to reduce blood-brain barrier (BBB) disruption in some neurological diseases. Since the brain is a complex organ, it makes the interpretation of in vivo data difficult, so BBB studies are often investigated using in vitro models. However, the investigation of hypoxia in cellular pathways is complex with physical hypoxia because HIF-1α (factor induced by hypoxia) has a short half-life. We had set up an innovative and original method of induction of hypoxic stress by hydralazine that was more reproducible, which allowed us to study its impact on an in vitro BBB model. Our results showed that hydralazine, a mimetic agent of the hypoxia pathway, had the same effect as physical hypoxia, with few cytotoxicity effects on our cells. Hypoxic stress led to an increase of BBB permeability which corresponded to an opening of our BBB model. Study of tight junction proteins revealed that this hypoxic stress decreased ZO-1 but not occludin expression. In contrast, cells established a defence mechanism by increasing expression and activity of their efflux transporters (Pgp and MRP-1). This induction method of hypoxic stress by hydralazine is simple, reproducible, controllable and suitable to understand the cellular and molecular mechanisms involved by hypoxia on the BBB. PMID:27337093

  9. Region-dependent disappearance of vinblastine in rat small intestine and characterization of its P-glycoprotein-mediated efflux system.

    PubMed

    Nakayama, A; Saitoh, H; Oda, M; Takada, M; Aungst, B J

    2000-10-01

    This study was aimed to characterize the absorption behavior of vinblastine (VLB), a well-known substrate of P-glycoprotein (P-gp), from rat small intestine, especially focusing on the regional-dependence of its efflux mediated by P-gp. VLB disappeared from duodenal and ileal loops of male Wistar rats fairly rapidly (30-60% in 30 min). In contrast, its disappearance from the jejunal loop was almost negligible and in some rats >100% of the jejunal dose was recovered. The radioactivity derived from [3H]VLB, which was absorbed from duodenum and ileum, was detected in the jejunal region. The jejunal appearance of radioactivity was increased when unlabeled VLB was present in the region in advance. The basolateral-to-apical transport of [3H]VLB across Caco-2 cell monolayers was greater when unlabeled VLB was added to the apical medium than when VLB-free buffer was applied to the apical side. When verapamil or cyclosporin A, potent modulators of P-gp, was added to the apical medium together with unlabeled VLB, enhanced basolateral-to-apical transport of [3H]VLB was disappeared. It is suggested that VLB absorption is strongly restricted by P-gp, especially in the jejunal region of the rat small intestine, and that the secretory transport via intestinal P-gp may be subject to trans-stimulation. Moreover, intravenously administered methylprednisolone and intramuscularly administered progesterone significantly enhanced the absorption of VLB, suggesting that parenterally administered P-gp modulators could influence the intestinal absorption of P-gp substrates. PMID:11033075

  10. Regulation of P-glycoprotein efflux activity by Z-guggulsterone of Commiphora mukul at the blood-brain barrier.

    PubMed

    Xu, Hong-Bin; Yu, Jing; Xu, Lu-Zhong; Fu, Jun

    2016-04-15

    The present study was to investigate whether Z-guggulsterone had the regulatory effect on the activity and expression of P-glycoprotein in rat brain microvessel endothelial cells (rBMECs) and in rat brain. Inorganic phosphate liberation assay, high performance liquid chromatography, and western blot analysis were performed to assess the P-glycoprotein ATPase activity, the accumulation of NaF and rhodamine 123, and P-glycoprotein and MRP1 expression. The results showed that Z-guggulsterone (0-100 μM) significantly enhanced basal P-glycoprotein ATPase activity in a concentration-dependent manner. Tetrandrine (0.1, 0.3, 1 μM) or cyclosporine A (0.1, 0.3, 1 μM) had non-competitively inhibitory manner on Z-guggulsterone-stimulated P-glycoprotein ATPase activity, suggesting that Z-guggulsterone might have unique binding site or regulating site on P-glycoprotein. However, Z-guggulsterone (30, 100 μM) had almost no influence on MRP1 expression in rBMECs. Further results revealed that Z-guggulsterone (50mg/kg) significantly increased the accumulation of rhodamine 123 by down-regulating P-glycoprotein expression in rat brain, as compared with control (P<0.05). Our studies suggested that Z-guggulsterone potentially inhibited the activity and expression of P-glycoprotein in rBMECs and in rat brain. PMID:27000241

  11. 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.

  12. 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

  13. Induction of Expression and Functional Activity of P-glycoprotein Efflux Transporter by Bioactive Plant Natural Products

    PubMed Central

    Abuznait, Alaa H.; Qosa, Hisham; O’Connell, Nicholas D.; Akbarian-Tefaghi, Jessica; Sylvester, Paul W.; El Sayed, Khalid A.; Kaddoumi, Amal

    2011-01-01

    The effect of bioactive plant natural products on the expression and functional activity of P-glycoprotein (P-gp) is poorly understood. Interactions of bioactive plant-based food and dietary supplements with P-gp can cause significant alteration of pharmacokinetic properties of P-gp substrate drugs when used in combination. This can augment toxicity and/or interfere with the drug’s therapeutic outcomes. This study investigated the effects of diverse commonly used plant natural products on the expression and activity of P-gp in human adenocarcinoma cells (LS-180). These natural products included the tobacco cembranoid (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (cembratriene), the palm oil-derived γ-tocotrienol, the extra-virgin olive oil-derived secoiridoid oleocanthal, and the triterpene acid asiatic acid derived from Melaleuca ericifolia and abundant in several other common plant dietary supplements. Treatment with 25 μM of cembratriene, oleocanthal, γ-tocotrienol, or asiatic acid showed 2.3-3.0-fold increase in P-gp expression as demonstrated by Western blotting. These results were consistent with those obtained by quantitative analysis of fluorescent micrographs for P-gp. Accumulation studies demonstrated 31-38% decrease in rhodamine 123 intracellular levels when LS-180 cells were treated with the investigated compounds as a result of P-gp induction. Bioactive natural products can up-regulate the P-gp expression and functionality, which may induce herb/food-drug interactions when concomitantly used with P-gp substrate drugs. PMID:21851848

  14. 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

  15. Expression of Efflux Pumps and Fatty Acid Activator One Genes in Azole Resistant Candida Glabrata Isolated From Immunocompromised Patients.

    PubMed

    Farahyar, Shirin; Zaini, Farideh; Kordbacheh, Parivash; Rezaie, Sassan; Falahati, Mehraban; Safara, Mahin; Raoofian, Reza; Hatami, Kamran; Mohebbi, Masoumeh; Heidari, Mansour

    2016-07-01

    Acquired azole resistance in opportunistic fungi causes severe clinical problems in immunosuppressed individuals. This study investigated the molecular mechanisms of azole resistance in clinical isolates of Candida glabrata. Six unmatched strains were obtained from an epidemiological survey of candidiasis in immunocompromised hosts that included azole and amphotericin B susceptible and azole resistant clinical isolates. Candida glabrata CBS 138 was used as reference strain. Antifungal susceptibility testing of clinical isolates was evaluated using Clinical and Laboratory Standards Institute (CLSI) methods. Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) technology, semi-quantitative RT-PCR, and sequencing were employed for identification of potential genes involved in azole resistance. Candida glabrata Candida drug resistance 1 (CgCDR1) and Candida glabrata Candida drug resistance 2 (CgCDR2) genes, which encode for multidrug transporters, were found to be upregulated in azole-resistant isolates (≥2-fold). Fatty acid activator 1 (FAA1) gene, belonging to Acyl-CoA synthetases, showed expression in resistant isolates ≥2-fold that of the susceptible isolates and the reference strain. This study revealed overexpression of the CgCDR1, CgCDR2, and FAA1 genes affecting biological pathways, small hydrophobic compounds transport, and lipid metabolism in the resistant clinical C.glabrata isolates. PMID:27424018

  16. A mass spectrometry-based assay for improved quantitative measurements of efflux pump inhibition.

    PubMed

    Brown, Adam R; Ettefagh, Keivan A; Todd, Daniel; Cole, Patrick S; Egan, Joseph M; Foil, Daniel H; Graf, Tyler N; Schindler, Bryan D; Kaatz, Glenn W; Cech, Nadja B

    2015-01-01

    Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC50 value of 75 μg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin), were also active, with IC50 values ranging from 19 μg/mL to 75 μg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence. PMID:25961825

  17. A Mass Spectrometry-Based Assay for Improved Quantitative Measurements of Efflux Pump Inhibition

    PubMed Central

    Brown, Adam R.; Ettefagh, Keivan A.; Todd, Daniel; Cole, Patrick S.; Egan, Joseph M.; Foil, Daniel H.; Graf, Tyler N.; Schindler, Bryan D.; Kaatz, Glenn W.; Cech, Nadja B.

    2015-01-01

    Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC50 value of 75 μg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin), were also active, with IC50 values ranging from 19 μg/mL to 75 μg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence. PMID:25961825

  18. 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

  19. Using the BacMam Baculovirus System to Study Expression and Function of Recombinant Efflux Drug Transporters in Polarized Epithelial Cell Monolayers

    PubMed Central

    Fung, King Leung; Kapoor, Khyati; Pixley, Jessica N.; Talbert, Darrell J.; Kwit, Alexandra D.T.; Ambudkar, Suresh V.

    2016-01-01

    The ATP-binding cassette (ABC) transporter superfamily includes several membrane-bound proteins that are critical to drug pharmacokinetics and disposition. Pharmacologic evaluation of these proteins in vitro remains a challenge. In this study, human ABC transporters were expressed in polarized epithelial cell monolayers transduced using the BacMam baculovirus gene transfer system. The purpose of the study was to evaluate the efficacy of BacMam baculovirus to transduce cells grown in monolayers. In a porcine kidney cell line, LLC-PK1 cells, baculoviral transduction is successful only via the apical side of a polarized monolayer. We observed that recombinant ABC transporters were expressed on the cell surface with post-translational modification. Furthermore, sodium butyrate played a critical role in recombinant protein expression, and preincubation in the presence of tunicamycin or thapsigargin enhanced protein expression. Cells overexpressing human P-glycoprotein (P-gp) showed vectorial basolateral-to-apical transport of [3H]-paclitaxel, which could be reversed by the inhibitor tariquidar. Similarly, coexpression of human P-gp and ABCG2 in LLC-PK1 cells resulted in higher transport of mitoxantrone, which is a substrate for both transporters, than in either P-gp– or ABCG2-expressing cells alone. Taken together, our results indicate that a high level of expression of efflux transporters in a polarized cell monolayer is technically feasible with the BacMam baculovirus system PMID:26622052

  20. Do Phytotropins Inhibit Auxin Efflux by Impairing Vesicle Traffic?1

    PubMed Central

    Petrášek, Jan; Černá, Adriana; Schwarzerová, Kateřina; Elčkner, Miroslav; Morris, David A.; Zažímalová, Eva

    2003-01-01

    Phytotropins such as 1-N-naphthylphthalamic acid (NPA) strongly inhibit auxin efflux, but the mechanism of this inhibition remains unknown. Auxin efflux is also strongly decreased by the vesicle trafficking inhibitor brefeldin A (BFA). Using suspension-cultured interphase cells of the BY-2 tobacco (Nicotiana tabacum L. cv Bright-Yellow 2) cell line, we compared the effects of NPA and BFA on auxin accumulation and on the arrangement of the cytoskeleton and endoplasmic reticulum (ER). The inhibition of auxin efflux (stimulation of net accumulation) by both NPA and BFA occurred rapidly with no measurable lag. NPA had no observable effect on the arrangement of microtubules, actin filaments, or ER. Thus, its inhibitory effect on auxin efflux was not mediated by perturbation of the cytoskeletal system and ER. BFA, however, caused substantial alterations to the arrangement of actin filaments and ER, including a characteristic accumulation of actin in the perinuclear cytoplasm. Even at saturating concentrations, NPA inhibited net auxin efflux far more effectively than did BFA. Therefore, a proportion of the NPA-sensitive auxin efflux carriers may be protected from the action of BFA. Maximum inhibition of auxin efflux occurred at concentrations of NPA substantially below those previously reported to be necessary to perturb vesicle trafficking. We found no evidence to support recent suggestions that the action of auxin transport inhibitors is mediated by a general inhibition of vesicle-mediated protein traffic to the plasma membrane. PMID:12529533

  1. Do phytotropins inhibit auxin efflux by impairing vesicle traffic?

    PubMed

    Petrásek, Jan; Cerná, Adriana; Schwarzerová, Katerina; Elckner, Miroslav; Morris, David A; Zazímalová, Eva

    2003-01-01

    Phytotropins such as 1-N-naphthylphthalamic acid (NPA) strongly inhibit auxin efflux, but the mechanism of this inhibition remains unknown. Auxin efflux is also strongly decreased by the vesicle trafficking inhibitor brefeldin A (BFA). Using suspension-cultured interphase cells of the BY-2 tobacco (Nicotiana tabacum L. cv Bright-Yellow 2) cell line, we compared the effects of NPA and BFA on auxin accumulation and on the arrangement of the cytoskeleton and endoplasmic reticulum (ER). The inhibition of auxin efflux (stimulation of net accumulation) by both NPA and BFA occurred rapidly with no measurable lag. NPA had no observable effect on the arrangement of microtubules, actin filaments, or ER. Thus, its inhibitory effect on auxin efflux was not mediated by perturbation of the cytoskeletal system and ER. BFA, however, caused substantial alterations to the arrangement of actin filaments and ER, including a characteristic accumulation of actin in the perinuclear cytoplasm. Even at saturating concentrations, NPA inhibited net auxin efflux far more effectively than did BFA. Therefore, a proportion of the NPA-sensitive auxin efflux carriers may be protected from the action of BFA. Maximum inhibition of auxin efflux occurred at concentrations of NPA substantially below those previously reported to be necessary to perturb vesicle trafficking. We found no evidence to support recent suggestions that the action of auxin transport inhibitors is mediated by a general inhibition of vesicle-mediated protein traffic to the plasma membrane. PMID:12529533

  2. 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.

  3. Helicobacter pylori uptake and efflux: basis for intrinsic susceptibility to antibiotics in vitro.

    PubMed

    Bina, J E; Alm, R A; Uria-Nickelsen, M; Thomas, S R; Trust, T J; Hancock, R E

    2000-02-01

    We previously demonstrated (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995) that Helicobacter pylori has at least one nonspecific porin, HopE, which has a low abundance in the outer membrane but forms large channels. H. pylori is relatively susceptible to most antimicrobial agents but less susceptible to the polycationic antibiotic polymyxin B. We demonstrate here that H. pylori is able to take up higher basal levels of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN) than Pseudomonas aeruginosa or Escherichia coli, consistent with its enhanced susceptibility to hydrophobic agents. Addition of polymyxin B led to a further increase in NPN uptake, indicative of a self-promoted uptake pathway, but it required a much higher amount of polymyxin B to yield a 50% increase in NPN uptake in H. pylori (6 to 8 microg/ml) than in P. aeruginosa or E. coli (0.3 to 0.5 microg/ml), suggesting that H. pylori has a less efficient self-promoted uptake pathway. Since intrinsic resistance involves the collaboration of restricted outer membrane permeability and secondary defense mechanisms, such as periplasmic beta-lactamase (which H. pylori lacks) or efflux, we examined the possible role of efflux in antibiotic susceptibility. We had previously identified in H. pylori 11637 the presence of portions of three genes with homology to potential restriction-nodulation-division (RND) efflux systems. It was confirmed that H. pylori contained only these three putative RND efflux systems, named here hefABC, hefDEF, and hefGHI, and that the hefGHI system was expressed only in vivo while the two other RND systems were expressed both in vivo and in vitro. In uptake studies, there was no observable energy-dependent tetracycline, chloramphenicol, or NPN efflux activity in H. pylori. Independent mutagenesis of the three putative RND efflux operons in the chromosome of H. pylori had no effect on the in vitro susceptibility of H. pylori to 19

  4. Lack of efflux mediated quinolone resistance in Salmonella enterica serovars Typhi and Paratyphi A.

    PubMed

    Baucheron, Sylvie; Monchaux, Isabelle; Le Hello, Simon; Weill, François-Xavier; Cloeckaert, Axel

    2014-01-01

    Salmonella enterica serovars Typhi and Paratyphi A isolates from human patients in France displaying different levels of resistance to quinolones or fluoroquinolones were studied for resistance mechanisms to these antimicrobial agents. All resistant isolates carried either single or multiple target gene mutations (i.e., in gyrA, gyrB, or parC) correlating with the resistance levels observed. Active efflux, through upregulation of multipartite efflux systems, has also been previously reported as contributing mechanism for other serovars. Therefore, we investigated also the occurrence of non-target gene mutations in regulatory regions affecting efflux pump expression. However, no mutation was detected in these regions in both Typhi and Paratyphi isolates of this study. Besides, no overexpression of the major efflux systems was observed for these isolates. Nevertheless, a large deletion of 2334 bp was identified in the acrS-acrE region of all S. Typhi strains but which did not affect the resistance phenotype. As being specific to S. Typhi, this deletion could be used for specific molecular detection purposes. In conclusion, the different levels of quinolone or FQ resistance in both S. Typhi and S. Paratyphi A seem to rely only on target modifications. PMID:24478769

  5. Trimerized apolipoprotein A-I (TripA) forms lipoproteins, activates lecithin: cholesterol acyltransferase, elicits lipid efflux, and is transported through aortic endothelial cells.

    PubMed

    Ohnsorg, Pascale M; Mary, Jean-Luc; Rohrer, Lucia; Pech, Michael; Fingerle, Jürgen; von Eckardstein, Arnold

    2011-12-01

    Apolipoprotein A-I (apoA-I) exerts many potentially anti-atherogenic properties and is therefore attractive for prevention and therapy of coronary heart disease. Since induction of apoA-I production by small molecules has turned out as difficult, application of exogenous apoA-I is pursued as an alternative therapeutic option. To counteract fast renal filtration of apoA-I, a trimeric high-molecular weight variant of apoA-I (TripA) was produced by recombinant technology. We compared TripA and apoA-I for important properties in reverse cholesterol transport. Reconstituted high-density lipoproteins (rHDL) containing TripA or apoA-I together with palmitoyl-2-oleyl-phosphatidylcholine (POPC) differed slightly by size. Compared to apoA-I, TripA activated lecithin:cholesterol acyltransferase (LCAT) with similar maximal velocity but concentration leading to half maximal velocity was slightly reduced (K(m)=2.1±0.3μg/mL vs. 0.59±0.06μg/mL). Both in the lipid-free form and as part of rHDL, TripA elicited cholesterol efflux from THP1-derived macrophages with similar kinetic parameters and response to liver-X-receptor activation as apoA-I. Lipid-free TripA is bound and transported by aortic endothelial cells through mechanisms which are competed by apoA-I and TripA and inhibited by knock-down of ATP-binding cassette transporter (ABC) A1. Pre-formed TripA/POPC particles were bound and transported by endothelial cells through mechanisms which are competed by excess native HDL as well as reconstituted HDL containing either apoA-I or TripA and which involve ABCG1 and scavenger receptor B1 (SR-BI). In conclusion, apoA-I and TripA show similar in vitro properties which are important for reverse cholesterol transport. These findings are important for further development of TripA as an anti-atherosclerotic drug. PMID:21930241

  6. The Sinorhizobium meliloti EmrAB efflux system is regulated by flavonoids through a TetR-like regulator (EmrR).

    PubMed

    Rossbach, Silvia; Kunze, Kati; Albert, Susann; Zehner, Susanne; Göttfert, Michael

    2014-04-01

    The divergently oriented Sinorhizobium meliloti emrAB (SMc03168 and SMc03167) and emrR (SMc03169) genes are predicted to encode an efflux system of the major facilitator superfamily and a TetR-like transcriptional regulator, respectively. The transcription of the emrA gene was found to be inducible by flavonoids, including luteolin and apigenin, which are known inducers of the nodulation genes in S. meliloti. Interestingly, quercetin, which does not induce nodulation genes, was also a potent inducer of emrA, indicating that NodD is not directly involved in regulation of emrA. The likely regulator of emrAB is EmrR, which binds to palindrome-like sequences in the intergenic region. Several modifications of the palindromes, including an increase of the spacing between the two half sites, prevented binding of EmrR. Binding was also impaired by the presence of luteolin. Mutations in emrA had no obvious effect on symbiosis. This was in contrast to the emrR mutant, which exhibited a symbiotic deficiency with Medicago sativa. Conserved binding sites for TetR-like regulators within the intergenic regions between the emrAB and emrR genes were identified in many symbiotic and pathogenic members of the order Rhizobiales. PMID:24224534

  7. Individual- and stand-level Stem CO2 efflux in a subtropical Schima superba plantation

    NASA Astrophysics Data System (ADS)

    Zhu, L. W.; Zhao, P.; Ni, G. Y.; Cao, Q. P.; Zhou, C. M.; Zeng, X. P.

    2012-03-01

    Stem CO2 efflux was investigated with an open gas exchange system while stand microclimate and stem temperature were continuously monitored in a Schima superba plantation in South China for several days in August and December, 2010. The temperature response of respiration over the different seasons, the vertical variation in stem CO2 efflux along the stem and the stand-level stem CO2 efflux were examined. Stem volume was identified as the better correlate for stem CO2 efflux and was used as scaling scalar for the stand-level estimates of stem CO2 efflux in this S. superba plantation. Volume-based stem CO2 efflux was higher at 2 m than at 1.3 m. Mean stem CO2 efflux was 268.9 and 104.6 μmol m-3 s-1 in August and December, respectively, indicating a dramatic seasonal variation of stem CO2 efflux. The temperature response of stem CO2 efflux was constant during our study period with Q10 values of 1.9 and 1.8. In this subtropical S. superba plantation, the averaged stem CO2 efflux per unit ground area was 3.36 and 1.26 μmol m-2 s-1 in August and December, respectively, which was underestimated due to the vertical variation of stem CO2 efflux along the stem. Our results suggest that stem CO2 efflux has a constant temperature response on the stand scale, and the seasonal variation in stem CO2 efflux is mainly controlled by stem temperature, and the vertical variation in stem CO2 efflux needs to be considered at the stand-level estimation.

  8. Carbon Dioxide Efflux from Soil with Poultry Litter Applications in Conventional and Conservation Tillage Systems in Northern Alabama

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increased carbon dioxide release from soils resulting from agricultural practices such as tillage and application of poultry litter in cotton production systems has generated concerns about contributions to global warming which negatively impacts natural ecosystems, weather patterns, and food pro...

  9. 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. PMID:26772887

  10. Clinically Relevant Chromosomally Encoded Multidrug Resistance Efflux Pumps in Bacteria

    PubMed Central

    Piddock, Laura J. V.

    2006-01-01

    Efflux pump genes and proteins are present in both antibiotic-susceptible and antibiotic-resistant bacteria. Pumps may be specific for one substrate or may transport a range of structurally dissimilar compounds (including antibiotics of multiple classes); such pumps can be associated with multiple drug (antibiotic) resistance (MDR). However, the clinical relevance of efflux-mediated resistance is species, drug, and infection dependent. This review focuses on chromosomally encoded pumps in bacteria that cause infections in humans. Recent structural data provide valuable insights into the mechanisms of drug transport. MDR efflux pumps contribute to antibiotic resistance in bacteria in several ways: (i) inherent resistance to an entire class of agents, (ii) inherent resistance to specific agents, and (iii) resistance conferred by overexpression of an efflux pump. Enhanced efflux can be mediated by mutations in (i) the local repressor gene, (ii) a global regulatory gene, (iii) the promoter region of the transporter gene, or (iv) insertion elements upstream of the transporter gene. Some data suggest that resistance nodulation division systems are important in pathogenicity and/or survival in a particular ecological niche. Inhibitors of various efflux pump systems have been described; typically these are plant alkaloids, but as yet no product has been marketed. PMID:16614254

  11. Case report: A novel apolipoprotein A-I missense mutation apoA-I (Arg149Ser)Boston associated with decreased lecithin-cholesterol acyltransferase activation and cellular cholesterol efflux.

    PubMed

    Anthanont, Pimjai; Asztalos, Bela F; Polisecki, Eliana; Zachariah, Benoy; Schaefer, Ernst J

    2015-01-01

    We report a novel heterozygous apolipoprotein A-I (apoA-I) missense mutation (c.517C>A, p.Arg149Ser, designated as apoA-IBoston) in a 67-year-old woman and her 2 sons, who had mean serum high-density lipoprotein (HDL) cholesterol, apoA-I, and apoA-I in very large α-1 HDL that were 10%, 35%, and 16% of normal, respectively (all P < .05). The percentage of HDL cholesterol in the esterified form was also significantly (P < .05) reduced to 52% of control values. Cholesteryl ester tranfer protein (CETP) activity was normal. The mean global, adenosine triphosphate (ATP)-binding cassette transporter A1 and scavenger receptor B type I-mediated cellular cholesterol efflux capacity in apoB-depleted serum from affected family members were 41%, 37%, 47%, 54%, and 48% of control values, respectively (all P < .05). lecithin-cholesterol acyltransferase (LCAT) activity in plasma was 71% of controls, whereas in the cell-based assay, it was 73% of control values (P < .05). The data indicate that this novel apoA-I missense is associated with markedly decreased levels of HDL cholesterol and very large α-1 HDL, as well as decreased serum cellular cholesterol efflux and LCAT activity, but not with premature coronary heart disease, similar to other apoA-I mutations that have been associated with decreased LCAT activity. PMID:26073399

  12. Effects of Elevated CO2 Atmospheric CO2 on Soil Efflux in Conventional and Conservation Cropping Systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Elevated atmospheric CO2 can affect both the quantity and quality of plant tissues, which will impact the cycling and storage of carbon within plant/soil systems and the rate of CO2 release back to the atmosphere. Research is needed to more accurately quantify the effects of elevated CO2 on soil CO...

  13. Effects of Elevated Atmospheric CO2 on Soil CO2 Efflux in Conventional and Conservation Cropping Systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Elevated atmospheric carbon dioxide (CO2) can affect both the quantity and quality of plant tissues produced, which will impact the cycling and storage of carbon (C) within plant/soil systems and thus the rate of CO2 release back to the atmosphere. Research is needed to more accurately quantify the...

  14. Cholesterol efflux is LXRα isoform-dependent in human macrophages

    PubMed Central

    2014-01-01

    Background The nuclear receptor liver X receptor (LXR) has two isoforms: LXRα and LXRβ. LXR activation promotes cholesterol efflux in macrophages, but the relative importance of each LXR isoform in mediating cholesterol efflux remains elusive. Methods We evaluated the ability of different doses of LXRs agonist T0901317 to affect cholesterol efflux in human macrophages and its relationship with mRNA and protein levels of several well-characterized proteins involved in cholesterol efflux, including ABCA1, ABCG1, SR-BI, LXRβ and LXRα, using quantitative real-time PCR, Western blotting, and siRNA techniques. Results Here we show that LXRα rather than LXRβ sustains baseline cholesterol efflux in human blood-derived macrophages. Treatment of human macrophages with a non-isoform-specific LXR agonist T0901317 substantially increased HDL- and apoA-I-mediated cholesterol efflux, which was associated with increased mRNA and protein expression levels of ABCA1, ABCG1, SR-BI, LXRα and LXRβ. The siRNA- mediated silencing of LXRα, but not LXRβ significantly reduced the protein levels of ABCA1,ABCG1, and SR-BI as wellas HDL- and ApoA1-mediated cholesterol in human macrophages. Conclusions These findings imply that LXRα- rather than LXRβ- specific agonists may promote reverse cholesterol transport in humans. PMID:24996838

  15. HCO3-stimulated Cl efflux in the gulf toadfish acclimated to sea water.

    PubMed

    Kormanik, G A; Evans, D H

    1979-04-01

    Unidirectional efflux of Cl was examined in the Gulf toadfish, Opsanus beta, in artificial seawater solutions with modified concentrations of Cl and HCO3. Removal of Cl HCO3 reduced Cl efflux. Addition of HCO3 at typical seawater concentrations stimulated Cl efflux, independent of changes in the transepithelial potential. This active, HCO3-stimulated Cl efflux is saturable, with a Km of 2.4 mM, typical of the concentration of HCO3 found in sea water, and independent of external pH. Active extrusion of Cl offsets the net diffusional and oral gain of Cl faced by O. beta in sea water. PMID:469477

  16. Current Advances in Developing Inhibitors of Bacterial Multidrug Efflux Pumps.

    PubMed

    Mahmood, Hannah Y; Jamshidi, Shirin; Sutton, J Mark; Rahman, Khondaker M

    2016-01-01

    Antimicrobial resistance represents a significant challenge to future healthcare provision. An acronym ESKAPEE has been derived from the names of the organisms recognised as the major threats although there are a number of other organisms, notably Neisseria gonorrhoeae, that have become equally challenging to treat in the clinic. These pathogens are characterised by the ability to rapidly develop and/or acquire resistance mechanisms in response to exposure to different antimicrobial agents. A key part of the armoury of these pathogens is a series of efflux pumps, which effectively exclude or reduce the intracellular concentration of a large number of antibiotics, making the pathogens significantly more resistant. These efflux pumps are the topic of considerable interest, both from the perspective of basic understanding of efflux pump function, and its role in drug resistance but also as targets for the development of novel adjunct therapies. The necessity to overcome antimicrobial resistance has encouraged investigations into the characterisation of resistance-modifying efflux pump inhibitors to block the mechanisms of drug extrusion, thereby restoring antibacterial susceptibility and returning existing antibiotics into the clinic. 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. This review discusses different bacterial EPIs originating from both natural source and chemical synthesis and examines the challenges to designing successful EPIs that can be useful against multidrug resistant bacteria. PMID:26947776

  17. 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

  18. Individual- and stand-level stem CO2 efflux in a subtropical Schima superba plantation

    NASA Astrophysics Data System (ADS)

    Zhu, L. W.; Zhao, P.; Ni, G. Y.; Cao, Q. P.; Zhou, C. M.; Zeng, X. P.

    2012-10-01

    Stem respiration is an important, but poorly studied component of total forest ecosystem respiration. Stem CO2 efflux was investigated with an open gas exchange system while stand microclimate and stem temperature were continuously monitored in a Schima superba plantation in South China for several days in August and December 2010. The temperature response of respiration in the different seasons, the vertical variation in stem CO2 efflux along the stem, and the stand-level stem CO2 efflux were examined. Stem volume was identified as the better correlate for stem CO2 efflux and was used as the scalar for the stand-level estimates of stem CO2 efflux in this S. superba plantation. Volume-based stem CO2 efflux was higher at 2 m than at 1.3 m. Mean stem CO2 efflux was 268.9 and 104.6 μmol m-3 s-1 in August and December, respectively, indicating a dramatic seasonal variation of stem CO2 efflux. The temperature response of stem CO2 efflux remained constant during our study period with Q10 values of 1.9 and 1.8. In this subtropical S. superba plantation, stem CO2 efflux per unit ground area averaged 3.36 and 1.26 μmol m-2 s-1 based on the measurement data at 1.3-m height of the stem in August and December, respectively. Our results suggest that stem CO2 efflux has a constant temperature response, and the seasonal variation in stem CO2 efflux is mainly controlled by stem temperature, and the vertical variation in stem CO2 efflux needs to be considered in the stand-level estimation.

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

    PubMed

    Kapoor, Vikram; Wendell, David

    2013-05-01

    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. PMID:23581993

  20. Nanostructured lipid-carrageenan hybrid carriers (NLCCs) for controlled delivery of mitoxantrone hydrochloride to enhance anticancer activity bypassing the BCRP-mediated efflux.

    PubMed

    Ling, Guixia; Zhang, Tianhong; Zhang, Peng; Sun, Jin; He, Zhonggui

    2016-08-01

    Novel nanostructured lipid-carrageenan hybrid carriers (NLCCs) were exploited for controlled delivery of water soluble chemotherapeutic agent mitoxantrone hydrochloride (MTO) with high loading capacity, sustained release property, and potential for improving oral bioavailability and antitumor efficacy. By introducing the negative polymer of carrageenan, MTO was highly incorporated into NLCCs with encapsulation efficiency of 95.8% by electrostatic interaction. In vivo pharmacokinetics of MTO solution (MTO-Sol) and MTO-NLCCs in rats demonstrated that the apparent bioavailability of MTO-NLCCs was increased to approximate 3.5-fold compared to that of MTO-Sol. The cytotoxicity investigations by MTT method indicated that NLCCs could significantly enhanced the antitumor efficacy against resistant MCF-7/MX cells. The relative cellular association of MTO-NLCCs was 9.2-fold higher than that of MTO-Sol in breast cancer resistance protein (BCRP) over-expressing MCF-7/MX cells, implying that BCRP-mediated drug efflux was diminished by the introduction of NLCCs. The endocytosis inhibition study implied that the NLCCs entered the MCF-7/MX cells by clathrin-mediated endocytosis process, which can bypass the efflux of MTO mediated by BCRP. The new developed NLCCs provide an effective strategy for oral delivery of water-soluble MTO with improved encapsulation efficiency, oral bioavailability, and cytotoxicity against resistant breast cancer cells. PMID:26754913

  1. 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

  2. Pharmacophore generation of 2-substituted benzothiazoles as AdeABC efflux pump inhibitors in A. baumannii.

    PubMed

    Yilmaz, S; Altinkanat-Gelmez, G; Bolelli, K; Guneser-Merdan, D; Over-Hasdemir, M U; Yildiz, I; Aki-Yalcin, E; Yalcin, I

    2014-01-01

    RND family efflux pumps are important for multidrug resistance in Gram-negative bacteria. To date no efflux pump inhibitors for clinical use have been found, so developing the specific inhibitors of this pump system will be beneficial for the treatment of infections caused by these multidrug-resistant pathogens. A set of BSN-coded 2-substituted benzothiazoles were tested alone and in combination with ciprofloxacin (CIP) against the RND family efflux pump AdeABC overexpressor Acinetobacter baumannii SbMox-2 strain. The results indicated that the BSN compounds did not have antimicrobial activity when tested alone. However, if they were applied in combination with CIP, it was observed that the antibiotic had antimicrobial activity against the tested pathogen, possessing a minimum inhibitory concentration value that could be utilized in clinical treatment. A 3D-common features pharmacophore model was applied by using the HipHop method and the generated pharmacophore hypothesis revealed that the hydrogen bond acceptor property of nitrogen in the thiazole ring and the oxygen of the amide substituted at the second position of the benzothiazole ring system were significant for binding to the target protein. Moreover, three hydrophobic aromatic features were found to be essential for inhibitory activity. PMID:24905472

  3. Low-Level Resistance of Staphylococcus aureus to Thrombin-Induced Platelet Microbicidal Protein 1 In Vitro Associated with qacA Gene Carriage Is Independent of Multidrug Efflux Pump Activity

    PubMed Central

    Bayer, A. S.; Kupferwasser, L. I.; Brown, M. H.; Skurray, R. A.; Grkovic, S.; Jones, T.; Mukhopadhay, K.; Yeaman, M. R.

    2006-01-01

    Thrombin-induced platelet microbial protein 1 (tPMP-1), a cationic antimicrobial polypeptide released from thrombin-stimulated rabbit platelets, targets the Staphylococcus aureus cytoplasmic membrane to initiate its microbicidal effects. In vitro resistance to tPMP-1 correlates with survival advantages in vivo. In S. aureus, the plasmid-carried qacA gene encodes a multidrug transporter, conferring resistance to organic cations (e.g., ethidium [Et]) via proton motive force (PMF)-energized export. We previously showed that qacA also confers a tPMP-1-resistant (tPMP-1r) phenotype in vitro. The current study evaluated whether (i) transporters encoded by the qacB and qacC multidrug resistance genes also confer tPMP-1r and (ii) tPMP-1r mediated by qacA is dependent on efflux pump activity. In contrast to tPMP-1r qacA-bearing strains, the parental strain and its isogenic qacB- and qacC-containing strains were tPMP-1 susceptible (tPMP-1s). Efflux pump inhibition by cyanide m-chlorophenylhydrazone abrogated Etr, but not tPMP-1r, in the qacA-bearing strain. In synergy assays, exposure of the qacA-bearing strain to tPMP-1 did not affect the susceptibility of Et (ruling out Et-tPMP-1 cotransport). The following cytoplasmic membrane parameters did not differ significantly between the qacA-bearing and parental strains: contents of the major phospholipids; asymmetric distributions of the positively charged species, lysyl-phosphotidylglycerol; fatty acid composition; and relative surface charge. Of note, the qacA-bearing strain exhibited greater membrane fluidity than that of the parental, qacB-, or qacC-bearing strain. In conclusion, among these families of efflux pumps, only the multidrug transporter encoded by qacA conferred a tPMP-1r phenotype. These data suggest that qacA-encoded tPMP-1r results from the impact of a specific transporter upon membrane structure or function unrelated to PMF-dependent peptide efflux. PMID:16801425

  4. The MexJK Efflux Pump of Pseudomonas aeruginosa Requires OprM for Antibiotic Efflux but Not for Efflux of Triclosan

    PubMed Central

    Chuanchuen, Rungtip; Narasaki, Craig T.; Schweizer, Herbert P.

    2002-01-01

    Using the biocide triclosan as a selective agent, several triclosan-resistant mutants of a susceptible Pseudomonas aeruginosa strain were isolated. Cloning and characterization of a DNA fragment conferring triclosan resistance from one of these mutants revealed a hitherto uncharacterized efflux system of the resistance nodulation cell division (RND) family, which was named MexJK and which is encoded by the mexJK operon. Expression of this operon is negatively regulated by the product of mexL, a gene located upstream of and transcribed divergently from mexJK. The triclosan-resistant mutant contained a single nucleotide change in mexL, which caused an amino acid change in the putative helix-turn-helix domain of MexL. The MexL protein belongs to the TetR family of repressor proteins. The MexJK system effluxed tetracycline and erythromycin but only in the presence of the outer membrane protein channel OprM; OprJ and OprN did not function with MexJK. Triclosan efflux required neither of the outer membrane protein channels tested but necessitated the MexJ membrane fusion protein and the MexK inner membrane RND transporter. The results presented in this study suggest that MexJK may function as a two-component RND pump for triclosan efflux but must associate with OprM to form a tripartite antibiotic efflux system. Furthermore, the results confirm that triclosan is an excellent tool for the study of RND multidrug efflux systems and that this popular biocide therefore readily selects mutants which are cross-resistant with antibiotics. PMID:12193619

  5. Regulation of ABC efflux transporters at blood-brain barrier in health and neurological disorders.

    PubMed

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

    2015-12-01

    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. This article is part of a Special Issue entitled SI: Neuroprotection. PMID:26187753

  6. 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

  7. Cysteines Introduced into Extracellular Loops 1 and 4 of Human P-Glycoprotein That Are Close Only in the Open Conformation Spontaneously Form a Disulfide Bond That Inhibits Drug Efflux and ATPase Activity*

    PubMed Central

    Loo, Tip W.; Clarke, David M.

    2014-01-01

    P-glycoprotein (P-gp) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. The protein is organized into two halves. The halves contain a transmembrane domain (TMD) with six transmembrane segments and a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the TMD1/TMD2 and NBD1/NBD2 interfaces, respectively. ATP-dependent drug efflux involves changes between the open inward-facing (NBDs apart, extracellular loops (ECLs) close together) and the closed outward-facing (NBDs close together, ECLs apart) conformations. It is controversial, however, whether the open conformation only exists transiently in intact cells because of the presence of high levels of ATP. To test for the presence of an open conformation in intact cells, reporter cysteines were placed in extracellular loops 1 (A80C, N half) and 4 (R741C, C half). The rationale was that cysteines A80C/R741C would only come close enough to form a disulfide bond in an open conformation (6.9 Å apart) because they are separated widely (30.4 Å apart) in the closed conformation. It was observed that the mutant A80C/R741C cross-linked spontaneously (>90%) when expressed in cells. In contrast to previous reports showing that trapping P-gp in a closed conformation highly activated ATPase activity, here we show that A80C/R741C cross-linking inhibited ATPase activity and drug efflux. Both activities were restored when the cross-linked mutant was treated with a thiol-reducing agent. The results show that an open conformation can be readily detected in cells and that cross-linking of cysteines placed in ECLs 1 and 4 inhibits activity. PMID:25053414

  8. The role of efflux pumps in macrolide resistance in Mycobacterium avium complex.

    PubMed

    Rodrigues, Liliana; Sampaio, Daniela; Couto, Isabel; Machado, Diana; Kern, Winfried V; Amaral, Leonard; Viveiros, Miguel

    2009-12-01

    Mycobacteriumavium complex (MAC) is clinically important since it can cause severe infections in acquired immune deficiency syndrome (AIDS) patients and other immunocompromised individuals. Use of the macrolides clarithromycin and azithromycin has improved the outcome of MAC infections, but therapeutic failure is still a major problem. In this work, we studied efflux pump activity in MAC clinical strains and evaluated the contribution of active efflux to macrolide resistance. Eighteen clinical strains isolated from AIDS patients were evaluated for macrolide resistance in the presence and absence of the efflux pump inhibitors (EPIs) thioridazine, chlorpromazine and verapamil. The efflux activity of these strains was then assessed by a semi-automated fluorometric method that detects extrusion of ethidium bromide (EtBr), a known efflux pump substrate. Resistance to clarithromycin was significantly reduced in the presence of thioridazine, chlorpromazine and verapamil. The same EPIs were effective in decreasing the efflux of EtBr from MAC cells. Moreover, increased retention of [(14)C]-erythromycin in the presence of these EPIs further demonstrated that active efflux contributes to MAC resistance to macrolides. This study demonstrates that efflux pumps play an important role in MAC resistance to antibiotics. PMID:19740629

  9. Tripartite assembly of RND multidrug efflux pumps

    PubMed Central

    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

    2016-01-01

    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. PMID:26867482

  10. 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.

  11. 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. PMID:27111147

  12. 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

  13. 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

  14. Some effects of way-120,491 on electrical and mechanical activity and on sup 42 K/ sup 85 Rb efflux in rat blood vessels

    SciTech Connect

    Edwards, G.; Weston, A.H. ); Oshiro, G. )

    1990-02-26

    The effects of WAY-120,491 (-)-(3A,4R-trans)-2-(2,3-dihydro-3-hydroxy-2,2-dimethyl-6-(trifluoromethoxy)-2H-1-benzopyran-4-yl)-2,3-dihydro-1H-isoindol-1-one have been examined in vitro using portal veins and aortic segments from male Sprague-Dawley rats. In aorta, WAY-120,491 (3.3-33nM) produced a dose-dependent relaxation of 20mM KC1 contractions, an effect antagonized by glibenclamide. WAY-120,491 had no effect on tension induced by 80mM CK1. When segments of aorta were loaded with {sup 86}Rb and {sup 42}K, WAY-120,491 (330nM-33{mu}M) increased the efflux of both isotopes, an effect antagonized by glibenclamide. In portal vein, WAY 120-491 (3.3-330nM) inhibited spontaneous tension development by abolishing electrical multispike complexes. In tissues loaded with {sup 86}Rb, WAY-120,491 (330nM-33{mu}M) increased the rate of {sup 86}Rb exchange. It is concluded that these effects of WAY-120,491 in rat blood vessels are consistent with the opening of plasmalemmal K-channels and that WAY-120,491 belongs to the pharmacological grouping known as the K-channel openers.

  15. 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.

  16. 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. PMID:23183443

  17. 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 Central

    Kothary, Vishesh; Scherl, Ellen J.; Bosworth, Brian; Jiang, Zhi-Dong; DuPont, Herbert L.; Harel, Josee

    2013-01-01

    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. PMID:23183443

  18. Characteristics of spatial variability in soil CO2 efflux in a Moso bamboo (Phyllostachys pubescens) forest

    NASA Astrophysics Data System (ADS)

    Lin, Meng Ying; Hsieh, Yi-Fang; Kume, Tomonori; Cheng, Chih-Hsin

    2014-05-01

    The expansion of bamboo forest to surrounding ecosystems in eastern Asian countries such as Taiwan can alter the carbon balance, in which soil CO2 efflux is an essential component. Spatial heterogeneity of soil CO2 efflux in forested ecosystems is essential not only for understanding CO2 dynamics but also for suitable sampling design to estimate annual soil CO2 efflux and the response to environmental changes. The aim of this study is to understand characteristics of spatial variability of soil CO2 efflux in a bamboo forest, situated in a montane cloud forest of central Taiwan. To this end, this study 1) evaluated the seasonal changes in spatial variability in soil respiration in Moso bamboo (Phyllostachys pubescens) forest through one year and 2) quantified the spatial variation of soil CO2 efflux in this stand using the semivariance. We measured soil CO2 efflux using the closed dynamic chamber method with an infrared gas analyzer (PP system, EGM-4) once a month from April 2012 to November 2013. The semivariance was used to quantify the spatial variability of soil CO2 efflux. In this study, we found spatially averaged soil CO2 efflux during each measurement campaign varied from 1.54 to 4.98 µmol CO2 m-2 s-1, which was larger in bamboo sprouting period (April to August) than other months (September to March). The average coefficient of variation (CV) of the soil CO2 effluxes was 46.4%. Our CV was comparable to that in other tropical forests. The semivariogram revealed that there was autocorrelation of soil CO2 effluxes, and the scale was significantly different between bamboo sprouting period and the other periods. The spatial distribution map showed temporal changes in soil CO2 efflux, and the fairly conservative spatial patterns in soil CO2 effluxes were found through the year in our site. On the other the hand, the range of autocorrelation was much larger in April (33.4 m - 123 m) than other months (2.4m - 5.5m). This indicated that considering a long distance

  19. 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

  20. 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. PMID:27225657

  1. Spatial variation in spring CO2 efflux along the trans-Alaska pipeline, Alaska: Contribution of spring carbon

    NASA Astrophysics Data System (ADS)

    Kim, Y.

    2013-12-01

    Spring soil CO2 efflux-measurement was conducted in representative sites along the trans-Alaska pipeline during 2010 to 2012 for the understanding of spatial variation in spring CO2 efflux response to change in snow-melting timing. The sites is 3 tundra sites (coastal tundra, upland tundra, upland tundra, and sub-alpine tundra), 2 white spruce sites in tundra-boreal forest ecotone, and Gold Creek, and 3 black spruce sites in Coldfoot, upper and lower reaches of the Yukon River. Soil CO2 efflux-measurement, which is a portable manual chamber CO2 efflux system, was conducted during snow-covered and snow-melting periods, minimizing artificial effects. CO2 effluxes in snow-covered and exposed soils showed a significantly difference, suggesting that spring CO2 efflux is much higher than that in snow-covered soil. The efflux was measured at 4-direction due to the difference of exposed extent, implying the magnitude of CO2 production. Average diameter in breast height (DBH: 85 × 11 cm) of white spruce is much thicker than black spruce (DBH: 33 × 5 cm), suggesting the difference of heat uptake and emission capacity between both forests. Soil temperature at 5 cm below the surface is one of significant keys in determining soil CO2 efflux. The magnitude of spring CO2 efflux showed white spruce, black spruce, and tundra in turn, suggesting that spring CO2 efflux (> 8 gC/m2/day) of corresponds to summer soil CO2 efflux. Then, spring soil CO2 efflux should be not overlooked the contribution of annual soil carbon efflux in spite of difficulties in snow-disappeared timing and springtime.

  2. 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.

  3. 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

  4. 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. PMID:25462452

  5. Multidrug efflux pumps of Gram-positive bacteria.

    PubMed

    Schindler, Bryan D; Kaatz, Glenn W

    2016-07-01

    Gram-positive organisms are responsible for some of the most serious of human infections. Resistance to front-line antimicrobial agents can complicate otherwise curative therapy. These organisms possess multiple drug resistance mechanisms, with drug efflux being a significant contributing factor. Efflux proteins belonging to all five transporter families are involved, and frequently can transport multiple structurally unrelated compounds resulting in a multidrug resistance (MDR) phenotype. In addition to clinically relevant antimicrobial agents, MDR efflux proteins can transport environmental biocides and disinfectants which may allow persistence in the healthcare environment and subsequent acquisition by patients or staff. Intensive research on MDR efflux proteins and the regulation of expression of their genes is ongoing, providing some insight into the mechanisms of multidrug recognition and transport. Inhibitors of many of these proteins have been identified, including drugs currently being used for other indications. Structural modifications guided by structure-activity studies have resulted in the identification of potent compounds. However, lack of broad-spectrum pump inhibition combined with potential toxicity has hampered progress. Further work is required to gain a detailed understanding of the multidrug recognition process, followed by application of this knowledge in the design of safer and more highly potent inhibitors. PMID:27449594

  6. Efflux transporter engineering markedly improves amorphadiene production in Escherichia coli.

    PubMed

    Zhang, Congqiang; Chen, Xixian; Stephanopoulos, Gregory; Too, Heng-Phon

    2016-08-01

    Metabolic engineering aims at altering cellular metabolism to produce valuable products at high yields and titers. Achieving high titers and productivity can be challenging if final products are largely accumulated intracellularly. A potential solution to this problem is to facilitate the export of these substances from cells by membrane transporters. Amorphadiene, the precursor of antimalarial drug artemisinin, is known to be secreted from Escherichia coli overexpressing the biosynthetic pathway. In order to assess the involvement of various endogenous efflux pumps in amorphadiene transport, the effects of single gene deletion of 16 known multidrug-resistant membrane efflux transporters were examined. The outer membrane protein TolC was found to be intimately involved in amorphadiene efflux. The overexpression of tolC together with ABC family transporters (macAB) or MFS family transporters (emrAB or emrKY) enhanced amorphadiene titer by more than threefold. In addition, the overexpression of transporters in the lipopolysaccharide transport system (msbA, lptD, lptCABFG) was found to improve amorphadiene production. As efflux transporters often have a wide range of substrate specificity, the multiple families of transporters were co-expressed and synergistic benefits were observed in amorphadiene production. This strategy of screening and then rationally engineering transporters can be used to improve the production of other valuable compounds in E. coli. Biotechnol. Bioeng. 2016;113: 1755-1763. © 2016 Wiley Periodicals, Inc. PMID:26804325

  7. Harmine augments electrically evoked dopamine efflux in the nucleus accumbens shell.

    PubMed

    Brierley, Daniel I; Davidson, Colin

    2013-01-01

    Harmine is a β-carboline alkaloid and major component of ayahuasca, a traditional South American psychoactive tea with anecdotal efficacy for treatment of cocaine dependence. Harmine is an inhibitor of monoamine oxidase A (MAO-A) and interacts in vitro with several pharmacological targets which modulate dopamine (DA) neurotransmission. In vivo studies have demonstrated dopaminergic effects of harmine, attributed to monoamine oxidase inhibitor (MAOI) activity, however none have directly demonstrated a pharmacological mechanism. This study investigated the acute effects, and pharmacological mechanism(s), of harmine on electrically evoked DA efflux parameters in the nucleus accumbens both in the absence and presence of cocaine. Fast cyclic voltammetry in rat brain slices was used to measure electrically evoked DA efflux in accumbens core and shell. Harmine (300 nM) significantly augmented DA efflux (148±8% of baseline) in the accumbens shell. Cocaine augmented efflux in shell additive to harmine (260±35%). Harmine had no effect on efflux in the accumbens core or on reuptake in either sub-region. The effect of harmine in the shell was attenuated by the 5-HT(2A/2C) antagonist ketanserin. The MAOI moclobemide (10 µM) had no effect on DA efflux. These data suggest that harmine augments DA efflux via a novel, shell-specific, presynaptic 5-HT(2A) receptor-dependent mechanism, independent of MAOI activity. A DA-releasing 'agonist therapy' mechanism may thus contribute to the putative therapeutic efficacy of ayahuasca for cocaine dependence. PMID:23076833

  8. 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

  9. Cultured human vascular smooth muscle cells with functional thromboxane A2 receptors: measurement of U46619-induced /sup 45/calcium efflux

    SciTech Connect

    Dorn, G.W. II; Sens, D.; Chaikhouni, A.; Mais, D.; Halushka, P.V.

    1987-06-01

    Thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) are potent vasoconstrictors whose contractile effects are mediated by increases in cellular calcium. Stable analogues of these compounds have shown calcium ionophore activity at high concentrations. To determine if effects of TXA2/PGH2 analogues on /sup 45/Ca/sup 2 +/ fluxes are receptor mediated, the effects of the stable TXA2/PGH2 mimetic U46619 and the TXA2/PGH2 receptor antagonist I-PTA-OH on /sup 45/Ca/+ fluxes in cultured human vascular smooth muscle cells were studied. The smooth muscle cells were cultured from human saphenous vein explants, and they retained the morphologic and immunologic characteristics of vascular smooth muscle cells. U46619 stimulated /sup 45/Ca/sup 2 +/ efflux in a dose-dependent manner with an EC50 of 398 +/- 26 nM (n = 4). The maximal /sup 45/Ca/sup 2 +/ efflux in response to U46619 (5 microM) was significantly greater (p = 0.006) than the /sup 45/Ca/sup 2 +/ efflux induced by KCl (40 mM). I-PTA-OH inhibited the U46619-induced /sup 45/Ca/sup 2 +/ efflux but had no effect on KCl-induced /sup 45/Ca/sup 2 +/ efflux. These results suggest that the effects of U46619 in increasing vascular smooth muscle cell calcium efflux are receptor mediated. Furthermore, vascular smooth muscle cells with functional TXA2/PGH2 receptors were cultured from human saphenous veins and provide a potentially useful in vitro system for the further study of TXA2/PGH2 receptor-mediated phenomena in human vascular tissue.

  10. Mechanism of ligand-gated potassium efflux in bacterial pathogens

    PubMed Central

    Roosild, Tarmo P.; Castronovo, Samantha; Healy, Jess; Miller, Samantha; Pliotas, Christos; Rasmussen, Tim; Bartlett, Wendy; Conway, Stuart J.; Booth, Ian R.

    2010-01-01

    Gram negative pathogens are protected against toxic electrophilic compounds by glutathione-gated potassium efflux systems (Kef) that modulate cytoplasmic pH. We have elucidated the mechanism of gating through structural and functional analysis of Escherichia coli KefC. The revealed mechanism can explain how subtle chemical differences in glutathione derivatives can produce opposite effects on channel function. Kef channels are regulated by potassium transport and NAD-binding (KTN) domains that sense both reduced glutathione, which inhibits Kef activity, and glutathione adducts that form during electrophile detoxification and activate Kef. We find that reduced glutathione stabilizes an interdomain association between two KTN folds, whereas large adducts sterically disrupt this interaction. F441 is identified as the pivotal residue discriminating between reduced glutathione and its conjugates. We demonstrate a major structural change on the binding of an activating ligand to a KTN-domain protein. Analysis of the regulatory interactions suggests strategies to disrupt pathogen potassium and pH homeostasis. PMID:21041667

  11. 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

  12. 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.

  13. 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.

  14. Solar cell activation system

    SciTech Connect

    Apelian, L.

    1983-07-05

    A system for activating solar cells involves the use of phosphorescent paint, the light from which is amplified by a thin magnifying lens and used to activate solar cells. In a typical system, a member painted with phosphorescent paint is mounted adjacent a thin magnifying lens which focuses the light on a predetermined array of sensitive cells such as selenium, cadmium or silicon, mounted on a plastic board. A one-sided mirror is mounted adjacent the cells to reflect the light back onto said cells for purposes of further intensification. The cells may be coupled to rechargeable batteries or used to directly power a small radio or watch.

  15. 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. PMID:27242375

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

    PubMed

    Rasgado-Flores, H; Santiago, E M; Blaustein, M P

    1989-06-01

    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

  17. Novel effect of paeonol on the formation of foam cells: promotion of LXRα-ABCA1-dependent cholesterol efflux in macrophages.

    PubMed

    Zhao, Jin-Feng; Jim Leu, Shr-Jeng; Shyue, Song-Kun; Su, Kuo-Hui; Wei, Jeng; Lee, Tzong-Shyuan

    2013-01-01

    Paeonol, a phenolic component purified from Paeonia suffruticosa (Cortex Moutan), is used in traditional Chinese medicine to treat inflammatory diseases. However, little is known about the effect of paeonol on cholesterol metabolism. We investigated the efficacy of paeonol on cholesterol metabolism and the underlying mechanism in macrophages and apolipoprotein E deficient (apoE(-/-)) mice. Treatment with paeonol markedly attenuated cholesterol accumulation induced by oxidized LDL in macrophages, which was due to increased cholesterol efflux. Additionally, paeonol enhanced the mRNA and protein expression of ATP-binding membrane cassette transport protein A1 (ABCA1) but did not alter the protein level of ABCG1 or other scavenger receptors. Inhibition of ABCA1 activity with a pharmacological inhibitor, neutralizing antibody or small interfering RNA (siRNA), negated the effects of paeonol on cholesterol efflux and cholesterol accumulation. Furthermore, paeonol induced the nuclear translocation of liver X receptor α (LXRα) by increasing its activity. siRNA knockdown of LXRα abolished the paeonol-induced upregulation of ABCA1, promotion of cholesterol efflux and suppression of cholesterol accumulation. Moreover, atherosclerotic lesions, hyperlipidemia and systemic inflammation were reduced and the protein expression of ABCA1 was increased in aortas of paeonol-treated apoE(-/-) mice. Paeonol may alleviate the formation of foam cells by enhancing LXRα-ABCA1-dependent cholesterol efflux. PMID:24117070

  18. Bovicin HC5, a lantibiotic produced by Streptococcus bovis HC5, catalyzed the efflux of intracellular potassium but not ATP

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovicin HC5, a broad spectrum lantibiotic produced by Streptococcus bovis HC5, catalyzed the efflux of intracellular potassium from Streptococcus bovis JB1, a sensitive strain. ATP also decreased, but this decline appeared to be caused by the activity of the F1FO ATPase rather than efflux per se....

  19. Multidrug Efflux Pumps in the Genus Erwinia: Physiology and Regulation of Efflux Pump Gene Expression.

    PubMed

    Thekkiniath, J; Ravirala, R; San Francisco, M

    2016-01-01

    Plant pathogens belonging to the genus Erwinia cause diseases in several economically important plants. Plants respond to bacterial infection with a powerful chemical arsenal and signaling molecules to rid themselves of the microbes. Although our understanding of how Erwinia initiate infections in plants has become clear, a comprehensive understanding of how these bacteria rid themselves of noxious antimicrobial agents during the infection is important. Multidrug efflux pumps are key factors in bacterial resistance toward antibiotics by reducing the level of antimicrobial compounds in the bacterial cell. Erwinia induce the expression of efflux pump genes in response to plant-derived antimicrobials. The capability of Erwinia to co-opt plant defense signaling molecules such as salicylic acid to trigger multidrug efflux pumps might have developed to ensure bacterial survival in susceptible host plants. In this review, we discuss the developments in Erwinia efflux pumps, focusing in particular on efflux pump function and the regulation of efflux pump gene expression. PMID:27571694

  20. 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…

  1. 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…

  2. Oxysterols trigger ABCA1-mediated basolateral surfactant efflux.

    PubMed

    Agassandian, Marianna; Mathur, Satya N; Zhou, Jiming; Field, F Jeffrey; Mallampalli, Rama K

    2004-08-01

    Surfactant is an apically-secreted surface-active material containing primarily disaturated phosphatidylcholine (DSPtdCho) that is released from alveolar epithelia into the alveolus. Surfactant deficiency is an important aspect of inflammatory lung disease and may result from extravasation of serum lipoproteins into the alveolus. We investigated whether one bioactive component of modified lipoproteins, oxysterols, might reduce surfactant PtdCho availability by altering its trafficking. The oxysterol, 22-hydroxycholesterol (22HC), in combination with its obligate partner, 9 cis-retinoic acid (RA), decreased surfactant PtdCho levels, in part, by stimulating basolateral phospholipid export in murine lung epithelia. 22HC/RA stimulated basolateral PtdCho efflux in cells via transcriptional activation of the ATP-binding cassette transporter 1 (ABCA1) gene. This effect was mediated by a DR-4 locus within the ABCA1 promoter. ABCA1 knockdown studies using ABCA1 siRNA or the ABCA1 inhibitor, glyburide, selectively attenuated 22HC/RA-driven basolateral PtdCho efflux. 22HC/RA significantly increased export of PtdCho molecular species containing saturated (16:0) fatty-acyl species typical of DSPtdCho. Overexpression of ABCA1 mimicked 22HC/RA effects by increasing cellular PtdCho efflux, whereas mutagenesis of ABCA1 at Trp590 attenuated PtdCho release. The results indicate the existence of an oxysterol-activated basolateral exit pathway for surfactant that might impact the availability of phospholipid destined for apical secretion. PMID:15039140

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

    PubMed

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

    2015-12-01

    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

  4. 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

  5. All0809/8/7 is a DevBCA-like ABC-type efflux pump required for diazotrophic growth in Anabaena sp. PCC 7120.

    PubMed

    Staron, Peter; Maldener, Iris

    2012-10-01

    Efflux pumps export a wide variety of proteinaceous and non-proteinaceous substrates across the Gram-negative cell wall. For the filamentous cyanobacterium Anabaena sp. strain PCC 7120, the ATP-driven glycolipid efflux pump DevBCA-TolC has been shown to be crucial for the differentiation of N(2)-fixing heterocysts from photosynthetically active vegetative cells. In this study, a homologous system was described. All0809/8/7-TolC form a typical ATP-driven efflux pump as shown by surface plasmon resonance. This putative exporter is also involved in diazotrophic growth of Anabaena sp. PCC 7120. A mutant in all0809 encoding the periplasmic membrane fusion protein of the pump was not able to grow without combined nitrogen. Although heterocysts of this mutant were not distinguishable from those of the wild-type in light and electron micrographs, they were impaired in providing the microoxic environment necessary for N(2) fixation. RT-PCR of all0809 transcripts and localization studies on All0807-GFP revealed that All0809/8/7 was initially downregulated during heterocyst maturation and upregulated at later stages of heterocyst formation in all cells of the filament. A substrate of the efflux pump could not be identified in ATP hydrolysis assays. We discuss a role for All0809/8/7-TolC in maintaining the continuous periplasm and how this would be of special importance for heterocyst differentiation. PMID:22859614

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

    PubMed

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

    2013-01-30

    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 [(3)H]Gly-Sar were conducted on transfected MDCKII and wild-type cells to investigate functional differences. Cellular [(3)H]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

  7. 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

  8. A Comparative Analysis of Synthetic Quorum Sensing Modulators in Pseudomonas aeruginosa: New Insights into Mechanism, Active Efflux Susceptibility, Phenotypic Response, and Next-Generation Ligand Design

    PubMed Central

    2015-01-01

    Quorum sensing (QS) is a chemical signaling mechanism that allows bacterial populations to coordinate gene expression in response to social and environmental cues. Many bacterial pathogens use QS to initiate infection at high cell densities. Over the past two decades, chemical antagonists of QS in pathogenic bacteria have attracted substantial interest for use both as tools to further elucidate QS mechanisms and, with further development, potential anti-infective agents. Considerable recent research has been devoted to the design of small molecules capable of modulating the LasR QS receptor in the opportunistic pathogen Pseudomonas aeruginosa. These molecules hold significant promise in a range of contexts; however, as most compounds have been developed independently, comparative activity data for these compounds are scarce. Moreover, the mechanisms by which the bulk of these compounds act are largely unknown. This paucity of data has stalled the choice of an optimal chemical scaffold for further advancement. Herein, we submit the best-characterized LasR modulators to standardized cell-based reporter and QS phenotypic assays in P. aeruginosa, and we report the first comprehensive set of comparative LasR activity data for these compounds. Our experiments uncovered multiple interesting mechanistic phenomena (including a potential alternative QS-modulatory ligand binding site/partner) that provide new, and unexpected, insights into the modes by which many of these LasR ligands act. The lead compounds, data trends, and mechanistic insights reported here will significantly aid the design of new small molecule QS inhibitors and activators in P. aeruginosa, and in other bacteria, with enhanced potencies and defined modes of action. PMID:26491787

  9. 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

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

    PubMed

    Hürlimann, Lea M; Corradi, Valentina; Hohl, Michael; Bloemberg, Guido V; Tieleman, D Peter; Seeger, Markus A

    2016-09-01

    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

  11. 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.

  12. 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.

  13. Modifications of laccase activities of copper efflux oxidase, CueO by synergistic mutations in the first and second coordination spheres of the type I copper center.

    PubMed

    Kataoka, Kunishige; Kogi, Hiroki; Tsujimura, Seiya; Sakurai, Takeshi

    2013-02-15

    The redox potential of type I copper in the Escherichia coli multicopper oxidase CueO was shifted in the positive or negative direction as a result of the single, double, and triple mutations in the first and second coordination spheres: the formation of the NH···S(-)(Cys500 ligand) hydrogen bond, the breakdown of the NH(His443 ligand)···O(-)(Asp439) hydrogen bond, and the substitution of the Met510 ligand for the non-coordinating Leu or coordinating Gln. Laccase activities of CueO were maximally enhanced 140-fold by virtue of the synergistic effect of mild mutations at and at around the ligand groups to type I copper. PMID:23337502

  14. 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

  15. 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. PMID:25863936

  16. Radiocarbon in Tree STEM CO2 Efflux

    NASA Astrophysics Data System (ADS)

    Muhr, J.; Czimczik, C. I.; Angert, A.; Trumbore, S.

    2011-12-01

    Carbon dioxide efflux from tree stems can be a significant component of the stand-level carbon balance. Recent studies have demonstrated that tree stem CO2 efflux may reflect more than just in-situ respiration but also transport from other locations and it has been suggested that it may also include C originally respired in roots or even uptake of soil CO2. We report measurements of the radiocarbon signature of carbon emitted from a range of mature tree stems in tropical and temperate forest ecosystems. Comparison of the radiocarbon signature of respired CO2 with the observed rate of decline in atmsopheric 14C-CO2 provides a measure of the time elapsed between C fixation by the plant and its return to the atmosphere as stem CO2 efflux. In all investigated trees, we observed that stem CO2 efflux had higher radiocarbon signatures than the contemporary atmospheric 14C-CO2, and therefore was derived from C fixed one to several years earlier. In tropical forest trees, we found that the 14C signature of CO2 within the stem (~4-5 cm depth) had even higher radiocarbon signatures than the stem CO2 efflux. In one of the investigated tree species, the in-stem CO2 was derived from C sources fixed on average ~20 years previously. These results confirm observations of root-respired CO2 that also have shown contributions of C substrates older than recent photosynthetic products, and the presence of extracable C reserves in wood that reflect the presence of older C sources. Our results imply that stem CO2 efflux is not only derived from respiration of recent photosynthetic products but includes contributions from older, stored C pools. Ongoing investigations will enable us to compare CO2 efflux for trees subjected to experimental drought, and using different life strategies (deciduous versus evergreen oaks) to determine if the use of these older C stores varies with stress.

  17. Expression of the RND-type efflux pump AdeABC in Acinetobacter baumannii is regulated by the AdeRS two-component system.

    PubMed

    Marchand, Isabelle; Damier-Piolle, Laurence; Courvalin, Patrice; Lambert, Thierry

    2004-09-01

    The AdeABC pump of Acinetobacter baumannii BM4454, which confers resistance to various antibiotic classes including aminoglycosides, is composed of the AdeA, AdeB, and AdeC proteins; AdeB is a member of the RND superfamily. The adeA, adeB, and adeC genes are contiguous and adjacent to adeS and adeR, which are transcribed in the opposite direction and which specify proteins homologous to sensors and regulators of two-component systems, respectively (S. Magnet, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 45:3375-3380, 2001). Analysis by Northern hybridization indicated that the three genes were cotranscribed, although mRNAs corresponding to adeAB and adeC were also present. Cotranscription of the two regulatory genes was demonstrated by reverse transcription-PCR. Inactivation of adeS led to aminoglycoside susceptibility. Transcripts corresponding to adeAB were not detected in susceptible A. baumannii CIP 70-10 but were present in spontaneous gentamicin-resistant mutants obtained in vitro. Analysis of these mutants revealed the substitutions Thr153-->Met in AdeS downstream from the putative His-149 site of autophosphorylation, which is presumably responsible for the loss of phosphorylase activity by the sensor, and Pro116-->Leu in AdeR at the first residue of the alpha(5) helix of the receiver domain, which is involved in interactions that control the output domain of response regulators. These mutations led to constitutive expression of the pump and, thus, to antibiotic resistance. These data indicate that the AdeABC pump is cryptic in wild A. baumannii due to stringent control by the AdeRS two-component system. PMID:15328088

  18. Expression of the RND-Type Efflux Pump AdeABC in Acinetobacter baumannii Is Regulated by the AdeRS Two-Component System

    PubMed Central

    Marchand, Isabelle; Damier-Piolle, Laurence; Courvalin, Patrice; Lambert, Thierry

    2004-01-01

    The AdeABC pump of Acinetobacter baumannii BM4454, which confers resistance to various antibiotic classes including aminoglycosides, is composed of the AdeA, AdeB, and AdeC proteins; AdeB is a member of the RND superfamily. The adeA, adeB, and adeC genes are contiguous and adjacent to adeS and adeR, which are transcribed in the opposite direction and which specify proteins homologous to sensors and regulators of two-component systems, respectively (S. Magnet, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 45:3375-3380, 2001). Analysis by Northern hybridization indicated that the three genes were cotranscribed, although mRNAs corresponding to adeAB and adeC were also present. Cotranscription of the two regulatory genes was demonstrated by reverse transcription-PCR. Inactivation of adeS led to aminoglycoside susceptibility. Transcripts corresponding to adeAB were not detected in susceptible A. baumannii CIP 70-10 but were present in spontaneous gentamicin-resistant mutants obtained in vitro. Analysis of these mutants revealed the substitutions Thr153→Met in AdeS downstream from the putative His-149 site of autophosphorylation, which is presumably responsible for the loss of phosphorylase activity by the sensor, and Pro116→Leu in AdeR at the first residue of the α5 helix of the receiver domain, which is involved in interactions that control the output domain of response regulators. These mutations led to constitutive expression of the pump and, thus, to antibiotic resistance. These data indicate that the AdeABC pump is cryptic in wild A. baumannii due to stringent control by the AdeRS two-component system. PMID:15328088

  19. Increased efflux of glutathione conjugate in acutely diabetic cardiomyocytes.

    PubMed

    Ghosh, Sanjoy; Ting, Simon; Lau, Howard; Pulinilkunnil, Thomas; An, Ding; Qi, Dake; Abrahani, Mohammed A; Rodrigues, Brian

    2004-10-01

    In diabetes, cell death and resultant cardiomyopathy have been linked to oxidative stress and depletion of antioxidants like glutathione (GSH). Although the de novo synthesis and recycling of GSH have been extensively studied in the chronically diabetic heart, their contribution in modulating cardiac oxidative stress in acute diabetes has been largely ignored. Additionally, the possible contribution of cellular efflux in regulating GSH levels during diabetes is unknown. We used streptozotocin to make Wistar rats acutely diabetic and after 4 days examined the different processes that regulate cardiac GSH. Reduction in myocyte GSH in diabetic rats was accompanied by increased oxidative stress, excessive reactive oxygen species, and an elevated apoptotic cell death. The effect on GSH was not associated with any change in either synthesis or recycling, as both gamma-glutamylcysteine synthetase gene expression (responsible for bio syn thesis) and glutathione reductase activity (involved with GSH recycling) remained unchanged. However, gene expression of multidrug resistance protein 1, a transporter implicated in effluxing GSH during oxidative stress, was elevated. GSH conjugate efflux mediated by multidrug resistance protein 1 also increased in diabetic cardiomyocytes, an effect that was blocked using MK-571, a specific inhibitor of this transporter. As MK-571 also decreased oxidative stress in diabetic cardiomyocytes, an important role can be proposed for this transporter in GSH and reactive oxygen species homeostasis in the acutely diabetic heart. PMID:15573148

  20. 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

  1. 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)

  2. 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

  3. 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.

  4. Stimulation of H+ Efflux and Inhibition of Photosynthesis by Esters of Carboxylic Acids 1

    PubMed Central

    Duhaime, Donna E.; Bown, Alan W.

    1983-01-01

    Suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used to investigate the influence of various carboxyester compounds on rates of net H+ efflux in the dark or light and photosynthetic O2 production. Addition of 0.15 to 1.5 millimolar malathion, α-naphthyl acetate, phenyl acetate, or p-nitrophenyl acetate stimulated H+ efflux and inhibited photosynthesis within 1 minute. In contrast, the more polar esters methyl acetoacetate or ethyl p-aminobenzoate had little or no effect on either of these two processes. A 0.15 millimolar concentration of α-naphthylacetate stimulated the normal rate of H+ efflux, 0.77 nanomoles H+ per 106 cells per minute by 750% and inhibited photosynthesis by 100%. The four active carboxyester compounds also stimulated H+ efflux after the normal rate of H+ efflux was eliminated with 0.01 milligrams per milliliter oligomycin or 100% N2. Oligomycin reduced the ATP level by 70%. Incubation of cells with malathion, α-naphthyl acetate, or p-nitrophenyl acetate resulted in the generation of the respective hydrolysis products ethanol, α-naphthol, and p-nitrophenol. It is proposed that inhibition of photosynthesis and stimulation of H+ efflux result when nonpolar carboxyester compounds enter the cell and generate acidic carboxyl groups when hydrolyzed by esterase enzymes. PMID:16663308

  5. Characterization of MATE-Type Multidrug Efflux Pumps from Klebsiella pneumoniae MGH78578

    PubMed Central

    Ogawa, Wakano; Minato, Yusuke; Dodan, Hayata; Onishi, Motoyasu; Tsuchiya, Tomofusa; Kuroda, Teruo

    2015-01-01

    We previously described the cloning of genes related to drug resistance from Klebsiella pneumoniae MGH78578. Of these, we identified a putative gene encoding a MATE-type multidrug efflux pump, and named it ketM. Escherichia coli KAM32 possessing ketM on a plasmid showed increased minimum inhibitory concentrations for norfloxacin, ciprofloxacin, cefotaxime, acriflavine, Hoechst 33342, and 4',6-diamidino-2-phenyl indole (DAPI). The active efflux of DAPI was observed in E. coli KAM32 possessing ketM on a plasmid. The expression of mRNA for ketM was observed in K. pneumoniae cells, and we subsequently disrupted ketM in K. pneumoniae ATCC10031. However, no significant changes were observed in drug resistance levels between the parental strain ATCC10031 and ketM disruptant, SKYM. Therefore, we concluded that KetM was a multidrug efflux pump, that did not significantly contribute to intrinsic resistance to antimicrobial chemicals in K. pneumoniae. MATE-type transporters are considered to be secondary transporters; therefore, we investigated the coupling cations of KetM. DAPI efflux by KetM was observed when lactate was added to produce a proton motive force, indicating that KetM effluxed substrates using a proton motive force. However, the weak efflux of DAPI by KetM was also noted when NaCl was added to the assay mixture without lactate. This result suggests that KetM may utilize proton and sodium motive forces. PMID:25807080

  6. Directed evolution of an E. coli inner membrane transporter for improved efflux of biofuel molecules

    PubMed Central

    2013-01-01

    Background The depletion of fossil fuels and the rising need to meet global energy demands have led to a growing interest in microbial biofuel synthesis, particularly in Escherichia coli, due to its tractable characteristics. Besides engineering more efficient metabolic pathways for synthesizing biofuels, efforts to improve production yield by engineering efflux systems to overcome toxicity problems is also crucial. This study aims to enhance hydrocarbon efflux capability in E. coli by engineering a native inner membrane transporter, AcrB, using the directed evolution approach. Results We developed a selection platform based on competitive growth using a toxic substrate surrogate, which allowed rapid selection of AcrB variants showing enhanced efflux of linear and cyclic fuel molecule candidates, n-octane and α-pinene. Two mutants exhibiting increased efflux efficiency for n-octane and α-pinene by up to 47% and 400%, respectively, were isolated. Single-site mutants based on the mutations found in the isolated variants were synthesized and the amino acid substitutions N189H, T678S, Q737L and M844L were identified to have conferred improvement in efflux efficiency. The locations of beneficial mutations in AcrB suggest their contributions in widening the substrate channel, altering the dynamics of substrate efflux and promoting the assembly of AcrB with the outer membrane channel protein TolC for more efficient substrate export. It is interesting to note that three of the four beneficial mutations were located relatively distant from the known substrate channels, thus exemplifying the advantage of directed evolution over rational design. Conclusions Using directed evolution, we have isolated AcrB mutants with improved efflux efficiency for n-octane and α-pinene. The utilization of such optimized native efflux pumps will increase productivity of biofuels synthesis and alleviate toxicity and difficulties in production scale-up in current microbial platforms. PMID

  7. European Neutron Activation System.

    2013-01-11

    Version 03 EASY-2010 (European Activation System) consists of a wide range of codes, data and documentation all aimed at satisfying the objective of calculating the response of materials irradiated in a neutron flux. The main difference from the previous version is the upper energy limit, which has increased from 20 to 60 MeV. It is designed to investigate both fusion devices and accelerator based materials test facilities that will act as intense sources of high-energymore » neutrons causing significant activation of the surrounding materials. The very general nature of the calculational method and the data libraries means that it is applicable (with some reservations) to all situations (e.g. fission reactors or neutron sources) where materials are exposed to neutrons below 60 MeV. EASY can be divided into two parts: data and code development tools and user tools and data. The former are required to develop the latter, but EASY users only need to be able to use the inventory code FISPACT and be aware of the contents of the EAF library (the data source). The complete EASY package contains the FISPACT-2007 inventory code, the EAF-2003, EAF-2005, EAF-2007 and EAF-2010 libraries, and the EASY User Interface for the Window version. The activation package EASY-2010 is the result of significant development to extend the upper energy range from 20 to 60 MeV so that it is capable of being used for IFMIF calculations. The EAF-2010 library contains 66,256 reactions, almost five times more than in EAF-2003 (12,617). Deuteron-induced and proton-induced cross section libraries are also included, and can be used with EASY to enable calculations of the activation due to deuterons and proton [2].« less

  8. Top consumer abundance influences lake methane efflux

    PubMed Central

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

    2015-01-01

    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. PMID:26531291

  9. Top consumer abundance influences lake methane efflux.

    PubMed

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

    2015-01-01

    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. PMID:26531291

  10. Glucocorticoid receptors in the prefrontal cortex regulate dopamine efflux to stress via descending glutamatergic feedback to the ventral tegmental area.

    PubMed

    Butts, Kelly A; Phillips, Anthony G

    2013-09-01

    Enhanced dopamine (DA) efflux in the medial prefrontal cortex (mPFC) is a well-documented response to acute stress. We have previously shown that glucocorticoid receptors in the mPFC regulate stress-evoked DA efflux but the underlying mechanism is unknown. DA neurons in the ventral tegmental area (VTA) receive excitatory input from and send reciprocal projections to the mPFC. We hypothesize that blockade of prefrontal glucocorticoid receptors can reduce activity of descending glutamatergic input to the VTA, thereby attenuating stress-evoked DA efflux in the mPFC. Using in vivo microdialysis, we demonstrate that acute tail-pinch stress leads to a significant increase in glutamate efflux in the VTA. Blockade of prefrontal glucocorticoid receptors with the selective antagonist CORT 108297 attenuates stress-evoked glutamate efflux in the VTA together with DA efflux in the mPFC. Furthermore, blockade of ionotrophic glutamate receptors in the VTA attenuates stress-evoked DA efflux in the mPFC. We also examine the possible role of glucocorticoid-induced synthesis and release of endocannabinoids acting presynaptically via cannabinoid CB1 receptors to inhibit GABA release onto prefrontal pyramidal cells, thus enhancing descending glutamatergic input to the VTA leading to an increase in mPFC DA efflux during stress. However, administration of the cannabinoid CB1 receptor antagonist into the mPFC does not attenuate stress-evoked DA efflux in the mPFC. Taken together, our data indicate that glucocorticoids act locally within the mPFC to modulate mesocortical DA efflux by potentiation of glutamatergic drive onto DA neurons in the VTA. PMID:23590841

  11. Toxin sensitivity of the calcium-dependent rubidium efflux in Madin-Darby canine kidney cells.

    PubMed

    Tauc, M; Gastineau, M; Poujeol, P

    1993-01-29

    86Rb+ efflux was measured on polarized Madin-Darby canine kidney cells under A23187 or ATP stimulation. This efflux, inhibited by barium, Leiurus quinquestriatus hebraeus venom and charybdotoxin was attributed to the stimulation of Ca(++)-activated maxi K+ channels. Snake venom from Dendroaspis polylepis did not alter the stimulation as well as did apamine. ATP was effective on both the apical and basolateral membranes and the Ca(++)-activated maxi K+ channels were predominantly found on the basolateral membrane. This study presents the physiological evidence that dendrotoxin is ineffective on the epithelial Ca(++)-activated maxi K+ channel present in MDCK cells. PMID:7678959

  12. 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.

  13. Efflux Pump, the Masked Side of ß-Lactam Resistance in Klebsiella pneumoniae Clinical Isolates

    PubMed Central

    Pages, Jean-Marie; Lavigne, Jean-Philippe; Leflon-Guibout, Véronique; Marcon, Estelle; Bert, Frédéric; Noussair, Latifa; Nicolas-Chanoine, Marie-Hélène

    2009-01-01

    Background β-lactamase production and porin decrease are the well-recognized mechanisms of acquired ß-lactam resistance in Klebsiella pneumoniae isolates. However, such mechanisms proved to be absent in K. pneumoniae isolates that are non susceptible to cefoxitin (FOX) and succeptible to amoxicillin+clavulanic acid in our hospital. Assessing the role of efflux pumps in this β-lactam phenotype was the aim of this study. Methodology/Findings MICs of 9 β-lactams, including cloxacillin (CLX), and other antibiotic families were tested alone and with an efflux pump inhibitor (EPI), then with both CLX (subinhibitory concentrations) and EPI against 11 unique bacteremia K. pneumoniae isolates displaying the unusual phenotype, and 2 ATCC strains. CLX and EPI-dose dependent effects were studied on 4 representatives strains. CLX MICs significantly decreased when tested with EPI. A similar phenomenon was observed with piperacillin+tazobactam whereas MICs of the other β-lactams significantly decreased only in the presence of both EPI and CLX. Thus, FOX MICs decreased 128 fold in the K. pneumoniae isolates but also16 fold in ATCC strain. Restoration of FOX activity was CLX dose-dependent suggesting a competitive relationship between CLX and the other β-lactams with regard to their efflux. For chloramphenicol, erythromycin and nalidixic acid whose resistance was also due to efflux, adding CLX to EPI did not increase their activity suggesting differences between the efflux process of these molecules and that of β-lactams. Conclusion This is the first study demonstrating that efflux mechanism plays a key role in the β-lactam susceptibility of clinical isolates of K. pneumoniae. Such data clearly evidence that the involvement of efflux pumps in ß-lactam resistance is specially underestimated in clinical isolates. PMID:19279676

  14. Cooperation between prokaryotic (Lde) and eukaryotic (MRP) efflux transporters in J774 macrophages infected with Listeria monocytogenes: studies with ciprofloxacin and moxifloxacin.

    PubMed

    Lismond, Ann; Tulkens, Paul M; Mingeot-Leclercq, Marie-Paule; Courvalin, Patrice; Van Bambeke, Françoise

    2008-09-01

    Antibiotic efflux is observed in both eukaryotic and prokaryotic cells, modulating accumulation and resistance. The present study examines whether eukaryotic and prokaryotic fluoroquinolone transporters can cooperate in the context of an intracellular infection. We have used (i) J774 macrophages (comparing a ciprofloxacin-resistant cell line overexpressing an MRP-like transporter with wild-type cells with basal expression), (ii) Listeria monocytogenes (comparing a clinical isolate [CLIP21369] displaying ciprofloxacin resistance associated with overexpression of the Lde efflux system with a wild-type strain [EGD]), (iii) ciprofloxacin (substrate of both Lde and MRP) and moxifloxacin (nonsubstrate), and (iv) probenecid and reserpine (preferential inhibitors of MRP and Lde, respectively). The ciprofloxacin MICs for EGD were unaffected by reserpine, while those for CLIP21369 were decreased approximately fourfold (and made similar to those of EGD). Neither probenecid nor reserpine affected the moxifloxacin MICs against EGD or CLIP21369. In dose-response studies (0.01x to 100x MIC) in broth, reserpine fully restored the susceptibility of CLIP21369 to ciprofloxacin (no effect on EGD) but did not influence the activity of moxifloxacin. In studies with intracellular bacteria, reserpine, probenecid, and their combination increased the activity of ciprofloxacin in wild-type and ciprofloxacin-resistant macrophages in parallel with an increase in ciprofloxacin accumulation in macrophages for EGD and an increase in accumulation and decrease in MIC (in broth) for CLIP21369. Moxifloxacin accumulation and intracellular activity were consistently not affected by the inhibitors. A bacterial efflux pump may thus actively cooperate with a eukaryotic efflux transporter to reduce the activity of a common substrate (ciprofloxacin) toward an intracellular bacterial target. PMID:18573933

  15. 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

  16. Efflux-Mediated Antibiotic Resistance in Acinetobacter spp. ▿

    PubMed Central

    Coyne, Sébastien; Courvalin, Patrice; Périchon, Bruno

    2011-01-01

    Among Acinetobacter spp., A. baumannii is the most frequently implicated in nosocomial infections, in particular in intensive care units. It was initially thought that multidrug resistance (MDR) in this species was due mainly to horizontal acquisition of resistance genes. However, it has recently become obvious that increased expression of chromosomal genes for efflux systems plays a major role in MDR. Among the five superfamilies of pumps, resistance-nodulation-division (RND) systems are the most prevalent in multiply resistant A. baumannii. RND pumps typically exhibit a wide substrate range that can include antibiotics, dyes, biocides, detergents, and antiseptics. Overexpression of AdeABC, secondary to mutations in the adeRS genes encoding a two-component regulatory system, constitutes a major mechanism of multiresistance in A. baumannii. AdeIJK, intrinsic to this species, is responsible for natural resistance, but since overexpression above a certain threshold is toxic for the host, its contribution to acquired resistance is minimal. The recently described AdeFGH, probably regulated by a LysR-type transcriptional regulator, also confers multidrug resistance when overexpressed. Non-RND efflux systems, such as CraA, AmvA, AbeM, and AbeS, have also been characterized for A. baumannii, as have AdeXYZ and AdeDE for other Acinetobacter spp. Finally, acquired narrow-spectrum efflux pumps, such as the major facilitator superfamily (MFS) members TetA, TetB, CmlA, and FloR and the small multidrug resistance (SMR) member QacE in Acinetobacter spp., have been detected and are mainly encoded by mobile genetic elements. PMID:21173183

  17. 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,…

  18. 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.

  19. The ins and outs of RND efflux pumps in Escherichia coli

    PubMed Central

    Anes, João; McCusker, Matthew P.; Fanning, Séamus; Martins, Marta

    2015-01-01

    Infectious diseases remain one of the principal causes of morbidity and mortality in the world. Relevant authorities including the WHO and CDC have expressed serious concern regarding the continued increase in the development of multidrug resistance among bacteria. They have also reaffirmed the urgent need for investment in the discovery and development of new antibiotics and therapeutic approaches to treat multidrug resistant (MDR) bacteria. The extensive use of antimicrobial compounds in diverse environments, including farming and healthcare, has been identified as one of the main causes for the emergence of MDR bacteria. Induced selective pressure has led bacteria to develop new strategies of defense against these chemicals. Bacteria can accomplish this by several mechanisms, including enzymatic inactivation of the target compound; decreased cell permeability; target protection and/or overproduction; altered target site/enzyme and increased efflux due to over-expression of efflux pumps. Efflux pumps can be specific for a single substrate or can confer resistance to multiple antimicrobials by facilitating the extrusion of a broad range of compounds including antibiotics, heavy metals, biocides and others, from the bacterial cell. To overcome antimicrobial resistance caused by active efflux, efforts are required to better understand the fundamentals of drug efflux mechanisms. There is also a need to elucidate how these mechanisms are regulated and how they respond upon exposure to antimicrobials. Understanding these will allow the development of combined therapies using efflux inhibitors together with antibiotics to act on Gram-negative bacteria, such as the emerging globally disseminated MDR pathogen Escherichia coli ST131 (O25:H4). This review will summarize the current knowledge on resistance-nodulation-cell division efflux mechanisms in E. coli, a bacteria responsible for community and hospital-acquired infections, as well as foodborne outbreaks worldwide

  20. Efflux of Creatine Kinase from Isolated Soleus Muscle Depends on Age, Sex and Type of Exercise in Mice

    PubMed Central

    Baltusnikas, Juozas; Venckunas, Tomas; Kilikevicius, Audrius; Fokin, Andrej; Ratkevicius, Aivaras

    2015-01-01

    Elevated plasma creatine kinase (CK) activity is often used as an indicator of exercise-induced muscle damage. Our aim was to study effects of contraction type, sex and age on CK efflux from isolated skeletal muscles of mice. The soleus muscle (SOL) of adult (7.5-month old) female C57BL/6J mice was subjected to either 100 passive stretches, isometric contractions or eccentric contractions, and muscle CK efflux was assessed after two-hour incubation in vitro. SOL of young (3-month old) male and female mice was studied after 100 eccentric contractions. For adult females, muscle CK efflux was larger (p < 0.05) after eccentric contractions than after incubation without exercise (698 ± 344 vs. 268 ± 184 mU·h−1, respectively), but smaller (p < 0.05) than for young females after the same type of exercise (1069 ± 341 mU·h−1). Eccentric exercise-induced CK efflux was larger in muscles of young males compared to young females (2046 ± 317 vs 1069 ± 341 mU · h−1, respectively, p < 0.001). Our results show that eccentric contractions induce a significant increase in muscle CK efflux immediately after exercise. Isolated muscle resistance to exercise-induced CK efflux depends on age and sex of mice. Key points Muscle lengthening contractions induce the highest CK efflux in vitro compared with similar protocol of isometric contractions or passive stretches. Muscle CK efflux in vitro is applicable in studying changes of sarcolemma permeability/integrity, a proxy of muscle damage, in response to muscle contractile activity. Isolated muscle resistance to exercise-induced CK efflux is greater in female compared to male mice of young age and is further increased in adult female mice. PMID:25983588

  1. 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

  2. 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

  3. 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

  4. Chalcone inhibitors of the NorA efflux pump in Staphylococcus aureus whole cells and enriched everted membrane vesicles.

    PubMed

    Holler, Jes Gitz; Slotved, Hans-Christian; Mølgaard, Per; Olsen, Carl Erik; Christensen, Søren Brøgger

    2012-07-15

    A library of 117 chalcones was screened for efflux pump inhibitory (EPI) activity against NorA mediated ethidium bromide efflux. Five of the chalcones (5-7, 9, and 10) were active and two chalcones (9 and 10) were equipotent to reserpine with IC(50)-values of 9.0 and 7.7 μM, respectively. Twenty chalcones were subsequently proved to be inhibitors of the NorA efflux pump in everted membrane vesicles. Compounds 5, 7, and 9 synergistically increased the effect of ciprofloxacin on Staphylococcus aureus. Our results suggest that chalcones might be developed into drugs for overcoming multidrug resistance based on efflux transporters of microorganisms. PMID:22682300

  5. Development of efflux pump inhibitors in antituberculosis therapy.

    PubMed

    Song, Lele; Wu, Xueqiong

    2016-06-01

    Resistance and tolerance to antituberculosis (anti-TB) drugs, especially the first-line drugs, has become a serious problem in anti-TB therapy. Efflux of antimicrobial agents via bacterial efflux pumps is one of the main reasons for drug resistance. Efflux pump inhibitors (EPIs) bind to efflux pumps to inhibit drug efflux and thus enhance the drug effect and reduce drug resistance. Studies on EPIs targeting the efflux pumps of Mycobacterium tuberculosis (Mtb) help to understand Mtb resistance and to identify the potential drug target and are of significance in guiding the development of new anti-TB drugs and optimal combinations. Currently, there are many potential EPIs under study, but none of them has been used clinically for anti-TB therapy. In this article, we will provide an overview on the current development of EPIs targeting the efflux pumps of Mtb and discuss their potential clinical applications. PMID:27211826

  6. Alterations in the intestine of Patagonian silverside (Odontesthes hatcheri) exposed to microcystin-LR: Changes in the glycosylation pattern of the intestinal wall and inhibition of multidrug resistance proteins efflux activity.

    PubMed

    Bieczynski, Flavia; Torres, Walter D C; Painefilu, Julio C; Castro, Juan M; Bianchi, Virginia A; Frontera, Jimena L; Paz, Dante A; González, Carolina; Martín, Alejandro; Villanueva, Silvina S M; Luquet, Carlos M

    2016-09-01

    )), calcein efflux was inhibited by MCLR (2.3μmolL(-1)) and MK571 (3μmolL(-1)) by 38 and 27%, respectively (p<0.05). Finally, middle intestine segments were incubated with different concentrations of MCLR applied alone or together with 3μM MK571. After one hour, protein phosphatase 1 (PP1) activity, the main target of MCLR, was measured. 2.5μM MCLR did not produce any significant effect, while the same amount plus MK571 inhibited PP1 activity (p<0.05). This effect was similar to that of 5μM MCLR. Our results suggest that in O. hatcheri enterocytes MCLR is conjugated with GSH via GST and then exported to the intestinal lumen through Abcc-like transporters. This mechanism would protect the cell from MCLR toxicity, limiting toxin transport into the blood, which is probably mediated by basolateral Abccs. From an ecotoxicological point of view, elimination of MCLR through this mechanism would reduce the amount of toxin available for trophic transference. PMID:27474942

  7. 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.

  8. 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. PMID:24140409

  9. Differential Regulation of ABCA1 and Macrophage Cholesterol Efflux By Elaidic and Oleic Acids

    PubMed Central

    Shao, Fei; Ford, David A.

    2013-01-01

    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 sho that cis and trans structural differences in eighteen carbon n-9 monoenoic fatty acids variably impact cholesterol efflux through disparate effects on ABCA1 protein degradation. PMID:23800855

  10. Alkylaminoquinolines inhibit the bacterial antibiotic efflux pump in multidrug-resistant clinical isolates.

    PubMed Central

    Malléa, Monique; Mahamoud, Abdallah; Chevalier, Jacqueline; Alibert-Franco, Sandrine; Brouant, Pierre; Barbe, Jacques; Pagès, Jean-Marie

    2003-01-01

    Over the last decade, MDR (multidrug resistance) has increased worldwide in microbial pathogens by efflux mechanisms, leading to treatment failures in human infections. Several Gram-negative bacteria efflux pumps have been described. These proteinaceous channels are capable of expelling structurally different drugs across the envelope and conferring antibiotic resistance in various bacterial pathogens. Combating antibiotic resistance is an urgency and the blocking of efflux pumps is an attractive response to the emergence of MDR phenotypes in infectious bacteria. In the present study, various alkylaminoquinolines were tested as potential inhibitors of drug transporters. We showed that alkylaminoquinolines are capable of restoring susceptibilities to structurally unrelated antibiotics in clinical isolates of MDR Gram-negative bacteria. Antibiotic efflux studies indicated that 7-nitro-8-methyl-4-[2'-(piperidino)ethyl]aminoquinoline acts as an inhibitor of the AcrAB-TolC efflux pump and restores a high level of intracellular drug concentration. Inhibitory activity of this alkylaminoquinoline is observed on clinical isolates showing different resistance phenotypes. PMID:12959639

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

    PubMed

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

    2015-11-01

    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. PMID:26379107

  12. Ascorbate efflux as a new strategy for iron reduction and transport in plants.

    PubMed

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

    2014-01-31

    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 (55)Fe 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

  13. 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

  14. Recent advances in the brain-to-blood efflux transport across the blood-brain barrier.

    PubMed

    Hosoya, Ken-ichi; Ohtsuki, Sumio; Terasaki, Tetsuya

    2002-11-01

    Elucidating the details of the blood-brain barrier (BBB) transport mechanism is a very important step towards successful drug targeting to the brain and understanding what happens in the brain. Although several brain uptake methods have been developed to characterize transport at the BBB, these are mainly useful for investigating influx transport across the BBB. In 1992, P-glycoprotein was found to act as an efflux pump for anti-cancer drugs at the BBB using primary cultured bovine brain endothelial cells. In order to determine the direct efflux transport from the brain to the circulating blood of exogenous compounds in vivo, the Brain Efflux Index method was developed to characterize several BBB efflux transport systems. Recently, we have established conditionally immortalized rat (TR-BBB) and mouse (TM-BBB) brain capillary endothelial cell lines from transgenic rats and mice harboring temperature-sensitive simian virus 40 large T-antigen gene to characterize the transport mechanisms at the BBB in vitro. TR-BBB and TM-BBB cells possess certain in vivo transport functions and express mRNAs for the BBB. Using a combination of newly developed in vivo and in vitro methods, we have elucidated the efflux transport mechanism at the BBB for neurosteroids, excitatory neurotransmitters, suppressive neurotransmitters, amino acids, and other organic anions to understand the physiological role played by the BBB as a detoxifying organ for the brain. PMID:12429456

  15. Energy-dependent efflux of cadmium coded by a plasmid resistance determinant in Staphylococcus aureus.

    PubMed Central

    Tynecka, Z; Gos, Z; Zajac, J

    1981-01-01

    Resistance of Staphylococcus aureus strain 17810R to Cd2+ appears to be due to a plasmid-coded Cd2+ efflux system. Complete efflux of Cd2+ after transfer of preloaded cells into Cd2+-free medium occurred in the resistant strain 17810R, but not in the plasmidless derivative strain 17810S. Net efflux was blocked by 2,4-dinitrophenol, N,N,-dicyclohexylcarbodiimide (DCCD), and incubation at 4 degrees C. The inhibition of Cd2+ efflux by DCCD paralleled a stimulation of net uptake in the resistant cells by this agent. Cd2+ efflux by the resistant strain was accompanied by a reversal of inhibition of respiration, whereas in the sensitive strain, inhibition of respiration was not reversed after transfer to Cd2+-free medium. Net Cd2+ uptake by strain 17810R was inhibited by p-chloromercuribenzoate. In Cd2+ contrast, Cd2+ uptake by the plasmidless strain 17810S was affected neither by p-chloromercuribenzoate nor by DCCD when added alone, but was blocked by a combination of these two agents. Valinomycin had no effect on the reduced Cd2+ uptake by the resistant strain, whereas nigericin stimulated uptake to values comparable to those of the untreated sensitive cells. With sensitive cells, valinomycin reduced Cd2+ uptake by about 50%, whereas nigericin was without effect. A possible mechanism of Cd2+ movements in both strains is discussed. PMID:7263609

  16. 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

  17. Cholesterol efflux and reverse cholesterol transport.

    PubMed

    Favari, Elda; Chroni, Angelika; Tietge, Uwe J F; Zanotti, Ilaria; Escolà-Gil, Joan Carles; Bernini, Franco

    2015-01-01

    Both alterations of lipid/lipoprotein metabolism and inflammatory events contribute to the formation of the atherosclerotic plaque, characterized by the accumulation of abnormal amounts of cholesterol and macrophages in the artery wall. Reverse cholesterol transport (RCT) may counteract the pathogenic events leading to the formation and development of atheroma, by promoting the high-density lipoprotein (HDL)-mediated removal of cholesterol from the artery wall. Recent in vivo studies established the inverse relationship between RCT efficiency and atherosclerotic cardiovascular diseases (CVD), thus suggesting that the promotion of this process may represent a novel strategy to reduce atherosclerotic plaque burden and subsequent cardiovascular events. HDL plays a primary role in all stages of RCT: (1) cholesterol efflux, where these lipoproteins remove excess cholesterol from cells; (2) lipoprotein remodeling, where HDL undergo structural modifications with possible impact on their function; and (3) hepatic lipid uptake, where HDL releases cholesterol to the liver, for the final excretion into bile and feces. Although the inverse association between HDL plasma levels and CVD risk has been postulated for years, recently this concept has been challenged by studies reporting that HDL antiatherogenic functions may be independent of their plasma levels. Therefore, assessment of HDL function, evaluated as the capacity to promote cell cholesterol efflux may offer a better prediction of CVD than HDL levels alone. Consistent with this idea, it has been recently demonstrated that the evaluation of serum cholesterol efflux capacity (CEC) is a predictor of atherosclerosis extent in humans. PMID:25522988

  18. 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.

  19. 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. PMID:27298617

  20. Targeting mitochondrial 18 kDa translocator protein (TSPO) regulates macrophage cholesterol efflux and lipid phenotype.

    PubMed

    Taylor, Janice M W; Allen, Anne-Marie; Graham, Annette

    2014-11-01

    The aim of the present study was to establish mitochondrial cholesterol trafficking 18 kDa translocator protein (TSPO) as a potential therapeutic target, capable of increasing macrophage cholesterol efflux to (apo)lipoprotein acceptors. Expression and activity of TSPO in human (THP-1) macrophages were manipulated genetically and by the use of selective TSPO ligands. Cellular responses were analysed by quantitative PCR (Q-PCR), immunoblotting and radiolabelling, including [3H]cholesterol efflux to (apo)lipoprotein A-I (apoA-I), high-density lipoprotein (HDL) and human serum. Induction of macrophage cholesterol deposition by acetylated low-density lipoprotein (AcLDL) increased expression of TSPO mRNA and protein, reflecting findings in human carotid atherosclerosis. Transient overexpression of TSPO enhanced efflux (E%) of [3H]cholesterol to apoA-I, HDL and human serum compared with empty vector (EV) controls, whereas gene knockdown of TSPO achieved the converse. Ligation of TSPO (using PK11195, FGIN-1-27 and flunitrazepam) triggered increases in [3H]cholesterol efflux, an effect that was amplified in TSPO-overexpressing macrophages. Overexpression of TSPO induced the expression of genes [PPARA (peroxisome-proliferator-activated receptor α), NR1H3 (nuclear receptor 1H3/liver X receptor α), ABCA1 (ATP-binding cassette A1), ABCG4 (ATP-binding cassette G4) and APOE (apolipoprotein E)] and proteins (ABCA1 and PPARα) involved in cholesterol efflux, reduced macrophage neutral lipid mass and lipogenesis and limited cholesterol esterification following exposure to AcLDL. Thus, targeting TSPO reduces macrophage lipid content and prevents macrophage foam cell formation, via enhanced cholesterol efflux to (apo)lipoprotein acceptors. PMID:24814875

  1. 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

  2. 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

  3. 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.

  4. Stress responses as determinants of antimicrobial resistance in Pseudomonas aeruginosa: multidrug efflux and more.

    PubMed

    Poole, Keith

    2014-12-01

    Pseudomonas aeruginosa is a notoriously antimicrobial-resistant organism that is increasingly refractory to antimicrobial chemotherapy. While the usual array of acquired resistance mechanisms contribute to resistance development in this organism a multitude of endogenous genes also play a role. These include a variety of multidrug efflux loci that contribute to both intrinsic and acquired antimicrobial resistance. Despite their roles in resistance, however, it is clear that these efflux systems function in more than just antimicrobial efflux. Indeed, recent data indicate that they are recruited in response to environmental stress and, therefore, function as components of the organism's stress responses. In fact, a number of endogenous resistance-promoting genes are linked to environmental stress, functioning as part of known stress responses or recruited in response to a variety of environmental stress stimuli. Stress responses are, thus, important determinants of antimicrobial resistance in P. aeruginosa. As such, they represent possible therapeutic targets in countering antimicrobial resistance in this organism. PMID:25388098

  5. Efflux and uptake transporters involved in the disposition of bazedoxifene.

    PubMed

    Lušin, Tina Trdan; Mrhar, Aleš; Stieger, Bruno; Kristl, Albin; Berginc, Katja; Trontelj, Jurij

    2016-06-01

    Bazedoxifene, a novel selective estrogen receptor modulator, has complex pharmacokinetics with rapid absorption, high metabolic clearance, low oral bioavailability (6.25 %) and a slow elimination phase. Our hypothesis is that drug uptake and efflux transporters may play an important role in its disposition. To adequately cover all aspects of bazedoxifene transport, several approaches were undertaken: PAMPA assay, ATPase assay, membrane inside-out vesicles and Caco-2 and CHO cell lines. The results obtained from PAMPA experiments showed moderate passive permeability of bazedoxifene (P app ≈ 2 × 10(-6)cm/s), suggesting the existence of an active transport during the rapid absorption phase. The Caco-2 transport assay showed large and significant changes in the measured efflux ratios of bazedoxifene when selective transporter inhibitors were applied: verapamil (a Pgp inhibitor), MK571 (an MRP inhibitor), Ko143 (a BCRP inhibitor) and DIDS (an OATP inhibitor). Additionally, membrane preparation experiments demonstrated the interaction of bazedoxifene with P-gp, MRP2 and BCRP. CHO experiments did not show any interactions of bazedoxifene with OATP1B1 or OATP1B3; therefore, bazedoxifene may be a substrate of other OATP isoform(s). The comprehensive in vitro study indicates a strong involvement of Pgp, MRP, BCRP and OATP in bazedoxifene disposition. PMID:25631963

  6. 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. PMID:26996926

  7. 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.

  8. MAPK1 of Leishmania donovani Modulates Antimony Susceptibility by Downregulating P-Glycoprotein Efflux Pumps

    PubMed Central

    Garg, Mansi

    2015-01-01

    Emergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) in the Indian subcontinent. Mitogen-activated protein kinases (MAPKs) are well-known mediators of signal transduction of eukaryotes, regulating important processes, like proliferation, differentiation, stress response, and apoptosis. In Leishmania, MAPK1 has been shown to be consistently downregulated in antimony-resistant field isolates, suggesting that it has a role in antimony resistance. The present work investigates the molecular mechanism of MAPK1 in antimony resistance in Leishmania donovani. The L. donovani MAPK1 (LdMAPK1) single-allele replacement mutants exhibited increased resistance to Sb(III) (5.57-fold) compared to wild-type promastigotes, while overexpressing parasites became much more susceptible to antimony. The LdMAPK1-mediated drug sensitivity was directly related to antimony-induced apoptotic death of the parasite, as was evidenced by a 4- to 5-fold decrease in cell death parameters in deletion mutants and a 2- to 3-fold increase in MAPK1-overexpressing cells. LdMAPK1-underexpressing parasites also exhibited increased P-glycoprotein (P-gp)-mediated efflux pump activity, while a significant decrease in pump activity was observed in overexpressing cells. This change in efflux pump activity was directly related to expression levels of P-gp in all cell lines. However, episomal complementation of the gene restored normal growth, drug sensitivity, P-gp expression, and efflux pump activity. The data indicate that LdMAPK1 negatively regulates the expression of P-glycoprotein-type efflux pumps in the parasite. The decrease in efflux pump activity with an increase in LdMAPK1 expression may result in increased antimony accumulation in the parasite, making it more vulnerable to the drug. PMID:25870075

  9. 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. PMID:26337618

  10. 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

  11. Functionally Cloned pdrM from Streptococcus pneumoniae Encodes a Na+ Coupled Multidrug Efflux Pump

    PubMed Central

    Hashimoto, Kohei; Ogawa, Wakano; Nishioka, Toshihiro; Tsuchiya, Tomofusa; Kuroda, Teruo

    2013-01-01

    Multidrug efflux pumps play an important role as a self-defense system in bacteria. Bacterial multidrug efflux pumps are classified into five families based on structure and coupling energy: resistance−nodulation−cell division (RND), small multidrug resistance (SMR), major facilitator (MF), ATP binding cassette (ABC), and multidrug and toxic compounds extrusion (MATE). We cloned a gene encoding a MATE-type multidrug efflux pump from Streptococcus pneumoniae R6, and designated it pdrM. PdrM showed sequence similarity with NorM from Vibrio parahaemolyticus, YdhE from Escherichia coli, and other bacterial MATE-type multidrug efflux pumps. Heterologous expression of PdrM let to elevated resistance to several antibacterial agents, norfloxacin, acriflavine, and 4′,6-diamidino-2-phenylindole (DAPI) in E. coli KAM32 cells. PdrM effluxes acriflavine and DAPI in a Na+- or Li+-dependent manner. Moreover, Na+ efflux via PdrM was observed when acriflavine was added to Na+-loaded cells expressing pdrM. Therefore, we conclude that PdrM is a Na+/drug antiporter in S. pneumoniae. In addition to pdrM, we found another two genes, spr1756 and spr1877,that met the criteria of MATE-type by searching the S. pneumoniae genome database. However, cloned spr1756 and spr1877 did not elevate the MIC of any of the investigated drugs. mRNA expression of spr1756, spr1877, and pdrM was detected in S. pneumoniae R6 under laboratory growth conditions. Therefore, spr1756 and spr1877 are supposed to play physiological roles in this growth condition, but they may be unrelated to drug resistance. PMID:23555691

  12. Sediment CO2 efflux from cleared and intact temperate mangroves and tidal flats

    NASA Astrophysics Data System (ADS)

    Bulmer, R. H.; Schwendenmann, L.; Lundquist, C. J.

    2015-02-01

    Temperate mangroves in Southern Australia and New Zealand have been increasing in area over the past 50 years, whereas tropical mangroves have declined by 30-50% over a similar time frame. Tropical mangroves are understood to be an important carbon sink and carbon dioxide (CO2) emissions following clearance are estimated to be comparable or greater than CO2 emissions following the clearance of many terrestrial forest systems. Recreational and amenity values or perceived loss of other estuarine habitats due to expanding temperate mangrove forests have resulted in clearing of temperate mangroves. In this study, we investigated the impact of temperate mangrove clearance on CO2 efflux from the sediment to the atmosphere along with a range of other biotic and abiotic factors. Significantly higher CO2 efflux rates were measured in cleared (1.34 ± 0.46 μmol m2 s-1) and intact mangrove sites (2.31 ± 0.72 μmol m2 s-1) than in tidal flats (-0.23 ± 0.27 μmol m2 s-1). Site and sediment characteristics such as sediment carbon and nitrogen concentration, chlorophyll α concentration, grain size, mangrove height, macrofaunal abundance, sediment temperature and moisture were strongly correlated with sediment CO2 efflux. Our results suggest that carbon stored within temperate mangrove sediment is released over a period of years to decades after mangrove clearance. CO2 efflux from intact and cleared temperate mangroves was found to be comparable to rates observed in the tropics. Disturbance of the surface biofilm resulted in elevated CO2 efflux across all habitats, suggesting the important role of surface biofilm communities in mediating CO2 efflux.

  13. Inflammatory remodeling of the HDL proteome impairs cholesterol efflux capacity.

    PubMed

    Vaisar, Tomáš; Tang, Chongren; Babenko, Ilona; Hutchins, Patrick; Wimberger, Jake; Suffredini, Anthony F; Heinecke, Jay W

    2015-08-01

    Recent studies demonstrate that HDL's ability to promote cholesterol efflux from macrophages associates strongly with cardioprotection in humans independently of HDL-cholesterol (HDL-C) and apoA-I, HDL's major protein. However, the mechanisms that impair cholesterol efflux capacity during vascular disease are unclear. Inflammation, a well-established risk factor for cardiovascular disease, has been shown to impair HDL's cholesterol efflux capacity. We therefore tested the hypothesis that HDL's impaired efflux capacity is mediated by specific changes of its protein cargo. Humans with acute inflammation induced by low-level endotoxin had unchanged HDL-C levels, but their HDL-C efflux capacity was significantly impaired. Proteomic analyses demonstrated that HDL's cholesterol efflux capacity correlated inversely with HDL content of serum amyloid A (SAA)1 and SAA2. In mice, acute inflammation caused a marked impairment of HDL-C efflux capacity that correlated with a large increase in HDL SAA. In striking contrast, the efflux capacity of mouse inflammatory HDL was preserved with genetic ablation of SAA1 and SAA2. Our observations indicate that the inflammatory impairment of HDL-C efflux capacity is due in part to SAA-mediated remodeling of HDL's protein cargo. PMID:25995210

  14. Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB

    PubMed Central

    Eicher, Thomas; Seeger, Markus A; Anselmi, Claudio; Zhou, Wenchang; Brandstätter, Lorenz; Verrey, François; Diederichs, Kay; Faraldo-Gómez, José D; Pos, Klaas M

    2014-01-01

    Membrane transporters of the RND superfamily confer multidrug resistance to pathogenic bacteria, and are essential for cholesterol metabolism and embryonic development in humans. We use high-resolution X-ray crystallography and computational methods to delineate the mechanism of the homotrimeric RND-type proton/drug antiporter AcrB, the active component of the major efflux system AcrAB-TolC in Escherichia coli, and one most complex and intriguing membrane transporters known to date. Analysis of wildtype AcrB and four functionally-inactive variants reveals an unprecedented mechanism that involves two remote alternating-access conformational cycles within each protomer, namely one for protons in the transmembrane region and another for drugs in the periplasmic domain, 50 Å apart. Each of these cycles entails two distinct types of collective motions of two structural repeats, coupled by flanking α-helices that project from the membrane. Moreover, we rationalize how the cross-talk among protomers across the trimerization interface might lead to a more kinetically efficient efflux system. DOI: http://dx.doi.org/10.7554/eLife.03145.001 PMID:25248080

  15. 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). PMID:27057677

  16. Purple perilla extracts with α-asarone enhance cholesterol efflux from oxidized LDL-exposed macrophages.

    PubMed

    Park, Sin-Hye; Paek, Ji Hun; Shin, Daekeun; Lee, Jae-Yong; Lim, Soon Sung; Kang, Young-Hee

    2015-04-01

    The cellular accumulation of cholesterol is critical in the development and progression of atherosclerosis. ATP-binding cassette (ABC) transporters play an essential role in mediating the efflux of excess cholesterol. In the current study, we investigated whether purple Perilla frutescens extracts (PPE) at a non-toxic concentration of 1-10 µg/ml stimulate the induction of the ABC transporters, ABCA1 and ABCG1, and cholesterol efflux from lipid-laden J774A.1 murine macrophages exposed to 50 ng/ml oxidized low-density lipoprotein (LDL). Purple perilla, an annual herb in the mint family and its constituents, have been reported to exhibit antioxidant and cytostatic activity, as well as to exert anti-allergic effects. Our results revealed that treatment with oxidized LDL for 24 h led to the accumulation of lipid droplets in the macrophages. PPE suppressed the oxidized LDL-induced foam cell formation by blocking the induction of scavenger receptor B1. However, PPE promoted the induction of the ABC transporters, ABCA1 and ABCG1, and subsequently accelerated cholesterol efflux from the lipid-loaded macrophages. The liver X receptor (LXR) agonist, TO-091317, and the peroxisome proliferator-activated receptor (PPAR) agonist, pioglitazone, increased ABCA1 expression and treatment with 10 µg/ml PPE further enhanced this effect. PPE did not induce LXRα and PPARγ expression per se, but enhanced their expression in the macrophages exposed to oxidized LDL. α-asarone was isolated from PPE and characterized as a major component enhancing the induction of ABCA1 and ABCG1 in macrophages exposed to oxidized LDL. α-asarone, but not β-asarone was effective in attenuating foam cell formation and enhancing cholesterol efflux, revealing an isomeric difference in their activity. The results from the present study demonstrate that PPE promotes cholesterol efflux from macrophages by activating the interaction of PPARγ-LXRα-ABC transporters. PMID:25673178

  17. Structure of the AcrAB-TolC multidrug efflux pump

    PubMed Central

    Du, Dijun; Wang, Zhao; James, Nathan R.; Voss, Jarrod E.; Klimont, Ewa; Ohene-Agyei, Thelma; Venter, Henrietta; Chiu, Wah; Luisi, Ben F.

    2015-01-01

    The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative bacteria, many of these transporters form multicomponent ‘pumps’ that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component1-7. A model system for such a pump is the acridine resistance complex of Escherichia coli1. This pump assembly comprises the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA, which bridges these two integral membrane proteins. The AcrAB-TolC efflux pump is able to vectorially transport a diverse array of compounds with little chemical similarity, and accordingly confers resistance to a broad spectrum of antibiotics. Homologous complexes are found in many Gram-negative species, including pathogens of animals and plants. Crystal structures are available for the individual pump components2-7 and these have provided insights into substrate recognition, energy coupling and the transduction of conformational changes associated with the transport process. How the subunits are organised in the pump, their stoichiometry and the details of their interactions are not known and are under debate. In this manuscript, we present the pseudoatomic structure of a complete multidrug efflux pump in complex with a modulatory protein partner8. The model defines the quaternary organization of the pump, identifies key domain interactions, and suggests a cooperative process for channel assembly and opening. These findings illuminate the basis for drug resistance in numerous pathogenic bacterial species. PMID:24747401

  18. Structure of the AcrAB-TolC multidrug efflux pump.

    PubMed

    Du, Dijun; Wang, Zhao; James, Nathan R; Voss, Jarrod E; Klimont, Ewa; Ohene-Agyei, Thelma; Venter, Henrietta; Chiu, Wah; Luisi, Ben F

    2014-05-22

    The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative bacteria, many of these transporters form multicomponent 'pumps' that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component. A model system for such a pump is the acridine resistance complex of Escherichia coli. This pump assembly comprises the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA, which bridges these two integral membrane proteins. The AcrAB-TolC efflux pump is able to transport vectorially a diverse array of compounds with little chemical similarity, thus conferring resistance to a broad spectrum of antibiotics. Homologous complexes are found in many Gram-negative species, including in animal and plant pathogens. Crystal structures are available for the individual components of the pump and have provided insights into substrate recognition, energy coupling and the transduction of conformational changes associated with the transport process. However, how the subunits are organized in the pump, their stoichiometry and the details of their interactions are not known. Here we present the pseudo-atomic structure of a complete multidrug efflux pump in complex with a modulatory protein partner from E. coli. The model defines the quaternary organization of the pump, identifies key domain interactions, and suggests a cooperative process for channel assembly and opening. These findings illuminate the basis for drug resistance in numerous pathogenic bacterial species. PMID:24747401

  19. Efflux as a glutaraldehyde resistance mechanism in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms.

    PubMed

    Vikram, Amit; Bomberger, Jennifer M; Bibby, Kyle J

    2015-01-01

    A major challenge in microbial biofilm control is biocide resistance. Phenotypic adaptations and physical protective effects have been historically thought to be the primary mechanisms for glutaraldehyde resistance in bacterial biofilms. Recent studies indicate the presence of genetic mechanisms for glutaraldehyde resistance, but very little is known about the contributory genetic factors. Here, we demonstrate that efflux pumps contribute to glutaraldehyde resistance in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms. The RNA-seq data show that efflux pumps and phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis metabolic pathways were induced upon glutaraldehyde exposure. Furthermore, chemical inhibition of efflux pumps potentiates glutaraldehyde activity, suggesting that efflux activity contributes to glutaraldehyde resistance. Additionally, induction of known modulators of biofilm formation, including phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis, may contribute to biofilm resistance and resilience. Fundamental understanding of the genetic mechanism of biocide resistance is critical for the optimization of biocide use and development of novel disinfection strategies. Our results reveal genetic components involved in glutaraldehyde resistance and a potential strategy for improved control of biofilms. PMID:25824217

  20. Characterization of Zebrafish Abcc4 as an Efflux Transporter of Organochlorine Pesticides

    PubMed Central

    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. PMID:25478949

  1. Simultaneous Influx and Efflux of Nitrate during Uptake by Perennial Ryegrass 1

    PubMed Central

    Morgan, M. A.; Volk, R. J.; Jackson, W. A.

    1973-01-01

    Experiments with intact plants of Lolium perenne previously grown with 14NO3− revealed significant efflux of this isotopic species when the plants were transferred to solutions of highly enriched 15NO3−. The exuded 14NO3− was subsequently reabsorbed when the ambient solutions were not replaced. When they were frequently replaced, continual efflux of the 14NO3− was observed. Influx of 15NO3− was significantly greater than influx of 14NO3− from solutions of identical NO3− concentration. Transferring plants to 14NO3− solutions after a six-hour period in 15NO3− resulted in efflux of the latter. Presence of Mg2+, rather than Ca2+, in the ambient 15NO3− solution resulted in a decidedly increased rate of 14NO3− efflux and a slight but significant increase in 15NO3− influx. Accordingly, net NO3− influx was slightly depressed. A model in accordance with these observations is presented; its essential features include a passive bidirectional pathway, an active uptake mechanism, and a pathway for recycling of endogenous NO3− within unstirred layers from the passive pathway to the active uptake site. PMID:16658313

  2. 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. PMID:26907376

  3. Efflux as a Glutaraldehyde Resistance Mechanism in Pseudomonas fluorescens and Pseudomonas aeruginosa Biofilms

    PubMed Central

    Vikram, Amit; Bomberger, Jennifer M.

    2015-01-01

    A major challenge in microbial biofilm control is biocide resistance. Phenotypic adaptations and physical protective effects have been historically thought to be the primary mechanisms for glutaraldehyde resistance in bacterial biofilms. Recent studies indicate the presence of genetic mechanisms for glutaraldehyde resistance, but very little is known about the contributory genetic factors. Here, we demonstrate that efflux pumps contribute to glutaraldehyde resistance in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms. The RNA-seq data show that efflux pumps and phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis metabolic pathways were induced upon glutaraldehyde exposure. Furthermore, chemical inhibition of efflux pumps potentiates glutaraldehyde activity, suggesting that efflux activity contributes to glutaraldehyde resistance. Additionally, induction of known modulators of biofilm formation, including phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis, may contribute to biofilm resistance and resilience. Fundamental understanding of the genetic mechanism of biocide resistance is critical for the optimization of biocide use and development of novel disinfection strategies. Our results reveal genetic components involved in glutaraldehyde resistance and a potential strategy for improved control of biofilms. PMID:25824217

  4. 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.

  5. Dopamine inhibits maitotoxin-stimulated pituitary /sup 45/Ca/sup 2 +/ efflux and prolactin release

    SciTech Connect

    Login, I.S.; Judd, A.M.; MacLeod, R.M.

    1986-06-01

    The authors examined the hypothesis that dopaminergic inhibition of prolactin release is coupled to modulation of cellular calcium flux. Dispersed female rat pituitary cells were prelabeled in /sup 45/Ca/sup 2 +/ and perifused to determine simultaneously fractional calcium efflux and prolactin release, as stimulated by maitotoxin, a calcium channel activator. The integrated response of each parameter to 5 ng/ml maitotoxin was obtained in individual perifusion columns in the absence or presence of various concentrations of dopamine. Maitotoxin-stimulated calcium efflux was suppressed by dopamine concentrations of 0.01 ..mu..M and greater and achieved a maximal effect at approx.0.1 ..mu..M, at which calcium efflux was reduced by 50%. Maitotoxin-stimulated prolactin release was inhibited by 0.03 ..mu..M dopamine and greater concentrations, and at a concentration of approx.10.0 ..mu..M dopamine the effect became maximal at approx.85% suppression. Haloperidol (0.1 ..mu..M) blocked the effects of 0.1 ..mu..M dopamine on both parameters. Simultaneous suppression of maitotoxin-stimulated calcium efflux and prolactin release by concentrations of dopamine within the nonomolar range suggests that dopamine receptor activation is negatively coupled to modulation of calcium flux in the physiological regulation of prolactin secretion.

  6. 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. PMID:25478949

  7. 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. PMID:27113203

  8. Proteomic analysis of HDL from inbred mouse strains implicates APOE associated with HDL in reduced cholesterol efflux capacity via the ABCA1 pathway[S

    PubMed Central

    Pamir, Nathalie; Hutchins, Patrick; Ronsein, Graziella; Vaisar, Tomas; Reardon, Catherine A.; Getz, Godfrey S.; Lusis, Aldons J.; Heinecke, Jay W.

    2016-01-01

    Cholesterol efflux capacity associates strongly and negatively with the incidence and prevalence of human CVD. We investigated the relationships of HDL’s size and protein cargo with its cholesterol efflux capacity using APOB-depleted serum and HDLs isolated from five inbred mouse strains with different susceptibilities to atherosclerosis. Like humans, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. HDL’s content of specific proteins strongly correlated with its size and cholesterol efflux capacity, suggesting that its protein cargo regulates its function. Cholesterol efflux capacity with macrophages strongly and positively correlated with retinol binding protein 4 (RBP4) and PLTP, but not APOA1. In contrast, ABCA1-specific cholesterol efflux correlated strongly with HDL’s content of APOA1, APOC3, and APOD, but not RBP4 and PLTP. Unexpectedly, APOE had a strong negative correlation with ABCA1-specific cholesterol efflux capacity. Moreover, the ABCA1-specific cholesterol efflux capacity of HDL isolated from APOE-deficient mice was significantly greater than that of HDL from wild-type mice. Our observations demonstrate that the HDL-associated APOE regulates HDL’s ABCA1-specific cholesterol efflux capacity. These findings may be clinically relevant because HDL’s APOE content associates with CVD risk and ABCA1 deficiency promotes unregulated cholesterol accumulation in human macrophages. PMID:26673204

  9. Medial Septum-Diagonal Band of Broca (MSDB) GABAergic Regulation of Hippocampal Acetylcholine Efflux Is Dependent on Cognitive Demands

    PubMed Central

    Roland, Jessica J.; Stewart, Amanda L.; Janke, Kellie L.; Gielow, Matthew R.; Kostek, John A.; Savage, Lisa M.; Servatius, Richard J.

    2014-01-01

    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. PMID:24403150

  10. Perspective on plasma membrane cholesterol efflux and spermatozoal function.

    PubMed

    Sheriff, Dhastagir Sultan; Ali, Elshaari Farag

    2010-05-01

    The process of sperm maturation, capacitation, and fertilization occur in different molecular milieu provided by epididymis and female reproductive tract including oviduct. The different tissue environment with different oxygen tension and temperature may still influence the process of sperm maturation and capacitation. Reactive oxygen species (ROS) is reported to be an initial switch that may activate the molecular process of capacitation. Therefore, the generation of reactive oxygen species and its possible physiological role depends upon a balance between its formation and degradation in an open environment provided by female reproductive tract. The sensitivity of the spermatozoa to the action of ROS may be due to its exposure for the first time to an oxygen rich external milieu compared to its internal milieu in the male reproductive tract. Reduced temperature in testicular environment coupled with reduced oxygen tension may be the right molecular environment for the process of spermatogenesis and spermiogenesis. The morphologically mature spermatozoa then may attain its motility in an environment provided by the caput epididymis wherein, the dyenin motor can become active. This ability to move forward will make the spermatozoa physiologically fit to undertake its sojourn in the competitive race of fertilization in a new oxygen rich female reproductive tract. The first encounter may be oxygen trigger or preconditioning of the spermatozoa with reactive oxygen species that may alter the spermatozoal function. Infertility is still one of the major global health problems that need medical attention. Apart from the development of artificial methods of reproduction and development of newer techniques in the field of andrology focuses attention on spermatozoal structure and metabolism. Therefore, understanding the molecular mechanisms involved in fertilization in general and that of sperm capacitation in particular may help lead to new and better techniques for

  11. 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.

  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). PMID:25038543

  13. Insight into determinants of substrate binding and transport in a multidrug efflux protein

    PubMed Central

    Alegre, Kamela O.; Paul, Stephanie; Labarbuta, Paola; Law, Christopher J.

    2016-01-01

    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. PMID:26961153

  14. Insight into determinants of substrate binding and transport in a multidrug efflux protein.

    PubMed

    Alegre, Kamela O; Paul, Stephanie; Labarbuta, Paola; Law, Christopher J

    2016-01-01

    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. PMID:26961153

  15. 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

  16. Dietary ellagic acid attenuates oxidized LDL uptake and stimulates cholesterol efflux in murine macrophages.

    PubMed

    Park, Sin-Hye; Kim, Jung-Lye; Lee, Eun-Sook; Han, Seon-Young; Gong, Ju-Hyun; Kang, Min-Kyung; Kang, Young-Hee

    2011-11-01

    Foam cell formation is the hallmark of early atherosclerosis. Lipid uptake by scavenger receptors (SR) in macrophages initiates chronic proinflammatory cascades linked to atherosclerosis. It has been reported that the upregulation of cholesterol efflux may be protective in the development of atherosclerosis. Ellagic acid, a polyphenolic compound mostly found in berries, walnuts, and pomegranates, possesses antioxidative, growth-inhibiting and apoptosis-promoting activities in cancer cells. However, the antiatherogenic actions of ellagic acid are not well defined. The current study elucidated oxidized LDL handling of ellagic acid in J774A1 murine macrophages. Noncytotoxic ellagic acid suppressed SR-B1 induction and foam cell formation within 6 h after the stimulation of macrophages with oxidized LDL, confirmed by Oil red O staining of macrophages. Ellagic acid at ≤5 μmol/L upregulated PPARγ and ATP binding cassette transporter-1 in lipid-laden macrophages, all responsible for cholesterol efflux. In addition, 5 μmol/L ellagic acid accelerated expression and transcription of the nuclear receptor of liver X receptor-α highly implicated in the PPAR signaling. Furthermore, ellagic acid promoted cholesterol efflux in oxidized LDL-induced foam cells. These results provide new information that ellagic acid downregulated macrophage lipid uptake to block foam cell formation of macrophages and boosted cholesterol efflux in lipid-laden foam cells. Therefore, dietary and pharmacological interventions with berries rich in ellagic acid may be promising treatment strategies to interrupt the development of atherosclerosis. PMID:21940512

  17. 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).

  18. Impact of heme oxygenase-1 on cholesterol synthesis, cholesterol efflux and oxysterol formation in cultured astroglia.

    PubMed

    Hascalovici, Jacob R; Song, Wei; Vaya, Jacob; Khatib, Soliman; Fuhrman, Bianca; Aviram, Michael; Schipper, Hyman M

    2009-01-01

    Up-regulation of heme oxygenase-1 (HO-1) and altered cholesterol (CH) metabolism are characteristic of Alzheimer-diseased neural tissues. The liver X receptor (LXR) is a molecular sensor of CH homeostasis. In the current study, we determined the effects of HO-1 over-expression and its byproducts iron (Fe(2+)), carbon monoxide (CO) and bilirubin on CH biosynthesis, CH efflux and oxysterol formation in cultured astroglia. HO-1/LXR interactions were also investigated in the context of CH efflux. hHO-1 over-expression for 3 days ( approximately 2-3-fold increase) resulted in a 30% increase in CH biosynthesis and a two-fold rise in CH efflux. Both effects were abrogated by the competitive HO inhibitor, tin mesoporphyrin. CO, released from administered CORM-3, significantly enhanced CH biosynthesis; a combination of CO and iron stimulated CH efflux. Free iron increased oxysterol formation three-fold. Co-treatment with LXR antagonists implicated LXR activation in the modulation of CH homeostasis by heme degradation products. In Alzheimer's disease and other neuropathological states, glial HO-1 induction may transduce ambient noxious stimuli (e.g. beta-amyloid) into altered patterns of glial CH homeostasis. As the latter may impact synaptic plasticity and neuronal repair, modulation of glial HO-1 expression (by pharmacological or other means) may confer neuroprotection in patients with degenerative brain disorders. PMID:19046352

  19. 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

  20. The JET Neutron Activation System

    NASA Astrophysics Data System (ADS)

    Roquemore, A. L.; Bertalot, L.; Esposito, B.; Jarvis, O. N.; Loughlin, M. J.; Sadler, G.; van Belle, P.

    1997-11-01

    The JET activation system provides the absolute value of the neutron yields as well as a check on the linearity of other neutron detector systems. The total neutron yield is standardized to one irradiation end reentrant in the top of the vessel, while the results from the other seven irradiation ends are normalized to this standard end and provide redundancy as well as information on the plasma position. A pneumatic transfer system is used to transfer up to five capsules containing elemental foils for a single discharge on JET. Eleven different elemental foils have been utilized to determine the yields from both DD and DT plasmas. By placing several different foils with different activation energy thresholds in a single capsule for one DT discharge, neutron spectral information has been obtained by use of the SAND-II unfolding code. A description of the activation system hardware and calibration of the activation detector system will be presented along with the results from the DT neutron calibration campaign.

  1. Modeling approaches for active systems

    NASA Astrophysics Data System (ADS)

    Herold, Sven; Atzrodt, Heiko; Mayer, Dirk; Thomaier, Martin

    2006-03-01

    To solve a wide range of vibration problems with the active structures technology, different simulation approaches for several models are needed. The selection of an appropriate modeling strategy is depending, amongst others, on the frequency range, the modal density and the control target. An active system consists of several components: the mechanical structure, at least one sensor and actuator, signal conditioning electronics and the controller. For each individual part of the active system the simulation approaches can be different. To integrate the several modeling approaches into an active system simulation and to ensure a highly efficient and accurate calculation, all sub models must harmonize. For this purpose, structural models considered in this article are modal state-space formulations for the lower frequency range and transfer function based models for the higher frequency range. The modal state-space formulations are derived from finite element models and/or experimental modal analyses. Consequently, the structure models which are based on transfer functions are directly derived from measurements. The transfer functions are identified with the Steiglitz-McBride iteration method. To convert them from the z-domain to the s-domain a least squares solution is implemented. An analytical approach is used to derive models of active interfaces. These models are transferred into impedance formulations. To couple mechanical and electrical sub-systems with the active materials, the concept of impedance modeling was successfully tested. The impedance models are enhanced by adapting them to adequate measurements. The controller design strongly depends on the frequency range and the number of modes to be controlled. To control systems with a small number of modes, techniques such as active damping or independent modal space control may be used, whereas in the case of systems with a large number of modes or with modes that are not well separated, other control

  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. The activation system EASY-2007

    NASA Astrophysics Data System (ADS)

    Forrest, R. A.; Kopecky, J.

    2009-04-01

    Safety and waste management of materials for ITER, IFMIF and future power plants require knowledge of the activation caused by irradiation with neutrons, or in the case of IFMIF, deuterons. The European Activation System has been developed for such calculations and a new version was released earlier this year. This contains a large amount of nuclear data in the European Activation File covering neutron-, deuteron- and proton-induced cross sections. These data are input to the FISPACT code for activation calculations. EASY-2007 is being validated using integral and differential measurements. However, only a minority of reactions have experimental support and a statistical method is described that can test the complete library. Importance diagrams are useful in finding the dominant nuclides formed following irradiation and the reactions responsible for their production. These diagrams now cover energies above 20 MeV and examples of new dominant nuclides and reactions relevant to IFMIF are given.

  4. 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. PMID:26151644

  5. Role of Efflux Pumps and Intracellular Thiols in Natural Antimony Resistant Isolates of Leishmania donovani

    PubMed Central

    Rai, Smita; Bhaskar; Goel, Sudhir K.; Nath Dwivedi, Upendra; Sundar, Shyam; Goyal, Neena

    2013-01-01

    Background In view of the recent upsurge in the phenomenon of therapeutic failure, drug resistance in Leishmania, developed under natural field conditions, has become a great concern yet little understood. Accordingly, the study of determinants of antimony resistance is urgently warranted. Efflux transporters have been reported in Leishmania but their role in clinical resistance is still unknown. The present study was designed to elucidate the mechanism of natural antimony resistance in L. donovani field isolates by analyzing the functionality of efflux pump(s) and expression profiles of known genes involved in transport and thiol based redox metabolism Methodology/Principal Findings We selected 7 clinical isolates (2 sensitive and 5 resistant) in addition to laboratory sensitive reference and SbIII resistant mutant strains for the present study. Functional characterization using flow cytometry identified efflux pumps that transported substrates of both P-gp and MRPA and were inhibited by the calmodulin antagonist trifluoperazine. For the first time, verapamil sensitive efflux pumps for rhodamine 123 were observed in L. donovani that were differentially active in resistant isolates. RT-PCR confirmed the over-expression of MRPA in isolates with high resistance index only. Resistant isolates also exhibited consistent down regulation of AQP1 and elevated intracellular thiol levels which were accompanied with increased expression of ODC and TR genes. Interestingly, γ-GCS is not implicated in clinical resistance in L. donovani isolates. Conclusions/Significance Here we demonstrate for the first time, the role of P-gp type plasma membrane efflux transporter(s) in antimony resistance in L. donovani field isolates. Further, decreased levels of AQP1 and elevated thiols levels have emerged as biomarkers for clinical resistance. PMID:24069359

  6. 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

  7. 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

  8. Efflux pump inhibitors (EPIs) as new antimicrobial agents against Pseudomonas aeruginosa

    PubMed Central

    Askoura, Momen; Mottawea, Walid; Abujamel, Turki; Taher, Ibrahim

    2011-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen and one of the leading causes of nosocomial infections worldwide. The difficulty in treatment of pseudomonas infections arises from being multidrug resistant (MDR) and exhibits resistance to most antimicrobial agents due to the expression of different mechanisms overcoming their effects. Of these resistance mechanisms, the active efflux pumps in Pseudomonas aeruginosa that belong to the resistance nodulation division (RND) plays a very important role in extruding the antibiotics outside the bacterial cells providing a protective means against their antibacterial activity. Beside its role against the antimicrobial agents, these pumps can extrude biocides, detergents, and other metabolic inhibitors. It is clear that efflux pumps can be targets for new antimicrobial agents. Peptidomimetic compounds such as phenylalanine arginyl β-naphthylamide (PAβN) have been introduced as efflux pump inhibitors (EPIs); their mechanism of action is through competitive inhibition with antibiotics on the efflux pump resulting in increased intracellular concentration of antibiotic, hence, restoring its antibacterial activity. The advantage of EPIs is the difficulty to develop bacterial resistance against them, but the disadvantage is their toxic property hindering their clinical application. The structure activity relationship of these compounds showed other derivatives from PAβN that are higher in their activity with higher solubility in biological fluids and decreased toxicity level. This raises further questions on how can we compact Pseudomonas infections. Of particular importance, the recent resurgence in the use of older antibiotics such as polymyxins and probably applying stricter control measures in order to prevent their spread in clinical sittings. PMID:21594004

  9. The photochemical thiol–ene reaction as a versatile method for the synthesis of glutathione S-conjugates targeting the bacterial potassium efflux system Kef† †Electronic supplementary information (ESI) available: Further experimental details and NMR spectra. See DOI: 10.1039/c5qo00436e Click here for additional data file.

    PubMed Central

    Rasmussen, Tim; Miller, Samantha; Booth, Ian R.

    2016-01-01

    The thiol–ene coupling reaction is emerging as an important conjugation reaction that is suitable for use in a biological setting. Here, we explore the utility of this reaction for the synthesis of glutathione-S-conjugates (GSX) and present a general, operationally simple, protocol with a wide substrate scope. The GSX afforded are an important class of compounds and provide invaluable molecular tools to study glutathione-binding proteins. In this study we apply the diverse library of GSX synthesised to further our understanding of the structural requirements for binding to the glutathione-binding protein, Kef, a bacterial K+ efflux system, found in many bacterial pathogens. This system is vital to the survival of bacteria upon exposure to electrophiles, and plays an essential role in the maintenance of intracellular pH and K+ homeostasis. Consequently, Kef is an appealing target for the development of novel antibacterial drugs. PMID:27110363

  10. New phenotypes of functional expression of the mKir2.1 channel in potassium efflux-deficient Saccharomyces cerevisiae strains.

    PubMed

    Kolacna, Lucie; Zimmermannova, Olga; Hasenbrink, Guido; Schwarzer, Sarah; Ludwig, Jost; Lichtenberg-Fraté, Hella; Sychrova, Hana

    2005-12-01

    The functional expression of the mouse Kir2.1 potassium channel in yeast cells lacking transport systems for potassium and sodium efflux (ena1-4delta nha1delta) resulted in increased cell sensitivity to high external concentrations of potassium. The phenotype depended on the level of Kir2.1 expression and on the external pH. The activity of Kir2.1p in the yeast cells was almost negligible at pH 3.0 and the highest at pH 7.0. Kir2.1p was permeable for both potassium and rubidium cations, but neither sodium nor lithium were transported via the channel. Measurements of the cation contents in cells confirmed the higher concentration of potassium in cells with Kir2.1p. Specific inhibition of the mKir2.1 channel activity by Ba2+ cations was observed. The use of a mutant strain lacking both potassium efflux and uptake transporters (ena1-4delta nha1delta trk1delta trk2delta) enabled the monitoring of channel activity on two levels--the provision of the necessary amount of intracellular K+ in media with low potassium concentrations, and simultaneously, the channel's contribution to cell potassium sensitivity in the presence of high external K+. This combination of mutations proved to be a new, sensitive and practical tool for characterizing the properties of heterologously expressed transporters mediating both the efflux and influx of alkali-metal-cations. PMID:16358319

  11. Sarothrin from Alkanna orientalis is an antimicrobial agent and efflux pump inhibitor

    PubMed Central

    Bame, Jessica R.; Graf, Tyler N.; Junio, Hiyas A.; Bussey, R. Owen; Jarmusch, Scott A.; El-Elimat, Tamam; Falkinham, Joseph O.; Oberlies, Nicholas H.; Cech, Richard A.; Cech, Nadja B.

    2015-01-01

    An Alkanna orientalis leaf and flower extract inhibited the growth of Staphylococcus aureus, a pathogen that causes an estimated 478,000 hospitalizations in the US annually. Bioassay-guided fractionation of A. orientalis resulted in isolation of the flavonoid sarothrin (5,7,4′-trihydroxy-3,6,8-trimethoxyflavone), which inhibited the growth of Mycobacterium smegmatis (MIC 75 μM) and S. aureus (MIC >800 μM), and possessed efflux pump inhibitory activity. This is the first report of antimicrobial or efflux pump inhibitory activity of sarothrin, and of its presence in A. orientalis. Our findings suggest that the effectiveness of A. orientalis extracts is due to a combination of multiple constituents, including sarothrin. PMID:23468310

  12. 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.

  13. Cadmium induced potassium efflux from Scenedesmus quadricauda

    SciTech Connect

    Reddy, G.N.; Prasad, M.N.V.

    1992-10-01

    Plants, algae and bacteria respond to heavy metal toxicity by inducing different enzymes, ion influx/efflux for ionic balance and synthesize small peptides such as poly({gamma}-glutamyl cysteinyl) glycines called phytochelatins (PCs) mainly consisting of glutamate, cysteine and glycine. These peptides bind metal ions and reduce toxicity. The uptake of metal ions comprises two phases. The first phase consists of a quick and nonspecific binding of the cations to negatively-charged membrane components located at the cell surface. The second phase consists of energy-dependent intracellular uptake of the metal ions. During uptake of Co{sup 2+} by yeast cells, an electroneutral 2:1 exchange with K{sup +} was found. Cd{sup 2+} uptake by yeast also caused loss of cell K{sup +}, however, there was no electroneutral exchange of K{sup +}. The molar ratio of K{sup +} released and Cd{sup 2+} accumulated by yeast in the initial stage of incubation is 22 and seems to be independent of the Cd concentration. Disruption of the cell membrane of part of the cells, according to an all-or-none process, by Cd{sup 2+} may explain the disproportional loss of cell K{sup +} during Cd{sup 2+} uptake. This paper examines the exchange of K{sup +} with Cd{sup 2+} uptake in Scenedesmus quadricauda, and whether it follows an electroneutral 2:1 exchange or an all-or-none process. 11 refs., 2 figs.

  14. 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.

  15. 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

    2016-05-01

    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. PMID:26961872

  16. Seasonal Variation in CO2 Efflux of Stems and Branches of Norway Spruce Trees

    PubMed Central

    Acosta, Manuel; Pavelka, Marian; Pokorný, Radek; Janouš, Dalibor; Marek, Michal V.

    2008-01-01

    Background and Aims Stem and branch respiration, important components of total forest ecosystem respiration, were measured on Norway spruce (Picea abies) trees from May to October in four consecutive years in order (1) to evaluate the influence of temperature on woody tissue CO2 efflux with special focus on variation in Q10 (change in respiration rate resulting from a 10 °C increase in temperature) within and between seasons, and (2) to quantify the contribution of above-ground woody tissue (stem and branch) respiration to the carbon balance of the forest ecosystem. Methods Stem and branch CO2 efflux were measured, using an IRGA and a closed gas exchange system, 3–4 times per month on 22-year-old trees under natural conditions. Measurements of ecosystem CO2 fluxes were also determined during the whole experiment by using the eddy covariance system. Stem and branch temperatures were monitored at 10-min intervals during the whole experiment. Key Results The temperature of the woody tissue of stems and branches explained up to 68 % of their CO2 efflux. The mean annual Q10 values ranged from 2·20 to 2·32 for stems and from 2·03 to 2·25 for branches. The mean annual normalized respiration rate, R10, for stems and branches ranged from 1·71 to 2·12 µmol CO2 m−2s −1 and from 0·24 to 0·31 µmol CO2 m−2 s−1, respectively. The annual contribution of stem and branch CO2 efflux to total ecosystem respiration were, respectively, 8·9 and 8·1 % in 1999, 9·2 and 9·2 % in 2000, 7·6 and 8·6 % in 2001, and 8·6 and 7·9 % in 2002. Standard deviation for both components ranged from 3 to 8 % of the mean. Conclusions Stem and branch CO2 efflux varied diurnally and seasonally, and were related to the temperature of the woody tissue and to growth. The proportion of CO2 efflux from stems and branches is a significant component of the total forest ecosystem respiration, approx. 8 % over the 4 years, and predictive models must take their contribution into account

  17. Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae

    PubMed Central

    Peters, Theodore W.; Miller, Aaron W.; Tourette, Cendrine; Agren, Hannah; Hubbard, Alan; Hughes, Robert E.

    2015-01-01

    Adenosine triphosphate (ATP) plays an important role as a primary molecule for the transfer of chemical energy to drive biological processes. ATP also functions as an extracellular signaling molecule in a diverse array of eukaryotic taxa in a conserved process known as purinergic signaling. Given the important roles of extracellular ATP in cell signaling, we sought to comprehensively elucidate the pathways and mechanisms governing ATP efflux from eukaryotic cells. Here, we present results of a genomic analysis of ATP efflux from Saccharomyces cerevisiae by measuring extracellular ATP levels in cultures of 4609 deletion mutants. This screen revealed key cellular processes that regulate extracellular ATP levels, including mitochondrial translation and vesicle sorting in the late endosome, indicating that ATP production and transport through vesicles are required for efflux. We also observed evidence for altered ATP efflux in strains deleted for genes involved in amino acid signaling, and mitochondrial retrograde signaling. Based on these results, we propose a model in which the retrograde signaling pathway potentiates amino acid signaling to promote mitochondrial respiration. This study advances our understanding of the mechanism of ATP secretion in eukaryotes and implicates TOR complex 1 (TORC1) and nutrient signaling pathways in the regulation of ATP efflux. These results will facilitate analysis of ATP efflux mechanisms in higher eukaryotes. PMID:26585826

  18. The selective delta opioid agonist SNC80 enhances amphetamine-mediated efflux of dopamine from rat striatum.

    PubMed

    Bosse, Kelly E; Jutkiewicz, Emily M; Gnegy, Margaret E; Traynor, John R

    2008-10-01

    The highly selective delta opioid agonist, SNC80, elicits dopamine-related behaviors including locomotor stimulation and conditioned place-preference. In contrast, it has been reported that SNC80 fails to promote dopamine efflux from the striatum of freely moving rats. However, SNC80 does enhance behavioral responses to the stimulants, amphetamine and cocaine, suggesting an interaction between delta opioids and psychostimulants. Since the increase in locomotor activity elicited by amphetamine and related stimulants acting at the dopamine transporter is associated with increases in extracellular concentrations of dopamine within the striatum, we hypothesized that SNC80 enhances this activity by potentiating the overflow of dopamine through the transporter. To test this hypothesis, striatal preparations from Sprague Dawley rats were assayed for dopamine efflux in response to amphetamine challenge. SNC80 was given either in vivo or in vitro directly to rat striatal tissue, prior to in vitro amphetamine challenge. Both in vivo and in vitro administration of SNC80 enhanced amphetamine-mediated dopamine efflux in a concentration- and time-dependent manner. However, SNC80 in either treatment paradigm produced no stimulation of dopamine efflux in the absence of amphetamine. The effect of SNC80 on amphetamine-mediated dopamine overflow, but not the effect of amphetamine alone, was blocked by the delta selective antagonist, naltrindole and was also observed with other delta agonists. The results of this study demonstrate that even though SNC80 does not stimulate dopamine efflux alone, it is able to augment amphetamine-mediated dopamine efflux through a delta opioid receptor mediated action locally in the striatum. PMID:18602932

  19. Pathways for Ca2+ efflux in heart and liver mitochondria.

    PubMed Central

    Rizzuto, R; Bernardi, P; Favaron, M; Azzone, G F

    1987-01-01

    1. Two processes of Ruthenium Red-insensitive Ca2+ efflux exist in liver and in heart mitochondria: one Na+-independent, and another Na+-dependent. The processes attain maximal rates of 1.4 and 3.0 nmol of Ca2+.min-1.mg-1 for the Na+-dependent and 1.2 and 2.0 nmol of Ca2+.min-1.mg-1 for the Na+-independent, in liver and heart mitochondria, respectively. 2. The Na+-dependent pathway is inhibited, both in heart and in liver mitochondria, by the Ca2+ antagonist diltiazem with a Ki of 4 microM. The Na+-independent pathway is inhibited by diltiazem with a Ki of 250 microM in liver mitochondria, while it behaves as almost insensitive to diltiazem in heart mitochondria. 3. Stretching of the mitochondrial inner membrane in hypo-osmotic media results in activation of the Na+-independent pathway both in liver and in heart mitochondria. 4. Both in heart and liver mitochondria the Na+-independent pathway is insensitive to variations of medium pH around physiological values, while the Na+-dependent pathway is markedly stimulated parallel with acidification of the medium. The pH-activated, Na+-dependent pathway maintains the diltiazem sensitivity. 5. In heart mitochondria, the Na+-dependent pathway is non-competitively inhibited by Mg2+ with a Ki of 0.27 mM, while the Na+-independent pathway is less affected; similarly, in liver mitochondria Mg2+ inhibits the Na+-dependent pathway more than it does the Na+-independent pathway. In the presence of physiological concentrations of Na+, Ca2+ and Mg2+, the Na+-independent and the Na+-dependent pathways operate at rates, respectively, of 0.5 and 1.0 nmol of Ca2+.min-1.mg-1 in heart mitochondria and 0.9 and 0.2 nmol of Ca2+.min-1.mg-1 in liver mitochondria. It is concluded that both heart and liver mitochondria possess two independent pathways for Ca2+ efflux operating at comparable rates. PMID:3689311

  20. 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

  1. CFTR mediates noradrenaline-induced ATP efflux from DRG neurons

    PubMed Central

    2011-01-01

    In our earlier study, noradrenaline (NA) stimulated ATP release from dorsal root ganglion (DRG) neurons as mediated via β3 adrenoceptors linked to Gs protein involving protein kinase A (PKA) activation, to cause allodynia. The present study was conducted to understand how ATP is released from DRG neurons. In an outside-out patch-clamp configuration from acutely dissociated rat DRG neurons, single-channel currents, sensitive to the P2X receptor inhibitor PPADS, were evoked by approaching the patch-electrode tip close to a neuron, indicating that ATP is released from DRG neurons, to activate P2X receptor. NA increased the frequency of the single-channel events, but such NA effect was not found for DRG neurons transfected with the siRNA to silence the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In the immunocytochemical study using acutely dissociated rat DRG cells, CFTR was expressed in neurons alone, but not satellite cells, fibroblasts, or Schwann cells. It is concluded from these results that CFTR mediates NA-induced ATP efflux from DRG neurons as an ATP channel. PMID:21943397

  2. 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

  3. Constraint of soil moisture on CO2 efflux from tundra lichen, moss, and tussock in Council, Alaska, using a hierarchical Bayesian model

    NASA Astrophysics Data System (ADS)

    Kim, Y.; Nishina, K.; Chae, N.; Park, S. J.; Yoon, Y. J.; Lee, B. Y.

    2014-10-01

    The tundra ecosystem is quite vulnerable to drastic climate change in the Arctic, and the quantification of carbon dynamics is of significant importance regarding thawing permafrost, changes to the snow-covered period and snow and shrub community extent, and the decline of sea ice in the Arctic. Here, CO2 efflux measurements using a manual chamber system within a 40 m × 40 m (5 m interval; 81 total points) plot were conducted within dominant tundra vegetation on the Seward Peninsula of Alaska, during the growing seasons of 2011 and 2012, for the assessment of driving parameters of CO2 efflux. We applied a hierarchical Bayesian (HB) model - a function of soil temperature, soil moisture, vegetation type, and thaw depth - to quantify the effects of environmental factors on CO2 efflux and to estimate growing season CO2 emissions. Our results showed that average CO2 efflux in 2011 was 1.4 times higher than in 2012, resulting from the distinct difference in soil moisture between the 2 years. Tussock-dominated CO2 efflux is 1.4 to 2.3 times higher than those measured in lichen and moss communities, revealing tussock as a significant CO2 source in the Arctic, with a wide area distribution on the circumpolar scale. CO2 efflux followed soil temperature nearly exponentially from both the observed data and the posterior medians of the HB model. This reveals that soil temperature regulates the seasonal variation of CO2 efflux and that soil moisture contributes to the interannual variation of CO2 efflux for the two growing seasons in question. Obvious changes in soil moisture during the growing seasons of 2011 and 2012 resulted in an explicit difference between CO2 effluxes - 742 and 539 g CO2 m-2 period-1 for 2011 and 2012, respectively, suggesting the 2012 CO2 emission rate was reduced to 27% (95% credible interval: 17-36%) of the 2011 emission, due to higher soil moisture from severe rain. The estimated growing season CO2 emission rate ranged from 0.86 Mg CO2 in 2012 to 1

  4. In squid axons intracellular Mg2+ is essential for ATP-dependent Na+ efflux in the absence and presence of strophanthidin.

    PubMed

    Beaugé, L; Rojas, H; DiPolo, R

    1983-03-01

    The effect on Na+ efflux of removal of intracellular Mg2+ was studied in squid giant axons dialyzed without internal Ca2+. In the absence of Mg2i+, ATP was unable to stimulate any efflux of Na+ above the baseline of about 1 pmol . cm-2 . s-1. This behavior was observed in otherwise normal axons and in axons poisoned with 50 microM strophanthidin in the sea water. Reinstatement of 4 mM MgCl2 in excess to ATP in the dialysis solution brought about the usual response of Na+ efflux to ATP, external K+ and strophanthidin. The present experiments show that, regardless of the mechanism for the ATP-dependent Na+ efflux in strophanthidin-poisoned axons, this type of flux shares with the active Na+ extrusion the need for the simultaneous presence of intracellular ATP and Mg2+. PMID:6402014

  5. Endoplasmic reticulum stress impairs cholesterol efflux and synthesis in hepatic cells[S

    PubMed Central

    Röhrl, Clemens; Eigner, Karin; Winter, Katharina; Korbelius, Melanie; Obrowsky, Sascha; Kratky, Dagmar; Kovacs, Werner J.; Stangl, Herbert

    2014-01-01

    Metabolic disorders such as type 2 diabetes cause hepatic endoplasmic reticulum (ER) stress, which affects neutral lipid metabolism. However, the role of ER stress in cholesterol metabolism is incompletely understood. Here, we show that induction of acute ER stress in human hepatic HepG2 cells reduced ABCA1 expression and caused ABCA1 redistribution to tubular perinuclear compartments. Consequently, cholesterol efflux to apoA-I, a key step in nascent HDL formation, was diminished by 80%. Besides ABCA1, endogenous apoA-I expression was reduced upon ER stress induction, which contributed to reduced cholesterol efflux. Liver X receptor, a key regulator of ABCA1 in peripheral cells, was not involved in this process. Despite reduced cholesterol efflux, cellular cholesterol levels remained unchanged during ER stress. This was due to impaired de novo cholesterol synthesis by reduction of HMG-CoA reductase activity by 70%, although sterol response element-binding protein-2 activity was induced. In mice, ER stress induction led to a marked reduction of hepatic ABCA1 expression. However, HDL cholesterol levels were unaltered, presumably because of scavenger receptor class B, type I downregulation under ER stress. Taken together, our data suggest that ER stress in metabolic disorders reduces HDL biogenesis due to impaired hepatic ABCA1 function. PMID:24179149

  6. 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

  7. Inflammatory Regulation of ATP Binding Cassette Efflux Transporter Expression and Function in Microglia

    PubMed Central

    Gibson, Christopher J.; Hossain, Muhammad M.; Richardson, Jason R.

    2012-01-01

    ATP-binding cassette (ABC) efflux transporters, including multidrug resistance protein 1 (Mdr1), breast cancer resistance protein (Bcrp), and multidrug resistance-associated proteins (Mrps) extrude chemicals from the brain. Although ABC transporters are critical for blood-brain barrier integrity, less attention has been placed on the regulation of these proteins in brain parenchymal cells such as microglia. Prior studies demonstrate that inflammation after lipopolysaccharide (LPS) treatment alters transporter expression in the livers of mice. Here, we sought to determine the effects of inflammation on the expression and function of transporters in microglia. To test this, the expression and function of ABC efflux transport proteins were quantified in mouse BV-2 microglial cells in response to activation with LPS. Intracellular retention of fluorescent rhodamine 123, Hoechst 33342, and calcein acetoxymethyl ester was increased in LPS-treated microglia, suggesting that the functions of Mdr1, Bcrp, and Mrps were decreased, respectively. LPS reduced Mdr1, Bcrp, and Mrp4 mRNA and protein expression between 40 and 70%. Conversely, LPS increased expression of Mrp1 and Mrp5 mRNA and protein. Immunofluorescent staining confirmed reduced Bcrp and Mrp4 and elevated Mrp1 and Mrp5 protein in activated microglia. Pharmacological inhibition of nuclear factor κB (NF-κB) transcriptional signaling attenuated down-regulation of Mdr1a mRNA and potentiated up-regulation of Mrp5 mRNA in LPS-treated cells. Together, these data suggest that LPS stimulates microglia and impairs efflux of prototypical ABC transporter substrates by altering mRNA and protein expression, in part through NF-κB signaling. Decreased transporter efflux function in microglia may lead to the retention of toxic chemicals and aberrant cell-cell communication during neuroinflammation. PMID:22942241

  8. Control of Cl− Efflux in Chara corallina by Cytosolic pH, Free Ca2+, and Phosphorylation Indicates a Role of Plasma Membrane Anion Channels in Cytosolic pH Regulation1

    PubMed Central

    Johannes, Eva; Crofts, Alan; Sanders, Dale

    1998-01-01

    Enhanced Cl− efflux during acidosis in plants is thought to play a role in cytosolic pH (pHc) homeostasis by short-circuiting the current produced by the electrogenic H+ pump, thereby facilitating enhanced H+ efflux from the cytosol. Using an intracellular perfusion technique, which enables experimental control of medium composition at the cytosolic surface of the plasma membrane of charophyte algae (Chara corallina), we show that lowered pHc activates Cl− efflux via two mechanisms. The first is a direct effect of pHc on Cl− efflux; the second mechanism comprises a pHc-induced increase in affinity for cytosolic free Ca2+ ([Ca2+]c), which also activates Cl− efflux. Cl− efflux was controlled by phosphorylation/dephosphorylation events, which override the responses to both pHc and [Ca2+]c. Whereas phosphorylation (perfusion with the catalytic subunit of protein kinase A in the presence of ATP) resulted in a complete inhibition of Cl− efflux, dephosphorylation (perfusion with alkaline phosphatase) arrested Cl− efflux at 60% of the maximal level in a manner that was both pHc and [Ca2+]c independent. These findings imply that plasma membrane anion channels play a central role in pHc regulation in plants, in addition to their established roles in turgor/volume regulation and signal transduction. PMID:9733536

  9. 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. PMID:27190287

  10. 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.

  11. Use of a yeast-based membrane protein expression technology to overexpress drug resistance efflux pumps.

    PubMed

    Lamping, Erwin; Cannon, Richard D

    2010-01-01

    Azole antifungal drugs are used widely to treat people with oral fungal infections. Unfortunately, fungi can develop resistance to these drugs. This resistance can be due to the overexpression or mutation of cytochrome P450 14alpha-lanosterol demethylase, also known as ERG11 or CYP51, and/or the overexpression of membrane-located multidrug efflux pumps. We have developed a heterologous membrane protein expression system that can be used to study the structure and function of these proteins in the non-pathogenic, genetically stable, and versatile eukaryotic model organism, Saccharomyces cerevisiae. In this chapter we describe the techniques used to express the Candida albicans efflux pump Cdr1p in S. cerevisiae. PMID:20717788

  12. 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

  13. Sediment CO2 efflux from cleared and intact temperate mangrove and tidal flat habitat

    NASA Astrophysics Data System (ADS)

    Bulmer, Richard; Lundquist, Carolyn; Schwendenmann, Luitgard

    2015-04-01

    Temperate mangroves in Southern Australia and New Zealand have been increasing in extent over the past 50 years, whereas tropical mangroves have declined by 30-50% over a similar time frame to support development of aquaculture, land development and timber production. Tropical mangroves are understood to be an important carbon sink and carbon emissions following clearance are estimated to be significant; comparable or greater than clearance of many terrestrial forest systems. As temperate mangrove clearance is proposed and has already occurred at some locations, it is important to determine potential carbon emissions from temperate mangroves, as well as exploring the factors which may influence emission rates. Here, we investigated the impact of temperate mangrove clearance on CO2 efflux from the sediment to the atmosphere along with a range of other biotic and abiotic factors. Higher CO2 efflux rates were observed within cleared (1.34

  14. 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

  15. Oxidative stress and alterations in actin cytoskeleton trigger glutathione efflux in Saccharomyces cerevisiae.

    PubMed

    Bradamante, Silvia; Villa, Alessandro; Versari, Silvia; Barenghi, Livia; Orlandi, Ivan; Vai, Marina

    2010-12-01

    A marked deficiency in glutathione (GSH), the most abundant antioxidant in living systems, plays a major role in aging and the pathogenesis of diseases ranging from neurological disorders to early atherosclerosis and the impairment of various immunological functions. In an attempt to shed light on GSH homeostasis, we carried out the space experiment SCORE (Saccharomyces cerevisiae oxidative stress response evaluation) during the FOTON-M3 mission. Microgravity and hyperoxic conditions induced an enormous extracellular release of GSH from S. cerevisiae cells (≈40% w/dw), changed the distribution of the buds, and activated the high osmolarity glycerol (HOG) and cell integrity/PKC pathways, as well as protein carbonylation. The results from the single spaceflight experiment were validated by a complete set of experiments under conditions of simulated microgravity and indicate that cytoskeletal alterations are mainly responsible for the observed effects. The results of ground experiments in which we induced cytoskeletal modifications by means of treatment with dihydrocytochalasin B (DHCB), a potent inhibitor of actin polymerisation, or (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), a selective ROCK (Rho-associated coiled-coil forming protein serine/threonine kinase) inhibitor, confirmed the role of actin in GSH efflux. We also found that the GSH release can be inhibited using the potent chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). PMID:20708643

  16. Challenges in measuring the δ13C of the soil surface CO2 efflux.

    PubMed

    Midwood, Andrew J; Millard, Peter

    2011-01-15

    The δ13C of the soil surface efflux of carbon dioxide (δ13CRS) has emerged as a powerful tool enabling investigation of a wide range of soil processes from characterising entire ecosystem respiration to detailed compound-specific isotope studies. δ13CRS can be used to trace assimilated carbon transfer below ground and to partition the overall surface efflux into heterotrophic and autotrophic components. Despite this wide range of applications no consensus currently exists on the most appropriate method of sampling this surface efflux of CO2 in order to measure δ13CRS. Here we consider and compare the methods which have been used, and examine the pitfalls. We also consider a number of analysis options, isotope ratio mass spectrometry (IRMS), tuneable diode laser spectroscopy (TDLS) and cavity ring-down laser spectroscopy (CRDS). δ13CRS is typically measured using chamber systems, which fall into three types: closed, open and dynamic. All are imperfect. Closed chambers often rely on Keeling plots to estimate δ13CRS, which may not be appropriate without free turbulent air mixing. Open chambers have the advantage of being able to maintain steady-state conditions but analytical errors may become limiting with low efflux rates. Dynamic chambers like open chambers are complex, and controlling pressure fluctuations caused by air movement is a key concern. Both open and dynamic chambers in conjunction with field portable TDLS and CRDS analysis systems have opened up the possibility of measuring δ13CRS in real time permitting new research opportunities and are on balance the most suited to this type of measurement. PMID:21181784

  17. Responses of methane effluxes and soil methane concentrations to compaction.

    NASA Astrophysics Data System (ADS)

    Plain, C.; Delogu, E.; Longdoz, B.; Epron, D.; Ranger, J.

    2015-12-01

    Forest soils host methanotrophic bacterial communities that make them a major methane sink worldwide. Soil compaction resulting from mechanization of forest operations is first affecting soil macroporosity, and thus gas and water transfer within the soil, leading to a reduced oxygenation of the soil. This reduction of soil aeration is expected to reduce the methanotrophic activity leading thus to less CH4 oxidation and more CH4 production, affecting the overall soil CH4budget. Compaction was applied in 2007 and had created linear ruts. We measured continuously since September 2014, in three different situations (compacted-mound, compacted hollow and control), soil CO2 and CH4 effluxes using closed chamber coupled to a cavity ring down spectrometer in an young oak plantation. Since December 2015, in addition to these measurements, we have implanted hydrophobic tubes to measure vertical soil profiles of CH4, O2 and CO2 concentrations in the 3 situations. The soil acts as CH4 sink, with no significant difference in net CH4uptake between control and both hollow and mound in the compacted treatment. However, the uptake of CH4 was significantly lower for the hollows than for the mounds resulting from both a lower diffusion of CH4 within soil and a higher production of CH4 in deeper layer when the soil is water saturated.

  18. Polyamines interact with hydroxyl radicals in activating Ca(2+) and K(+) transport across the root epidermal plasma membranes.

    PubMed

    Zepeda-Jazo, Isaac; Velarde-Buendía, Ana María; Enríquez-Figueroa, René; Bose, Jayakumar; Shabala, Sergey; Muñiz-Murguía, Jesús; Pottosin, Igor I

    2011-12-01

    Reactive oxygen species (ROS) are integral components of the plant adaptive responses to environment. Importantly, ROS affect the intracellular Ca(2+) dynamics by activating a range of nonselective Ca(2+)-permeable channels in plasma membrane (PM). Using patch-clamp and noninvasive microelectrode ion flux measuring techniques, we have characterized ionic currents and net K(+) and Ca(2+) fluxes induced by hydroxyl radicals (OH(•)) in pea (Pisum sativum) roots. OH(•), but not hydrogen peroxide, activated a rapid Ca(2+) efflux and a more slowly developing net Ca(2+) influx concurrent with a net K(+) efflux. In isolated protoplasts, OH(•) evoked a nonselective current, with a time course and a steady-state magnitude similar to those for a K(+) efflux in intact roots. This current displayed a low ionic selectivity and was permeable to Ca(2+). Active OH(•)-induced Ca(2+) efflux in roots was suppressed by the PM Ca(2+) pump inhibitors eosine yellow and erythrosine B. The cation channel blockers gadolinium, nifedipine, and verapamil and the anionic channel blockers 5-nitro-2(3-phenylpropylamino)-benzoate and niflumate inhibited OH(•)-induced ionic currents in root protoplasts and K(+) efflux and Ca(2+) influx in roots. Contrary to expectations, polyamines (PAs) did not inhibit the OH(•)-induced cation fluxes. The net OH(•)-induced Ca(2+) efflux was largely prolonged in the presence of spermine, and all PAs tested (spermine, spermidine, and putrescine) accelerated and augmented the OH(•)-induced net K(+) efflux from roots. The latter effect was also observed in patch-clamp experiments on root protoplasts. We conclude that PAs interact with ROS to alter intracellular Ca(2+) homeostasis by modulating both Ca(2+) influx and efflux transport systems at the root cell PM. PMID:21980172

  19. Winter and early spring CO2 efflux from tundra communities of northern Alaska

    NASA Astrophysics Data System (ADS)

    Fahnestock, J. T.; Jones, M. H.; Brooks, P. D.; Walker, D. A.; Welker, J. M.

    1998-11-01

    Carbon dioxide concentrations through snow were measured in different arctic tundra communities on the North Slope of Alaska during winter and early spring of 1996. Subnivean CO2 concentrations were always higher than atmospheric CO2. A steady state diffusion model was used to generate conservative estimates of CO2 flux to the atmosphere. The magnitude of CO2 efflux differed with tundra community type, and rates of carbon release increased from March to May. Winter CO2 efflux was highest in riparian and snow bed communities and lowest in dry heath, upland tussock, and wet sedge communities. Snow generally accrues earlier in winter and is deeper in riparian and snow bed communities compared with other tundra communities, which are typically windswept and do not accumulate much snow during the winter. These results support the hypothesis that early and deep snow accumulation may insulate microbial populations from very cold temperatures, allowing sites with earlier snow cover to sustain higher levels of activity throughout winter compared to communities that have later developing snow cover. Extrapolating our estimates of CO2 efflux to the entire snow-covered season indicates that total carbon flux during winter in the Arctic is 13-109 kg CO2-C ha-1, depending on the vegetation community type. Wintertime CO2 flux is a potentially important, yet largely overlooked, part of the annual carbon cycle of tundra, and carbon release during winter should be accounted for in estimates of annual carbon balance in arctic ecosystems.

  20. Focus on the Outer Membrane Factor OprM, the Forgotten Player from Efflux Pumps Assemblies.

    PubMed

    Phan, Gilles; Picard, Martin; Broutin, Isabelle

    2015-01-01

    Antibiotics have been used extensively during several decades and we are now facing the emergence of multidrug resistant strains. It has become a major public concern, urging the need to discover new strategies to combat them. Among the different ways used by bacteria to resist antibiotics, the active efflux is one of the main mechanisms. In Gram-negative bacteria the efflux pumps are comprised of three components forming a long edifice crossing the complete cell wall from the inside to the outside of the cell. Blocking these pumps would permit the restoration of the effectiveness of the current antibiotherapy which is why it is important to increase our knowledge on the different proteins involved in these complexes. A tremendous number of experiments have been performed on the inner membrane protein AcrB from Escherichia coli and, to a lesser extent, the protein partners forming the AcrAB-TolC pump, but less information is available concerning the efflux pumps from other virulent Gram-negative bacteria. The present review will focus on the OprM outer membrane protein from the MexAB-OprM pump of Pseudomonas aeruginosa, highlighting similarities and differences compare to the archetypal AcrAB-TolC in terms of structure, function, and assembly properties. PMID:27025640

  1. Retinoid regulated macrophage cholesterol efflux involves the steroidogenic acute regulatory protein

    PubMed Central

    Manna, Pulak R.

    2016-01-01

    Elimination of excess cholesteryl esters from macrophage-derived foam cells is known to be a key process in limiting plaque stability and progression of atherosclerotic lesions. We have recently demonstrated that regulation of retinoid mediated cholesterol efflux is influenced by liver X receptor (LXR) signaling in mouse macrophages (Manna, P.R. et al., 2015, Biochem. Biophys. Res. Commun., 464:312-317). The data presented in this article evaluate the importance of the steroidogenic acute regulatory protein (StAR) in retinoid mediated macrophage cholesterol efflux. Overexpression of StAR in mouse RAW 264.7 macrophages increased the effects of both all-trans retinoic acid (atRA) and 9-cis RA on cholesterol efflux, suggesting StAR enhances the efficacy of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR) ligands. Additional data revealed that atRA enhances (Bu)2cAMP induced StAR and ATP-binding cassette transporter A1 protein levels. Treatment of macrophages transfected with an LXRE reporter plasmid (pLXREx3-Luc) was found to induce the effects of RAR and RXR analogs on LXR activity. PMID:27081671

  2. Retinoid regulated macrophage cholesterol efflux involves the steroidogenic acute regulatory protein.

    PubMed

    Manna, Pulak R

    2016-06-01

    Elimination of excess cholesteryl esters from macrophage-derived foam cells is known to be a key process in limiting plaque stability and progression of atherosclerotic lesions. We have recently demonstrated that regulation of retinoid mediated cholesterol efflux is influenced by liver X receptor (LXR) signaling in mouse macrophages (Manna, P.R. et al., 2015, Biochem. Biophys. Res. Commun., 464:312-317). The data presented in this article evaluate the importance of the steroidogenic acute regulatory protein (StAR) in retinoid mediated macrophage cholesterol efflux. Overexpression of StAR in mouse RAW 264.7 macrophages increased the effects of both all-trans retinoic acid (atRA) and 9-cis RA on cholesterol efflux, suggesting StAR enhances the efficacy of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR) ligands. Additional data revealed that atRA enhances (Bu)2cAMP induced StAR and ATP-binding cassette transporter A1 protein levels. Treatment of macrophages transfected with an LXRE reporter plasmid (pLXREx3-Luc) was found to induce the effects of RAR and RXR analogs on LXR activity. PMID:27081671

  3. Down-regulation of lipoprotein lipase increases ABCA1-mediated cholesterol efflux in THP-1 macrophages.

    PubMed

    Kawashima, Ryoko L; Medh, Jheem D

    2014-08-01

    The ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of excess cholesterol from foam cells to lipid-poor apolipoprotein A-I, in a process called reverse cholesterol transport. Lipoprotein lipase (LPL) is a lipolytic enzyme expressed by macrophages within atherosclerotic lesions. Lentivirus-mediated RNA interference was used to genetically knock-down (KD) the expression of LPL in THP-1 macrophages. Silencing of the LPL gene was confirmed by end-point PCR, real time PCR, and protein analysis. Suppression of LPL expression correlated with a 1.6-fold up-regulation of ABCA1 mRNA levels, and resulted in a 4.5-fold increase in ABCA1-dependent cholesterol efflux. Replenishing LPL by addition of purified bovine LPL to the cell culture media resulted in down-regulation of ABCA1-mediated cholesterol efflux in both wild-type and LPL knockdown cells. These findings suggest an inverse correlation between macrophage LPL levels and ABCA1 cholesterol transport activity. PMID:25017912

  4. Increased H+ efflux is sufficient to induce dysplasia and necessary for viability with oncogene expression

    PubMed Central

    Grillo-Hill, Bree K; Choi, Changhoon; Jimenez-Vidal, Maite; Barber, Diane L

    2015-01-01

    Intracellular pH (pHi) dynamics is increasingly recognized as an important regulator of a range of normal and pathological cell behaviors. Notably, increased pHi is now acknowledged as a conserved characteristic of cancers and in cell models is confirmed to increase proliferation and migration as well as limit apoptosis. However, the significance of increased pHi for cancer in vivo remains unresolved. Using Drosophila melanogaster, we show that increased pHi is sufficient to induce dysplasia in the absence of other transforming cues and potentiates growth and invasion with oncogenic Ras. Using a genetically encoded biosensor we also confirm increased pHi in situ. Moreover, in Drosophila models and clonal human mammary cells we show that limiting H+ efflux with oncogenic Raf or Ras induces acidosis and synthetic lethality. Further, we show lethality in invasive primary tumor cell lines with inhibiting H+ efflux. Synthetic lethality with reduced H+ efflux and activated oncogene expression could be exploited therapeutically to restrain cancer progression while limiting off-target effects. DOI: http://dx.doi.org/10.7554/eLife.03270.001 PMID:25793441

  5. Novel epitopes identified from efflux pumps of Mycobacterium tuberculosis could induce cytotoxic T lymphocyte response

    PubMed Central

    Zhai, Ming-xia; Chen, Fei; Zhao, Yuan-yuan; Wu, Ya-hong; Li, Guo-dong; Qi, Yuan-ming

    2015-01-01

    Overcoming drug-resistance is one of the major challenges to control tuberculosis (TB). The up-regulation of efflux pumps is one common mechanism that leads to drug-resistance. Therefore, immunotherapy targeting these efflux pump antigens could be promising strategy to be combined with current chemotherapy. Considering that CD8+ cytotoxic T lymphocytes (CTLs) induced by antigenic peptides (epitopes) could elicit HLA-restricted anti-TB immune response, efflux pumps from classical ABC family (Mycobacterium tuberculosis, Mtb) were chosen as target antigens to identify CTL epitopes. HLA-A2 restricted candidate peptides from Rv2937, Rv2686c and Rv2687c of Mycobacterium tuberculosis were predicted, synthesized and tested. Five peptides could induce IFN-γ release and cytotoxic activity in PBMCs from HLA-A2+ PPD+ donors. Results from HLA-A2/Kb transgenic mice immunization assay suggested that four peptides Rv2937-p168, Rv2937-p266, Rv2686c-p151, and Rv2686c-p181 could induce significant CTL response in vivo. These results suggested that these novel epitopes could be used as immunotherapy candidates to TB drug-resistance. PMID:26417538

  6. Focus on the Outer Membrane Factor OprM, the Forgotten Player from Efflux Pumps Assemblies

    PubMed Central

    Phan, Gilles; Picard, Martin; Broutin, Isabelle

    2015-01-01

    Antibiotics have been used extensively during several decades and we are now facing the emergence of multidrug resistant strains. It has become a major public concern, urging the need to discover new strategies to combat them. Among the different ways used by bacteria to resist antibiotics, the active efflux is one of the main mechanisms. In Gram-negative bacteria the efflux pumps are comprised of three components forming a long edifice crossing the complete cell wall from the inside to the outside of the cell. Blocking these pumps would permit the restoration of the effectiveness of the current antibiotherapy which is why it is important to increase our knowledge on the different proteins involved in these complexes. A tremendous number of experiments have been performed on the inner membrane protein AcrB from Escherichia coli and, to a lesser extent, the protein partners forming the AcrAB-TolC pump, but less information is available concerning the efflux pumps from other virulent Gram-negative bacteria. The present review will focus on the OprM outer membrane protein from the MexAB-OprM pump of Pseudomonas aeruginosa, highlighting similarities and differences compare to the archetypal AcrAB-TolC in terms of structure, function, and assembly properties. PMID:27025640

  7. 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

  8. Role of fatty acyl coenzyme A oxidase in the efflux of oxidized glutathione from perfused livers of rats treated with the peroxisome proliferator nafenopin.

    PubMed

    Conway, J G; Neptun, D A; Garvey, L K; Popp, J A

    1987-09-15

    arachidonate had no effect on GSSG efflux from livers from fasted, vehicle-treated rats. Infusion of 2-bromooctanoate (600 microM) completely blocked lauric acid-induced increases in GSSG efflux and acetoacetate and beta-hydroxybutyrate production in livers from fasted, nafenopin-treated rats. Infusion of 1-3-bis(2-chloroethyl)-1-nitrosourea reduced glutathione reductase activity by 90% but did not alter lauric acid-induced increases in GSSG efflux or ketogenesis in livers from fasted, nafenopin-treated rats.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:3621175

  9. Amyloid-β efflux from the CNS into the plasma

    PubMed Central

    Roberts, Kaleigh Filisa; Elbert, Donald L.; Kasten, Tom P.; Patterson, Bruce W.; Sigurdson, Wendy C.; Connors, Rose E.; Ovod, Vitaliy; Munsell, Ling Y.; Mawuenyega, Kwasi G.; Miller-Thomas, Michelle M.; Moran, Christopher J.; Cross, Dewitte T.; Derdeyn, Colin P.; Bateman, Randall J.

    2015-01-01

    Objective The aim of this study was to measure the flux of amyloid-β (Aβ) across the human cerebral capillary bed in order to determine if transport into the blood is a significant mechanism of clearance for Aβ produced in the central nervous system (CNS). Methods Time-matched blood samples were simultaneously collected from a cerebral vein (including the sigmoid sinus, inferior petrosal sinus, and the internal jugular vein), femoral vein, and radial artery of patients undergoing Inferior Petrosal Sinus Sampling (IPSS). For each plasma sample, Aβ concentration was assessed by three assays and the venous to arterial Aβ concentration ratios were determined. Results Aβ concentration was increased by ~7.5% in venous blood leaving the CNS capillary bed compared to arterial blood, indicating efflux from the CNS into the peripheral blood (p < 0.0001). There was no difference in peripheral venous Aβ concentration compared to arterial blood concentration. Interpretation Our results are consistent with clearance of CNS-derived Aβ into the venous blood supply with no increase from a peripheral capillary bed. Modeling these results suggests that direct transport of Aβ across the blood-brain barrier accounts for ~25% of Aβ clearance, and reabsorption of cerebrospinal fluid Aβ accounts for ~25% of the total CNS Aβ clearance in humans. PMID:25205593

  10. 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

  11. Effects of neurotransmitters on calcium efflux from cultured glioma cells

    SciTech Connect

    Lazarewicz, J.W.; Kanje, M.

    1981-01-01

    The effects of various neurotransmitters and cyclic nucleotides on 45Ca2+ efflux in cultured human glioma cells were investigated. Glutamate and glycine, but not GABA, stimulated 45Ca2+ release from the cells. Stimulation of beta-adrenergic receptors but not alpha-adrenergic receptors also increased 45Ca2+ efflux. Cholinergic receptor stimulation by carbachol had the same effect. The stimulatory effect of carbachol was abolished in the presence of either atropine or hexamethonium. C-AMP and c-GMP increased the 45Ca2+ efflux, suggesting that these agents are involved in the transmitter-stimulated release of 45Ca2+ from the cell. Kinetic analysis of the efflux revealed four calcium compartments. The carbachol-stimulated efflux represented a net release of calcium and could be ascribed to the slowest compartment. The physiological role of the transmitter-stimulated calcium release is discussed in terms of calcium-regulated stimulus-response coupling in glial-neural interaction during excitation.

  12. 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

  13. Arsenic Efflux from Microcystis aeruginosa under Different Phosphate Regimes

    PubMed Central

    Yan, Changzhou; Wang, Zhenhong; Luo, Zhuanxi

    2014-01-01

    Phytoplankton plays an important role in arsenic speciation, distribution, and cycling in freshwater environments. Little information, however, is available on arsenic efflux from the cyanobacteria Microcystis aeruginosa under different phosphate regimes. This study investigated M. aeruginosa arsenic efflux and speciation by pre-exposing it to 10 µM arsenate or arsenite for 24 h during limited (12 h) and extended (13 d) depuration periods under phosphate enriched (+P) and phosphate depleted (−P) treatments. Arsenate was the predominant species detected in algal cells throughout the depuration period while arsenite only accounted for no greater than 45% of intracellular arsenic. During the limited depuration period, arsenic efflux occurred rapidly and only arsenate was detected in solutions. During the extended depuration period, however, arsenate and dimethylarsinic acid (DMA) were found to be the two predominant arsenic species detected in solutions under −P treatments, but arsenate was the only species detected under +P treatments. Experimental results also suggest that phosphorus has a significant effect in accelerating arsenic efflux and promoting arsenite bio-oxidation in M. aeruginosa. Furthermore, phosphorus depletion can reduce arsenic efflux from algal cells as well as accelerate arsenic reduction and methylation. These findings can contribute to our understanding of arsenic biogeochemistry in aquatic environments and its potential environmental risks under different phosphorus levels. PMID:25549253

  14. 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. PMID:17938115

  15. Correlation between rhodamine 123 accumulation and azole sensitivity in Candida species: possible role for drug efflux in drug resistance.

    PubMed Central

    Clark, F S; Parkinson, T; Hitchcock, C A; Gow, N A

    1996-01-01

    A wide variety of prokaryotic and eukaryotic cells exhibit a multidrug resistance (MDR) phenotype, indicating that resistance to potentially toxic compounds is mediated by their active efflux from the cell. We have sought to determine whether resistance to azoles in some strains of Candida species may be due in part to active drug efflux. Rhodamine 123 (Rh123) is a fluorescent compound that is transported by a wide variety of MDR cell types. We have shown that certain azole-resistant strains of Candida albicans, C. glabrata, and C. krusei accumulate less Rh123 than azole-susceptible ones. In C. albicans, Rh123 accumulation was growth phase and temperature dependent and was increased by proton uncouplers and by reserpine, an MDR modulator. This is consistent with an energy-dependent efflux mechanism for Rh123, mediated by an MDR transporter. In C. glabrata, but not in C. albicans, there was competition between Rh123 and fluconazole for efflux. Thus, in C. glabrata, Rh123 and fluconazole appear to be transported via a common MDR-like transporter, whereas in C. albicans, the Rh123 transporter does not appear to transport azoles. PMID:8834890

  16. Isotopic rubidium ion efflux assay for the functional characterization of nicotinic acetylcholine receptors on clonal cell lines

    SciTech Connect

    Lukas, R.J.; Cullen, M.J.

    1988-11-15

    An isotopic rubidium ion efflux assay has been developed for the functional characterization of nicotinic acetylcholine receptors on cultured neurons. This assay first involves the intracellular sequestration of isotopic potassium ion analog by the ouabain-sensitive action of a sodium-potassium ATPase. Subsequently, the release of isotopic rubidium ion through nicotinic acetylcholine receptor-coupled monovalent cation channels is activated by application of nicotinic agonists. Specificity of receptor-mediated efflux is demonstrated by its sensitivity to blockade by nicotinic, but not muscarinic, antagonists. The time course of agonist-mediated efflux, within the temporal limitations of the assay, indicates a slow inactivation of receptor function on prolonged exposure to agonist. Dose-response profiles (i) have characteristic shapes for different nicotinic agonists, (ii) are described by three operationally defined parameters, and (iii) reflect different affinities of agonists for binding sites that control receptor activation and functional inhibition. The rubidium ion efflux assay provides fewer hazards but greater sensitivity and resolution than isotopic sodium or rubidium ion influx assays for functional nicotinic receptors.

  17. 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. PMID:6199026

  18. Manually controlled neutron-activation system

    NASA Astrophysics Data System (ADS)

    Johns, R. A.; Carothers, G. A.

    1982-01-01

    A manually controlled neutron activation system, the Manual Reactor Activation System, was designed and built and has been operating at one of the Savannah River Plant's production reactors. With this system, samples can be irradiated for up to 24 hours and pneumatically transferred to a shielded repository for decay until their activity is low enough for them to be handled at a radiobench. The Manual Reactor Activation System was built to provide neutron activation of solid waste forms for the Alternative Waste Forms Leach Testing Program. Neutron activation of the bulk sample prior to leaching permits sensitive multielement radiometric analyses of the leachates.

  19. Wave activated power generation system

    SciTech Connect

    Ono, Y.

    1983-08-09

    A wave activated power generation system of the float type is disclosed, comprising at least one piston-cylinder device having an anchored cylinder and a piston slidable in the cylinder and cooperating with the cylinder to form a pumping chamber above the piston and a low pressure chamber below the piston. The cylinder has an intake port and an exhaust port both formed at an upper port thereof to communicate with the pumping chamber and each provided with a check valve. A float is connected through a cable to the piston of the piston- cylinder device. A pair of fluid storages are connected to the intake port and the exhaust port of the pumping chamber, respectively. A waterwheel generator is driven by the fluid flowing from one of the fluid storages to another. A pressure regulating device is connected to the low pressure chamber so as to maintain the low pressure chamber at a pressure lower than the pressure in the pumping chamber, the difference in pressure ceaselessly applying a downward force on the piston to keep the cable in a tensed condition.

  20. 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

  1. 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. PMID:23939894

  2. Altered regulation of hepatic efflux transporters disrupts acetaminophen disposition in pediatric nonalcoholic steatohepatitis.

    PubMed

    Canet, Mark J; Merrell, Matthew D; Hardwick, Rhiannon N; Bataille, Amy M; Campion, Sarah N; Ferreira, Daniel W; Xanthakos, Stavra A; Manautou, Jose E; A-Kader, H Hesham; Erickson, Robert P; Cherrington, Nathan J

    2015-06-01

    Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, representing a spectrum of liver pathologies that include simple hepatic steatosis and the more advanced nonalcoholic steatohepatitis (NASH). The current study was conducted to determine whether pediatric NASH also results in altered disposition of acetaminophen (APAP) and its two primary metabolites, APAP-sulfate and APAP-glucuronide. Pediatric patients with hepatic steatosis (n = 9) or NASH (n = 3) and healthy patients (n = 12) were recruited in a small pilot study design. All patients received a single 1000-mg dose of APAP. Blood and urine samples were collected at 1, 2, and 4 hours postdose, and APAP and APAP metabolites were determined by high-performance liquid chromatography. Moreover, human liver tissues from patients diagnosed with various stages of NAFLD were acquired from the Liver Tissue Cell Distribution System to investigate the regulation of the membrane transporters, multidrug resistance-associated protein 2 and 3 (MRP2 and MRP3, respectively). Patients with the more severe disease (i.e., NASH) had increased serum and urinary levels of APAP-glucuronide along with decreased serum levels of APAP-sulfate. Moreover, an induction of hepatic MRP3 and altered canalicular localization of the biliary efflux transporter, MRP2, describes the likely mechanism for the observed increase in plasma retention of APAP-glucuronide, whereas altered regulation of sulfur activation genes may explain decreased sulfonation activity in NASH. APAP-glucuronide and APAP-sulfate disposition is altered in NASH and is likely due to hepatic membrane transporter dysregulation as well as altered intracellular sulfur activation. PMID:25788542

  3. Astaxanthin enhances ATP-binding cassette transporter A1/G1 expressions and cholesterol efflux from macrophages.

    PubMed

    Iizuka, Maki; Ayaori, Makoto; Uto-Kondo, Harumi; Yakushiji, Emi; Takiguchi, Shunichi; Nakaya, Kazuhiro; Hisada, Tetsuya; Sasaki, Makoto; Komatsu, Tomohiro; Yogo, Makiko; Kishimoto, Yoshimi; Kondo, Kazuo; Ikewaki, Katsunori

    2012-01-01

    ATP-binding cassette transporters (ABC) A1 and G1 are key molecules in cholesterol efflux from macrophages, which is an initial step of reverse cholesterol transport (RCT), a major anti-atherogenic property of high-density lipoprotein (HDL). Astaxanthin is one of the naturally occurring carotenoids responsible for the pink-red pigmentation in a variety of living organisms. Although astaxanthin is known to be a strong antioxidant, it remains unclear through what mechanism of action it affects cholesterol homeostasis in macrophages. We therefore investigated the effects of astaxanthin on cholesterol efflux and ABCA1/G1 expressions in macrophages. Astaxanthin enhanced both apolipoprotein (apo) A-I- and HDL-mediated cholesterol efflux from RAW264.7 cells. In supporting these enhanced cholesterol efflux mechanisms, astaxanthin promoted ABCA1/G1 expression in various macrophages. In contrast, peroxisome proliferator-activated receptor γ, liver X receptor (LXR) α and LXRβ levels remained unchanged by astaxanthin. An experiment using actinomycin D demonstrated that astaxanthin transcriptionally induced ABCA1/G1 expression, and oxysterol depletion caused by overexpression of cholesterol sulfotransferase further revealed that these inductions in ABCA1/G1 were independent of LXR-mediated pathways. Finally, we performed luciferase assays using human ABCA1/G1 promoter-reporter constructs to reveal that astaxanthin activated both promoters irrespective of the presence or absence of LXR-responsive elements, indicating LXR-independence of these activations. In conclusion, astaxanthin increased ABCA1/G1 expression, thereby enhancing apoA-I/HDL-mediated cholesterol efflux from the macrophages in an LXR-independent manner. In addition to the anti-oxidative properties, the potential cardioprotective properties of astaxanthin might therefore be associated with an enhanced anti-atherogenic function of HDL. PMID:22790567

  4. Characterization of a Novel Pyranopyridine Inhibitor of the AcrAB Efflux Pump of Escherichia coli

    PubMed Central

    Kwasny, Steven M.; Kim, Hong-Suk; Nguyen, Son T.; Houseweart, Chad; D'Souza, Sanjay; Walker, Graham C.; Peet, Norton P.; Nikaido, Hiroshi; Bowlin, Terry L.

    2014-01-01

    Members of the resistance-nodulation-division (RND) family of efflux pumps, such as AcrAB-TolC of Escherichia coli, play major roles in multidrug resistance (MDR) in Gram-negative bacteria. A strategy for combating MDR is to develop efflux pump inhibitors (EPIs) for use in combination with an antibacterial agent. Here, we describe MBX2319, a novel pyranopyridine EPI with potent activity against RND efflux pumps of the Enterobacteriaceae. MBX2319 decreased the MICs of ciprofloxacin (CIP), levofloxacin, and piperacillin versus E. coli AB1157 by 2-, 4-, and 8-fold, respectively, but did not exhibit antibacterial activity alone and was not active against AcrAB-TolC-deficient strains. MBX2319 (3.13 μM) in combination with 0.016 μg/ml CIP (minimally bactericidal) decreased the viability (CFU/ml) of E. coli AB1157 by 10,000-fold after 4 h of exposure, in comparison with 0.016 μg/ml CIP alone. In contrast, phenyl-arginine-β-naphthylamide (PAβN), a known EPI, did not increase the bactericidal activity of 0.016 μg/ml CIP at concentrations as high as 100 μM. MBX2319 increased intracellular accumulation of the fluorescent dye Hoechst 33342 in wild-type but not AcrAB-TolC-deficient strains and did not perturb the transmembrane proton gradient. MBX2319 was broadly active against Enterobacteriaceae species and Pseudomonas aeruginosa. MBX2319 is a potent EPI with possible utility as an adjunctive therapeutic agent for the treatment of infections caused by Gram-negative pathogens. PMID:24247144

  5. MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 7: highly soluble and in vivo active quaternary ammonium analogue D13-9001, a potential preclinical candidate.

    PubMed

    Yoshida, Ken-Ichi; Nakayama, Kiyoshi; Ohtsuka, Masami; Kuru, Noriko; Yokomizo, Yoshihiro; Sakamoto, Atsunobu; Takemura, Makoto; Hoshino, Kazuki; Kanda, Hiroko; Nitanai, Hironobu; Namba, Kenji; Yoshida, Kumi; Imamura, Yuichiro; Zhang, Jason Z; Lee, Ving J; Watkins, William J

    2007-11-15

    A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, substituted at the 2-position with piperidines bearing quaternary ammonium salt side chains, were synthesized and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin (LVFX) and the beta-lactam aztreonam (AZT) in Pseudomonas aeruginosa. Attachment of the charged entity using an N-ethylcarbamoyloxy linker led to the discovery of the highly soluble compound 22 (D13-9001), which maintained good potency in vitro and displayed excellent activity in vivo in a rat pneumonia model of P. aeruginosa. PMID:17869116

  6. The Listeria monocytogenes Fur-regulated virulence protein FrvA is an Fe(II) efflux P1B4 -type ATPase.

    PubMed

    Pi, Hualiang; Patel, Sarju J; Argüello, José M; Helmann, John D

    2016-06-01

    Listeria monocytogenes FrvA (Lmo0641) is critical for virulence in the mouse model and is an ortholog of the Bacillus subtilis Fur- and PerR-regulated Fe(II) efflux P1B4 -type ATPase PfeT. Previously, FrvA was suggested to protect against heme toxicity. Here, we demonstrate that an frvA mutant is sensitive to iron intoxication, but not to other metals. Expression of frvA is induced by high iron and this induction requires Fur. FrvA functions in vitro as a divalent cation specific ATPase most strongly activated by ferrous iron. When expressed in B. subtilis, FrvA increases resistance to iron both in wild-type and in a pfeT null strain. FrvA is a high affinity Fe(II) exporter and its induction imposes severe iron limitation in B. subtilis resulting in derepression of both Fur- and PerR-regulated genes. FrvA also recognizes Co(II) and Zn(II) as substrates and can complement B. subtilis strains defective in the endogenous export systems for these cations. Building on these results, we conclude that FrvA functions in the efflux of Fe(II), and not heme during listerial infection. PMID:26946370

  7. Cholesterol efflux from THP-1 macrophages is impaired by the fatty acid component from lipoprotein hydrolysis by lipoprotein lipase.

    PubMed

    Yang, Yanbo; Thyagarajan, Narmadaa; Coady, Breanne M; Brown, Robert J

    2014-09-01

    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. PMID:25130461

  8. Leoligin, the Major Lignan from Edelweiss (Leontopodium nivale subsp. alpinum), Promotes Cholesterol Efflux from THP-1 Macrophages.

    PubMed

    Wang, Limei; Ladurner, Angela; Latkolik, Simone; Schwaiger, Stefan; Linder, Thomas; Hošek, Jan; Palme, Veronika; Schilcher, Nicole; Polanský, Ondřej; Heiss, Elke H; Stangl, Herbert; Mihovilovic, Marko D; Stuppner, Hermann; Dirsch, Verena M; Atanasov, Atanas G

    2016-06-24

    Leoligin is a natural lignan found in Edelweiss (Leontopodium nivale ssp. alpinum). The aim of this study was to examine its influence on cholesterol efflux and to address the underlying mechanism of action. Leoligin increases apo A1- as well as 1% human plasma-mediated cholesterol efflux in THP-1 macrophages without affecting cell viability as determined by resazurin conversion. Western blot analysis revealed that the protein levels of the cholesterol efflux transporters ABCA1 and ABCG1 were upregulated, whereas the SR-B1 protein level remained unchanged upon treatment with leoligin (10 μM, 24 h). Quantitative reverse transcription PCR further uncovered that leoligin also increased ABCA1 and ABCG1 mRNA levels without affecting the half-life of the two mRNAs in the presence of actinomycin D, a transcription inhibitor. Proteome analysis revealed the modulation of protein expression fingerprint in the presence of leoligin. Taken together, these results suggest that leoligin induces cholesterol efflux in THP-1-derived macrophages by upregulating ABCA1 and ABCG1 expression. This novel activity suggests leoligin as a promising candidate for further studies addressing a possible preventive or therapeutic application in the context of atherosclerosis. PMID:27220065

  9. Leoligin, the Major Lignan from Edelweiss (Leontopodium nivale subsp. alpinum), Promotes Cholesterol Efflux from THP-1 Macrophages

    PubMed Central

    2016-01-01

    Leoligin is a natural lignan found in Edelweiss (Leontopodium nivale ssp. alpinum). The aim of this study was to examine its influence on cholesterol efflux and to address the underlying mechanism of action. Leoligin increases apo A1- as well as 1% human plasma-mediated cholesterol efflux in THP-1 macrophages without affecting cell viability as determined by resazurin conversion. Western blot analysis revealed that the protein levels of the cholesterol efflux transporters ABCA1 and ABCG1 were upregulated, whereas the SR-B1 protein level remained unchanged upon treatment with leoligin (10 μM, 24 h). Quantitative reverse transcription PCR further uncovered that leoligin also increased ABCA1 and ABCG1 mRNA levels without affecting the half-life of the two mRNAs in the presence of actinomycin D, a transcription inhibitor. Proteome analysis revealed the modulation of protein expression fingerprint in the presence of leoligin. Taken together, these results suggest that leoligin induces cholesterol efflux in THP-1-derived macrophages by upregulating ABCA1 and ABCG1 expression. This novel activity suggests leoligin as a promising candidate for further studies addressing a possible preventive or therapeutic application in the context of atherosclerosis. PMID:27220065

  10. miR-206 controls LXRα expression and promotes LXR-mediated cholesterol efflux in macrophages.

    PubMed

    Vinod, Manjula; Chennamsetty, Indumathi; Colin, Sophie; Belloy, Loic; De Paoli, Federica; Schaider, Helmut; Graier, Wolfgang F; Frank, Saša; Kratky, Dagmar; Staels, Bart; Chinetti-Gbaguidi, Giulia; Kostner, Gerhard M

    2014-06-01

    Liver X receptors (LXRα and LXRβ) are key transcription factors in cholesterol metabolism that regulate cholesterol biosynthesis/efflux and bile acid metabolism/excretion in the liver and numerous organs. In macrophages, LXR signaling modulates cholesterol handling and the inflammatory response, pathways involved in atherosclerosis. Since regulatory pathways of LXR transcription control are well understood, in the present study we aimed at identifying post-transcriptional regulators of LXR activity. MicroRNAs (miRs) are such post-transcriptional regulators of genes that in the canonical pathway mediate mRNA inactivation. In silico analysis identified miR-206 as a putative regulator of LXRα but not LXRβ. Indeed, as recently shown, we found that miR-206 represses LXRα activity and expression of LXRα and its target genes in hepatic cells. Interestingly, miR-206 regulates LXRα differently in macrophages. Stably overexpressing miR-206 in THP-1 human macrophages revealed an up-regulation and miR-206 knockdown led to a down-regulation of LXRα and its target genes. In support of these results, bone marrow-derived macrophages (BMDMs) from miR-206 KO mice also exhibited lower expression of LXRα target genes. The physiological relevance of these findings was proven by gain- and loss-of-function of miR-206; overexpression of miR-206 enhanced cholesterol efflux in human macrophages and knocking out miR-206 decreased cholesterol efflux from MPMs. Moreover, we show that miR-206 expression in macrophages is repressed by LXRα activation, while oxidized LDL and inflammatory stimuli profoundly induced miR-206 expression. We therefore propose a feed-back loop between miR-206 and LXRα that might be part of an LXR auto-regulatory mechanism to fine tune LXR activity. PMID:24603323

  11. miR-206 controls LXRα expression and promotes LXR-mediated cholesterol efflux in macrophages

    PubMed Central

    Vinod, Manjula; Chennamsetty, Indumathi; Colin, Sophie; Belloy, Loic; De Paoli, Federica; Schaider, Helmut; Graier, Wolfgang F.; Frank, Saša; Kratky, Dagmar; Staels, Bart; Chinetti-Gbaguidi, Giulia; Kostner, Gerhard M.

    2014-01-01

    Liver X receptors (LXRα and LXRβ) are key transcription factors in cholesterol metabolism that regulate cholesterol biosynthesis/efflux and bile acid metabolism/excretion in the liver and numerous organs. In macrophages, LXR signaling modulates cholesterol handling and the inflammatory response, pathways involved in atherosclerosis. Since regulatory pathways of LXR transcription control are well understood, in the present study we aimed at identifying post-transcriptional regulators of LXR activity. MicroRNAs (miRs) are such post-transcriptional regulators of genes that in the canonical pathway mediate mRNA inactivation. In silico analysis identified miR-206 as a putative regulator of LXRα but not LXRβ. Indeed, as recently shown, we found that miR-206 represses LXRα activity and expression of LXRα and its target genes in hepatic cells. Interestingly, miR-206 regulates LXRα differently in macrophages. Stably overexpressing miR-206 in THP-1 human macrophages revealed an up-regulation and miR-206 knockdown led to a down-regulation of LXRα and its target genes. In support of these results, bone marrow-derived macrophages (BMDMs) from miR-206 KO mice also exhibited lower expression of LXRα target genes. The physiological relevance of these findings was proven by gain- and loss-of-function of miR-206; overexpression of miR-206 enhanced cholesterol efflux in human macrophages and knocking out miR-206 decreased cholesterol efflux from MPMs. Moreover, we show that miR-206 expression in macrophages is repressed by LXRα activation, while oxidized LDL and inflammatory stimuli profoundly induced miR-206 expression. We therefore propose a feed-back loop between miR-206 and LXRα that might be part of an LXR auto-regulatory mechanism to fine tune LXR activity. PMID:24603323

  12. What Is an Activity? Appropriating an Activity-Centric System

    NASA Astrophysics Data System (ADS)

    Yarosh, Svetlana; Matthews, Tara; Moran, Thomas P.; Smith, Barton

    Activity-Centric Computing (ACC) systems seek to address the fragmentation of office work across tools and documents by allowing users to organize work around the computational construct of an Activity. Defining and structuring appropriate Activities within a system poses a challenge for users that must be overcome in order to benefit from ACC support. We know little about how knowledge workers appropriate the Activity construct. To address this, we studied users’ appropriation of a production-quality ACC system, Lotus Activities, for everyday work by employees in a large corporation. We contribute to a better understanding of how users articulate their individual and collaborative work in the system by providing empirical evidence of their patterns of appropriation. We conclude by discussing how our findings can inform the design of other ACC systems for the workplace.

  13. Modulation of 5-hydroxytryptamine efflux from rat cortical synaptosomes by opioids and nociceptin

    PubMed Central

    Sbrenna, S; Marti, M; Morari, M; Calo', G; Guerrini, R; Beani, L; Bianchi, C

    2000-01-01

    The modulation of [3H]-5-hydroxytryptamine ([3H]-5-HT) efflux from superfused rat cortical synaptosomes by delta, kappa, mu and ORL1 opioid receptor agonists and antagonists was studied. Spontaneous [3H]-5-HT efflux was reduced (20% inhibition) by either 0.5 μM tetrodotoxin or Ca2+-omission. Ten mM K+-evoked [3H]-5-HT overflow was largely Ca2+-dependent (90%) and tetrodotoxin-sensitive (50%). The delta receptor agonist, deltorphin-I, failed to modulate the K+-evoked neurotransmitter efflux up to 0.3 μM. The kappa and the mu receptor agonists, U-50,488 and endomorphin-1, inhibited K+-evoked [3H]-5-HT overflow (EC50=112 and 7 nM, respectively; Emax=28 and 29% inhibition, respectively) in a norBinaltorphimine- (0.3 μM) and naloxone- (1 μM) sensitive manner, respectively. None of these agonists significantly affected spontaneous [3H]-5-HT efflux. The ORL1 receptor agonist nociceptin inhibited both spontaneous (EC50=67 nM) and K+-evoked (EC50=13 nM; Emax=52% inhibition) [3H]-5-HT efflux. The effect of NC was insensitive to naloxone (up to 10 μM), but was antagonized by [Nphe1]nociceptin(1-13)NH2 (a novel selective ORL1 receptor antagonist; pA2=6.7) and by naloxone benzoylhydrazone (pA2=6.3). The ORL1 ligand [Phe1ψ(CH2-NH)Gly2]nociceptin(1-13)NH2 also inhibited K+ stimulated [3H]-5-HT overflow (EC50=64 nM; Emax=31% inhibition), but its effect was partially antagonized by 10 μM naloxone. It is concluded that the ORL1 receptor is the most important presynaptic modulator of neocortical 5-HT release within the opioid receptor family. This suggests that the ORL1/nociceptin system may have a powerful role in the control of cerebral 5-HT-mediated biological functions. PMID:10807682

  14. Soil CO2 efflux in a sand grassland: contribution by root, mycorrhizal and basal respiration components

    NASA Astrophysics Data System (ADS)

    Papp, Marianna; Balogh, János; Pintér, Krisztina; Cserhalmi, Dóra; Nagy, Zoltán

    2014-05-01

    Grasslands play an important role in global carbon cycle because of their remarkable extension and carbon storage capacity. Soil respiration takes a major part in the carbon cycle of the ecosystems; ratio of its autotrophic and heterotrophic components is important also when considering their sensitivity to environmental drivers. The aim of the study was to estimate the contribution by root, mycorrhizal and basal components to total soil CO2 efflux. The study was carried out in the semi-arid sandy grassland dominated by Festuca pseudovina at the Kiskunság National Park in Hungary (Bugac site) where C-flux measurements have been going on since 2002. The soil CO2 effluxes were measured in the following treatments: a./ control, b./ root-exclusion, c./ root and mycorrhiza exclusion by using 80 cm long 15 cm inner diameter PVC tubes and micro-pore inox meshes. Inox mesh was used to exclude roots, but let the mycorrhiza filaments to grow into the tubes. 10 soil cores were excavated, sieved, then root-free soil was packed back layer by layer into the cores giving 6 and 4 repetitions in b and c treatments respectively. Basal respiration is referred to as the heterotrophic respiration without influence of roots or mycorrhiza. Difference between root-exclusion and root and mycorrhiza exclusion treatment gave the value of mycorrhizal respiration and control (non-disturbed) plots the total soil CO2 efflux. The contribution by the above components was evaluated. Soil CO2 efflux was measured continuously by using an automated open system of 10 soil respiration chambers. Data was collected in every two hours from each treatment (one of the chambers recorded basal respiration, 3 chambers were settled on root-excluded treatments and 6 chambers measured control plots). Chambers were moved in every 2 weeks between the repetitions of the treatments. Soil CO2 efflux (mycorrhiza-free, root free, control) data were fitted using a soil respiration model, where soil temperature, soil

  15. Measurement of sputtered efflux from 5-, 8-, and 30-cm diameter mercury ion thrusters

    NASA Technical Reports Server (NTRS)

    Weigand, A. J.; Mirtich, M. J.

    1975-01-01

    A technique has been developed which uses spectral transmittance of samples exposed to thruster efflux to determine and characterize the effect of the efflux on spacecraft surfaces and optical devices. An investigation of facility backsputter revealed that efflux samples must be protected (e.g., by small shield boxes) from materials from tank walls and targets. The composition of the sputter efflux deposited on the samples was mostly molybdenum with trace amounts of tantalum, iron and/or mercury. The efflux from a 5-cm diameter thruster was deposited on samples located in the plane of the accelerator grid; the 8-cm diameter thruster efflux results showed that the location of ion beam sputtering and efflux deposition equilibrium occurred at 57 deg with respect to the thruster axis; and the 30-cm diameter thruster had an ion beam erosion-efflux deposition equilibrium at 45 deg.

  16. Nitrogen monoxide (NO)-mediated iron release from cells is linked to NO-induced glutathione efflux via multidrug resistance-associated protein 1

    PubMed Central

    Watts, Ralph N.; Hawkins, Clare; Ponka, Prem; Richardson, Des R.

    2006-01-01

    Nitrogen monoxide (NO) plays a role in the cytotoxic mechanisms of activated macrophages against tumor cells by inducing iron (Fe) release. We have shown that NO-mediated Fe efflux from cells required glutathione (GSH), and we have hypothesized that a GS–Fe–NO complex was released. Hence, we studied the role of the GSH-conjugate transporter multidrug resistance-associated protein 1 (MRP1) in NO-mediated Fe efflux. MCF7-VP cells overexpressing MRP1 exhibited a 3- to 4-fold increase in NO-mediated 59Fe and GSH efflux compared with WT cells (MCF7-WT) over 4 h. Similar results were found for other MRP1-overexpressing cell types but not those expressing another drug efflux pump, P-glycoprotein. NO-mediated 59Fe and GSH efflux were temperature- and energy-dependent and were significantly decreased by the GSH-depleting agent and MRP1 transport inhibitor l-buthionine-[S,R]-sulfoximine. Other MRP1 inhibitors, MK571, probenecid, and difloxacin, significantly inhibited NO-mediated 59Fe release. EPR spectroscopy demonstrated the dinitrosyl-dithiol-Fe complex (DNIC) peak in NO-treated cells was increased by MRP1 inhibitors, indicating inhibited DNIC transport from cells. The extent of DNIC accumulation correlated with the ability of MRP1 inhibitors to prevent NO-mediated 59Fe efflux. MCF7-VP cells were more sensitive than MCF7-WT cells to growth inhibition by effects of NO, which was potentiated by l-buthionine-[S,R]-sulfoximine. These data indicate the importance of GSH in NO-mediated inhibition of proliferation. Collectively, NO stimulates Fe and GSH efflux from cells via MRP1. Active transport of NO by MRP1 overcomes diffusion that is inefficient and nontargeted, which has broad ramifications for understanding NO biology. PMID:16679408

  17. 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.

  18. Anti-Campylobacter Activities and Resistance Mechanisms of Natural Phenolic Compounds in Campylobacter

    PubMed Central

    Klančnik, Anja; Možina, Sonja Smole; Zhang, Qijing

    2012-01-01

    Background Campylobacter is a major foodborne pathogen and alternative antimicrobials are needed to prevent or decrease Campylobacter contamination in foods or food producing animals. The objectives of this study are to define the anti-Campylobacter activities of natural phenolic compounds of plant origin and to determine the roles of bacterial drug efflux systems in the resistance to these natural phenolics in Campylobacter jejuni. Methodology/Principal Findings Anti-Campylobacter activities were evaluated by an MIC assay using microdilution coupled with ATP measurement. Mutants of the cmeB and cmeF efflux genes and the cmeR transcriptional repressor gene were compared with the wild-type strain for their susceptibilities to phenolics in the absence and presence of efflux-pump inhibitors (EPIs). The phenolic compounds produced significant, but variable activities against both antibiotic-susceptible and antibiotic resistant Campylobacter. The highest anti-Campylobacter activity was seen with carnosic and rosmarinic acids in their pure forms or in enriched plant extracts. Inactivation of cmeB rendered C. jejuni significantly more susceptible to the phenolic compounds, while mutation of cmeF or cmeR only produced a moderate effect on the MICs. Consistent with the results from the efflux pump mutants, EPIs, especially phenylalanine-arginine β-naphthylamide and NMP, significantly reduced the MICs of the tested phenolic compounds. Further reduction of MICs by the EPIs was also observed in the cmeB and cmeF mutants, suggesting that other efflux systems are also involved in Campylobacter resistance to phenolic compounds. Conclusion/Significance Natural phenolic compounds of plant origin have good anti-Campylobacter activities and can be further developed for potential use in controlling Campylobacter. The drug efflux systems in Campylobacter contribute significantly to its resistance to the phenolics and EPIs potentiate the anti-Campylobacter activities of plant phenolic

  19. HDL-Mediated Cellular Cholesterol Efflux Assay Method.

    PubMed

    Hafiane, Anouar; Genest, Jacques

    2015-01-01

    Biomarkers of high-density lipoprotein (HDL) function may provide mechanistic insights and better cardiovascular risk discrimination than HDL-cholesterol mass. The purpose of this work is to describe a simplified experimental protocol that can be used in the determination of cholesterol efflux from macrophages cultured cells and be brought to a medium throughput volume. The cellular cholesterol efflux assay is designed to quantify the rate of cholesterol efflux from cultured cells to an acceptor particle or to plasma. This assay is multi step, cell based assay. Various factors, if not carefully controlled may influence the accuracy and reproducibility of the assay. Attempts were made to address factors influencing this assay and to provide a standardized method that is relatively rapid and scalable. We demonstrate that further centrifugation of the HDL fraction is necessary to avoid apolipoprotein B contamination when using polyethylene glycol (PEG) method. We demonstrate also no effect on cholesterol efflux efficiency when using PEG with plasma or serum. This method has been previously applied in our laboratory in context of cardiovascular research, cardiovascular disease and pharmacologic therapies. PMID:26663796

  20. Expression of Multidrug Resistance Efflux Pump Gene norA Is Iron Responsive in Staphylococcus aureus

    PubMed Central

    Deng, Xin; Sun, Fei; Ji, Quanjiang; Liang, Haihua; Missiakas, Dominique; Lan, Lefu

    2012-01-01

    Staphylococcus aureus utilizes efflux transporter NorA to pump out a wide range of structurally dissimilar drugs, conferring low-level multidrug resistance. The regulation of norA expression has yet to be fully understood although past studies have revealed that this gene is under the control of the global transcriptional regulator MgrA and the two-component system ArlRS. To identify additional regulators of norA, we screened a transposon library in strain Newman expressing the transcriptional fusion norA-lacZ for altered β-galactosidase activity. We identify a transposon insertion in fhuB, a gene that encodes a ferric hydroxamate uptake system permease, and propose that the norA transcription is iron responsive. In agreement with this observation, addition of FeCl3 repressed the induction of norA-lacZ, suggesting that bacterial iron uptake plays an important role in regulating norA transcription. In addition, a fur (ferric uptake regulator) deletion exhibited compromised norA transcription and reduced resistance to quinolone compared to the wild-type strain, indicating that fur functions as a positive regulator of norA. A putative Fur box identified in the promoter region of norA was confirmed by electrophoretic mobility shift and DNase I footprint assays. Finally, by employing a siderophore secretion assay, we reveal that NorA may contribute to the export of siderophores. Collectively, our experiments uncover some novel interactions between cellular iron level and norA regulation in S. aureus. PMID:22267518

  1. 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.

  2. Lack of an effect of static magnetic field on calcium efflux from isolated chick brains

    SciTech Connect

    Bellossi, A.

    1986-01-01

    /sup 45/Ca2+ efflux from neonatal isolated chick brains was measured. The brains were exposed to uniform or nonuniform static magnetic fields. The field intensity ranged from 200-900 mT. The exposure took place during incubation and/or when efflux was being measured. No difference appeared in the /sup 45/Ca2+ efflux between controls and exposed brains.

  3. 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.

  4. 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.

  5. Constraint of soil moisture on CO2 efflux from tundra lichen, moss, and tussock in Council, Alaska using a hierarchical Bayesian model

    NASA Astrophysics Data System (ADS)

    Kim, Y.; Nishina, K.; Chae, N.; Park, S.; Yoon, Y.; Lee, B.

    2014-04-01

    The tundra ecosystem is quite vulnerable to drastic climate change in the Arctic, and the quantification of carbon dynamics is of significant importance in response to thawing permafrost, changes in the snow-covered period and snow and shrub community extent, and the decline of sea ice in the Arctic. Here, CO2 efflux measurements using a manual chamber system within a 40 m × 40 m (5 m interval; 81 total points) plot were conducted in dominant tundra vegetation on the Seward Peninsula of Alaska, during the growing seasons of 2011 and 2012, for the assessment of the driving parameters of CO2 efflux. We applied a hierarchical Bayesian (HB) model - which is a function of soil temperature, soil moisture, vegetation type and thaw depth - to quantify the effect of environmental parameters on CO2 efflux, and to estimate growing season CO2 emission. Our results showed that average CO2 efflux in 2011 is 1.4-fold higher than in 2012, resulting from the distinct difference in soil moisture between the two years. Tussock-dominated CO2 efflux is 1.4 to 2.3 times higher than those measured in lichen and moss communities, reflecting tussock as a significant CO2 source in the Arctic, with wide area distribution on a circumpolar scale. CO2 efflux followed soil temperature nearly exponentially from both the observed data and the posterior medians of the HB model. This reveals soil temperature as the most important parameter in regulating CO2 efflux, rather than soil moisture and thaw depth. Obvious changes in soil moisture during the growing seasons of 2011 and 2012 resulted in an explicit difference in CO2 efflux - 742 and 539 g CO2 m-2 period-1 in 2011 and 2012, respectively, suggesting that the 2012 CO2 emission rate was constrained by 27% (95% credible interval: 17-36%) compared to 2011, due to higher soil moisture from severe rain. Estimated growing season CO2 emission rate ranged from 0.86 Mg CO2 period-1 in 2012 to 1.2 Mg CO2 period-1 in 2011 within a 40 m × 40 m plot

  6. Doxorubicin induces drug efflux pumps in Candida albicans.

    PubMed

    Kofla, Grzegorz; Turner, Vincent; Schulz, Bettina; Storch, Ulrike; Froelich, Daniela; Rognon, Bénédicte; Coste, Alix T; Sanglard, Dominique; Ruhnke, Markus

    2011-02-01

    Candida albicans is one of the most important opportunistic fungal pathogens. It can cause serious fungal diseases in immunocompromised patients, including those with cancer. Treatment failures due to the emergence of drug-resistant C. albicans strains have become a serious clinical problem. Resistance incidents were often mediated by fungal efflux pumps which are closely related to the human ABC transporter P-glycoprotein (P-gp). P-gp is often overexpressed in cancer cells and confers resistance to many cytotoxic drugs. We examined whether cytotoxic drugs commonly used for cancer treatment (doxorubicin and cyclophosphamide) could alter the expression of genes responsible for the development of fluconazole resistance in Candida cells in the way they can influence homologous genes in cancer cell lines. ABC transporters (CDR1 and CDR2) and other resistance genes (MDR1 and ERG11) were tested by real-time PCR for their expression in C. albicans cells at the mRNA level after induction by antineoplastic drugs. The results were confirmed by a lacZ gene reporter system and verified at the protein level using GFP and immunoblotting. We showed that doxorubicin is a potent inducer of CDR1/CDR2 expression in C. albicans at both the mRNA and protein level and thus causes an increase in fluconazole MIC values. However, cyclophosphamide, which is not a substrate of human P-gp, did not induce ABC transporter expression in C. albicans. Neither doxorubicin nor cyclophosphamide could influence the expression of the other resistance genes (MDR1 and ERG11). The induction of CDR1/CDR2 by doxorubicin in C. albicans and the resulting alteration of antifungal susceptibility might be of clinical relevance for the antifungal treatment of Candida infections occurring after anticancer chemotherapy with doxorubicin. PMID:20818920

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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. PMID:24644297

  12. Experiment system of LAMOST active optics

    NASA Astrophysics Data System (ADS)

    Cui, Xiangqun; Su, Ding; Li, Guoping; Yao, Zhengqiu; Zhang, Zhengcao; Li, Yeping; Zhang, Yong; Wang, You; Xu, Xinqi; Wang, Hai

    2004-10-01

    Active optics is the most difficult part in LAMOST project. Especially for the segmented reflecting Schmidt plate Ma, in which both segmented mirror active optics and thin mirror (or deformable mirror) active optics are applied. To test and optimize the thin mirror active optics of Ma, and to approach the reality of operating environment of the telescope, an outdoor experiment system has been established. This experiment system is also a `small LAMOST" with one sub-mirror of the primary mirror Mb and one sub-mirror of the Schmidt plate Ma, and with full scale in spacing (40 meters) between Ma and Mb. many parts of LAMOST were tested in the experiment system except segmented mirror active optics. Especially for force actuators, thin mirror support system, friction driving of the alt-azimuth mounting and its control system, wave front test along such a long optical path. This paper presents the experiment system, research and developments, and some experiment results.

  13. 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.

  14. High speed hybrid active system

    NASA Astrophysics Data System (ADS)

    Gonzalez, Ignacio F.; Chang, Fu-Kuo; Qing, Peter X.; Kumar, Amrita; Zhang, David

    2005-05-01

    A novel piezoelectric/fiber-optic system is developed for long-term health monitoring of aerospace vehicles and structures. The hybrid diagnostic system uses the piezoelectric actuators to input a controlled excitation to the structure and the fiber optic sensors to capture the corresponding structural response. The aim of the system is to detect changes in structures such as those found in aerospace applications (damage, cracks, aging, etc.). This system involves the use of fiber Bragg gratings, which may be either bonded to the surface of the material or embedded within it in order to detect the linear strain component produced by the excitation waves generate by an arbitrary waveform generator. Interrogation of the Bragg gratings is carried out using a high speed fiber grating demodulation unit and a high speed data acquisition card to provide actuation input. With data collection and information processing; is able to determine the condition of the structure. The demands on a system suitable for detecting ultrasonic acoustic waves are different than for the more common strain and temperature systems. On the one hand, the frequency is much higher, with typical values for ultrasonic frequencies used in non-destructive testing ranging from 100 kHz up to several MHz. On the other hand, the related strain levels are much lower, normally in the μstrain range. Fiber-optic solutions for this problem do exist and are particularly attractive for ultrasonic sensing as the sensors offer broadband detection capability.

  15. IRAK regulates macrophage foam cell formation by modulating genes involved in cholesterol uptake and efflux.

    PubMed

    Rana, Minakshi; Kumar, Amit; Tiwari, Rajiv Lochan; Singh, Vishal; Chandra, Tulika; Dikshit, Madhu; Barthwal, Manoj Kumar

    2016-07-01

    Interleukin-1 receptor-associated kinase-1 (IRAK1) is linked to the pathogenesis of atherosclerosis; however, its role in macrophage foam cell formation is not known. Therefore, the present study investigated the role of IRAK1 in lipid uptake, biosynthesis, and efflux in THP-1 derived macrophages and human monocyte-derived macrophages (HMDMs). Ox-LDL (40 μg/mL, 15 minutes-48 hours) treatment induced time-dependent increase in IRAK1, IRAK4, and Stat1 activation in THP-1 derived macrophages. IRAK1/4 inhibitor (INH) or IRAK1 siRNA significantly attenuated cholesterol accumulation, DiI-Ox-LDL binding, and uptake while cholesterol efflux to apoAI and HDL was enhanced in THP-1 derived macrophages and HMDMs. Ox-LDL treatment significantly increased the mRNA expression of CD36, LOX-1, SR-A, ABCA1, ABCG1, Caveolin-1, CYP27A1 while that of SR-BI was decreased. IRAK1/4 inhibition or IRAK1 knockdown, however, attenuated Ox-LDL-induced CD36 expression; augmented ABCA1 and ABCG1 expression while expression of others was unaffected in THP-1 derived macrophages and HMDMs. Moreover, IRAK1/4 inhibition had no significant effect on genes involved in lipid biosynthesis. In IRAK1/4 INH pre-treated THP-1 derived macrophages Ox-LDL-induced Stat1 phosphorylation and its binding to CD36 promoter was significantly attenuated while LXRα expression and its binding to the ABCA1/ABCG1 locus, NFATc2 activation and its binding to ABCA1 locus was enhanced. The present study thus demonstrates that IRAK regulates lipid accumulation by modulating CD36-mediated uptake and ABCA1-, ABCG1-dependent cholesterol efflux. Therefore, IRAK1 can be a potential target for preventing macrophage foam cell formation. PMID:27270491

  16. A novel nanoparticle formulation overcomes multiple types of membrane efflux pumps in human breast cancer cells.

    PubMed

    Prasad, Preethy; Cheng, Ji; Shuhendler, Adam; Rauth, Andrew M; Wu, Xiao Yu

    2012-04-01

    Multidrug resistance (MDR) in cancer cells can involve overexpression of different types of membrane drug efflux pumps and other drug resistance mechanisms. Hence, inhibition of one resistance mechanism may not be therapeutically effective. Previously we demonstrated a new polymer lipid hybrid nanoparticle (PLN) system was able to circumvent drug resistance of P-glycoprotein (P-gp) overexpressing breast cancer cells. The objectives of the present study were 2-fold: (1) to evaluate the ability of the PLN system to overcome two other membrane efflux pumps-multidrug resistance protein 1 (MRP1+) and breast cancer resistance protein (BCRP+) overexpressed on human breast cancer cell lines MCF7 VP (MRP1+) and MCF7 MX (BCRP+); and (2) to evaluate possible synergistic effects of doxorubicin (Dox)-mitomycin C (MMC) in these cell lines. These objectives were accomplished by measuring in vitro cellular uptake, intracellular trafficking, and cytotoxicity (using a clonogenic assay and median effect analysis), of Dox, MMC, or Dox-MMC co-loaded PLN. Treatment of MDR cells with PLN encapsulating single anticancer agents significantly enhanced cell kill compared to free Dox or MMC solutions. Dox-MMC co-loaded PLN were 20-30-folds more effective in killing MDR cells than free drugs. Co-encapsulated Dox-MMC was more effective in killing MDR cells than single agent-encapsulated PLN. Microscopic images showed perinuclear localization of fluorescently labelled PLN in all cell lines. These results are consistent with our previous results for P-gp overexpressing breast cancer cells suggesting the PLN system can overcome multiple types of membrane efflux pumps increasing the cytotoxicity of Dox-MMC at significantly lower doses than free drugs. PMID:25786718

  17. [Analysis of the spatial and temporal changes in soil CO2 efflux and its related factors from alpine meadow in Qilian Mountains].

    PubMed

    Chang, Zong-qiang; Feng, Qi; Si, Jian-hua; Su, Yong-hong; Xi, Hai-yang; Guo, Rui

    2007-10-01

    Continuous field experiment was carried out on alpine meadow along different elevation gradient in Qilian Mountains during the growing season of 2004 using a soil respiration chamber (Li-6400-09) connected to a portable photosynthesis system (Li-6400). The possible effect of water-heat factors and root parameter on characteristics of alpine meadow soil respiration was statistically analyzed. The results indicate that soil CO2 efflux has apparent spatial variation laws. Spatial variation patterns of soil respiration from different alpine meadow along elevation gradient are gradually decreased and its coefficient of variation increases gently. The curves of day change of soil CO2 efflux have a simple peak value, and it is low at night, with lowest at 02:00-06:00, and starts to rise rapidly during 07:00-08:30, and then descends during 16:00 -18:30. The peak CO2 efflux appears during 11:00-16:00. The diurnal average of soil CO2 efflux is between (0.56 +/- 0.32) - (2.53 +/- 0.76) micromol x (m2 x s)(-1). As for seasonal variation, soil CO2 fluxes of various are relatively high in summer and autumn but relatively low in spring and winter. The maximum of average soil CO2 efflux occurrs in July and August [4.736 micromol (m2 x s)(-1), and the second is in June and September, and the third is in May and October. Soil CO2 efflux of alpine meadow during growth season is positively correlated with soil temperature, root biomass and soil water content at depths of 10 cm to different degrees. These results indicate that the temperature, soil moisture and root biomass have great effects on the spatial variation of soil CO2 efflux in the area. PMID:18269010

  18. Application of Human Placental Villous Tissue Explants to Study ABC Transporter Mediated Efflux of 2,4-Dinitrophenyl-S-Glutathione

    PubMed Central

    Vaidya, Soniya S.; Walsh, Scott W.; Gerk, Phillip M.

    2011-01-01

    Objective The purpose of this study was to characterize the human term placental villous tissue explant culture model as a tool to study the formation and efflux of 1-chloro-2,4-dinitrobenzene (CDNB) conjugate 2,4-dinitrophenyl-S-glutathione (DNP-SG) as a model system for phase II metabolism and ATP-binding cassette (ABC) transporter-mediated cellular efflux. Methods Placental tissue samples were obtained after cesarean section following normal pregnancies (n=9). Cultured villous tissue was monitored up to 48 h to study the effect of time in culture on biochemical parameters, formation and efflux of DNP-SG in the absence or presence of ATPase inhibitor sodium orthovanadate and the protein expression of ABC transporters - multidrug resistance associated protein 2 (MRP2), P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and enzyme glutathione-S-transferase isoform P1-1 (GSTP1-1). Results Villous tissue structure, tissue viability and expression of BCRP, GSTP1-1 remained unchanged, while expression of MRP2, P-gp and total tissue glutathione decreased with time in culture. Tissue integrity was unchanged up to 24 h but declined at 48 h. However, DNP-SG formation, DNP-SG efflux, and the extent of inhibition of DNP-SG efflux by sodium orthovanadate showed only minor changes through 48 h. Sodium orthovanadate decreased DNP-SG efflux, consistent with inhibition of apical ABC transporters. Conclusion The results support the use of the cultured human term placental villous tissue explants model to study coordinated function of GSTP1-1 and apical ABC transporters in the formation and efflux of the model substrate DNP-SG. PMID:21342117

  19. 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

  20. 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. PMID:26727205

  1. The pseudo-atomic structure of an RND-type tripartite multidrug efflux pump.

    PubMed

    Du, Dijun; Voss, Jarrod; Wang, Zhao; Chiu, Wah; Luisi, Ben F

    2015-09-01

    Microorganisms encode several classes of transmembrane molecular pumps that can expel a wide range of chemically distinct toxic substances. These machines contribute to the capacity of the organisms to withstand harsh environments, and they help to confer resistance against clinical antimicrobial agents. In Gram-negative bacteria, some of the pumps comprise tripartite assemblies that actively transport drugs and other harmful compounds across the cell envelope. We describe recent structural and functional data that have provided insights into the architecture and transport mechanism of the AcrA-AcrB-TolC pump of Escherichia coli. This multidrug efflux pump is powered by proton electrochemical gradients through the activity of AcrB, a member of the resistance/nodulation/cell division (RND) transporter family. Crystallographic data reveal how the small protein AcrZ binds to AcrB in a concave surface of the transmembrane domain, and we discuss how this interaction may affect the efflux activities of the transporter. PMID:25803077

  2. Beta amyloid effects on expression of multidrug efflux transporters in brain endothelial cells.

    PubMed

    Kania, Katarzyna D; Wijesuriya, Hasini C; Hladky, Stephen B; Barrand, Margery A

    2011-10-18

    ABC (ATP Binding Cassette) efflux transporters at the blood-brain barrier, P-glycoprotein (ABCB1), multidrug resistance associated protein 4 (ABCC4) and breast cancer resistance protein (ABCG2), are important for protecting the brain from circulating xenobiotics. Their expression is regulated by signals from surrounding brain tissue that may alter in CNS pathologies. Differences have been reported in transporter expression on brain vasculature of Alzheimer's subjects where raised levels of β-amyloid (Aβ) occur. The present study examines in vitro the effects of Aβ using immortalised brain endothelial cells (hCMEC/D3). Significantly lower expression of ABCB1 but not ABCC4 or ABCG2 was found following exposure to Aβ(1-42) peptide but not its scrambled equivalent. This was evident at both protein and transcript level and was reflected in lower transcriptional activity of the ABCB1 promoter as judged from the luciferase reporter gene assay and in decreases in ABCB1-mediated efflux of rhodamine 123. Aβ exposure also affected Wnt/β-catenin signalling, decreasing levels of β-catenin protein, reducing activation of TOPFLASH and increasing transcript levels of endogenous inhibitor, Dkk-1. Application of Wnt3a reversed the Aβ-induced changes to ABCB1 protein. These results suggest that Aβ may impair Wnt/β-catenin signalling at the blood-brain barrier but that activation of this pathway may restore ABCB1. PMID:21920506

  3. Phytoestrogen Suppresses Efflux of the Diagnostic Marker Protoporphyrin IX in Lung Carcinoma.

    PubMed

    Fujita, Hirofumi; Nagakawa, Keisuke; Kobuchi, Hirotsugu; Ogino, Tetsuya; Kondo, Yoichi; Inoue, Keiji; Shuin, Taro; Utsumi, Toshihiko; Utsumi, Kozo; Sasaki, Junzo; Ohuchi, Hideyo

    2016-04-01

    One promising method to visualize cancer cells is based on the detection of the fluorescent photosensitizer protoporphyrin IX (PpIX) synthesized from 5-aminolevulinic acid (ALA), but this method cannot be used in cancers that exhibit poor PpIX accumulation. PpIX appears to be pumped out of cancer cells by the ABC transporter G2 (ABCG2), which is associated with multidrug resistance. Genistein is a phytoestrogen that appears to competitively inhibit ABCG2 activity. Therefore, we investigated whether genistein can promote PpIX accumulation in human lung carcinoma cells. Here we report that treatment of A549 lung carcinoma cells with genistein or a specific ABCG2 inhibitor promoted ALA-mediated accumulation of PpIX by approximately 2-fold. ABCG2 depletion and overexpression studies further revealed that genistein promoted PpIX accumulation via functional repression of ABCG2. After an extended period of genistein treatment, a significant increase in PpIX accumulation was observed in A549 cells (3.7-fold) and in other cell lines. Systemic preconditioning with genistein in a mouse xenograft model of lung carcinoma resulted in a 1.8-fold increase in accumulated PpIX. Long-term genistein treatment stimulated the expression of genes encoding enzymes involved in PpIX synthesis, such as porphobilinogen deaminase, uroporphyrinogen decarboxylase, and protoporphyrinogen oxidase. Accordingly, the rate of PpIX synthesis was also accelerated by genistein pretreatment. Thus, our results suggest that genistein treatment effectively enhances ALA-induced PpIX accumulation by preventing the ABCG2-mediated efflux of PpIX from lung cancer cells and may represent a promising strategy to improve ALA-based diagnostic approaches in a broader set of malignancies. Cancer Res; 76(7); 1837-46. ©2016 AACR. PMID:26837765

  4. Seasonal, daily and diel N2 effluxes in permeable carbonate sediments

    NASA Astrophysics Data System (ADS)

    Eyre, B. D.; Santos, I. R.; Maher, D. T.

    2013-04-01

    Benthic metabolism and inorganic nitrogen and N2 flux rates (denitrification) were measured in permeable carbonate sands from Heron Island (Great Barrier Reef). Some of the N2 flux rates were among the highest measured in sediments. All benthic fluxes showed a significant difference between seasons with higher rates in summer and late summer. There was no distinct response of the benthic system to mass coral spawning. Instead, changes in benthic fluxes over 12 days in summer appear to be driven by tidal changes in water depth and associated changes in phytosynthetically active radiation reaching the sediments. Dark N2 fluxes were strongly correlated to benthic oxygen consumption across all sites and seasons (r2 = 0.63; p < 0.005; slope = 0.035). However, there were seasonal differences with a steeper slope in summer than winter, reflecting either more efficient coupling between respiration and nitrification-denitrification at higher temperatures or different sources of organic matter. Adding data from published studies on carbonate sands revealed two slopes in the dark N2 flux versus benthic oxygen consumption relationship. The lower slope (0.035) was most likely due to high carbon : nitrogen (C : N) organic matter from coral reefs, and associated assimilation of nitrogen by heterotrophic bacteria including enhanced heterotrophic N-fixation, but competition by benthic microalgae or inefficient coupling between respiration and nitrification-denitrification cannot be excluded. The steeper slope (0.089) was most likely due to respiration being driven by low C : N phytodetritus. If the different slopes were driven by the sources of organic matter, then global estimates of continental shelf denitrification are probably about right. In contrast, global estimates of continental shelf denitrification may be over-estimated if the low slope was due to inefficient coupling between respiration and nitrification-denitrification and also due to reduced N2 effluxes in the light

  5. 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 proton-translocating ATPase inhibitor. These results support the hypothesis that galactose efflux in Gal{sup {minus}} S. thermophilus is electrogenic and that the exchange reaction (lactose uptake and galactose efflux) probably occurs via an antiporter system.

  6. Spatial and temporal variability of soil CO2 efflux in southeastern forests

    NASA Astrophysics Data System (ADS)

    Oishi, A. C.; Palmroth, S.; Butnor, J. R.; Johnsen, K.; Oren, R.

    2011-12-01

    The combination of biotic and abiotic factors that contribute to soil CO2 efflux can lead to large variability across spatial and temporal scales. This variability can result in uncertainty in respiration estimates; however, it can also provide insight into the primary processes affecting CO2 emissions from soil. In order to quantify the magnitude and identify the sources of variability in soil CO2 efflux across spatial and temporal scales, we deployed several automated, multi-chamber sampling systems in forest stands in the southeastern U.S. We measured fluxes from a medium-age loblolly pine stand for 10 years (2001-2010), which spanned two severe droughts and one very wet year. During this time period, we also operated three systems in additional plots within the pine stand for 5 years, one system in an adjacent mixed-species broadleaf deciduous stand for 4 years, and one system in a nearby mature loblolly pine stand for 2 years. The high frequency of measurements (~2 hours per chamber) allows us to examine diurnal trends and precipitation responses while longevity of this study provides information on interannual variability and effects of stand age. Additionally, we quantify spatial variability at the scale of plot-level (1-10 m), stand-level (~100 m), and regionally (1-50 km) to examine the effects of local heterogeneity, species composition, and soil type. Chamber-based measurements are compared with leaf litter fall, trenched plot data, and eddy covariance measurements.

  7. Cross-Resistance between Triclosan and Antibiotics in Pseudomonas aeruginosa Is Mediated by Multidrug Efflux Pumps: Exposure of a Susceptible Mutant Strain to Triclosan Selects nfxB Mutants Overexpressing MexCD-OprJ

    PubMed Central

    Chuanchuen, Rungtip; Beinlich, Kerry; Hoang, Tung T.; Becher, Anna; Karkhoff-Schweizer, RoxAnn R.; Schweizer, Herbert P.

    2001-01-01

    Triclosan is an antiseptic frequently added to items as diverse as soaps, lotions, toothpaste, and many commonly used household fabrics and plastics. Although wild-type Pseudomonas aeruginosa expresses the triclosan target enoyl-acyl carrier protein reductase, it is triclosan resistant due to expression of the MexAB-OprM efflux system. Exposure of a susceptible Δ(mexAB-oprM) strain to triclosan selected multidrug-resistant bacteria at high frequencies. These bacteria hyperexpressed the MexCD-OprJ efflux system due to mutations in its regulatory gene, nfxB. The MICs of several drugs for these mutants were increased up to 500-fold, including the MIC of ciprofloxacin, which was increased 94-fold. Whereas the MexEF-OprN efflux system also participated in triclosan efflux, this antimicrobial was not a substrate for MexXY-OprM. PMID:11158736

  8. Cross-resistance between triclosan and antibiotics in Pseudomonas aeruginosa is mediated by multidrug efflux pumps: exposure of a susceptible mutant strain to triclosan selects nfxB mutants overexpressing MexCD-OprJ.

    PubMed

    Chuanchuen, R; Beinlich, K; Hoang, T T; Becher, A; Karkhoff-Schweizer, R R; Schweizer, H P

    2001-02-01

    Triclosan is an antiseptic frequently added to items as diverse as soaps, lotions, toothpaste, and many commonly used household fabrics and plastics. Although wild-type Pseudomonas aeruginosa expresses the triclosan target enoyl-acyl carrier protein reductase, it is triclosan resistant due to expression of the MexAB-OprM efflux system. Exposure of a susceptible Delta(mexAB-oprM) strain to triclosan selected multidrug-resistant bacteria at high frequencies. These bacteria hyperexpressed the MexCD-OprJ efflux system due to mutations in its regulatory gene, nfxB. The MICs of several drugs for these mutants were increased up to 500-fold, including the MIC of ciprofloxacin, which was increased 94-fold. Whereas the MexEF-OprN efflux system also participated in triclosan efflux, this antimicrobial was not a substrate for MexXY-OprM. PMID:11158736

  9. The effect of chronic and acute ethanol treatment on morphology, lipid peroxidation, enzyme activities and Na+ transport systems on WRL-68 cells.

    PubMed

    Gutiérrez-Ruiz, M C; Bucio, L; Souza, V; Cárabez, A

    1995-04-01

    In this study we measured some parameters that are associated with ethanol damage to the liver. The method allowed us to determine the injury that chronic and acute ethanol treatments produce at the cellular level without interference from homeostatic or compensatory mechanisms. The system used is a hepatic fetal human cell line, WRL-68, which retains, in culture, many of the liver-specific functions. WRL-68 cells do not metabolise ethanol, and consequently we could evaluate the effect of ethanol alone. We explored two different conditions: 30 days with 0.1 M ethanol (chronic treatment) and 24 h in the presence of 0.5 M ethanol (acute treatment). 1. The transmission electron microscopy studies revealed, in both treatments, the presence of granules not usually present in the cytoplasm of control cells and morphological mitochondrial alterations in chronically treated cells. 2. Lipid peroxidation, measured as the rate of malondialdehyde production, increased three a